IDENTIFICATION OF A VIRULENCE FUNCTION IN HERPES SIMPLEX VIRUS-
TYPE I MAPPING TO THE LATENCY ASSOCIATED TRANSCRIPT LOCUS
LEE WILLIAM GARY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
Lee William Gary
I would like to thank my parents, Bill and Barbara, for their endless support and
encouragement. I also appreciate the support of Jia Liu, Karen Johnstone, Jason Liem,
Chuck Peek and many other friends at the University of Florida. My advisor, David
Bloom, made this work possible and I greatly appreciate his expertise and patience.
TABLE OF CONTENTS
LIST OF TABLES .................. ....................... ................................vi
LIST OF FIGURES.......................................................................... vii
ABSTRACT......................................... ...... .......................................viii
1 INTRODUCTION......... ...................................... ...... ....... .... 1
General Characteristics of HSV-1........................... ..................... 1
Viral Infections in Human Host................................................... 3
Animal Models ................................ ...................... ........... 5
LAT Mutants and Phenotypes..................................... ............. 8
Establishment ......................................... ........................ 9
Reactivation............ ... ..................................................... 10
Other Regulatory Regions of LAT........... ... ............. .................. 12
Neuronal Survival and Apoptosis.................................................. 13
2 METHODS............ .................................................................21
LAT Recombinant Viruses ......................... ....... ...................... 26
17A480R.......... ...................................................... 26
17APstA480....................................................... ....... 27
Analyses In Vivo and Cell Culture................................................29
Virulence Determination in Mouse Model............................. 29
Tissue Restriction......................................................... 30
Time Course Experiments................................................. 30
3 R E SU LT S ................................................. .......................... 35
LDso/pfu Evaluation................................................................ 36
Tissue Restriction................................................................. 38
Cell Specific Replication Deficiency.......................................... 39
Additional LAT Recombinant Viruses........................................... 41
Virulence Phenotype and Nervous System Restriction..................... 57
Cell Culture Phenotype................................................................58
LAT Mutants and Multiple Regulatory Functions............................59
Lytic Promoter in the LAT Region ................................... 62
Transcriptional Permissivity.............................................. 63
ICPO Enhancer................. ....................................... 64
LIST OF REFERENCES..................................................................66
BIOGRAPHICAL SKETCH............................................................. 79
LIST OF TABLES
1-1. Analysis of the 5' LAT deletions on reactivation in the rabbit eye model........ 19
1-2. The ratio of HSV to actin DNA in rabbit trigeminal ganglia latently infected with
17A307 and 17A307R mutants of HSV-1......................................... 20
3-1. LD 50 values............................................................................... 44
LIST OF FIGURES
1-1. Schematic of the LAT gene and the position of 17APst, 17A307, 17A480 and
17A489 recombinant viruses.......................................................... 17
1-2. Large LAT deletions used for virulence indications...............................18
2-1. 17A480R construction.................................................... ......... 32
2-2. Dot blot hybridization of second round plaque purification of 17A480R........ 33
2-3. Dot blot hybridization of second round plaque purification of 17APstA480......34
3-1. Reduced corneal pathology of 17A307 in the rabbit eye model........ ....... 43
3-2. Infectious virus recovered from mouse feet at 2 days post-infection.............. 45
3-3. Virus production in dorsal root ganglia at 3 and 5 days post-infection ..........46
3-4. Virus production in spinal cord at 5 and 7 days post-infection..................... 47
3-5. Virus production in brain at 7 days post-infection....................................48
3-6. 17A307 and 17A480 multi-step time course in PC-12 cells...................... 49
3-7. 17A480R multi-step time course in PC-12 cells......................................50
3-8. Multi-step time course in rabbit skin cells............................................51
3-9. Single step time course in PC-12 cells................................................52
4-1. Separate genetic regions of the LAT locus............................................65
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IDENTIFICATION OF A VIRULENCE FUNCTION IN HERPES SIMPLEX VIRUS-
TYPE I MAPPING TO THE LATENCY ASSOCIATED TRANSCRIPT LOCUS
Lee William Gary
Chair: David C. Bloom
Major Department: Molecular Genetics and Microbiology
Two Herpes Simplex Virus type 1 (HSV-1) strain 17syn+ recombinants (17A307
and 17A480) possessing contiguous deletions spanning the 3' region of the Latency
Associated Transcript (LAT) exon 1 and the 5' portion of the LAT intron were analysed
for virulence in mouse footpad (f.p.) model. While the 17syn+ (parent) and 17APst (LAT
promoter mutant) yielded LD5o/pfu values of 3.25 x 102 and 5.11 x 102 respectively,
17A307 and 17A480 had LDso/pfu values of>7.0 x 104. A second independent
recombinant and a rescue (17A307a and 17A307aR) were constructed and yielded f.p.
LD50 values of>7.0 x 104 and 3.17 x 102 respectively, confirming that deletions within
this region of the LAT locus resulted in a dramatic reduction in virulence. Replication of
the recombinants was indistinguishable from wild-type 17syn+ and rescue viruses when
tested on rabbit skin cells but demonstrate 10-fold reduction in viral yields on PC-12 cells
as measured by multi-step growth curves. Significantly, the attenuation in virulence
displayed by 17A307 and 17A480 is not observed for the LAT promoter mutant, 17APst,,
suggesting that the phenotype is independent of the LAT latent promoter (LAP1). In
addition, 17A480 possesses a wild-type reactivation in the rabbit eye model suggesting
that the virulence and reactivation phenotypes are genetically separable.
General Characteristics of HSV-1
Herpes Simplex Virus-type I (HSV-1) is a widely prevalent human pathogen that is
nuclear replicating, enveloped particle within an icosahedral capsid containing an
amorphous tegument (Roizman et al., 1974). The genome of HSV-1 is a double stranded
DNA molecule of 152 kb, encoding more than 70 protein products (Becker et al., 1968;
Kieffet al., 1971; McGeoch et al., 1985; McGeoch et al., 1988). Regions of the genome
are rich in repetitive sequence, whereas other areas contain mostly unique sequence. The
HSV-1 genome also has a high GC content in comparison to other viruses (Becker et al.,
1968; Kieffet al., 1971; McGeoch et al., 1988). HSV-1 is an alphaherpesvirus and as
such is neurotropic with rapid replication capabilities and exhibits a broad host and cell
range (Jamieson et al., 1974). While HSV-1 is able to infect a wide spectrum of different
mammalian cell types, the ability to replicate and establish a latent infection within
neurons is central to HSV-1's biology (Dotti et al., 1987; Dotti et al., 1990). Because
most neurons divide infrequently and consequently rarely express proteins necessary for
DNA replication, HSV-1 encodes specialized functions for existing within the highly
differentiated cells (Caradonna et al., 1981; Honess et al., 1974; Honess et al., 1975;
Huszer et al., 1981; Preston et al., 1984).
Attachment and entry of infectious viral particles involve interaction between
several cellular membrane glycoproteins and receptors (Baines et al., 1991; Cai et al.,
1988; Forrester et al., 1992; Javier et al., 1988; Johnson et al., 1986; Longnecker et al.,
1987; Longnecker et al., 1987; Roop et al., 1993). Once viral particles have entered the
cell, the nucleocapsid is transported to the nucleus to deposit viral DNA (Batterson et al.,
1983; Tognan et al., 1981). Along with the viral genome is the VP16 protein, which
functions in enhancing viral immediate-early gene transcription (Campbell et al., 1984;
Heine et al., 1974; Spear et al., 1972). HSV-1 encodes five immediate-early genes that
are expressed prior to viral protein production: ICPO, ICP4, ICP22, ICP27 and ICP47
(Mackem et al., 1982; Mackem et al., 1982; Post et al., 1981). The ICPO and ICP4
proteins initiate and regulate viral transcription (Everett et al., 1984; Everett et al., 1984;
Everett et al., 1987; Gelman et al., 1985; Gelman et al., 1986; Gelman et al., 1987;
O'Hare et al., 1985). ICP27 is also active in gene expression but acts at the level of RNA
transport from the nucleus into the cytoplasm (Sandri-Goldman et al., 1994). In order for
HSV-1 to replicate in neurons, which are non-dividing cells, the virus encodes many of
its own enzymes for DNA replication (Caradonna et al., 1981; Honess et al., 1974;
Honess et al., 1975; Huszer et al., 1981; Preston et al., 1984). These products are
generally included among the early genes of HSV-1.
Seven early genes are required for viral replication in all cell types and conditions:
DNA polymerase, DNA binding proteins, ORI binding protein and the helicase/primer
trimer (Chartrand et al., 1980; Coen et al., 1984; Hay et al., 1976; Honess et al., 1984).
A few other key early genes that include thymidine kinase, ribonucleotide reductase and
dUTPase are encoded and allow for replication in the neuronal environment (Caradonna
et al., 1981; Honess et al., 1974; Honess et al., 1975; Huszer et al., 1981; Preston et al.,
1984). Late genes begin to be expressed during DNA replication and are divided into 2
subclasses: leaky-late and strict late (Costa et al., 1985; Dennis et al., 1984; Everett et al.,
1984; Guzowski et al., 1993). The leaky-late gene products can be transcribed prior to
the conclusion of replication but strict late genes are only transcribed after replication has
completed. Regardless of these differences, late genes are mainly the structural products
of HSV-1 and include glycoproteins and capsid proteins.
Viral Infections in the Human Host
The majority of the U.S. and World population is infected with HSV-1. In most
cases, the virus does not induce a severe acute infection, but for a small percentage, it can
cause blindness or encephalitic death. Individuals infected with HSV-1 harbor a lifelong
latent infection in sensory and autonomic neurons, with occasional recurrences of an
acute infection at the site of initial infection. Latent infection of neurons results in the
replicative silencing of the HSV-1 genome. For the establishment of latency, after entry
of virus, a dramatic repression of gene expression must occur. Transcription of most
HSV-1 genes must be silenced, or restricted to very low levels with few transcripts being
expressed or detectable. During the maintenance stage of latency, the genome is present
as an episome within the nucleus of the cell (Mellerick et al., 1987; Rock et al., 1983;
Rock et al., 1985). Maintenance of HSV-1 latency involves the potential for latent
genomes to reactivate and produce infectious virus upon appropriate stimulus.
Reactivation from latency is believed to be a rare event and results in the reappearance of
infectious virus from sensory ganglia that travels back to the initial site of infection in the
epithelia. Clinical reactivation results in a lesion with detectable infectious virus.
Reactivation also has been determined to occur subclinically, with HSV-1 genomes
detected by PCR in the epithelia. It is unclear whether reactivation is lytic to neurons, but
the multiple occurrences of reactivation in the human population makes neuronal
destruction upon reactivation unlikely (Corey et al., 1996). It is more plausible that
reactivating neurons produce very low levels of infectious virus with high levels of
replication occurring in other cell types of the nervous system, or exclusively in the
epithelia. Lesions in the periphery form at a rate and with cytopathology that coincides
with rapid virus replication in cell culture (Stevens et al., 1975; Whitley et al., 1993).
In a suitable host, the immune system is an important regulator of HSV-1 severity.
