JOURNAL OF VIROLOGY, May 2010, p. 5303–5313
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 10
Sequence Variability in Clinical and Laboratory Isolates of
Herpes Simplex Virus 1 Reveals New Mutations?†
Moriah L. Szpara,1Lance Parsons,2and L. W. Enquist1*
Department of Molecular Biology1and Lewis-Sigler Institute for Integrative Genomics,2
Princeton University, Princeton, New Jersey 08544
Received 9 February 2010/Accepted 4 March 2010
Herpes simplex virus 1 (HSV-1) is a well-adapted human pathogen that can invade the peripheral nervous
system and persist there as a lifelong latent infection. Despite their ubiquity, only one natural isolate of HSV-1
(strain 17) has been sequenced. Using Illumina high-throughput sequencing of viral DNA, we obtained the
genome sequences of both a laboratory strain (F) and a low-passage clinical isolate (H129). These data
demonstrated the extent of interstrain variation across the entire genome of HSV-1 in both coding and
noncoding regions. We found many amino acid differences distributed across the proteome of the new strain
F sequence and the previously known strain 17, demonstrating the spectrum of variability among wild-type
HSV-1 proteins. The clinical isolate, strain H129, displays a unique anterograde spread phenotype for which
the causal mutations were completely unknown. We have defined the sequence differences in H129 and propose
a number of potentially causal genes, including the neurovirulence protein ICP34.5 (RL1). Further studies will
be required to demonstrate which change(s) is sufficient to recapitulate the spread defect of strain H129.
Unexpectedly, these data also revealed a frameshift mutation in the UL13 kinase in our strain F isolate,
demonstrating how deep genome sequencing can reveal the full complement of background mutations in any
given strain, particularly those passaged or plaque purified in a laboratory setting. These data increase our
knowledge of sequence variation in large DNA viruses and demonstrate the potential of deep sequencing to
yield insight into DNA genome evolution and the variation among different pathogen isolates.
Herpes simplex virus 1 (HSV-1) is among the most wide-
spread pathogens of the herpesvirus family, with about 60%
seroprevalence, indicating exposure or ongoing infection,
among adults in the United States (83). HSV-1 infection be-
gins at epithelial surfaces but can progress to the peripheral
nervous system, where a lifelong latency is established in neu-
rons (60). HSV-2 is closely related and presents a major public
health concern in developing nations, where it is a risk factor
for the acquisition of HIV/AIDS (10, 23). Despite the clinical
importance of these viruses, only one wild-type genome se-
quence is available for HSV-1, that of strain 17, which was
completed over 20 years ago (41, 42). Remarkably, most of our
understanding of HSV-1 biology comes from experiments uti-
lizing just a few common laboratory strains or recent clinical
isolates. The only other HSV-1 genome sequence published in
the last 2 decades is that of HF10, an oncolytic mutant strain
harboring several large genomic deletions and rearrangement
relative to the reference strain 17 (78). Since HF10 was itself
derived from the nonneuroinvasive and highly attenuated
strain HF, the HF10 genome is informative for mutation-based
variation but provides little insight into the sequence variation
of virulent strains (45, 70). Several studies of specific genes or
genomic regions cloned in Escherichia coli have shed more
light on interstrain variation in HSV-1, but these studies can-
not address variation on a genome-wide scale encompassing
every protein in the HSV genome (48, 57, 74, 75). High-
throughput sequencing techniques have the potential to ad-
dress the entire genome of a population without resorting to
recombinant DNA techniques and have already enabled
substantial inroads into novel pathogen discovery and the
genetic characterization of other viruses and pathogens (13,
34, 54, 79, 81).
The genome of HSV-1 is a large double-stranded DNA
molecule of 152 kb, with a G/C content of 68%. The HSV
genome contains 77 annotated protein-coding sequences, ar-
ranged into two unique regions, each of which are flanked by
long terminal repeats (9.2 kb and 6.6 kb) (genome diagram in
Fig. 1A). In addition to these large repeats, the genome also
contains small microsatellite repeats (?100 bp each) and short
tandemly reiterated sequences (?500 bp each), also known as
variable-number tandem repeats (VNTRs) (14, 41, 42). The
large terminal repeats contain a higher concentration of
VNTRs and a lower percentage of coding regions than elsewhere
in the genome. The VNTRs are highly variable, with the num-
ber of repeated units varying both between strains and during
replication and repeated passages of the same strain (48, 74–
76). The large number of mononucleotide repeats in the
HSV-1 reference genome suggested that Illumina’s deep se-
quencing technology, which detects single bases at a time by
using reversible chain termination chemistry, would be a useful
technology for sequencing these genomes (14, 36).
