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Journal of Virology, January 2000, p. 965-974, Vol. 74, No. 2
Department of Molecular Genetics,
Microbiology, and Biochemistry, University of Cincinnati Medical
Center, Cincinnati, Ohio 45267-0524,1 and
Division of Infectious Diseases, Children's Hospital
Medical Center, Cincinnati, Ohio 45229-30392
Received 12 May 1999/Accepted 7 October 1999
The replication properties of a thymidine kinase-negative
(TK Experimental herpes simplex virus
type 1 (HSV-1) infection in mice parallels that which occurs in humans
(13, 53). During primary infection, viral replication at the
surface is detectable for a 10- to 12-day period. Within 24 h
postinfection (p.i.) virus is transported to the innervating sensory
ganglia, where peak titers are achieved around 3 to 4 days p.i., and
infectious virus becomes undetectable within the ganglia by 7 to 9 days
p.i. The establishment of latent infections occurs during this
replicative phase. Once latency is established, the ~75 viral genes
expressed during the lytic phase are extremely repressed and only the
latency-associated transcripts (LATs) are abundantly transcribed
(1, 25, 26, 52, 54, 63).
Viral replication is not an absolute prerequisite for the establishment
of latency, as replication-deficient or -incompetent viral mutants can
establish latent infections (3, 4, 8-11, 14, 20, 22, 27, 33, 34,
51). Indeed, lethal mutations that preclude most viral gene
expression do not completely prevent the establishment of latency
(8, 46). From these observations it has been hypothesized
that the lytic and latent pathways must diverge at a very early stage
in the virus replication cycle (8, 46). If the pathways
diverge very early, i.e., prior to DNA replication, it would follow
that DNA replication enzymes, such as the viral thymidine kinase (TK),
would have no effect on establishment. Indeed, TK null mutants do
establish latent infections (4, 28, 56). Additional studies
have demonstrated that following footpad inoculation the number of
neurons expressing LAT RNAs was only slightly reduced (28),
and LAT-positive neurons were also detected following corneal
inoculation (4). These findings led to the current
hypothesis that the viral TK (e.g., viral DNA replication in neurons)
is required primarily for reactivation from the latent state and not
for the establishment or maintenance of latency (for a review, see
reference 55). The work of Maroglis et al. supports
this hypothesis. When mice were infected by direct injection of virus
into the sciatic nerve trunk, treatment with antiviral nucleoside
analogs did not significantly reduce the amount of latent DNA detected
in dorsal root ganglia (DRG) (29). However, the opposite
conclusion was reached by Slobedman et al., who compared the
establishment of latency by the HSV-1 strain SC16 and a TK-negative
(TK The importance of this issue resides in the fact that current antiviral
therapies for HSV target the viral TK and/or DNA polymerase (DNA Pol)
and are based on the disruption of viral DNA replication. Drug-resistant mutants are rapidly selected in vivo but are thought to
be at a disadvantage in the wild. It is not yet clear whether the
increased use of such drugs will contribute to a significant increase
of drug-resistant mutants within the human population. Understanding
more fully the impact of the loss of TK function on the ability of the
virus to establish latent infections and to reactivate will be of
predictive value. Several recent advances suggested to us that the role
of the viral TK, and viral DNA replication in ganglia, on the
establishment of latency should be revisited. First, Horsburgh et al.
demonstrated that TK-negative mutants are reactivation competent when
the mutation resides in a clinical isolate (18). Thus,
drug-resistant variants of HSV may establish latency, subsequently
reactivate, and spread through the population. Second, assays to
directly determine the number of neurons containing the viral genome
and the viral genome copy number in these neurons have recently been
developed (40, 41, 43, 60). Since the earlier studies relied
on indirect measurements of establishment, a direct measure could
provide additional insight. Third, although it has been recognized that
the replication capacity of TK Mice were inoculated on either the snout (permissive for TK null
mutants) or the cornea (less permissive) with 17/tBTK Cells and viruses.