During an HSV-1 infection in peripheral tissue, CD4+ cells function to activate and
recruit macrophages (Nash et al., 1993). CD4+ cells have also been shown to serve as
cytotoxic lymphocytes (CTLs) in culture. Important immunological players in the
nervous system are significantly different than other infected regions of the host during a
HSV-1 infection. CD8+ cells and IgG function to limit viral spread and severity of
infection (Kapoor et al., 1982; Simmons et al., 1987). It is important to note that infected
neurons are not targeted by CTLs and that their action in the nervous tissue appears to be
limited to non-neuronal cells. Although the involvement of the immune system functions
to reduce the severity of acute infection, it has been difficult to determine how the stages
of latency are affected by immune regulators. In many ways, sensory neurons are
immune privileged cells. With regard to regulation of the latent infection, most attention
has been directed away from the immune system and toward viral gene regulation and
In a latent infection of neurons, the latency-associated transcript is the only viral
RNA produced at readily detectable levels (Figure 1-1). The latency-associated transcript
(LAT) gene is positioned in the long repeat regions of the viral genome and is thus
diploid. During latency, two poly(A)- LAT products can be observed and have molecular
sizes of 2.0 and 1.5 kb (Spivack et al., 1998a; Spivack et al., 1998b; Wagner et al., 1988;
Wechsler et al., 1988). These products have been shown to be nuclear-localized, stable
introns (Farrell et al., 1994; Zabolotny et al., 1997) and are spliced from an 8.3 kb
primary transcript (Farrell et al., 1991). Stability of the introns is hypothesized to be due
to nonconsensus A or G branch point sequence and inhibits debranching of the lariat in
vivo (Rodahl et al., 1997; Tahl-Singer et al., 1997; Zabolotny et al., 1997). The 1.5 kb
intron is only observed in neurons, and is believed to be an alternative splice product of
the 2 kb intron (Zabolotny et al., 1997). The LAT promoter itself contains several
neuron-specific expression elements (Farrell et al., 1994; Morrow et al., 1994; Zwaagstra
et al., 1991), consistent with its high-level of expression in these cells (Dobson et al.,
1989). Further, in animal models, the LAT promoter demonstrates greater activity in
neurons and is specifically more active in neurons not expressing acute antigens
(Margolis et al., 1992). Since the LAT is the only viral transcript abundantly transcribed
during latency, its role in the establishment, maintenance and reactivation from latency in
the mammalian host has been sought. Because latency can only be established in vivo,
the use of animal models is required.
Several animal models are used in HSV-1 research that mimic the acute and latent
infections occurring in the human population. The mouse footpad model allows the study
of both acute and latent infections. In this model, the epidermis of the footpad is infected
which allows acute replication in this tissue. Infectious virus enters the nervous system
by infiltration of nerve termini located in the epidermis. Virus then travels by fast axonal
transport into dorsal root ganglia (DRG). In the sensory neurons of the DRG the virus
progresses down one of two paths: 1) acute replication and virus production may ensue;
or 2) genomes are silenced and latency is established. With a high viral dose, highly
virulent viral strain, or a virulence sensitive mouse strain, acute infection will spread to
the central nervous system where death of the animal may occur by encephalitis. The
mouse footpad model is especially useful for acute virulence experiments due to efficient
infection of epidermis and progression into the peripheral and central nervous systems. It
allows the study of both neuroinvasion and neurovirulence of HSV-1 strains and mutants
(Izumi et al., 1988; Izumi et al., 1990; Thompson et al., 1986). Neuroinvasion is the
ability of a virus to enter into and travel through the nervous system and is measured by
peripheral inoculation of the virus. Neurovirulence measures the ability of the virus to
replicate within neurons and is assessed by the lethality of viruses and their ability to
The mouse footpad model is also utilized for latent analysis ofHSV-1, since the
virus efficiently establishes a latent infection in DRG. Reactivation can be studied in the
mouse model by the physical removal of DRG and incubation in cell culture media in a
process known as explant cultivation. Infectious virus is induced to emerge from these
sensory neurons and by placement upon a cell monolayer in culture the virus can be
readily detected (explant co-cultivation). Another mouse model for the study of HSV
latency and reactivation is the hyperthermic stress model. This model involves acute
infection of the cornea. After addition of virus on the cornea, HSV-1 then travels via
sensory axons to the trigeminal ganglion (TG) where a latent infection is established in
sensory neurons. Once a latent infection is established, latent virus can be reactivated by
placing the live infected mouse in a hyperthermic water bath set at 43C for 10 minutes.
Approximately 22 hours later, the TG are removed, sectioned and assayed for the
presence of virus by immunohistochemical methods (Sawtell and Thompson 1992). This
approach, and co-cultivation, does not adequately substitute for clinical reactivation since
virus detection in nervous tissue is very different from viral recovery in the periphery that
occurs in true reactivation. The mouse models have been useful in that they have
identified several aspects of the latent infection in vivo: identification of the neuron as
the site of latency, understanding of axonal transport of virus through the sciatic nerve,
the ability of non-replicating viruses to establish latent infections and characterization of
transcripts expressed during the stages of latency (Dobson et al., 1989; Kristensson et al.,
1971; Kwon et al., 1982; McLennan et al., 1980; Stevens et al., 1987; Wagner et al.,
Although the mouse models allow for the analysis of HSV-1 latency, the best
animal model would be one that allows reactivation to occur spontaneously but also
allows induction of reactivation with proper stress stimulus. Also, an ideal model would
result in the production of acute lesions at the periphery with the appearance of detectable
infectious virus. The mouse models require the physical removal of ganglia to detect
infectious virus. Latent infection and reactivation can also be induced in the TG of
rabbits upon corneal infection (Nesburn et al., 1967). From this discovery, the
spontaneous and inducible rabbit eye models were developed and function as superior
models for the study of reactivation since they allow for the detection of virus in the eye
after reactivation from the rabbit TG. The spontaneous model involves the detection of
random reactivation events by the sampling of tear film. The epinephrine-induced rabbit
eye model is one degree closer to the natural infection in humans than the spontaneous
model in that it is stress inducible. In this model, virus can be induced to reactivate by
application of current and epinephrine to the eye (Berman et al., 1985; Hill et al., 1986;
Hill et al., 1987a; Hill et al., 1987b; Kwon et al., 1981; Kwon et al., 1982). Viral
genomes within neurons of the TG reactivate and travel to the initial site of infection, the
eye. This model more closely mirrors the natural human infection in that infectious virus
can be recovered at the tissue initially infected with HSV-1 upon appropriate stress
stimuli. The rabbit models also are more discriminating than the mouse models in which
LAT mutants reactivate. Both the mouse model and the rabbit eye model have made it
possible to better understand the impact of the LAT upon the various stages of latency.
LAT Mutants and Phenotypes
From the use of animal models, the LAT has been shown to affect establishment
and reactivation phenotypes of latency. It has been proposed that the LAT may function
most directly on establishment and subsequently influence reactivation indirectly due to
decreased efficiency of establishment (Sawtell and Thompson 1992; Sawtell 1998;
Sawtell et al., 1998; Thompson and Sawtell 1997). When assayed with promoter reporter
viruses, it was determined that establishment of latent infections and the initiation of a
productive infection occur with the same kinetics (Margolis et al., 1992). This work also
identified four classes of neurons in the ophthalmic division of the trigeminal ganglion
that are susceptible to HSV-1 infection. These neuronal classes include substance P+,
CGRP+, LD2+ and SSEA-3+ neurons with the SSEA-3 (stage-specific embryonic
antigen 3) neurons to be the major class of these four to harbor a latent infection.
Additionally, with a similar reporter construct, the LAT promoter was shown to be most
active in both neuron number and intensity of staining during the acute stage of infection.
In this system, peak expression occurs in mouse trigeminal ganglia at 5-8 days post-
infection in concordance with the end of lytic infection and suggests a role in the
establishment of latency (Thompson and Sawtell 1997).
As proposed by Sawtell and Thompson, the LAT is hypothesized to act upon the
latent HSV-1 infection at the establishment stage of latency. It has been shown that some
LAT mutants exhibit a reduction in the ability to establish a latent infection. In latently
infected neurons, the number of viral genomes has been calculated to exist in the range of
10-100 (Cabrera et al., 1980; Efstathiou et al., 1986; Hill et al., 1996; Rock et al., 1983;
Sawtell et al., 1997), although it is important to point out that the number of latently
infected neurons that express LAT is less than 30% of the total neurons that contain latent
genomes (Ecob-Prince et al., 1995; Ecob-Prince et al., 1993; Gressens et al., 1994; Hill et
al., 1996; Maggioncalda et al., 1996; Mehta et al., 1995). A LAT negative mutant,
KOS/62 derived from the KOS strain of HSV-1, was reported to have a reduced
reactivation phenotype and was attributed to a minor impairment of this mutant to
establish a latent infection. The number of latently infected neurons with the KOS/62
mutant was measurably lower than mice infected with the parental KOS (Sawtell and
Thompson 1992). In the comparison of latent genome numbers in neurons between high
reactivating strains of HSV-1 and low reactivators, a low reactivating strain, KOS, was
present in lower amounts than the strains 17syn+ and McKrae (Sawtell et al., 1998). In
support of the establishment findings, other laboratories have reported slight decreases in
establishment with LAT-negative mutants in murine eye models (Devi-Rao et al., 1994).
Indirect support for the role of LAT in establishment comes from the finding that LAT
expression in mouse DRG occurs at the end of lytic replication in neurons (Sawtell and
Although some LAT mutations do alter the establishment of latency, these effects
are very low in magnitude. Also, most LAT mutants do not demonstrate measurable
establishment phenotypes and are not evident in both the mouse and the rabbit (Bloom et
al., 1994; Bloom et al., 1996; Jarman et al., 2002; Pemg et al., 1996). Specifically, the
establishment differences that have been reported are seen to occur only in the mouse,
and at most reflect 2 4 fold less viral DNA in the ganglia (Thompson and Sawtell
2001). Current understanding cannot rule out the action of the LAT on the establishment
of latency. It is possible that LAT does act in establishment and the lower levels of
establishment that have been observed may be due to an effect of LAT on down
regulating viral mRNA production in neurons (Garber et al., 1997).
The involvement of the LAT in any of the stages of HSV-1 latency has been shown
most conclusively for the reactivation phenotype in the epinephrine-induced rabbit eye
model. Initially, a HSV-1 mutant (X10-13) containing a large deletion encompassing the
LAT promoter and downstream region of the gene displayed a low level of reactivation in
the rabbit eye model (Hill et al., 1990). The reactivation phenotype was restored by use
of an EcoRI J+K subgenomic clone encompassing the LAT locus (Hill et al., 1990). A
smaller, engineered deletion of the core promoter (17APst) of LAT displayed a severe
reduction in reactivation (Bloom et al., 1994) and another group supported this finding
with a similar but independently constructed mutant (Pemg et al., 1994). In addition to
deletions that included the LAT promoter, it was shown that only a limited region of the
LAT primary transcript is required for efficient induced reactivation. Bloom et al.
(Bloom et al., 1996) showed that insertions of polyadenylation signals between the cap
site and the 3' end of LAT resulting in termination 1,500 base pairs from the cap site had
no impact upon reactivation (Bloom et al., 1996) and accumulation of the 2.0 kb LAT
intron has also been shown to not be required for reactivation (Jarman et al., 2002). The
17A348 mutant which contains a mutation engineered into the 5' region of exon 1 did
exhibit severe decreases in reactivation (Bloom et al., 1996). Another mutant, 17ASty,
partially overlaps the 17A348 mutant and is also reactivation impaired (Hill et al., 1996).