Historically, comparisons of phenotypic and genotypic vari-
ations among strains or species of related organisms have pro-
vided significant insights to the field of genetics. Similarly,
comparison of complete herpesviral genome sequences of clin-
ical and laboratory isolates would greatly facilitate studies of
sequence variation and conservation. Significant progress has
* Corresponding author. Mailing address: 314 Schultz Laboratory,
Princeton University, Princeton, NJ 08544. Phone: (609) 258-2415.
Fax: (609) 258-1035. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 10 March 2010.
already been demonstrated for varicella-zoster virus (VZV),
Marek’s disease virus (MDV), and human cytomegalovirus
(HCMV) (8, 12, 53, 58, 66, 67, 72, 85). Sequence analysis can
be used to highlight the most conserved, and thus functionally
important, domains of proteins, as well as to identify likely
regulatory regions in intergenic areas, based on their sequence
conservation in the absence of coding pressure. Sequencing the
entire genomes of HSV-1 strains with interesting phenotypes
will also allow identification of putative causative mutations
more comprehensively than single-gene cloning approaches.
The unique HSV-1 H129 strain presents one such opportunity;
it is the only virus known to transit neural circuits exclusively in
an anterograde or forward direction, a finding that has been
confirmed in both rodent and primate models (69, 86). H129
FIG. 1. Overview of genome coverage for HSV-1 strains F and H129, relative to strain 17 or to their own newly assembled genomes.
(A) Diagram of the HSV-1 genome structure, with two unique regions (long and short), each flanked by a pair of terminal repeats. The line graph
depicts the depth of sequence read coverage for the newly sequenced strains when each one is aligned to the reference, strain 17. The VNTRs,
or reiterations, of strain 17 are overlaid as green lines onto the coverage graphs. Reductions in coverage correlate with the reiterated sequences
and also occur more frequently in the terminal repeats that flank each unique region. Brown arrowheads highlight a dip in both new genomes when
aligned against strain 17; this is the location of an insertional frameshift in the UL17 gene of strain 17 that leads to alignment mismatch in the
nonframeshifted strains F and H129. (B and C) Colored boxes depict the blocks of continuous sequence, or contigs, assembled for each strain.
Boxes of the same color represent the same contig; these appear twice when they include sequence in the repeat regions. Sites of reiterations
(VNTRs) are overlaid in green. In some cases contigs assembled through a reiteration; in others the contigs terminated at the reiterations. The
line graph below the contigs for each strain depicts the improvement in depth of sequence read coverage when data from strain H129 were aligned
to the newly assembled H129 genome (B). A similar graph is depicted for strain F (C). Sharp drops in alignment coverage remain at the VNTR
sequences inserted from strain 17.
5304 SZPARA ET AL. J. VIROL.
was isolated from the brain of an encephalitic patient in 1977,
and the limited molecular characterizations thus far have not
shed light on any mutations to explain its unique phenotype
(17, 30, 33). The distinctive spread characteristics of this strain
makes it of great interest to the neuroscience community,
where it is used as a directional neural circuit tracer whose
spread is complementary to retrograde-limited tracing viruses,
such as the attenuated pseudorabies virus (PRV) strain Bartha
and various rhabdoviruses (3, 19, 24, 59, 73).
We demonstrate here the successful use of Illumina deep
sequencing technology and subsequent analyses to determine
the genome sequences of both the unique clinical isolate
HSV-1 H129 and a widely used laboratory isolate (strain F).
These strains differ in pathogenicity from the previously se-
quenced strain 17. After peripheral inoculation into mice,
strain 17 has a 50% lethal dose (LD50) of 103PFU, while the
LD50of strain H129 is 105PFU and for strain F it is ?107PFU
(17, 55). Our data demonstrate the extent of variation between
these strains across the entire genome of HSV-1, in both cod-
ing and noncoding regions. We found many protein-coding
variations between strain F and the current genome reference
strain 17 by which we can begin to define the spectrum of
variability among wild-type HSV-1 isolates. We have fully de-
fined the sequence differences in the unique anterograde
spread mutant strain H129 and propose a number of poten-
tially causal genes, including the neurovirulence protein
ICP34.5 (RL1). Unexpectedly, our data also revealed a frame-
shift mutation in the UL13 kinase in our isolate of HSV-1
strain F. This protein is dispensable in cell culture but is re-
quired for virulence and spread of infection in animal models
(11, 51, 71).