Rabbit skin cells (RSC) were cultured as
previously described (61). The wild-type HSV-1 strain
17syn+ was originally obtained from John H. Subak-Sharpe at
the Medical Research Council, Virology Unit, in Glasgow, Scotland, and
subsequently plaque purified as described previously (61,
62). The TK
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Replication of Herpes Simplex Virus Type 1 within Trigeminal
Ganglia Is Required for High Frequency but Not High Viral Genome
Copy Number Latency
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) mutant of herpes simplex virus type 1 (HSV-1) were
exploited to examine the relative contributions of replication at the
body surface and within trigeminal ganglia (TG) on the establishment of
latent infections. The replication of a TK mutant,
17/tBTK, was reduced by ~12-fold on the mouse cornea
compared to the rescued isolate 17/tBRTK+, and no
replication of 17/tBTK in the TG of these mice was
detected. About 1.8% of the TG neurons of mice infected with
17/tBTK harbored the latent viral genome compared to 23%
of those infected with 17/tBRTK+. In addition, the latent
sites established by the TK mutant contained fewer copies
of the HSV-1 genome (average, 2.3/neuron versus 28/neuron). On the
snout, sustained robust replication of 17tBTK in the
absence of significant replication within the TG resulted in a modest
increase in the number of latent sites. Importantly, these latently
infected neurons displayed a wild-type latent-genome copy number
profile, with some neurons containing hundreds of copies of the
TK mutant genome. As expected, the replication of the
TK mutant appeared to be blocked prior to DNA replication
in most ganglionic neurons in that (i) virus replication was severely restricted in ganglia, (ii) the number of neurons expressing HSV proteins was reduced 30-fold compared to the rescued isolate, (iii)
cell-to-cell spread of virus was not detected within ganglia, and (iv)
the proportion of infected neurons expressing late proteins was reduced
by 89% compared to the rescued strain. These results demonstrate that
the viral TK gene is required for the efficient establishment of
latency. This requirement appears to be primarily for efficient
replication within the ganglion, which leads to a sixfold increase in
the number of latent sites established. Further, latent sites with high
genome copy number can be established in the absence of significant
virus genome replication in neurons. This suggests that neurons can be
infected by many HSV virions and still enter the latent state.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) derivative, TKDM21 (50). Following
infection of the mouse flank, a comparison was made of the number of
LAT+ neurons detected and the total amount of HSV-1 DNA
that was present in the innervating DRG. The authors concluded that
both input (unreplicated) and progeny (replicated) viral genomes
contributed to the number of LAT-positive sites in DRG. Although not
directly measured, their data suggested that viral DNA replication
within neurons was required to establish latent sites that contain a high number of HSV-1 genomes (50). Thus, viral replication
(or viral DNA replication) within ganglia may increase the number of
latent sites established or increase the number of HSV-1 genomes contained within latently infected neurons.
mutants can vary depending
on the site of inoculation (11, 12), the effect of variable
surface replicative capacity on the establishment of latency and copy
number profile has not previously been examined.
or
a genomically rescued variant, 17/tBRTK+. The number
of latent infections established in the trigeminal ganglia (TG),
and the HSV-1 genome copy number profile within those neurons, was
determined. We conclude that while the increased viral replication at
the body surface is correlated with a modestly increased frequency of
latent sites, viral replication in sensory ganglia significantly
contributes to the number of latent infections established. The latent
infections that were established in mice infected on the cornea with
the TK mutant contained a universally low HSV-1 genome
copy number. In contrast, latently infected neurons in mice infected on
the snout contained a wild-type HSV genome copy profile, with some latent sites containing hundreds of HSV-1 genomes. It therefore appears
that individual neurons can be infected by hundreds of virions
originating at the body surface and still enter the latent state.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
mutant 17/tBTK was
generated by inserting a simian virus 40 promoter-Escherichia coli beta-galactosidase (
-Gal) minigene into the
SnaBI site present in the TK gene at bp 47560 on the viral
genome as previously detailed (36). HSV-1 genome base pair
designations are as described by McGeoch and colleagues (30,
35). This insertion totally obliterated enzyme function as
measured by antiviral drug resistance, plaque autoradiography, and a
sensitive in vitro enzyme assay (36). The mutant
17/tBTK was restored genomically and phenotypically to
wild type by recombination with sequences spanning bp 45055 to 48634 on
the viral genome to generate the rescued variant 17/tBRTK+
as described previously (59). The genomic structures of
these virus isolates are shown schematically in Fig.
1. Virus stocks were generated by routine
propagation in RSC monolayers. Infected cells were harvested and
sonicated, and the titer of the stock was determined by serial-dilution
plaque assay on RSC monolayers as previously described (61).
View larger version (30K):
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FIG. 1.
Genomic structures and transcriptional analysis. (Top) A
schematic representation of the HSV-1 genome is shown with the long
terminal and internal repeats (TRL and IRL) and internal and terminal
short repeats (IRS and TRS) shown as shaded bars. Below, the region
containing the TK gene is expanded with the ORFs for glycoprotein H
(gH), TK, UL24, and UL25 denoted by open arrows. The TK ORF was
disrupted by insertion of a -Gal minigene construct (36),
indicated by the hatched arrow. (Bottom) Representative RNA blots. The
blot on the left was hybridized with 32P-labeled sequences
specific for the TK mRNA, as described in Materials and Methods. The
similar blot on the right was hybridized to a probe specific for UL24
mRNA.
Transcription of TK and UL24 mRNA. Slightly subconfluent RSC monolayers in 10-cm-diameter dishes (Falcon) were infected at a multiplicity of infection (MOI) of 10 PFU/cell. Following adsorption for 1 h, the dishes were rinsed, fed with 10 ml of minimal essential medium supplemented with 5% newborn calf serum (Gibco-BRL), and incubated at 37°C for 5 h. Total RNA was isolated with Ultraspec (Biotex) according to the manufacturer's protocol. Ten micrograms of RNA was glyoxylated, electrophoresed, transferred to nylon membranes (GeneScreen), and probed as described previously (39).
Inoculation of Mice.
Male outbred Swiss Webster mice (Harlan
Laboratories) 4 to 5 weeks of age were used throughout these studies.