These mutations in exon 1 do not affect the transcriptional activity of the LAT promoter
and do not reduce the expression of the LAT introns. From these findings in the
epinephrine-induced rabbit eye model, the reactivation critical region (RCR) of LAT has
been mapped to the 5'region of exon 1 of the LAT gene.
Mutations in 5'region of exon 1 result in a dramatic reduction in the frequency of
induced, and spontaneous virus shedding in the tear film of infected rabbits. LAT null
mutants have also been shown to reactivate at reduced frequency in TG of mice (Sawtell
and Thompson 1992; Thompson and Sawtell 1997). With regard to mechanism, one
hypothesis is that the LAT gene may function directly to promote viral reactivation. LAT
has been shown to be actively transcribed in neurons during the latent state, which would
suggest a role in maintenance or reactivation. Examination of the sequence of LAT exon
1 found a surprisingly large occurrence of CpG motifs that suggested that LAT gene
transcription could be controlled by methylation (Bloom et al., 1996). It was shown,
however, that expression of the LAT gene is not influenced by a methylation mechanism
(Kubat et al., 2004). It is possible that LAT+ cells may only represent a small fraction of
latent neurons and could play a repressive role on reactivation. Recent data from our lab
suggest that the LAT is actively down-regulated 0.5-4.0 hours post-explant co-cultivation
and this may be due in part to a change in the acetylation profile of the region
(O'Neil/Kubat, in preparation). One could speculate that active LAT expression is not
compatible with reactivation, and its downregulation may in turn stimulate, or allow the
downstream, opposite strand regulatory protein, ICPO to be transcribed and initiate a lytic
cascade. Clearly, the LAT region has important regulatory implications for the regulation
of lytic and latent infections, and these will be discussed in further detail in the following
Other Regulatory Regions of LAT
The promoter responsible for expression of the 8.3 kb primary LAT transcript has
been shown to possess multiple control elements including cyclic AMP response
elements (CRE), CAT box, Spl, USF, YY1 and AP-2 sites (Kenny et al., 1994; Soares et
al., 1996; Wechsler et al., 1988; Wechsler et al., 1989; Zwaagstra et al., 1991). This
latent promoter has enhanced promoter activity in cells of neuronal origin (Zwaagstra et
al., 1990). It has been suggested that the LAT gene may encode an alternative, or
cryptic, downstream promoter element separate from the latent promoter that may be
active during acute viral replication (Nicosia et al., 1994; Goins et al., 1994). This
alternative promoter has been indicated by transient transfection assays and roughly
localizes to a region 5' of the 2.0 LAT intron splice donor site. The presence of a
neuronal enhancer approximately 60 base pairs downstream of the transcription start site
has been identified (Soares et al., 1996). This promoter has been incorrectly labeled as a
second latent promoter. It is clear that the promoter is active during the lytic infection
and not during latency (Soares et al., 1996). This region may play a very important role
in the acute phase of HSV-1 infection and the characterization of this downstream region
of the LAT gene may help to explain early events in latency or the reactivation from a
Neuronal Survival and Apoptosis
The region responsible for the reactivation phenotype of LAT has been well
mapped but the LAT gene may contain other regulatory motifs that control establishment
or functions in the acute infection. LAT may also play a role in neuronal survival and
apoptosis (for review see Bloom 2004). Portions of the LAT gene have been shown to
inhibit neuronal cell death and may function through suppression of receptor-mediated
(caspase-8) and mitochondrial-mediated (caspase-9) pathways. Results from in vivo
studies demonstrate that a large deletion of the promoter extending into the LAT intron
are reduced in establishment levels and cause a significant loss of sensory neurons (Perng
et al., 2000; Thompson and Sawtell 1997). Transient transfection of IMR-90 and CV-1
cells with a plasmid containing the 3' region of the LAT exon and most of the intron was
able to protect the majority of cells from ceramide, or fumonisin Bi-induced apoptosis
(Perng et al., 2000; Thompson et al., 2000). Expression of the bovine herpesvirus 1 LAT
(Perng et al., 2002) or portions of the HSV-1 LAT gene inhibit chemically induced
apoptosis in cell culture (Perng et al., 2002).
The anti-apoptotic effects of large portions of the LAT expressed by transfection
are also cell specific. In culture, the LAT is unable to protect 293, or COS-7 cells from
apoptosis (Inman et al., 2001). It is clear that the LAT does induce apoptosis in certain
cell lines using transient transfection assays with high multiplicities of infection and high
expression levels (Perng et al., 2000). These results indicate that the LAT may play a
role in apoptosis but it has not been conclusively shown to occur in vivo. It was reported
that LAT(-) viruses induce extensive apoptosis in rabbit TG (Perng et al., 2000) and that
the LAT gene promotes neuronal survival by reducing apoptosis. The apopotosis results
have been questioned due to incompatible reagents used in the p85 PARP and TUNEL
assays used to suggest an in vivo apoptotic role for the LAT (Thompson and Sawtell
2000). Another large LAT deletion mutant (17-AH) when used in the infection of the
mouse eye model showed a depletion of over half the TG neurons. Apoptosis in HSV-1
infected ganglia during lytic infection has been demonstrated. The 17N/H mutant
overlaps the impairment engineered in the 17-AH mutant but extends further into the
intron. Use of this mutant resulted in the loss of neurons as measured by an increase in
TUNEL-positive TG neurons (Ahmed et al., 2002), though the total numbers of TUNEL-
positive cells were low.
The LAT has been proposed to promote neuronal survival and consequently leads
to high levels of establishment (Thompson and Sawtell 2001). If this is true, then LAT(-)
viruses would be expected to demonstrate a reduced level of replication in sensory
ganglia and would have altered virulence phenotypes in the mouse. A possible reason
that this has not been observed is due to the impairment of several regulatory regions in
the construction of most of the relatively large LAT deletion mutants reported in the
literature. The LAT has been shown to influence reactivation, establishment, apoptosis in
cell culture and may affect neuronal survival. The reactivation phenotypes associated
with the LAT have been well mapped but the positions of other regulatory regions in the
LAT region are still uncertain. Mutants that have been used to show anti-apoptotic
include very large deletions that also affect the region of LAT known to function in
reactivation. To identify and characterize the motifs of LAT involved in the lytic
infection, it is necessary to construct discrete mutations beyond the reactivation critical
The 17A307, 17A307R, 17A480 and 17A489 mutants (Figure 1-1) were generated
in the lab of Dr. Ed Wagner (U. C. Irvine). Characterization of these viruses with respect
to latency was performed by Dr. Jim Hill (L.S.U. Eye Center) by use of the epinephrine-
induced rabbit eye model. The 17A307, 17A307R, 17A480 and 17A489 mutants were
tested for reactivation differences in comparion to 17syn+ (parental) and 17APst
(promoter mutant). From these experiments, it was determined that 17A307 was reduced
in its ability to reactivate in the rabbit model in comparison to its rescue, 17A307R, and
17syn+. Mutants 17A480 and 17A489 did not display a reactivation restriction. All
results are shown in Table 1-1. Establishment was also determined by quantitative PCR
in rabbit TG with no significant difference detected between 17A307 and 17A307R (see
Based on these data we hypothesized that a downstream portion of LAT outside of
the reactivation critical region influences the acute infection. We demonstrated here that
mutation of this region of LAT results in a decrease in virulence in the mouse model.
Additionally, we believe that the HSV-1 recombinants with the large LAT deletions used
by Sawtell and Thompson as well as Wechsler (Figure 1-2) mask dramatic virulence
effects due to the impairment of multiple regulatory elements of the LAT locus. To
examine the possibility that LAT influences acute infection and/or early times in the
latent infection, we generated an additional series of mutants in a region of LAT 3' of the
reactivation critical region. These mutants allowed us to test the involvement of the 3'
end ofexon 1 and the intron upon latent and acute phenotypes of HSV-1. From the
secondary promoter and apoptosis/neuronal protection findings described above, regions
of the LAT gene neighboring the reactivation critical region may act in the acute
infection ofHSV-1. This action may be through a trans-mediated effect from lytic
transcription, or it may be mediated in cis as an enhancer function. An enhancer scenario
is quite possible, since the reactivation function of the LAT gene is hypothesized to
operate in a similar manner (Wagner and Bloom 1997). The small, downstream deletions
of viruses 17A307, 17A307R, 17A480, 17A480R and a double mutant 17APstA480 allow
the determination of virulence properties of the LAT locus without also impairing other
defined regulatory functions of LAT that may mask a full virulence effect. The results
presented in the following chapters put forth a case for two distinct genetic elements
within the LAT gene: one (controlled by the LAP1 or latent promoter) that seems to act
as a facilitator of reactivation; and a second that may encompass the LAP2 promoter that
exerts its influence on virulence during the acute phase of the infection. The data
presented here demonstrating the dramatic reduction in virulence in the mouse following
deletion of a region spanning the 3' end of the 5'exon and the first several hundred bases
of the region encoding the LAT intron is the first description of a major virulence
phenotype mapping to the LAT gene. Our results also indicate that this phenotype is
independent of the LAT promoter (LAP1). Finally, as discussed in detail in the last
chapter, we hypothesize that the deletion of these two distinct genetic elements in vivo
results in a phenotype distinct from deletion of either element individually, suggesting
these elements work together in regulating some aspect of acute replication.
RL UL RL Rs Us Rs
--..-------------- 8.3 kb primary LAT transcript
---------------- -- ---_-------..--
exon 1 ------
Figure 1-1. Schematic of the LAT gene and the position of 17APst, 17A307, 17A480 and
17A489 recombinant viruses. The location of the LAT primary transcript is
shown along with its location in the HSV-1 genome. The promoter, exoni
and the 5' intron of LAT are depicted at the bottom of the figure along with
recombinant LAT viruses.
RL UL RL Rs Us Rs
S8.3 kb primary transcript
dLAT2903 (Pemg 2000)
17A-H (Thompson 2001) Region of Mutation
Region of Mutation |
Figure 1-2. Large LAT deletions used for virulence indications. An impact of the LAT
locus upon virulence has been implied by use of a couple of LAT deletion viruses. These
deletion viruses delete a large portion of the LAT locus and possibly remove several
regulatory regions in the LAT gene.
Table 1-1. Analysis of the 5' LAT deletions on reactivation in the rabbit eye modela
Positive Positive Total positive
Recombinant rabbits /total eyes/total eyes swabs/total swabs
17syn+ 5/5 (100%) 8/9 (89%) 24/72 (33%)
17APst 2/8 (25%) 2/16 (13%) 5/105 (5%)
17A307b 2/8(25%) 3/16 (19%) 6/112 (5.4%)
17A307R(Rescue) 4/4 (100%) 5/8 (63%) 14/64 (22%)
17A480 8/8 (100%) 12/16 (75%) 27/70 (39%)
17A489 4/5 (80%) 6/10 (60%) 29/70(41%)
aReactivation was measured by the level of infectious virus by plaque assay detected
from tear film of infected rabbits. In this model, the rabbit eye is infected with HSV-1
with the virus traveling to the TG and establishing a latent infection. Reactivation is
stimulated by the administration of epinephrine and current to the rabbit eye. Infectious
virus is then detectable at the initial site of infection, the eye.
bIontophoresis was performed once a day for three consecutive days. Eye swabs were
performed for 7 consecutive days.
c17A307 is significantly different from 17syn+.