MATERIALS AND METHODS
Virus stocks. HSV-1 strain F was originally isolated from a facial lesion and
maintained as a low-passage stock by B. Roizman and colleagues (18). We
received an aliquot from B. Roizman, which was passaged once in Vero cells and
then subjected to three rounds of plaque purification. HSV-1 strain H129 is a
low-passage clinical isolate received from Richard Dix (17); it is maintained as a
low-passage stock. All viral stocks were grown on monolayers of confluent Vero
(monkey kidney) cells (ATCC cell line CCL-81).
Nucleocapsid DNA preparation. Viral nucleocapsid DNA was isolated as
previously described (63). Briefly, confluent monolayers of Vero cells were in-
fected at a multiplicity of infection of 5 and harvested by scraping at 24 h
postinfection. Cell pellets were rinsed, resuspended, subjected to two rounds of
Freon extraction, and pelleted through a glycerol step gradient. Viral nucleo-
capsids were then lysed using SDS and proteinase K, extracted twice with phenol-
chloroform, and ethanol precipitated. Viral DNA was collected by a glass hook,
blotted dry, and resuspended in Tris-EDTA (10 mM Tris, pH 7.6; 1 mM EDTA).
Illumina sequencing. Five-microgram aliquots of HSV-1 strain F and H129
nucleocapsid DNA were processed for sequencing by the Microarray Core Fa-
cility at Princeton University’s Lewis-Sigler Institute for Integrative Genomics.
Two independent sequence libraries were generated by following the manufac-
turer’s protocol for sequencing of genomic DNA (Illumina genomic DNA sam-
ple prep kit; protocol part 1003806, revision A), with the slight modification that
the column for gel purification was not heated (56). Sequencing was carried out
using two lanes of a standard flow cell, using Illumina’s standard cluster gener-
ation and 36-cycle sequencing kits. The Illumina genome analyzer 2, with SCS 2.3
software, was run for either 36 (one H129 run) or 75 (all other runs) cycles of
data acquisition. Image analysis and base calling were performed using the
Illumina Pipeline v1.3 under default settings.
De novo and reference-guided assembly. De novo assembly of the short reads
was performed to generate new HSV-1 genomes from the sequence data, fol-
lowed by a reference-guided assembly of the resulting blocks of contiguous
sequences, or contigs. The short sequence reads were first passed through a
series of computational filters that removed (i) mononucleotide sequences, (ii)
host sequence contamination, and (iii) low-quality sequence. For step i, se-
quences that consisted of a single nucleotide or a single nucleotide with some N
(noncalled) bases were removed. (Step ii) Since virus stocks were prepared on
Vero cells, it was critical to identify and remove host DNA sequences. Because
the vervet monkey (Vero cell parent) genome sequence is not known, the se-
quence data were mapped to the human genome (version 36) using the Mapping
and Alignment with Qualities (MAQ) software package (32). Sequences homol-
ogous to human DNA varied from 0.2 to 15% of the data (see Table S1 in the
supplemental material); these were considered host contamination and removed
from the analysis. (Step iii) The sequences were then quality trimmed using a
modified version of the quality-trimming script supplied with the SSAKE assem-
bler (80). The process of quality trimming removed terminal bases below a
quality of 10 and then removed any sequences whose overall resulting length was
less than 20 bases. The 36-bp sequencing run for strain H129 (versus all others,
of 75-bp length) thus resulted in a net smaller number of sequences for de novo
assembly of strain H129 versus strain F. After these filtering procedures, the
SSAKE short read assembler was used to assemble the short sequences into
contigs, using default parameters.