The animals were housed in American Association for Laboratory Animal
Care-approved quarters with unlimited access to food and water. The
mice were anesthetized by intraperitoneal injection of sodium
pentobarbital (Nembutal [50 mg/kg of body weight]). Both corneas were
scarified or both sides of the snout were shaved and abraded to expose
a 5-mm2 surface area as described previously (44,
45). A total inoculation of 2 × 105 PFU of
17/tBTK or 17/tBRTK+ was applied to the
cornea or snout.
Replication kinetics in vitro and in vivo. Single- and multistep replication kinetic analysis was performed on slightly subconfluent RSC monolayers following infection at a high (10 PFU/cell) or low (0.001 PFU/cell) MOI. The cells and media were harvested from triplicate cultures at 4, 8, 12, 18, and 24 h p.i. (high MOI) or 4, 24, 48, and 72 h p.i. (low MOI) and subjected to three freeze-thaw cycles. Virus titers were determined on RSC monolayers.
A total of 80 mice (20 per group) were inoculated as described above. At each of the indicated times p.i., three mice from each of the four infected groups were euthanized and the appropriate tissues were removed, snap frozen, and stored at
80°C. Each group of three pairs
of eyes, snouts, or TG was homogenized in 1 ml of minimal essential
medium supplemented with 5% newborn calf serum (Gibco-BRL),
centrifuged for 5 min at 5,000 × g, and assayed for
infectious virus titer on RSC monolayers. The remaining mice were
maintained for latency studies as described below.
In an additional experiment, 50 mice were inoculated (10 to 15 per
group). On day 4 p.i., the mice were euthanized and the relevant
tissues were harvested from each mouse and placed in separate tubes.
The tissues from each of five mice from each group were homogenized,
and the viral titer was determined for each in order to confirm the
replication profiles and assess the extent of variability of the
individual mice within a group. The tissues from an additional five
mice per group were used to assess the amount of viral DNA by PCR as
described below. The remaining 10 mice were perfusion fixed for
immunohistochemistry as described below.
Quantitative PCR detection of HSV DNA. Quantitative PCR was carried out as described previously (22). Briefly, mice were infected as described above, and DNA was extracted from the relevant tissues (snout whisker pads, eyes, or TG). An average of 20 µg (range, 12 to 27 µg) of DNA was obtained from each TG pair, 50 µg (range, 23 to 62 µg) from each eye pair, and 100 µg (range, 64 to 135 µg) from the snouts. Aliquots of DNA were assessed for intactness and concentration by 0.8% agarose gel electrophoresis and quantified by comparison to commercial lambda DNA standards (Gibco-BRL).
One hundred nanograms of sample DNA or, as a standard, 100 ng of mouse liver DNA spiked with known concentrations of the cloned HSV-1 EcoRI N fragment, was analyzed by PCR with two primer pairs. One primer set was specific for the HSV-1 TK gene, and the second set was specific for the single-copy mouse adipsin gene as described previously (22). Only 10 ng of sample DNA was analyzed for the 17/tBRTK+ samples and the 17/tBTK snout
samples, as the amount of HSV-1 DNA present in 100 ng was beyond the
linear range of the assay. The reaction products were electrophoresed
on 12% polyacrylamide gels, transferred to nylon filters (GeneScreen;
NEN), and probed with 32P-labeled oligonucleotides internal
to the amplification primers. The resulting blots were scanned in a
Molecular Dynamics PhosphorImager for quantification with ImageQuant
software (Molecular Dynamics). The total amount of HSV-1 DNA in each
tissue was calculated by determining the number of HSV genomes in the
tested aliquot and normalizing this number to the average amount of DNA
obtained from that tissue.
Immunohistochemical detection of viral proteins.
Groups of
mice were inoculated on the snout with 2 × 105 PFU of
either 17/tBTK or 17/tBRTK+ as described
above. On day 4 p.i., the animals were anesthetized by
intraperitoneal injection of a 2.5-mg dose of sodium pentobarbital. The
deeply anesthetized animals were perfusion fixed with 0.5% paraformaldehyde, and the TG were removed and postfixed for 30 min in a
solution of 4% paraformaldehyde. The tissue was dehydrated through
graded ethanols and embedded in paraffin following routine procedures.
Eight-micron sections were cut with a rotary microtome, placed on
negatively charged glass slides (Superfrost Plus; Sigma), and incubated
in a dry oven for 12 h at 60°C. Paraffin was removed by
incubation in xylene followed by a graded ethanol series.
Tissue-sampling design.
Two experiments were performed. In
the first, 10 ganglia from 17/tBTK-infected mice or 8 ganglia from 17/tBRTK+-infected mice were randomly oriented
in a single paraffin block. Each block was serially sectioned, and five
consecutive sections were placed on a single slide until all sections
from the block were collected. This resulted in a total of 25 to 30 slides per block. Six nonconsecutive slides spanning the
17/tBRTK+ tissue block were examined; five were treated
with the anti-HSV antibody, and a sixth slide was used as the
no-primary-antibody control slide to assess background staining. Seven
slides were examined in an identical manner from the
17/tBTK tissue block. Thus, 40 and 60 ganglion profiles
per slide were examined for 17/tBRTK+ and
17/tBTK, respectively. The number of positively stained
neurons (based on morphology) in each ganglion profile in a section
from each of the slides was determined.