Table 1-2. The ratio of HSV to actin DNA in rabbit trigeminal ganglia
latently infected with 17A307 and 17A307R mutants of HSV-la
Rabbit infected with Ratio HSV to Rabbit infected with Ratio HSV
17A307 Tattoo(side) Actin DNA 17A307R Tattoo(side) to Actin
G2(od) 0.57 G23(od) 0.51
G2(os) 0.25 G23(os) 0.34
G3(od) 0.83 G24(od) 0.37
G3(os) 0.42 G24(os) 0.64
G4(od) 0.5 G28(od) 0.19
G4(os) 0.31 G28(os) 0.522
G6(od) 0.5 G32(od) 0.46
G6(os) 0.41 G32(os) 0.59
G8(od) 0.5 G34(od) 0.38
G8(os) 0.42 G34(os) 0.63
G9(od) 0.52 G35(od) 0.29
G9(os) 0.58 G35(os) 0.44
Average ratio = 0.480.14 Average ratio = 0.450.14
aThe 17A307 LAT recombinant virus was tested by semiquantitative PCR from
differences in establishment in comparison to its rescue, 17A307R. Values were plotted
for the HSV-1 gene DNA polymerase as a ratio to cellular rabbit actin DNA. From
measurement of the ratios between 17A307 and 17A307R, no detectable differences in
establishment were observed.
Technical details of the experiments used to generate the data presented in this
work are discussed in this chapter. The experiments were aimed at the generation of viral
recombinants and use of these mutants for characterization pertaining to virulence and
latency of HSV-1 in cell culture and animal models. This characterization involved
assessing virulence in the mouse model along with supportive replication data obtained
from cell culture assays. The examination of reactivation from and establishment of
latency were carried out at the LSU Eye Center by Drs. Jim Hill and Jeannette Loutsch,
and these results are summarized in chapter 1 (Table 1-1 and 1-2). In the determination
of virulence, the methods of creating viral recombinants by transfection, growth of
mutants in culture and infection and viral quantification from the animal models are
presented here in full.
Viral recombinants were constructed by use of homologous recombination in cell
culture. Recombinants were produced by either co-transfection of HSV-1 virion DNA
with a PCR fragment for recombining into full length genomic DNA (i.e. 17A480R), or
by co-infection with two HSV-1 LAT mutants (i.e. 17APstA480). The full length viral
genomic DNA was prepared in cell culture. A group of 8-10 T-150 flasks were infected
with the HSV-1 parental, or mutant strain of choice, at a multiplicity of infection (MOI)
of 0.01. Infections were maintained and monitored for 3 days until the cells were
completely detached. At this point, the suspension was removed and pelleted in a high
speed centrifuge at 16,000 x g for 40 minutes at 4C. The supernatant was removed and
the pellet was resuspended in 10 mL ofhypotonic lysis buffer (10 mM Tris pH 8.0, 10
mM EDTA, 0.5% NP-40 and 0.25% deoxycholic acid). This subsequent suspension was
vortexed, incubated briefly on ice and centrifuged at 3000 x g for 10 minutes at 40C to
pellet the nuclei from the fraction containing the viral DNA. After centrifugation, the
supernatant was removed and treated with a protein degradation solution (1% sodium
dodecyl sulfate and 1 mg/mL Proteinase K) at 500C for 1 hour. An additional 1 mg of
Proteinase K was added and the incubation was repeated an addition hour at 37"C to
further digest the protein in the sample. After this treatment, the solution was extracted
with sequentially with equal volumes of phenol, phenol/chloroform and chloroform. To
precipitate the full length viral DNA, the solution was treated with a 0.1 volume of 3M
sodium acetate and carefully layered with 25 mL of cold 95% ethanol. The DNA was
precipitated at the aqueous/ethanol mixing interface with moderate rotation by hand.
Once an appropriate amount of genomic DNA was visible, it was removed by spooling,
washed briefly with 70% ethanol, dried, resuspended in 100-500 pl of sterile water. The
DNA was quantified spectrophotometrically and the genomic quality of the DNA was
assessed by electrophoresis on an agarose gel. Full length viral DNA was then ready for
use in transfections to produce recombinant HSV-1 mutants.
Transfections utilized varying ratios of genomic DNA with a fixed amount of
plasmid, or PCR generated DNA where applicable. For this procedure, 60 mm2 dishes of
rabbit skins cells at 50% confluency were serum starved (1.5% calf serum, MEM)
overnight at 31.5"C in 5% CO2. The viral genomic DNA was used in a range of of 1, 2,
4, 8, 16 and 32 jg in addition to a 2 |jg amount ofrecombining DNA per dish. Each
transfection mix was prepared in a final volume of 225 jil of TNE buffer (10 mM Tris pH
7.4, 1 mM EDTA and 0.1 M NaCI). Upon dilution of the DNA into TNE, 25 ptl of CaCl2
was added per mix. DNA was precipitated by the administration of 250 pl of 2X HEPES
along with CO2 for pH adjustment. This DNA solution was incubated at room
temperature for 30 minutes. The transfection mix was then added to the dishes and
incubated at 25"C for 20-30 minutes. Media in a 5 mL volume was applied to the dishes
and transferred to 37C for 4 hours. The media was then aspirated and the cells were
hypotonically shocked with 1-2 mL of shock buffer (1X HEPES, 20% dextrose) for 60
seconds. The cells were briefly washed and 5 mL of media was added to the dishes.
Once these transfections were performed, viral infection was allowed to persist to full
cytopathic effect, usually 2-3 days post-infection. After the monolayer was depleted and
detached due to viral infection, any remaining cells were scraped and the entire solution
was vortexed and stored at -80C. This viral stock was frozen (-80C) and rapidly
thawed (37C) twice before isolating individual plaques.
Viruses produced from the transfections were detected and isolated by plaque
purification. Confluent 60 mm2 dishes of RS cells were infected with a series of dilutions
ranging from 10-3 to 10"8 in order to obtain dishes with well-isolated plaques. After a 1
hour inoculation, the infected dishes were covered with an overlay consisting of 1X 5%
calf serum MEM and 0.8% agarose. This solid overlay allowed the maintenance of the
monolayer during the infection and functioned to limit spread of virus through the
monolayer, allowing for discrete plaque formation and subsequent plaque isolation.
Infections were maintained and monitored for 2-3 days. Once plaques were visible, the
dishes were stained for 3 hours with neutral red at a 1:30 dilution. The neutral red stain
was absorbed by living cells and consequently left the plaques white. This contrast
allowed for easy visibility of plaques for picking with a Pasteur pipette. The plaques
were picked and each individual plaque was transferred to its own well of a 96 well plate
containing 200 pl of media per well. This plate was then frozen and thawed twice (-
800C/370C) and a 50 p volume was used per well to infect a 96 well plate that was
previously seeded with RS cells. Infection of this plate was allowed to proceed for 2-3
days (until 100% CPE) and was then frozen at -80C.
The recombinant viruses were screened by dot blot hybridization after each round
of plaque purification to determine the proper mutation has been engineered. In this
approach each 96 well plate that was infected with individual isolates per well was
thawed at 370C prior to the transfer for hybridization. On the bottom piece of the dot blot
apparatus, 3 layers of Whatman paper were applied followed by a section of Hybond
nitrocellulose membrane. The Whatman paper and the membrane were saturated with
solution C [(2X SSPE (0.36M sodium chloride, 0.02M sodium monophosphate, 0.002M
ethylene diaminotetraacetate)]. The dot blot apparatus was then sealed tightly and a
vacuum was applied. At this point, 50 pl of inoculum from each well of the 96 well plate
was transferred through the holes in the top of the dot blot apparatus and onto the
membrane. Each well in the dot blot manifold was subsequently washed with 200 pl of
fresh solution A (1.5M sodium chloride, 0.1N sodium hydroxide) followed by an equal
application of solution B (0.2M Tris, pH 7.5) and solution C. The nitrocellulose
membrane was then removed and baked in a vacuum oven for 1-2 hours at 80C. The
DNA was then covalently attached to the membrane and ready for labeling with
radioactive 32P [a-dCTP] probe.
Probes were created using the portion of DNA that identified the presence of the
proper mutation in the recombinant virus. To make the probe, 25 ng of DNA was diluted
to a total of 45 1l in TE (10mM Tris ImM EDTA, pH 8.0) and boiled in a water bath for
5 minutes. The DNA was then quickly transferred to ice and incubated for 5 minutes.
After this period, the DNA solution was added to a reaction tube from the RediprimeTM II
Random Prime Labelling System (Amersham Biosciences). A 50 LCi amount of32P [a-
dCTP] was also added and the reaction was performed at 37C for 10 minutes. The
reaction was quenched with the introduction of 5 ul of 0.2M EDTA. This probe was then
boiled for 5 minutes, placed on ice for 5 minutes and ready for use. Prior to the addition
of the probe to the hybridization, the blot itself was pre-hybridized at 63 C for 1 hour in a
shaking water bath to reduce nonspecific DNA labeling. After the pre-hybridization step,
the probe was added and the blot hybridized overnight at 630C. The next day, the blot
was washed twice with solution 1 (0.3M sodium chloride, 0.06M Tris-HCl pH 8.0,
0.002M EDTA) at 250C for 5 minutes, twice with solution 2 (0.3M sodium chloride,
0.06M Tris-HCl pH 8.0, 0.002M EDTA, 4% SDS) at 630C for 15 minutes and finally
twice with solution 3 (0.03M sodium chloride, 0.006M Tris-HCI pH 8.0, 0.0002M
EDTA) at 250C for 15 minutes. The blot was then dried briefly and covered with plastic
wrap before being exposed 8-12 hours on a Phosphor screen. Hybridization was then
visualized using a STORM phosphoimager (Molecular Dynamics, Sunnyvale, CA) in
order to identify the presence of the proper recombinant virus. This concludes the
general aspects of transfection and hybridization approaches used. The specific details of
the 17A480R and the 17APstA480 construction are described next.
LAT Recombinant Viruses
The HSV-1 recombinants 17APst, 17A307, 17A307a, 17A307R, 17A480, 17A480R
and 17APstA480 were all engineered by homologous recombination. Plasmids
containing the mutated regions were transfected into rabbit skin cells along with full
length 17syn+ HSV-1 genomic DNA. 17A307, 17A307a, 17A307R, 17A480 and 17A489
were constructed in the laboratory of Dr. Ed Wagner at U.C. Irvine. These recombinants
replaced portions of the LAT gene with stuffer DNA sequence and consequently retained
the size and spacing of the LAT gene. 17A480 and 17A489 contain inserted DNA from
the lacZ gene into the either the PpmuI-SfiI site for 17A480, or the SfiI-HpaI location for
17A489. The mutants 17A307 and 17A307a also contain stuffer sequences. 17A307 has
a polyadenylation sequence from SV-40, whereas the native DNA for 17A307a has been
replaced with a polyadenylation sequence from rabbit P-globin. The mutant 17APst is a
202 base pair clean deletion of DNA between the Pst I sites in the core latency promoter
of the LAT gene. This mutant was created in the Stevens lab at UCLA and has been
described previously (Bloom et al., 1994).
For in vivo and cell culture experiments, it was critical to make two additional LAT
mutants. First, the fact that the phenotypes for the 17A480 were the result of the 480 bp
deletion was confirmed by the construction of a rescue of this virus (17A480R). This
rescue was generated by homologous recombination using a 1.2 kb DNA PCR product.