Reference-guided assembly of the best contigs yielded the final reference
sequence. Those that were at least 100 bp long and had an average sequence
depth, or coverage, of at least 100 sequence reads were passed to the long read
assembler MINIMUS (65). All blocks of assembled sequence were surveyed by
BLAST to check for erroneous ends, and the most parsimonious and best-
supported sequence was accepted when there was disagreement at the ends of
joined segments (2). The resulting blocks of sequence, along with any contigs that
MINIMUS was unable to assemble further, were aligned to the strain 17 genome
using BLAST. The BLAST alignments provided guidance to position blocks of
sequence along the genome. Rarely, short mononucleotide runs caused BLAST
to place a contig at discontinuous locations. These anomalous breaks were
examined and accepted if supported by data from adjacent blocks of sequence.
The light orange and light green contigs on the right end of the strain H129
genome are one such example (Fig. 1; labeled minimus2_1 in GenBank and
Genome Browser). BLAST also allowed us to place data from assembled blocks
of sequence into both repeats when relevant (TRL/IRL and TRS/IRS); this can
be seen in Fig. 1 where contig colors match in the repeats.
Short reiterations, or VNTRs, are highly variable in length in both genomic
DNA preparations and in cloned DNA, making their assembly a challenge (37,
46, 72, 76, 77). In Illumina sequencing, the average number of repeats in a
population of DNA can be accurately estimated by de novo assembly only if the
short reads contain unique flanking sequence on one or both ends. This ability is
limited by the read length (75 bp in this case). The SSAKE program defaults to
assembling the shortest possible number of repeating units supported by the
sequence data and may thus underestimate the VNTR lengths for those exceed-
ing 75 bp. As was done for the currently available HSV-1 strain 17 transgenic
bacterial artificial chromosomes (BAC) sequence (accession number FJ59328),
we marked reiterations of uncertain lengths as such and expanded them to match
the published length of the original strain 17 reference sequence. This was done
for the following VNTRs: the a? reiterations, reiterations 1 and 4 in the long
repeats, reiterations 1 to 3 in the short repeats, the UL reiteration in UL36, and
the US reiteration 1. The exact boundaries of these VNTRs are annotated in the
GenBank nucleotide sequences for the corresponding accession numbers for
these genomes and are also visible at our genome browser, http://viro-genome
Coverage analysis by alignment to reference and new genomes. MAQ was
used to align short Illumina reads against the NCBI HSV-1 genome of strain 17
(RefSeq NC_001806) (44). The default parameters were used to produce an
alignment file as well as a consensus sequence. From the consensus, the SNPfilter
command was used with default parameters to filter out false-positive single-
nucleotide polymorphisms. Once a new genome was assembled for strains H129
and F, the reads were realigned to the new self-genome by using MAQ and
analyzed as above.
Determining DNA and amino acid variation. To determine overall DNA
sequence variation, we aligned each pair of genomes using BLAST and compiled
a list of differences using the MUMmer sequence analysis package (15). For
amino acid variation, we used BLAST to align each piece of coding sequence
from the strain 17 reference to the new genome. These coding sequence loca-
tions (see GenBank accession nos. GU734771 and GU734772 for exact posi-
tions) were used to generate amino acid translations from the new genome. Each
new amino acid sequence was aligned to the corresponding strain 17 protein
sequence by using BLAST, and differences were compiled as described above
(see Table S2 in the supplemental material). Finally, DNA sequence differences
in each coding region were tallied as above; these included both silent mutations
and nonsynonymous changes that led to protein-level differences (see Table S3).
VOL. 84, 2010SEQUENCE VARIATION IN HSV-1 GENOMES: F, H129, AND 175305
For both DNA and amino acid comparisons, we counted both the total number
of changes (e.g., three changes in a row were counted as three) and the number
of noncontiguous change events (e.g., three changes in a row were counted as
one change event).
PCR analysis of UL13 mutations. PCR for UL13 used the following primers:
forward, CTTACCGAGGTCCATGTCGT, and reverse, CTTTCTAACCGCA
CACCGAC. PCR products were not cloned but were directly sequenced using
internal primers, either CAGTTGGACTTCGCCGTATC in the forward direc-
tion or CTGGTCATGTGGCAGCTAAC in the reverse. This technique allowed
detection of a mixed population when present.