-infected mice on day
4 p.i.) were again serially sectioned. A group of serial sections
were placed in consecutive order on each of five slides such that slide
1 contained sections 1, 6, and 11, slide 2 contained sections 2, 7, and
12, etc. Four and five sets of these five-slide serially sectioned
ganglia were generated for analysis of 17/tBRTK+- and
17/tBTK-infected ganglia, respectively. These sets of
slides were used so that slides 1, 2, 3, 4, and 5 of each set were
incubated with an anti-HSV, anti-ICP4, anti-ICP5, and LP-1 (anti-VP16)
antibodies and no primary control, respectively. The staining of each
antibody could then be examined on multiple serial sections from
different regions of multiple ganglia. The total number of positively
stained neurons was determined for each antibody on profiles of the
same ganglia to avoid any bias of the regional variations in the number of infected cells. For 17/tBTK, 60 ganglion profiles were
counted for each antibody. Because the number of positive neurons was
much greater in the 17/tBRTK+ ganglia, fewer profiles were counted.
Contextual analysis of latency.
Mice were infected as
described above and maintained for at least 30 days p.i. Two sets of
three mice from each group were analyzed. Enriched neuron populations
were obtained exactly as described previously (40, 41, 43,
60). Briefly, animals were anesthetized with sodium pentobarbital
and perfused with Streck's tissue fixative. Fixed TG were dissociated
into single-cell suspensions with collagenase, and the neurons were
purified on Percoll (Pharmacia) gradients. The neurons were stained
with Ponceau S and aliquoted into 200-µl PCR tubes. The tube contents
were visualized microscopically; thus, the actual number of neurons analyzed in each sample is known. For the 17/tBRTK+
samples, only tubes containing a single neuron were employed. Preliminary analysis of the 17/tBTK samples revealed a
low frequency of positive neurons. Therefore, samples containing 10 neurons were employed as described previously (40).
Following treatment with immobilized DNase (Mobitec), intracellular DNA
was liberated with proteinase K and analyzed for the presence of the
HSV-1 genome by the quantitative PCR assay developed by Katz et al.
(22). The products were electrophoresed, blotted, probed
with an internal 32P-labeled oligonucleotide, and
quantified on a PhosphorImager with ImageQuant software as detailed
elsewhere (40).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characterization of 17/tBTK and
17/tBRTK+.
The generation and characterization of
17/tBTK was reported previously (36). In
brief, a simian virus 40 promoter--Gal reporter minigene was
inserted into the SnaBI site at bp 47560 in the viral
genome. This insertion disrupted the TK open reading frame (ORF) that
spans bp 47802 to 46674 and eliminated all detectable viral TK activity
as assayed by antiviral drug resistance, plaque autoradiography, and a
sensitive in vitro enzymatic assay for the viral TK enzyme
(36). A schematic representation of the genomic structure of
17/tBTK is shown in Fig. 1 (top). 17/tBTK
was rescued to wild type by recombination with wild-type sequences spanning bp 45055 to 48634 on the viral genome to generate
17/tBRTK+ as described in Materials and Methods. This
rescued variant was wild type in plaque morphology, replication in
vitro and in vivo in neural tissues, and sensitivity to antiviral drugs
(see below and data not shown).
-Gal minigene in 17/tBTK was inserted upstream of
UL24, a viral gene that is partially antisense to TK and is required for efficient viral replication (6, 19, 21). UL24 mRNA is
transcribed from three different promoters (23, 37), the most distal of which was disrupted by the insertion. To determine the
effect of the insertion on UL24 mRNA expression, RSC monolayers were
infected with 17/tBTK or 17/tBRTK+ and
employed as a source of mRNA for RNA blot analysis (Fig. 1, bottom). In
the left panel, a probe specific for the TK mRNA was employed. No
signal above background was detected in the 17/tBTK lane.
In the right panel, a similar blot was hybridized to a probe specific
for UL24 mRNA. As predicted by the genomic structure of
17/tBTK, the 1.4-kb UL24 mRNA was not produced by the
mutant. This form of the UL24 mRNA is transcribed from an upstream
promoter that was disrupted by the insertion in the TK gene. Normal
levels of the 1.2-kb UL24 mRNA were produced by 17/tBTK.
This mRNA species, transcribed from the second of the three UL24
promoters, represents a minor portion of the total transcription from
this locus (15). The 0.9-kb UL24 mRNA was also readily detectable in the 17/tBTK RNA samples at levels similar
to those of the wild type.
The total amount of UL24 mRNA produced by 17/tBTK in RSC
cells at 5 h p.i. was reduced by the loss of the 1.4-kb mRNA form. This could result in a reduced amount of UL24 protein. Null mutants at
the UL24 locus form small plaques and replicate poorly in cultured cells (21). As detailed below, 17/tBTK plaqued
normally and replicated with wild-type efficiency in RSC monolayers and
therefore did not display a UL24 null phenotype. This suggests that
transcription of the 1.2-kb mRNA that encodes the entire UL24 ORF
and/or the 0.9-kb mRNA (which produces a smaller version of UL24) was
adequate to provide UL24 function.
Replication properties in vitro and in vivo.