The PCR reaction was performed with oligonucleotides 17AB1R
(5'GAGATGAACACTCGGGGTTAC 3') and 17AB2R
(5'CGGACTBACCTGGCCTCTGG 3'). These primers begin at positions 119,181 and
120,347, respectively, in the 17syn+ genome and are shown schematically (Figure 2-1).
The amplified DNA was co-transfected with full length 17A480 DNA into RS cells. At
3-4 days post transfection, the cells were lysed by 2 rapid freeze/thaw (-800C/370C)
cycles and plated onto confluent 60 mm2 dishes of RS cells at varying dilutions of 10.3 to
10-8. These dishes were overlayed with a solution containing 0.8% SeaKem Agarose and
1X 5% calf serum MEM for 3 days. At this time, cells were stained with neutral red and
plaques were observed by absence of stain. Approximately 300 well-isolated plaques
were picked and tested by dot blot hybridization for the presence of the restored wild-
type sequence at the PpmuI-SfiI location (Figure 2-2). The HSV-1 viruses 17syn+ and
17A480 were included on the blots to serve as positive and negative controls,
respectively. Several positive plaques were detected after the first round of plaque
purification. Five of these positive samples were diluted and infected onto confluent 60
mm2 dishes ofRS cells. After the third round of purification, all plaques were positive.
To guarantee the purity of the 17A480R plaques, a couple of plaques were carried out for
a fourth round of purification which again yielded complete positives.
Another recombinant virus, 17APstA480, was engineered to complete the analysis
of the genetic basis of the virulence phenotype within the LAT locus in the mouse model.
As the name implies, this mutant contains mutations in both the latent promoter of the
LAT (APst) coupled with a mutation in the 5' portion of the LAT intron (A480). This
mutant was constructed by performing a co-infection of 17APst with 17A480 at an m.o.i.
of 3.0 for each virus in a T-75 flask of confluent rabbit skin cells. Plaque purified
isolates were tested by dot blot hybridization. In each round of plaque purification, each
96 well plate was blotted onto membrane in duplicate. One blot was tested with a probe
for the lacZ DNA present in the 480 region of the 17A480 mutation, and the other blot
was tested for the presence of the LAT promoter region (202 bp PstI fragment) from
wild-type HSV-1 strain 17syn+. If both mutations were correct in the 17APstA480 virus,
then plaque isolates would be positive when hybridized with the lacZ probe and negative
with the Pst region probe (Figure 2-3). Critical controls included in this analysis were the
inclusion of 17A480 and 17APst. In use of the lacZ probe, the 17A480 control produced a
positive signal and the 17APst remained negative. For the use of the Pst probe, the same
was also true, since the 17A480 mutant retained wild-type sequence at the latent promoter
and the 17APst mutant had the 202 bp PstI region removed.
In performing dual probing of 17APstA480 possible plaque isolates, a few viruses
were determined positive for the lacZ fragment and negative for the PstI fragment in
comparison to controls. After the first round of screening, a few properly hybridizing
isolates were selected and subjected to another round of plaque-purification. From the
second round of selection, approximately 30% of isolates produced desired hybridization
signals. Two of these samples were selected and put through another round of
purification. At this third round, 100% of samples were lacZ fragment-positive and PstI
fragment negative. To be certain of purity, two samples were taken into a fourth round of
purification and resulted again in 100% of isolates positive for lacZ and negative for the
PstI fragment. To prove that the PstI fragment-negative control contained adequate viral
DNA to be detected, it was included as a control in the final round of the 17A480R
plaque purification. This sample produced signal intensity equal to the other positive
Analyses In Vivo and Cell Culture
The LAT mutants 17APst, 17A307, 17A307a, 17A307R, 17A480, 17A480R and
17APstA480 were tested for virulence phenotypes in the mouse model. After determining
the phenotypes of these mutants in vivo, they were also characterized using a cell culture
system. The cell culture experiments involved two different cell lines, PC-12 cells and
RS cells. These cell types and their experimental usage is described later in this chapter.
Virulence Determination in Mouse Model
The virulence determination of the engineered LAT mutants was performed by
LD5o/pfu analysis. For these experiments, female 4-6 week old out-bred Swiss Webster
mice were used. Virulence was measured by footpad infection and by direct intracranial
inoculation. For the footpad infection, each footpad of anesthetized mice was injected 4
hours pre-infection with 50 pl 10% (w/v) sodium chloride. At the appropriate time, mice
were then sedated with 0.010-0.020 mL of a cocktail containing xylazine (7.5-11.5
mg/kg), acetamine (2.5-3.75 mg/kg) and ketamine (30-45 mg/kg). The keratinized layer
of the footpad was then removed with mild abrasion using an emery board, and viral
dilutions (in MEM with 5% CS) were applied onto the ventral footpad surface in volumes
of 50 pl. The mice were then placed back into their cages in an inverted manner to allow
virus absorption and monitored until they regained consciousness and became active.
Infected mice were then observed twice daily until hind limb paralysis ensued in which
case they were euthanized, or until they died from encephalitic HSV-1 infection. After
two weeks, mice were scored for lethality and LDso/pfu values were assessed by the Reed
and Muench method (Reed and Muench 1938). Surviving mice were assessed for the
presence of latent virus by PCR.
Mice were also tested for virulence phenotypes by direct intracranial infection with
the appointed LAT mutants. These mice were sedated in the same manner as the footpad
experiments. Infection was performed by direct administration of virus into the left
cerebral hemisphere in volumes of 20 L of inoculum by use of a 1.0 mL syringe and a 27
gauge needle. These mice were also observed until conscious and active and were
checked twice daily for signs of nervous system impairment, or encephalitic death.
The relative ability of the LAT recombinants to replicate in the feet, DRG, spinal
cord and brain was assessed following footpad inoculation. This analysis was performed
as a time course and for each time point, tissue and virus, samples were examined in
replicates of 3. The time points tested included 2, 3, 5 and 7 days post footpad
inoculation. At each of these times, all 4 of the designated tissues were removed and
homogenized in MEM containing 5% calf serum. The homogenates were diluted and
titered on confluent 24 well plates of RS cells in the presence of technical grade human
IgG to prevent viral spread through the monolayer. Plaques were counted at 2-3 days
post-infection and viral levels were calculated for each virus and tissue for the 4 time
Time Course Experiments
Mutants 17APst, 17A307, 17A307a, 17A307R, 17A480 and 17A480R were tested
for infectious virus production in cell culture along with parental virus 17syn+.
Infectious virus levels were assessed in RS and PC-12 cells (rat pheochromocytoma cells)
in both multi-step and single step growth curves. The PC-12 cells are developmentally
linked to neurons and represent a specific cell type of the nervous system (Pizer et al.,
1978; Rubenstein et al., 1983). For the multistep growth curves, cells were infected at a
multiplicity of infection of 0.001 and infections were terminated and quantified by plaque
titration at hours 8, 24, 48, 72 and 96 hours post-infection. Each time point virus-' was
performed in triplicate. The single step growth curves were carried out in a similar
manner but with a higher multiplicity of infection (3.0). The time points were also
different and included 0, 3, 6, 12 and 18 hours post-infection. For this experiment,
samples were also tested in triplicate with titrations being performed on RS cells in the
presence of human IgG. Resulting plaques were counted and infectious virus levels were
The experimental techniques described in this chapter were used to test the HSV-1
LAT mutants 17APst, 17A307, 17A307a, 17A307R, 17A480, 17A480R, RHA-6 and
17APstA480 along with parental control, 17syn+ in the mouse model. These experiments
were aimed at the determination of the role of the 3' portion of LAT exon 1, and to
determine if a phenotype can be attributed to the 5' intron. The next chapter is dedicated
to the report of virulence, tissue restriction and cell specific infectious virus reduction
phenotypes obtained for these mutants.
RL UL RL Rs Us Rs
119181 119502 119981 120347
PstI PstI 17A480 (Ppmu SftI)
....................... I ........................ I '
I I 480 bp
Sty I Sty I 321 bp 366 bp'
Figure 2-1. 17A480R construction. The 17A480R recombinant virus was made by
co-transfection of a PCR amplified portion of the parental virus, 17syn+,
between genomic base pairs 119,181 and 120,347 and genomic 17A480
DNA. The 17A480R recombinant was screened by dot blot
Figure 2-2. Dot blot hybridization of second round plaque purification of 17A480R. This
figure demonstrates that by the second round of plaque purification, most of
the viruses are positive for the region of the LAT gene corresponding to the
region that was altered in the 17A480 recombinant.
Figure 2-3. Dot blot hybridization of second round plaque purification of 17APstA480.
A) The blot at the top of the page was probed with a lacZ probe. B) The
bottom blot was probed with a Pst probe in order to identify 17APstA480
In our pursuit to further understand the function of the LAT locus in HSV
biology, we made recombinant virus 17A307 deleted of a 307 bp fragment in the 3'
region ofexon 1 (Figure 1-1). The native region of HSV-1 LAT exon 1 was removed by
homologous recombination and replaced with DNA stuffer from a SV40 polyadenylation
sequence. This recombinant virus contained alterations 3' of a region that mapped to the
general region that we had evidence was involved in reactivation. To test the impact of
this portion of the LAT on reactivation from latency, we utilized the epinephrine-induced
reactivation rabbit eye model. Results showed that the 17A307 recombinant virus
exhibits a reduced reactivation phenotype (Table 1-1).
Additionally, it was observed that the 17A307 displayed reduced cornea scarring in
comparison to 17syn+ (parental virus) and 17APst (promoter mutant) at early times post
infection (Figure 3-1). This indicated that the 17A307 recombinant was altered in
pathogenesis due to the 307 bp deletion, or due to secondary mutations outside of the
LAT region. To test this second possibility, a second 17A307 virus was engineered
(17A307a). This recombinant is the same as the original 17A307, except that it contained
a stuffer sequence from the rabbit j globin gene (rabbit B-globin poly A region). Along
with the 17A307a recombinant, a corresponding rescue was made, 17A307aR. These
viruses were once again tested in the epinephrine-induced rabbit eye model for
reactivation effects and demonstrated reduced reactivation (Table 1-1). These results
indicated that the 17A307 virus was not only restricted in reactivation, but unlike the
LAT promoter mutant 17APst, had alterations in corneal pathology suggesting that it
might possess attenuated virulence or pathogenesis properties.
The mouse footpad model was used to test the virulence profile of the 17A307
recombinants. The rabbit model is a superior model for reactivation due to its close
parallel to the natural infection as described in chapter 1. For virulence studies, however,
the mouse footpad model is more useful due to the ability to infect larger sample sizes,
while also conserving many characteristics of natural HSV infection. The footpad
infection allows initial infection of the epidermis of the foot. Virus then enters the
peripheral nervous system via nerve termini present in the epidermis. Once in the
nervous system, virions progress to the body of the neurons in the dorsal root ganglia
(drg). From the DRG, HSV can then enter the central nervous system and progress
through the spinal cord to the brain. Infection in the CNS can lead to death resulting
Initially, the 17A307 recombinant viruses were tested for virulence by LDso
(calculation of viral dose in plaque forming units required to kill 50% of animals in a
group) in comparison to 17syn+ (parental), 17APst and 17A307aR. Results in the mouse
showed that the 17A307 viruses did display a dramatic virulence phenotype. It is
important to note that the 17A307 mutation principally alters the 3' end of exon 1 but it
also changes the first 6 base pairs of the splice donor site. Additionally, the stuffer DNA
is a polyadenylation signal in the case of each 17A307 recombinant and the virulence
results could have been due to a RNA mediated effect. To answer these questions, I set
out to create additional 17A307 recombinants that do not mutate the splice donor site, or
any portion of the intron. I also planned to insert the polyadenylation sequences in each
orientation to test for a RNA directional effect. Prior to engineering and testing these
new 17A307 recombinants, we wanted to repeat the LDso experiments with the inclusion
of additional controls.