Nucleotide sequence accession numbers and online data repositories. Ge-
nome sequence data and all annotations described in the manuscript have been
deposited at GenBank under accession numbers GU734771 for strain F and
GU734772 for strain H129. Annotations include the locations of genes, coding
sequences (CDS), repeats, and reiterations. Boundaries of the contiguous se-
quence blocks (contigs) used to assemble each genome are also included so that
the boundaries can be reviewed by future users. Raw sequence reads have been
deposited at the NCBI Sequence Read Archive (SRA) under accession numbers
SRA010802.1 for strain F and SRA010966.2 for strain H129. These data are all
linked under NCBI Genome Project ID 43419. These data can also be viewed at
an interactive genome browser at http://viro-genome.princeton.edu. This site
includes data from this paper that were not incorporated by GenBank, such as
sequence coverage depth maps for each genome (Fig. 1), histograms of sequence
differences per 100 bp (see Fig. 2, below), and the location of insertions, dele-
tions, and single-nucleotide changes on each sequence relative to the reference
strain 17. Users can view data at the whole-genome scale or investigate the same
features at the level of individual genes (see, for example, Fig. S2 in the supple-
High-throughput sequencing of two viral genomes. Nucleo-
capsid DNA was used as the source material for high-throughput
deep sequencing of two new HSV-1 genomes. Two separate se-
quencing runs were carried out for each strain, providing a total
of 17.7 million short sequence reads for H129 and 14.1 million
for F (see Table S1 in the supplemental material). To provide
a general outline of genome coverage, we used MAQ software
to align these reads against the only currently available wild-
type HSV-1 genome of strain 17 (NCBI record NC_001806)
(32). This technique revealed an average coverage depth of
over 1,000 sequence reads per base pair in the unique regions
of the genome and revealed much lower and more variable
coverage depth in the terminal repeats that flank each unique
region (Fig. 1A). This variable coverage reflects more base
changes, insertions, and/or deletions in the repeat regions of
the new strains, relative to the reference sequence. Since align-
ment approaches are not well equipped computationally to
handle insertions, deletions, and repetitive sequences (22, 32),
we used de novo sequence assembly as a productive alternative
De novo assembly and reiterated sequences. In de novo as-
sembly, short sequence reads are assembled into larger blocks
by using overlapping stretches of homology between the reads.
This technique produces longer stretches of continuous se-
quence, termed contigs. To improve the de novo assembly
process, we identified and removed host DNA sequences that
always contaminate viral DNA preparations. Host sequences
amounted to 0.2 to 15% of the data (see Table S1 in the
supplemental material). We used BLAST to order the assem-
bled contigs along the reference genome. Many of these se-
quence blocks terminated at the VNTRs, or reiterations, found
throughout the HSV-1 genome (Fig. 1B and C) (2). We note
that all currently available high-throughput methods for se-
quence determination are unable to identify the length of a
VNTR unless the VNTR is within the actual sequence read
length (12, 22, 36). Among these data, only imperfect reitera-
tions or those less than the sequence read length of 75 bp could
be accurately sized by the presence of unique flanking se-
quence. Despite this, we were able to assemble the entire
genome as follows: we verified that the longer reiterations
contained sequence of the same repeating units, and then we
extended the VNTR length to match the number in the cur-
rently published reference strain 17 (see Materials and Meth-
ods for a list of expanded VNTRs). This method provides as
much consistency as possible in overall gene positions and
genome length. In summary, the new genome sequence assem-
bled for strain F is 152,151 bp, while that of strain H129 is
152,066 bp, both of which are similar to the length of strain 17
at 152,261 bp (see Fig. S1 in the supplemental material).
To confirm the accuracy of these new genome assemblies,
we realigned all of the sequence reads for each strain, and this
time we used the appropriate self-genome as an alignment
guide. This method revealed a more consistent, high level of
coverage across the genome, with significant reduction in cov-
erage only at the VNTRs where proxy sequence was inserted
from strain 17 (Fig. 1B and C). For strain F, 97.6% of the
nonreiteration portions of the genome have 100-fold or greater
sequence coverage and 95.6% have 1,000-fold or greater depth
of coverage. For strain H129, 97.5% of the nonreiteration
portions of the genome have 100-fold or greater sequence
coverage, with 93.4% of that at a coverage of 1,000-fold or
greater. The slightly lower coverage depth of strain H129 is
because one of the two sequencing runs had a shorter read
length, 36 bp, instead of the 75-bp length used for all other
runs. Even data from this short read data set could be assem-
bled into high-quality sequence. These newly assembled ge-
nomes were next used to assess DNA-level sequence variation
across the genome.