Replication
kinetic experiments were performed under low- and high-MOI conditions
in RSC monolayers as described previously (61). Both
17/tBTK and 17/tBRTK+ replicated as
efficiently as the parental strain, 17syn+, in the cells
following low- or high-multiplicity infection (data not shown). To
examine the replication properties of 17/tBTK and
17/tBRTK+ in vivo, groups of mice were infected on the
scarified cornea or the shaved and abraded snout as described above
(44, 45).
and 17/tBRTK+ replicated to
higher titers on the snout than on the eye (Fig.
2A). On the snout, the replication of the
two isolates was equivalent for the first 4 days p.i., after which the
titer of the TK mutant dropped rapidly.
17/tBTK was detectable through day 7 p.i. and was
cleared by 9 days p.i. The titer of the TK+ isolate did not
drop significantly through day 7 p.i. and was still about
104 PFU/snout on day 9 p.i. Integration of the areas
under the curves revealed a two-fold decrease in the total PFU produced
by 17/tBTK through day 9 p.i.
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was reduced by
~12-fold by day 4 p.i., and no virus was detected on day 7 p.i. The TK+ variant replicated efficiently through day
9 p.i. A comparison of the areas under the curves revealed a
>12-fold decrease in the replication of the mutant through day 9 p.i. The decreased replication efficiency of TK-negative mutants on the
cornea has been previously documented (4, 57). Some reports
have suggested that TK-negative viruses replicate with wild-type
efficiency on the mouse eye (24, 25). It should be noted
that these studies examined replication only on day 2 p.i. At day
2 p.i., the titers of 17/tBTK and
17/tBRTK+ were equivalent, but the titer present in
17/tBTK-infected animals rapidly diminished after this time.
No infectious 17/tBTK was detected in the TG following
infection of the cornea. The TK+ rescued isolate replicated
to 105 PFU in these ganglia (Fig. 2A). Replication of the
TK mutant in TG was detected following infection via the
snout. In these ganglia, less than 50 PFU of 17/tBTK (<8
PFU/ganglion tested) was present at day 4 p.i. It should be noted
that to detect this virus it was necessary to plate the entire
undiluted tissue homogenate on an RSC monolayer in a single 60-mm-diameter tissue culture plate. In other similar experiments, the
replication of 17/tBTK was not detected in ganglia
following snout inoculation (data not shown). It was therefore unclear
whether the replication detected in this experiment represented a
limited amount of replication in all ganglia or productive infection in
a subset of the ganglia. To address this question, additional animals
were infected with the mutant or rescued virus. Total infectious virus
or total viral DNA content was determined at day 4 p.i. in the
relevant tissues of five animals per group. The results of this
analysis are shown in Fig. 2B.
As can be seen in Fig. 2B, the variation in virus titer was not great
in any tissue examined regardless of the infecting strain. Therefore,
the infection of these animals was highly reproducible. It can also be
seen that in most cases the amount of viral DNA in the tissue was
directly correlated with the amount of infectious virus present. In the
eyes and snouts, the ratio of HSV-1 genomes to PFU was 103
for both 17/tBTK and 17/tBRTK+. As expected,
on the eye, where the titer of infectious virus was reduced about
15-fold in mice infected with the TK mutant, the amount
of HSV-1 DNA detected was also reduced about 15-fold. Likewise, on the
snout, where 17/tBTK replicated about twofold less well
than the wild-type, the amount of DNA present was also reduced about twofold.
The HSV DNA/PFU ratio was ~102 in TG infected with the
wild type regardless of the infection route. This correlation between the infectious virus titer and the HSV-1 genome content was not seen in
the TG infected with 17/tBTK. No infectious virus
was found in any of the 10 ganglia from mice infected on the cornea.
These ganglia contained between 103 and 5 × 103 HSV-1 genomes. Likewise, only one of five pairs of
ganglia from mice infected with 17/tBTK by the snout
contained detectable virus (total, 3 PFU). However, all of these
ganglia contained significantly more viral DNA than those infected on
the cornea (1.4 × 104 to 8.0 × 104
HSV genomes).
Analysis of viral protein expression in neurons.
The preceding
analysis demonstrated that there was about 10-fold more viral DNA
present in the ganglia of mice infected by the snout with
17/tBTK than in ganglia of mice infected on the cornea.
Since viral replication was not detected in the majority of these TG,
the origin of these additional genomes was not clear. The increased
levels of viral DNA may have come from limited viral genome replication
within the ganglia or from the increased transport and uptake of viral genomes from the snout, where far more virus was produced (Fig. 2). It
is not currently practical to quantify viral genome replication on a
cellular basis. Therefore, an analysis of the expression of viral
proteins of defined kinetic classes was employed to investigate whether
the genome of 17/tBTK was replicated in a significant
number of neurons.
or 17/tBRTK+
on their abraded snouts, and on day 4 p.i. TG were harvested and
processed for the immunohistochemical detection of viral proteins as
described in Materials and Methods. As shown in Fig.