The LAT recombinants 17A307, 17A307a, 17A307aR, 17A480 and 17A489
(Figure 1-1) were analyzed for virulence phenotypes by use of the mouse footpad model
(Table 3-1). These viruses were used to infect the rear footpad of out-bred Swiss
Webster mice along with control viruses, 17syn+ (parental), 17APst (promoter mutant)
and RHA-6, which contains a polyadenylation signal at a HpaI site in the middle of the
intron. The footpad of out-bred Swiss Webster mice was infected with mutants 17A307,
17A307a, 17A307aR, 17A480, 17A489 and RHA-6 in varying dilutions (see Methods)
with 10 mice virus -1 dilution"'. In comparison to 17syn+ (parental), 17APst (promoter
mutant) and each rescuant virus included in the experiment, mutants 17A307, 17A307a
and 17A480 expressed dramatically increased LDso/pfu levels by mouse footpad
infection. Each of these displayed a greater than 500 fold increase in LDso/pfu levels
(Table 3-1). The virulence phenotype for the 17A480 recombinant made the creation of
the new 17A307 recombinants described above unnecessary. The 17A480 mutation
resides entirely in the intron and retains the splice donor site. This virus also contains a
lacZ DNA stuffer and its effect argues against the orientation of the polyadenylation
sequence having an impact in the 17A307 recombinants. It is important to recognize that
the 17A489 mutant had an equal virulence profile to 17syn+ and 17APst, which identifies
the 3' boundary of the virulence region due to the lack of virulence phenotype for
17A489. Results from the footpad infections indicate that 17A307, 17A307a and 17A480
are impaired in either neuroinvasion, or neurovirulence.
To determine if 17A307 and 17A307a were deficient in neurovirulence, the LAT
mutants were used to infect the mouse by intracranial inoculation along with 17syn+ and
17APst. These results showed that the 17A307 and 17A307a viruses were also restricted
in virulence measured by LDso/pfu upon direct inoculation into the mouse brain (Table 3-
1). Taken together, the footpad and intracranial LDso/pfu values show that mutants
17A307, 17A307a and 17A480 are greatly impaired in virulence in the mouse and that the
results are due to a neurovirulence phenotype and not likely due to a defect in
neuroinvasion or an inability to enter the central nervous system.
To identify whether the 17A307, 17A307a and 17A480 mutants display a general
reduction in replication in the mouse causative of a virulence phenotype, several tissues
along the infection pathway from the foot were tested for production of infectious virus.
Tissues included the foot, DRG, spinal cord and brain with the inclusion of 17syn+,
17APst, 17A307a, 17A307aR, 17A480 viruses as controls. Infectious virus was assayed
for these tissues at 2, 3, 5 and 7 days post infection at an initial dose of 104 pfu virus"-
time-'. Each virus, time point and tissue was examined in triplicate.
Since the footpad is the initial site of infection in our model, it was critical to
determine if these mutants were impaired in virion production in this tissue. Upon
analyzing this tissue for plaque production, a significant difference in viral production
was not seen in the feet at any times tested (Figure 3-2). From previous data in our lab,
we know at the doses employed that 2 days post-infection is the peak of infectious virus
production in the feet (Jarman et al., 1999). The lack of a difference between 17A307
and 17A480 in comparison to 17syn+ (parental), 17APst and 17A307R demonstrates that
the mutants do not display a general replication defect in mouse tissue.
Infection in the footpad allows for the virus to enter the nervous system via nerve
termini in the epidermis. From the feet, virus progresses into the nervous system and
virus can be detected in the DRG. In examining the DRG, the 17A307 mutant does
display a reduction in infectious virus levels evident at 3 and 5 days post-infection (d.p.i.)
(Figure 3-3). Interestingly, 17A480 does not demonstrate a restriction at 3 d.p.i. and only
shows a possible reduction at 5 d.p.i. Both of these mutants, however, are restricted in
infectious virus production in the next tissue in the path of infection, the spinal cord.
Results from spinal cord show that both 17A307 and 17A480 have lower viral values in
comparison to 17syn+ (parental), 17APst, 17A307aR and 17A480R (Figure 3-4). Further
analysis reveals that 17A307 and 17A480 are also lower in abundance in brain tissue
(Figure 3-5). Values shown for these mutants only involve one positive sample for each
of the LAT virulence recombinant viruses, therefore, the brain restriction may be even
greater than depicted in the graph shown. Collectively, the tissue experiments display a
restriction in infectious virus production for 17A307 and 17A480 that is specific for
nervous tissue. These tissue restriction results show that the virulence deficient measured
by LD50 is due to a reduction of 17A307 and 17A480 for infectious virus production in
the nervous system.
Cell Specific Replication Deficiency
The tissue restriction results clearly demonstrate an impairment of infectious virus
production that is specific for nervous tissue. To identify a possible cell specific
phenotype for 17A307 and 17A480, replication of these viruses was tested by time course
in PC-12 cells and in rabbit skin cells. These LAT mutants were tested along with
17syn+, 17APst, 17A307R and 17A480R. The first experiment involved a multi-step time
course performed in PC-12 cells. The multi-step growth curves were performed with a
viral multiplicity of infection (m.o.i.) of 0.001 for each virus. A low dose of virus
administered, in addition to monitoring time points over several days, allows the
identification of subtle replication differences.
In the first experiments with 17A307 and 17A480, as early as 24 hours post-
infection, a 8-10 fold reduction in infectious virus was seen for both 17A307 and 17A480
in PC-12 cells (Figure 3-6). This decrease was also seen at 48, 72 and 96 hours post
infection. The experiment was repeated with 17A307 and 17A480 being tested in the
same experiment along with a few additional controls. As with the previous experiments,
17syn+ and 17A307R were included as controls but I additionally tested 17APst
(promoter mutant) along with 17A480R in the multi-step growth curve (Figure 3-7). The
17APst recombinant virus was shown to lack a virulence phenotype in the mouse but it
was important to compare a LAT recombinant with an alteration in a region of LAT that
is expected not to contain a cell specific replication function. In these experiments, an 8-
10 fold reduction was again observed for 17A307 and 17A480 (Figure 3-7). The 17syn+,
17APst and 17A480R viruses were not restricted in the production of infectious virus on
PC-12 cells. Multi-step growth curves were also performed in rabbit skin cells (Figure 3-
8) to test for a general growth restriction in cell culture. These results did not yield a
noticeable difference in virus production as compared with controls (Figure 3-8).
A single step growth curve was also conducted in PC-12 cells utilizing a higher
m.o.i (3.0) and shorter time points: 0, 3, 6, 12, 18 hours post-infection (Figure 3-9). This
experiment tested for dramatic replication differences due to the higher dose of virus and
the shorter time points. From these results, a difference between LAT mutants and
controls is not clearly evident. Together, these data show that a cell specific replication
effect is seen for the 17A307 and 17A480 LAT mutants at low multiplicities of infection.
The phenotype is only observed in PC-12 cells and is lacking in rabbit skin cells. From
these data, I hypothesize that the neuronal restriction of 17A307 and 17A480 in nervous
tissue is the result of growth restriction in neurons.
Additional LAT Recombinant Viruses
In addition to the LAT recombinants engineered by our collaborator, Dr. Wagner
(17A307, 17A307a, 17A307aR, 17A480 and 17A489), it was necessary to construct two
additional mutants. One necessary recombinant virus was 17A480R, which enables
validation of the findings for 17A480. Another mutant, 17APstA480, was also
constructed and is beneficial in understanding the different regulatory regions of the LAT
locus as described below.
To verify the results observed for the 17A480 mutant in vivo and in cell culture,
the mutation was restored in a 17A480R recombinant virus. Restoration of biological
function was observed by multi-step time course on PC-12 cells (Figure 3-7). This
mutant is also being characterized for virulence properties in the mouse footpad model.
Another recombinant, 17APstA480, was also engineered to compare the findings
for the 17A480 with a recombinant that included a deletion in a different region of the
LAT locus. As shown by LD5o and tissue restriction experiments, 17A480 is greatly
reduced in virulence, while a deletion of the core latent promoter (17APst) is
indistinguishable from parental virus (17syn+). This led us to believe that the LAT locus
contains different regulatory regions and the impairment of more than one of these
regions may mask a severe phenotype. The 17APstA480 virus was a critical recombinant
for testing this hypothesis. Our expectation is to see an intermediate virulence phenotype
residing between what we observed for 17A480 and 17APst. In this virus, as the name
implies, the 480 mutation was coupled with a clean deletion in the latent core promoter.
The resulting mutant has impairments in two potentially different regulatory regions of
LAT. This will enable us to compare its phenotypes with other mutants in the literature
that contain very large deletions in the LAT locus (Perng et al., 2000; Thompson and
Sawtell 2001). These experiments are currently in progress.
9 -V----- -17''
PI3 PI5 P17 P110 PI12 P114 PI17 P119 PI 21 P124 PI27
Mean Values ( Standard Error of the Mean) (data points as plotted above):
meansem 17syn+ 17APst 17A307
PI 3 1.310.079 1.310.082 1.060.038
PI 5 1.920.078 1.860.089 1.530.049
PI 7 2.630.168 2.50.171 1.670.09
PI 10 1.180.193 0.980.184 0.810.156
PI 12 1.060.193 0.710.146 0.780.16
P 14 0.880.171 0.60.144 0.770.186
PI 17 0.0.840.178 0.390.084 0.610.206
PI 19 0.70.155 0.490.072 0.540.217
PI 21 0.660.153 0.470.028 0.660.199
PI 24 0.60.128 0.390.043 0.630.202
PI 27 0.550.124 0.410.093 0.540.216
Figure 3-1. Reduced corneal pathology of 17A307 in the rabbit eye model. The graph at
the top of the figure is a measurement of the severity of pathology of HSV-1
infection in the rabbit cornea. This was measured by slit lamp which
measures the number of dendrites and the severity of their pathology in the
cornea. These values closely compare with infectious virus levels. Values
below the graph are the raw data that are plotted above. The graph indicates
that 17A307 may have a virulence deficient phenotype.
Table 3-1. LD5o values*a
LD50/pfu Values for LAT Mutants
"_I "Route of Infection
Virus Footpad Intracranial
17syn+ 3.25 x 102 6.30 x 10'
17APst 5.11 x 102 9.30 x 10'
17A307 >7.0 x 104 >1.17 x 103
17A307a >7.0 x 104 9.63 x 102
17A307aR 3.17 x 102 9.10 x 10'
17A480 >4.0 x 104
17A489 1.10x 102
RHA-6 3.67 x 102
*This is a modified LD5o analysis. Mice were euthanized once they demonstrated an
infection of the central nervous system and a consequent impairment of motor function.
aThe values were recorded as a dose sufficient to induce lethality for half the mice in a
group. Two routes of infection were used in this experiment: footpad and intracranial.