DNA-level sequence variation. Variation among viral ge-
nomes reflects the processes of mutation and recombination.
Subsequent selection pressures fix these changes in popula-
tions, and these pressures vary during replication in vivo and in
vitro. High-throughput sequencing is especially well suited to
reveal the full extent of overall genome variation between
strains, because it comprehensively surveys the entire genome
sequence in a given population of DNA. In pairwise align-
ments of each new genome against the reference, we found
that strain F had 961 bp changes relative to strain 17, while
H129 had 943 bp changes relative to strain 17 (Fig. 2A and B;
the figure shows changes by base type, A, C, G, and T) (see also
the summary in Fig. S1 of the supplemental material). Gaps
are created in the alignment whenever one strain has an inser-
tion or deletion relative to the reference strain. For strain F,
there were 332 bp of insertions and 431 bp of deletions relative
to strain 17. Strain H129 had 298 bp of insertions and 496 bp
of deletions relative to strain 17. Overall, these nucleotide
differences are dispersed throughout the genome, with a
slightly greater concentration of differences in the repeats rel-
ative to unique regions (Fig. 2A and B).
We also examined the number of evolutionary change events
in the DNA sequences, where contiguous variations, such as
deletions of several bases in a row, are considered one event.
These change events were examined for intergenic regions,
coding sequences, and untranslated regions (UTRs). Not sur-
5306 SZPARA ET AL.J. VIROL.
prisingly, the lowest rate of change from the strain 17 reference
was found in coding regions, where evolutionary pressure is
likely highest: six changes per kb in strain F or five per kb in
H129. In contrast, both new strains had three times more
changes (15/kb) in intergenic regions and a similarly high rate
in the UTRs (17/kb in F and 18/kb in H129). If we analyze the
large terminal repeats separately from the rest of the genome,
the most noticeable changes emerge in the intergenic repeat
regions, where the differences from the reference strain are at
17 per kb for both F and H129, versus just 10 (H129) or 11 (F)
per kb for intergenic, nonrepeat regions. However, all of these
changes together represent a ?1% deviation from the HSV-1
strain 17 genome sequence, indicating a high degree of overall
DNA sequence conservation among these three strains. We
likewise found that the relative positions of the open reading
frames are similar in all three genomes (Fig. 2C). Although the
positions of these coding sequences are largely conserved, we
next addressed the conservation and variation of the resulting
Amino acid coding-level changes. To a first approximation,
selection for function leads to maintenance of sequence fidel-
ity. Therefore, we determined which of the base pair changes,
insertions, and deletions affected the coding sequence. Overall,
we found 310 amino acid differences between wild-type strains
F and 17 and 281 amino acid differences between H129 and the
reference strain 17 (summarized in Fig. S1 of the supplemental
material). Strains F and H129 have fewer overall amino acid
differences with each other, totaling 231 across the proteome.
These amino acid differences occur throughout the comple-
ment of 77 proteins encoded by HSV-1 (Fig. 3) and can be
categorized as changes where strains F and H129 share the
same amino acid residue with each other, but differ from strain
17, versus those positions where only strain H129 or strain F
has a unique amino acid relative to the other two strains (see
Table S2 for a full list of all amino acid differences for each
protein). In a prior analysis using a limited number of genes,
Norberg and colleagues found that strain 17 and strain F were
divergent enough to fall into distinct clades (47, 48). Since
these clades are distinguishable based on restriction digest
patterns in the US4 and US7 genes (48), we applied this ap-
proach and found that strain F and strain H129 fall into the
same clade, while strain 17 does not (data not shown). This
similarity in clade may reflect the fact that strains F and H129
were both isolated from patients in the United States, while
strain 17 was isolated from a Scottish patient (17, 18, 42).
FIG. 2. DNA-level variation across the HSV-1 genome. (A) Bar graph summarizing the number of base pair differences for strain H129 relative
to reference strain 17, with differences averaged over a window of 100 bp. The x axis corresponds to the location along the HSV-1 genome (marked
in kbp in panel C). The y axis is plotted on a log scale to make small amounts of difference visible on the graph. Green boxes above the bar graph
show the location of the terminal repeats, to highlight the increased DNA sequence variation in these areas. Rows of vertical lines depict base pair
variations across the genome, with variation separated out by indels (insertions or deletions), and single nucleotide changes. Single base changes
are further subdivided by the identity of the base in the new strain: G, C, T, or A. (B) Similar bar graph and statistics for strain F. (C) Location
of coding sequences along the genome of HSV-1, which encodes 77 proteins. Plus- and minus-strand-encoded genes are drawn above and below
the genome position line. Repeats are denoted by green boxes. Horizontal lines connect the coding sequence boxes of the only spliced transcripts
in HSV-1: that of ICP0 (RL2), which is found in the long repeats TRL and IRL, and UL15, which is found around 30 kbp.