3A and B, there was a clear difference
between the numbers of HSV antigen-expressing cells in
17/tBRTK+ ganglia and in the 17/tBTK
group. In ganglia from mice infected with 17/tBRTK+,
viral-antigen-positive neurons and some associated satellite cells were
widespread and frequently clustered (Fig. 3A). The 17/tBTK ganglia were characterized by rare isolated
antigen-positive neurons (Fig. 3B). The number of HSV
antigen-expressing neurons was determined as described in Materials and
Methods. There were 30-fold-fewer anti-HSV-positive neurons per
ganglion profile in the 17/tBTK-infected ganglia than in
the 17/tBRTK+-infected ganglia, an average of
1.4 ± 0.1 and 44 ± 2.7, respectively (P < 0.0001; unpaired t test) (Table
1).
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-infected ganglia, most of the neurons
staining with the anti-HSV antibody also stained for ICP4 (Fig. 3C and
D). In the 17/tBRTK+-infected ganglia, 44 and 35% of the
anti-HSV-staining neurons expressed ICP5 and VP16, respectively (Table
2) (Fig. 3E and G). In contrast, while the number of ICP4 expressing
neurons in the 17/tBTK-infected ganglia was equal to the
number positive with anti-HSV, only extremely rare neurons expressed
either ICP5 or VP16 (5 and 2%, respectively) (Table 2) (Fig. 3F and
H). The differences in the relative percentages of positive neurons
expressing ICP5 and VP16 in 17/tBRTK+ and
17/tBTK were significant (P < 0.001;
Fisher's exact test). The numbers presented in Table 2 were generated
by counting all of the positive neurons on serial sections of the same
ganglia for each antibody. This was done to avoid any bias that would
be introduced by the regional variations within the ganglia of infected
cells. Thus, the results are not dependent on the identical neurons
being present in all of the serial sections of a given ganglion. Note
that while sections shown in Fig. 3F and H contain no positive neurons,
the insets show examples of the rare positive neurons in
17/tBTK-infected ganglia staining for ICP5 (Fig. 3F) and
VP16 (Fig. 3H).
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-infected
neurons. Consistent with previous reports of replication of
TK mutants in neural tissue, these findings suggest that
in most neurons the infection did not progress past the early stage of lytic viral gene expression (11, 24, 25).
Effect of the viral TK gene on percentage of neurons in which
latency was established.
A comparison was made of the efficiency
of the establishment of latency following inoculation of
17/tBTK or 17/tBRTK+ on the snout or on the
cornea. Additional mice from the groups infected above were maintained
for at least 30 days p.i. and processed for the quantification of
latently infected neurons by utilizing contextual analysis of DNA
(CXA-D) as described previously (40, 41, 43, 60).
Representative blots are shown in Fig. 4.
Two groups of three mice were analyzed for each virus and inoculation route. The numbers shown in Fig. 4 are totals from the individual groups analyzed. In all cases there was very close agreement
among groups: eye inoculations with 17/tBTK, 1.7%
(9 neurons positive of 531 tested) and 1.9% (12/635)
latently infected; eye inoculations with 17/tBRTK+, 22.5%
(25 of 111) and 23.5% (32 of 136); snout inoculations with
17/tBTK, 5.2% (15 of 291) and 4.8% (15 of 311); snout
inoculations with 17/tBRTK+, 27.4% (25 of 91) and 31.8%
(14 of 44).
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established
latency in 1.8% of the sensory neurons within the TG. Of the 1,166 neurons examined, only 21 were positive for the viral genome. The establishment of latency by the rescued virus 17/tBRTK+
was much greater, with 23.1% of the neurons harboring latent viral
genome (57 neurons positive of 247 tested; P < 0.0001;
Fisher's exact test). The frequency of latent infections established
by 17/tBRTK+ was indistinguishable from that established by
the wild-type strain, 17syn+, under similar conditions
(41, 60).
The number of latently infected neurons was significantly greater in
mice infected with 17/tBTK on the snout than in those
infected on the eye (P = 0.0003) but still less than
the number established in mice infected with 17/tBRTK+. In
17/tBTK-infected animals, 5% of the TG neurons harbored
latent virus (30 positive of 602 tested). In the
17/tBRTK+-infected animals, 29% of the TG neurons were
latently infected (P < 0.0001). The replication of
17/tBTK on the snout was reduced only about twofold
compared to that of 17/tBRTK+, and the percentage of
latently infected neurons was ~6-fold reduced. This suggests that
viral replication within the TG contributes significantly to the pool
of latent neurons. In addition, these data suggest that the extent of
replication at the body surface also impacts the number of latent sites
established within the TG.
Analysis of the HSV-1 genome copy number profile within individual
latently infected neurons.