17syn+ 17dPst 17d307 17d307R 17d480
Figure 3-2. Infectious virus recovered from mouse feet at 2 days post-infection. Feet
were homogenized and tested for infectious virus by plaque assay on RS cells.
Values are recorded as pfu/g tissue.
17syn+ 17dPst 17d307 17d307R 17d480
17syn+ 17dPst 17d307 17d307R 17d480
Figure 3-3. Virus production in dorsal root ganglia at 3 and 5 days post-infection. DRG
were homogenized and tested for infectious virus by plaque assay on RS cells.
Values are recorded as pfu/g tissue. A) The graph at the top is for 3 days post-
infection. B) The bottom is for DRG at 5 d.p.i. The difference between
17A307 and 17A307R is statistically significant P=0.0018 (unpaired t test,
17syn+ 17dPst 17d307 17d307R 17d480
17syn+ 17dPst 17d307 17d307R 17d480
Figure 3-4. Virus production in spinal cord at 5 and 7 days post-infection. A) Spinal
cord infectious virus levels at 5 dpi (P=0.002). B) Spinal cord at 7 dpi
17syn+ 17dPst 17d307 17d307R 17d480
Figure 3-5. Virus production in brain at 7 days post-infection. Mouse brains were
homogenized and tittered for infectious virus by plaque assay on RS cells.
Values are plotted as pfu/g tissue. The difference between 17A307 and
17A307R is statistically significant (P=0.009) and the difference between
17A480 and 17syn+ is also statistically significant (P-0.0022) as measured by
unpaired t test (Welsch corrected).
E 1.00E+04 -______ 017d307
8 24 48 72 96
8 24 48 72 96
Figure 3-6. 17A307 and 17A480 multi-step time courses in PC-12 cells. A) 17A307 is
statistically different than 17A307R (P=0.0019). B) 17A480 is statistically
different than 17syn+ (P0.0232).
I E04 7dPs
(parental) and 17APst (promoter deletion). 17A307 was statistically different
from 17A307R (P=0.02 at 24 hpi, P0.0069 at 48 hpi, P=0.0010 at 72 hpi) and
17A480 differed significantly from 17A480R (P=0.0364 at 24 hpi, Pm.0149
at 48 hpi, P=0.002 at 72 hpi).
Figure 3-8. Multi-step time course in rabbit skin cells. This is a comparison of infectious
virus production of 17A307, 17A307a and 17A307aR on RS cells. All 3
viruses produce high levels of infectious virus on RS cells.
8 24 48 72 96
Figure 3-8. Multi-step time course in rabbit skin cells. This is a comparison of infectious
virus production of 17A307, 17A307a and 17A307aR on RS cells. All 3
viruses produce high levels of infectious virus on RS cells.
0 3 6 12 18
Hours Pout-lnfMd l
Figure 3-9. Single step time course in PC-12 cells. 17A307 was compared to 17A307R
along with 17syn+ in a single step growth curve utilizing an initial MOI of
The influence of the LAT gene upon dictating the outcome of the latent phase of
the HSV-1 infection is clear. Uncertainty, however, begins with understanding: 1)
whether LAT acts primarily at the level of establishing a latent infection and helping to
either suppress viral replication and prevent cell death, or 2) whether it exerts its primary
influence as a mediator of reactivation from latency. Another difficulty in dissecting the
LAT gene's role has been linking the biologically diverse functions attributed to the LAT
region with the LAT RNA itself, and whether different regions of the gene may actually
have different functions. LAT promoter mutants have been seen to have different
phenotypes than larger deletions that include the promoter and downstream transcript-
encoding sequence. The goal of this study was to utilize recombinant virology to attempt
to define the genetic basis for LAT phenotypes that might go beyond just controlling
Viruses with alterations in the LAT region exhibit severe impairment of
reactivation in both the induced and the spontaneous rabbit eye models, and in mouse
models involving infection of either the footpad or the cornea. Specifically, mutations in
the promoter and/or the 5' region of exon 1 have most discriminately resulted in severe
reactivation deficiencies in rabbit models (Bloom et al., 1994; Bloom et al., 1996; Perng
et al., 1994; Perng et al., 1996; Trousdale et al., 1991). It has been debated whether these
effects are due to decreased reactivation, or are the result of decreased establishment of
LAT mutants. With respect to early events in HSV-1 latency, the LAT gene has been
proposed to enhance the efficiency of establishment (Pemg et al., 2000; Thompson and
Sawtell 1997). These studies were performed with relatively large LAT deletions, so
while one cannot rule out that a portion of the LAT gene may act upon establishment
these data are not consistent with the observation that LAT promoter mutants do not
show impaired establishment, and instead seem to act primarily at the level of
reactivation. In addition to LAT promoter mutants, concise, small mutations (200-400
bp) in the LAT promoter and/or the 5' region of exon 1 result in a dramatic reduction
(70-100%) in reactivating virus in the absence of any detectable differences in
establishment (Bloom et al., 1994; Bloom et al., 1996).
It has been argued by Sawtell and Thompson that the LAT mediates its effects on
the establishment phase of the latent infection (Sawtell and Thompson 1992; Thompson
and Sawtell 1997). To test for an establishment decrease, they tested 17syn+ (parental)
along with 17-AH, which is a deletion of the LAT promoter, exon 1 and half of the 2.0 kb
intron in comparison to its rescue, 17-AHR (Thompson and Sawtell 2001). These viruses
were tested for a measurable difference in establishment in the mouse TG after corneal
infection. In these experiments, they report a decrease of approximately 33% in viral
DNA detected in TG for the 17-AH mutant in comparison to parental virus and 17-AHR.
They argue that these results demonstrate an establishment decrease sufficient to
facilitate the reactivation reductions in their experiments and reported by others (Bloom
et al., 1994; Perng et al., 1994; Perng et al., 1996).
A biological mechanism hypothesized to result in establishment decreases is a
difference in neuronal survival, or apoptosis, for LAT mutants in comparison to parental
HSV-1. Sawtell and Thompson produced data suggesting a role of LAT in protecting
neurons during the establishment of latency (Thompson and Sawtell 2001). A difference
in viral DNA levels in ganglia between the LAT(-) mutant, 17-AH, and its rescue, 17-
AHR was reported. This difference is proposed to occur due to a inhibition of apoptosis
and an increase of surviving neurons. In the determination of neuronal survival, it was
reported that 17-AH has a 2-fold reduction in total latent neurons in comparison to its
rescue (Thompson and Sawtell 2001). These viruses were also tested for differences in
their relative replication in mouse TG following coreal infection. Interestingly, no
detectable replication differences were observed in cornea or ganglia. If these viruses do
express neuronal survival phenotypes, one would expect to see a replication difference
between LAT(-) and LAT(+) viruses. A virulence phenotype would also be expected if
LAT plays a role in neuronal survival and/or apoptosis in vivo.
A portion of the LAT gene has been shown by Wechsler (Pemg et al., 2000) to
stimulate apoptosis in transient transfection in cell culture through suppression of
receptor-mediated (caspase-8) and mitochondrial-mediated (caspase-9) pathways (Pemg
et al., 2000). Results in vivo with the large LAT mutants demonstrated a reduction in
establishment levels leading to a significant loss of sensory neurons (Perng et al., 2000;
Thompson and Sawtell 1997). Transient transfection of IMR-90 and CV-1 cells with a
plasmid containing the 3' region of the LAT exon and most of the intron was able to
protect the majority of cells from ceramide, or fumonisin Bi-induced apoptosis (Pemg et
al., 2000; Thompson and Sawtell 2000). Expression of the bovine herpesvirus 1 LAT
(Jones et al., 1996) or portions of the HSV-1 LAT gene inhibit chemically induced
apoptosis in cell culture (Jones et al., 2001; Perng et al., 2000).
The LAT effects on apoptosis appear to be cell specific. In culture, LAT is unable
to protect 293, or COS-7 cells from apoptosis (Inman et al., 2001). It is clear that LAT
does induce apoptosis in certain cell lines using transient transfection assays with high
multiplicities of infection and high expression levels (Pemg et al., 2000). These results
are very interesting but it is uncertain if LAT plays an apoptotic role in the context of
whole virus and in animal models. These findings were used to develop the hypothesis of
an apoptotic function of LAT in TG, and that LAT mutants result in greater levels of
apoptosis within sensory ganglia. Unfortunately, an apoptotic role of LAT has not been
adequately proven in whole virus or in animal models. The neuronal survival and
apoptosis results present a potential additional role of the LAT locus upon the acute
infection and the establishment of latency, although the mutants used in these
experiments do not adequately allow the identification of a virulence function distinct
from latency phenotypes.
Analysis of a series ofHSV recombinants containing deletions in the region known
to be required for reactivation identified a deletion that exhibited decreased pathology in
the rabbit eye during the acute infection. We hypothesized that this might represent a
separate function encoded by the LAT region and sought to study this recombinant's
virulence in the well-defined mouse model. LDso analysis and infectious virus production
in neuronal and non-neuronal tissues revealed that this mutant was dramatically restricted
in virulence. Additional analyses also included 17A480 and 17A489, which have been
described previously (Jarman et al., 2002) and involve mutations in the 5' region of the
LAT intron. Analysis of the LAT mutants 17A307 and 17A480, and their rescues, in the
mouse model revealed a dramatic impact of this region of the LAT gene on the acute
infection ofHSV-1. From the LDso/pfu values from both a footpad route of infection and
from an intracranial infection, a greater than 500 fold reduction occurs in the 17A307 and
17A480 mutants in comparison to 17syn+ (parental), 17APst (promoter mutant),
17A307R and 17A489. The dramatic reduction in virulence by each route of infection
proves a neurovirulence phenotype for these viruses and is not simply due to a
neuroinvasive effect. Some virulence effects have been reported for LAT mutants
(Thompson and Sawtell 2001; Perng et al., 2000) but none have exhibited such severe
deviations from wild-type and rescuant viruses. These data demonstrate a virulence
function of HSV-1 localized to the 3' region of exon 1 and the 5' intron of the latency
associated transcript locus.
Virulence Phenotype and Nervous Tissue Restriction
The LD5o/pfu determinations of 17A307 and 17A480 were a test of their ability to
induce encephalitis in the mouse model. In addressing the basis for this impairment,
spinal cord and brain were also analyzed for relative viral yields in these tissues by
plaque assay. Titrations of these tissues again showed restriction in replication in
nervous tissue for 17A307 and 17A480. Both of these LAT mutants were dramatically
restricted in viral yields in the spinal cord and in the brain. These results could possibly
be due to an initial restriction in the DRG, which would lead to lower seeding of the
spinal cord and consequently the brain. This is unlikely, though, because direct
intracranial inoculations and LDso/pfu determinations also showed a significant reduction
in virulence as occurred with the footpad route of infection, suggesting that the restriction
in the nervous tissue is more general.