With the CXA-D approach the number of
viral genomes present in individual latently infected neurons can be
determined to generate a profile of the latent HSV-1 genome copy number
(40, 43, 60). The amount of radioactivity present in each
positive sample discussed above was determined with ImageQuant software
and compared to that of a set of standard reactions run simultaneously
as described previously (40). The results are presented as a
scattergram in Fig. 5. Following corneal
inoculation with 17/tBTK, all positive neurons contained
very few HSV-1 genomes (average, 2.3 genomes/neuron; range, 1 to 9). By
comparison, those animals infected with 17/tBRTK+ displayed
a wild-type HSV-1 genome copy number profile (average, 28.4 genomes/neuron; P < 0.0001; Mann-Whitney test).
|
or 17/tBRTK+ were
indistinguishable. In 17/tBTK-infected mice, latent sites
contained between 1 and 250 latent genomes, with an average of 35.9 genomes/neuron. The genome copy numbers in 17/tBRTK+
latently infected neurons ranged between 1 and 300, with an average of
33.8 (P = 0.8224; Mann-Whitney test). TK
mutants do not efficiently replicate their genomes in ganglia (25). While limited 17/tBTK genome replication
within neurons that survive cannot be absolutely ruled out, the
immunohistochemical analysis presented above demonstrated that if this
occurred it was a rare event. Even so, viral DNA replication in rare
neurons could not result in a normal latent HSV-1 genome copy number
profile. Therefore, the multiple copies of the 17/tBTK
genome found in most of these latent sites most likely were transported to the neurons from the surface. This suggests that the efficiency of
viral replication at the body surface has a major impact on the latent
HSV-1 genome copy number, at least in the context of a TK null mutant.
| |
DISCUSSION |
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|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Our results support the following conclusions. First, the viral TK
gene is required for the establishment of latent infections at
wild-type frequency. There were 10-fold-fewer latent infections in the
TG of mice infected with 17/tBTK on the cornea compared
to the number established in the ganglia of mice infected with an
equivalent titer of the rescued isolate, 17/tBRTK+. In mice
infected on the snout, the TK-negative mutant established latency in
sixfold-fewer neurons. Second, viral replication within the TG is
required to establish latent infections at wild-type frequency. While
17/tBTK replicated significantly less well on the mouse
cornea, its replication on the mouse snout was nearly equivalent to
that of 17/tBRTK+. This increased surface replication
resulted in a 2.5-fold increase in the number of latent sites. This
number was still significantly less than the number in ganglia infected
with 17/tBRTK+, which replicated in the TG as well as on
the surface. Third, viral replication within ganglia, and in particular
viral DNA replication within sensory neurons, is not required to
establish latent sites that contain many copies of the HSV-1 genome.
Following infection on the snout, 17/tBTK established a
latent pool with a normal HSV-1 DNA genome copy number profile. Some of
these neurons contained hundreds of copies of the HSV-1 genome.
However, an analysis of viral replication within the TG, and of viral
protein expression in neurons, demonstrated that DNA replication did
not occur in most of the cells that expressed viral protein. In
addition, the number of cells that expressed any detectable viral
proteins at the peak of lytic infection was several orders of magnitude
less than the number of cells in which latency was established.
The viral TK gene is required for the efficient establishment of latency. From the earliest studies with viral TK-negative or -deficient mutants it was apparent that TK is required for reactivation from latency. An early hypothesis suggested that TK was absolutely required for the establishment of latent infections (55). However shortly after the LAT RNAs were discovered, several reports proved that TK-negative mutants could establish latent infections as measured by the detection of LATs by in situ hybridization (4, 28, 56). Work by Leist and colleagues suggested that TK-negative mutants could establish latent infections with near-wild-type efficiency in mouse DRG (28). Coen et al. demonstrated that TK null mutants could establish latent infections in mice following corneal inoculation (4).
Based on these observations it has been hypothesized that the role of TK in latency is primarily to facilitate reactivation and not to enhance the establishment of latency (for a review, see reference 55). However, our finding that TK is required for the efficient establishment of latency is consistent with several other studies. Leist et al. found a reduced number of LAT-positive DRG neurons in mice infected with a TK mutant (28), as did Slobedman et al. (50). In mouse TG, Katz and colleagues have also demonstrated a 100-fold decrease in the amount of latent HSV DNA present in mice latently infected with the TK-negative mutant (22). This is consistent with the 10-fold reduction in the frequency of latent sites multiplied by the 10-fold reduction in genome copy number we report here.Viral replication on the surface and its relationship to the number of latent sites established. These results are best viewed in the context of the physical relationship between the inoculation site and the site of latency. The axonal endings of sensory neurons project to the body surface, where many display significant dendritic branching (5, 7, 58). This network of neuronal endings is the portal for HSV to the neuronal cell bodies, where latency is established (for a review, see references 13 and 64). Therefore, it is likely that parameters that determine the number of infected neurons within the ganglia include (i) the total area of viral replication at the body surface, (ii) the efficiency of virion production, and (iii) the innervation density. Thus, the correlation between surface replication and the number of latent infections established is not surprising if more efficient replication results in a greater area of virus production and/or an increased density of virions at the site of infection. However, it should be noted that with the rescued isolate, 17/tBRTK+, the 100-fold increase in virion production on the snout compared to that on the cornea resulted in only a modest increase in the percentage of latently infected cells (from 23 to 28%). It seems most likely that replication within the TG is a dominant factor in determining the number of latent sites established. It is also possible that virus entry into the nervous system is not the same at these two inoculation sites.
Viral replication within ganglia and its relationship to the number of latent sites established. The mechanism underlying the correlation between viral replication in the ganglia and the increased number of latent sites is not straightforward. It is possible that lateral spread of virus within the ganglia increases the number of latent sites. While there is evidence that satellite and support cells are relatively nonpermissive and may limit spread within ganglia (16, 65), the focal distribution of acutely infected neurons in ganglia (Fig. 3) is consistent with such spread. This is a likely potential mechanism for an increase in the number of infected neurons.