Two possible explanations for the decreased virulence of 17A307 and 17A480 in
the mouse model are either a global restriction of these mutants to replicate in murine
tissue, or a tissue specific replication phenotype in neurons. In testing both epidermal
and nervous tissue infected in the mouse footpad model and the successive path of
infection in the nervous system, it was determined that the virulence phenotype is the
caused by a tissue specific restriction of the downstream LAT mutants. After footpad
infection, 17A307 and 17A480 are indistinguishable in infectious virus levels in
comparison to 17A307R, 17syn+ and 17APst proving that these mutations do not have a
general replication restriction in vivo. These LAT mutants were equal in p.f.u.
production at 2 days post-infection, which has been shown previously in our lab to be the
peak time of HSV-1 infection in the mouse footpad model (Jarman et al., 1999). After
infection of the foot, virus then enters the peripheral nervous system by entering
innervating nerve termini in the epidermis. Virus then travels via the sciatic nerve to the
dorsal root ganglia. In examing DRG for virus production, 17A307 was decreased in
infectious virus load at 3 days post-infection. Interestingly, 17A480 did not display a
detectable reduction in infectious levels at 3 days but did have a significant restriction by
5 days post-infection. 17A307 was restricted at this time point as well for dorsal root
ganglia. The virulence effects of 17A307 and 17A480 in the mouse are due to tissue
specific restriction of these mutants in nervous tissue.
Cell Culture Phenotype
A nervous tissue restriction was clearly observed in the mouse model but it was
uncertain whether this effect was caused by a neuronal cell type, or from supporting cells
in ganglia. To test for a cell specific replication impairment, 17A307 and 17A480
mutants were analyzed for replication in PC-12 cells (pheochromocytoma cells) and RS
cells in time course experiments. The PC-12 cells were chosen due their close
developmental relationship with neurons. Both the PC-12 cells and the RS cells were
tested for infection virus production in multistep and single step growth curves. From
these experiments, 17A307 and 17A480 each displayed an 8-10 fold restriction in plaque
forming unit production on PC-12 cells by 48 hours post-infection at low multiplicities of
infection in comparison to 17syn+, 17APst, 17A307R and 17A480R. When compared in
the RS cells, all viruses tested replicated to indistinguishable levels. This result was not
unexpected due to what was seen for replication values from feet in the tissue restriction
experiments. Together, these results indicate that the restriction specific for nervous
tissue is due to reduced replication, or at least decreased infectious virus production, in
As mentioned previously, from initial characterization of these mutants, a severe
reduction in epinephrine-induced reactivation in the rabbit eye model was evident for
17A307. In contrast, 17A480 did not exhibit a reactivation phenotype in comparison to
17syn+ (parental), or 17APst (latent promoter mutant). The region altered in the 17A307
mutant may include the 3' region of the reactivation critical portion of the LAT locus and
a virulence region. The 17A480 shows a separation of virulence from reactivation
providing evidence for another regulatory region of the LAT gene.
LAT Mutants and Multiple Regulatory Functions
Many of the LAT mutants described in the literature involve mutation of a very
large portion (>lkb) of the LAT locus. Two of these mutants are dLAT2903 and 17-AH
and each involves mutation of the latent promoter, exon 1 and half of the 2.0 intron
(Pemg et al., 1994; Thompson and Sawtell 2001). Examination of these mutants failed to
reveal dramatic virulence phenotypes. These viruses do show latent phenotypes as
described by other mutants that engineered changes into the 5' portion of exonl (Bloom
et al., 1994; Perng et al., 1994). The mutant dLAT2903 is indistinguishable from parental
or its rescue in cell culture replication, corneal replication or p.f.u. production in the TG
of the rabbit (Perng et al., 2001). Similarly, no replication differences were observed for
17-AH in cell culture, cornea and TG (Thompson and Sawtell 2001). The fact that these
recombinants do not display a clear virulence effect in animal models, coupled with the
data shown here for the 17A307 and 17A480 LAT recombinants, suggests that the LAT
locus contains several distinct regulatory regions (Figure 4-1). These regions are
involved in different stages of HSV-1 infection in the mouse model. The recombinant
viruses 17A307 and 17A480 display severe virulence reductions in the mouse footpad
Another interesting phenotype for these mutants is their difference in reactivation
abilities in the epinephrine-induced rabbit eye model. Reactivation is significantly
reduced for 17A307 and, as observed in this report, the mutant also shows decreased
virulence. From the establishment hypothesis of Sawtell and Thompson, it may seem
that 17A307 is simply inhibited in its ability to protect neurons and consequently results
in lower virulence in the acute infection and lower genome levels for the ability to
reactivate. The problem with this interpretation stems from the acute and latent
phenotypes of the 17A480 mutant. This virus does display a virulence phenotype as
observed for 17A307 but is devoid of a reactivation phenotype. Therefore, the virulence
phenotype cannot be explained by a lower amount of DNA seeding the ganglia.
Together, these data argue for the presence of a virulence region in the LAT locus clear
and distinct from a reactivation critical region (Figure 4-1). The virulence phenotypes for
the dLAT2903 and 17-AH viruses are not severe and the net result of deleting a large
region of LAT encompassing multiple genetic elements may be a phenotype that is less
severe than deleting single elements individually.
My hypothesis is that mutation of a combination of potentially opposing regulatory
regions may result in the masking of a clear acute phenotype. To address this possibility,
we engineered a mutant that removes the core element of the latent promoter along with
the exchange of a 480 bp region of the 5' intron of LAT with a lacZ stuffer sequence.
This mutant, 17APstA480, as the name suggests is simply the combination of the 17APst
and the 17A480 mutants. The virulence characteristics of this mutant are currently being
Establishment differences were seen with large LAT deletions but the regulatory
element was not well defined. The apoptosis and neuronal survival results were obtained
using mutants with a large region of shared sequence removed. From comparison of the
mutants, and what has been shown for the involvement of the 5' region ofexon 1 in
reactivation, it would seem that the region of the LAT locus involved in establishment,
neuronal survival and possibly apoptosis maps to the 3' region of exon 1 and the 5'
portion of the intron. Our data shows that small, concise mutations in this region of the
LAT locus do result in dramatic virulence results.
From the virulence data for the recombinants 17A307 and 17A480, it is clear that
a downstream region of LAT involving the end ofexon 1 and the beginning of the 2.0 kb
intron is acting upon the severity of acute infection and possibly the establishment of
latency. This region may function to protect neurons in vivo as proposed by Thompson
and Sawtell (Thompson and Sawtell 2001). On the molecular level, a few possible
mechanisms can be envisioned. First, it is possible that this region of the LAT locus
involves a secondary promoter. Another potential mechanism is through differential
transcriptional permissivity of the LAT region. Lastly, the LAT locus may encode an
enhancer/silencer region functioning to regulate the ICPO promoter.
Lytic Promoter in the LAT Region
A second promoter in the LAT gene was identified by use of transient
transfections in cell culture (Goins et al., 1994). Sequence involving exon 1 was able to
drive transcription of a lacZ reporter in Vero cells and was designated as LAP2 (Goins et
al., 1994). Discovery of this promoter resulted in its unfortunate designation as a latent
promoter. In its initial characterization, the secondary promoter was shown to be
transcriptionally active in Vero cells and consequently demonstrates its ability to express
RNA during a lytic infection.
Since the data for 17A480 demonstrates a function for this area of the LAT gene
upon the acute infection and does not result in a detectable reactivation phenotype, the
secondary promoter would be expected to be active during an acute infection. Therefore,
the LAT locus would have separate, opposing promoters: one functioning in a latent
infection to act upon reactivation and one operating in the acute infection in ganglia.
Multiple other transcripts have been proposed for the LAT region but have not been well
mapped and their kinetics and cell specific expression are poorly understood. Our lab has
detected expression of a RNA molecule in this region from a latent promoter mutant
(17APst) and mapping of this potential transcript is currently underway. The use of
another promoter in the LAT locus is possible due to the opposing virulence and
reactivation phenotypes seen for 17APst (LAP1 mutant) and the 17A480 mutant. If only
a primary 8.5 kb transcript is expressed, and a subsequently spliced intron, than it would
be unusual that removal of a downstream region (i.e. A480) would give a virulence
phenotype when removal of its core promoter (APst) does not affect the acute infection.
This argues strongly that they are separate and perhaps opposing elements.
It is also possible to model an action from a transcription accessibility hypothesis.
During latency, we know that LAT is the only abundantly expressed gene of HSV-1. The
other genes of HSV-1 are tightly repressed. In examination of the mechanism for this
dramatic difference, our lab showed that the LAT region is present in a transcriptionally
permissive state during latency as seen with histone acetylation data (Kubat et al., 2004).
The severity of acute infection may also result from alterations in the transcriptional
accessibility of the LAT and surrounding genes. It is possible that the region deleted in
the 17A307 and 17A480 mutant comprises a key site for the transcriptional permissivity
of LAT and possibly the neighboring, antisense gene, ICPO. Influence on ICPO is
especially likely, since is functions as a key transcriptional activator of all 3 classes of
HSV-1 genes. The LAT virulence locus could function to dock transcription factors,
which exert their effects upon either the latent LAT promoter, or possibly the ICPO
promoter. An impairment of this region to recruit the binding of cellular, or viral factors
present explicitly in ganglia, which are involved in ICPO expression, would result in a
decrease in replication and severity of infection.
Another possible mechanism for the virulence region of LAT is through
transcriptional regulation of the ICPO promoter. It may not simply function through a
transcriptional permissivity hypothesis as described above but could directly enhance the
expression of ICPO. During the lytic infection, the enhancer encoded in the LAT locus
would increase expression from the ICPO promoter directly by a looping mechanism.
Additionally, I hypothesize this occurs in a neuron-specific manner. In acute infection,
the enhancer would associate with transcription factors present in neurons to promote
expression of ICPO. The mutations introduced into the LAT locus in the 17A307 and
17A480 recombinant impair this enhancer function. ICPO is consequently lower in
neuronal tissue and leads to a general decrease in HSV-1 gene expression. This decrease
causes lower viral production and a milder infection in the mouse model. An enhancer
function involving the LAT locus and neuronal specific factors would explain why the
virulence effects were specific for nervous tissue. Future experiments are aimed at
determining if a secondary promoter is active in the LAT locus and mapping of
subsequent RNA molecules. Additionally, our lab will analyze the ICPO promoter region
for differences in transcription with the virulence mutants, and for possible changes in
SRegion of mutation
Figure 4-1. Separate genetic regions of the LAT locus. This is a representation of the
virulence data presented in this manuscript along with known reactivation data
for the LAT region. The combination of the reactivation and virulence
phenotypes identified for LAT recombinant viruses enabled the modeling of
separate regulatory regions ofthe LAT locus as shown in this figure.
17A*s 1T^y ".
separate regulatory regions of the LAT locus as shown in this figure.
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In the Fall of 1993, I started at Mars Hill College in Mars Hill, North Carolina. At
Mars Hill, I majored in biology with a chemistry minor and completed my Bachelor of
Science in May of 1997. I then entered into the Microbiology doctoral program at
Arizona State University in the Fall of 1997. After two years, my mentor accepted a
position at the University of Florida and I subsequently transferred to continue my
research. At the University of Florida, I entered into the Molecular Genetics and
Microbiology department and completed my Ph.D. in May of 2004.
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
David C. Bloom, Chair
Assistant Professor of Molecular Genetics
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Professor of Molecular Genetics and
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Professor of Molecular Genetics and
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope an ality, as a
dissertation for the degree of Doctor of Philosophy./
Associate Professor of Physiological
This dissertation was submitted to the College of Medicine and to the Graduate
School and was accepted as partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
May 2004 DM ed
Dean, College of Medicine
Dean, Gradte School