Ganglionic viral replication may also serve to increase the area of the body surface that supports virus replication. It is readily apparent from the zosteriform spread model that lytic replication within neurons results in anterograde transport (32, 38, 47, 48). The arborizing nature of sensory innervation at the surface could result in the deposition of virions at a surface site near, but spatially distinct from, the initial infection site (5, 7, 58). Alternatively, lateral spread to new neurons within ganglia and subsequent anterograde transport would result in expansion of the area of surface to include additional surface regions innervated by the infected ganglia. These new sites of infection can be spatially quite distant from the initial infection site. For example, evidence has been presented that following infection of the mouse snout, virus is transported through the nervous system and infects the eye (2, 31). Replication at such new sites of infection and subsequent cell-to-cell spread at the surface would lead to viral uptake into the neurons that innervate the extended surface area. Repetitive cycles of neuronal infection and spread at the surface would be expected until specific immune mechanisms become inhibitory (31, 47). Thus, it is likely that an increase in the infected surface area mediated by ganglionic viral replication plays a major role in increasing the number of latent sites established. In support of this we have noted that increasing the area of the inoculation site at the surface increases the number of both lytically and latently infected neurons within TG (unpublished observations).Viral thymidine kinase-mediated DNA replication and its
relationship to HSV latent genome copy number.
In the cornea
infection model, the mutant 17/tBTK established latent
infections of universally low HSV-1 genome copy number (<10
genomes/latently infected neuron), with an average of 2.3 genomes/latent site. In contrast, 17/tBRTK+ established
latent sites that contained between 1 and 1,000 HSV genome copies, with
an average of 28.4. Importantly, the HSV-1 genome copy number profile
of 17/tBTK and 17/tBRTK+ were
indistinguishable following snout inoculation. Consistent with previous
reports (11, 24, 25), infectious virus was not detected in
most ganglia of 17/tBTK-infected mice and when detected
it was just a few PFU. In addition, 1,000-fold less viral DNA was
present in these ganglia during the acute phase of infection
(25) (Fig. 2B). The possibility that some very limited
replication of the 17/tBTK genome occurred in neurons
that subsequently entered the latent state can not be absolutely ruled
out. Indirect evidence that some neurons which express detectable viral
lytic-phase proteins may survive to enter latency has been presented
(49). However, our analysis of viral protein expression in
neurons strongly suggested that the 17/tBTK genome was
not replicated in all but a very few neurons. HSV viral-protein-positive neurons were comparatively rare, and very few
neurons positive for ICP5 or VP16 were detected (Fig. 3 and Tables 1
and 2). Therefore, the most likely origin of the multiple genome copies
present in neurons latently infected with 17/tBTK is that
they were generated at the surface and transported to neurons.
and
17/tBRTK+ occurred after day 2 p.i. This suggests that
viral replication at the surface after this time is important to
increase the number of latent sites established and, importantly, to
increase the HSV-1 copy number within these latent sites. A hypothesis
as to when and how high-genome-copy-number latent sites are established and/or qualitatively modified in terms of viral DNA content must therefore account for the importance of surface viral replication after
day 2 p.i.
One factor that could regulate the outcome of a neuron's encounter
with HSV is the MOI. Although unproven, it seems likely those neurons
infected by a large number of virions would be prone to enter lytic
replication, whereas those receiving one or a few virus particles might
enter the latent state. The HSV-1 LAT gene has been shown to be
important for the establishment of latency (44, 60) and may
function to downregulate viral gene expression (1, 44).
Neurons that initially enter the latent pathway with a few viral genome
copies may begin to express the LAT locus. The entry of virus into a
given neuron can be protracted over many days. Thus, these LAT-positive
(and therefore less permissive) neurons may be superinfected, resulting
in an increased number of latent genomes. Since the replication of
17/tBTK on the mouse eye rapidly diminished after day 2, the probability of superinfection was reduced. The more productive and
prolonged replication of this mutant on the snout may have permitted
superinfection and a resulting increase in latent genome copies. This
hypothesis predicts that LAT null mutants would establish latent
infections that contain reduced viral genome copy numbers. Our
preliminary findings support this hypothesis.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by Public Health Service grant AI32121 from the National Institute of Allergy and Infectious Diseases.
We thank Anthony Minson for providing antibody LP-1 and C. Tansky and S. Goins for technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address for R. L. Thompson: University of Cincinnati Medical Center, Department of Molecular Genetics, Microbiology, and Biochemistry, 231 Bethesda Ave., Cincinnati, OH 45267-0524. Phone: (513) 558-0063. Fax: (513) 558-8474. E-mail: Richard.Thompson{at}UC.edu. Mailing address for N. M. Sawtell: Division of Infectious Diseases, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, Ohio 45229-3039. Phone: (513) 636-7880. Fax: (513) 636-7655. E-mail: Sawtn0{at}CHMCC.org.
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Materials and Methods
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Journal of Virology, January 2000, p. 965-974, Vol. 74, No. 2
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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