VOL. 84, 2010SEQUENCE VARIATION IN HSV-1 GENOMES: F, H129, AND 175307
Complete coding sequence conservation. The analysis of
amino acid differences across the HSV-1 proteome revealed 10
genes with complete conservation across strains F, H129, and
17: the capsid protein UL35; tegument protein UL16; the
envelope protein UL20 and glycoproteins gK (UL53) and gJ
(Us5); and the nonstructural proteins UL15, UL31, UL45,
UL55, and ICP22 (Us1). These proteins vary in coding se-
quence length, from 92 amino acids for glycoprotein J (US5) to
FIG. 3. Coding sequence variation across the HSV-1 proteome. Histogram depicting the total number of amino acid differences observed in
strains F and H129, relative to the reference strain 17, for every protein in the HSV-1 genome. Changes are color coded to depict the proportion
of changes that are different from strain 17 but common between strains F and H129, versus those that are unique to strain F or H129. Proteins
are listed in spatial order of occurrence along the HSV-1 genome. Names of the five immediate-early genes, expressed first upon herpesvirus
infection of a host cell, are highlighted in yellow (ICP0, ICP27, ICP4, ICP22, and ICP47). Genes in the repeats, ICP34.5 (RL1), ICP0 (RL2), and
ICP4 (RS1), are each listed only once, at their first position of occurrence. Ten genes have no amino acid changes at all. Protein functions are
summarized to the left of each gene name.
5308SZPARA ET AL.J. VIROL.
735 amino acids for the DNA terminase subunit protein UL15,
indicating that sequence length is not the primary criterion for
complete amino acid conservation. Several of the genes in this
group are known to be dispensable for growth in cell culture,
such as UL20, gK (UL53), ICP22 (Us1), gJ (Us5), UL45, and
UL55, but their conservation suggests an evolutionary advan-
tage to preserving their functions.
Amino acid differences unique to the mutant strain H129.
Although the complete conservation of coding sequences
across these strains is noteworthy, we were particularly inter-
ested in deducing the likely mutations behind the unique an-
terograde spread phenotype of the clinical isolate strain H129.
Rather than the typical HSV-1 bidirectional spread from in-
fected neurons, H129 appears to only exit via axonal connec-
tions from the presynaptic to postsynaptic cell, producing an
overall phenotype of exclusively anterograde-directed spread
along neural circuits in vivo. We searched for amino acid
changes unique to H129 relative to both reference strain 17
and to the newly sequenced strain F, to uncover the mutations
responsible for this directional spread phenotype. We first
examined genes with the largest number of amino acid changes
overall and highlighted those with many changes unique to
strain H129 (Fig. 4A). These included the large tegument
protein UL36, the neurovirulence protein ICP34.5 (RL1), the
ubiquitin E3 ligase ICP0 (RL2), and the envelope glycopro-
teins gI (US7) and gL (UL1). This analysis revealed that some
genes with large numbers of amino acid changes, such as the
transcriptional regulator ICP4 (RS1; see Fig. S2 in the supple-
mental material) and the uracil-DNA glycosylase UL2, have
changes that are largely shared with wild-type strain F, sug-
gesting that these are less likely candidates to explain the
unique phenotype of strain H129. Since gene length reflects
the target size for mutations accumulated over time, we also
normalized the number of amino acid changes observed for
gene length (Fig. 4B). Several of the same genes are high-
lighted again, including ICP34.5 (RL1), gI (US7), and gL
(UL1), while the short tegument protein UL11 now arises as
another potential candidate. Strain F has many amino acid
differences in several of the same genes: UL36, ICP0 (RL2),
and gI (US7) (see Fig. S3 in the supplemental material). The
FIG. 4. Potential causative mutations of the H129 spread defect. (A) Scatter plot of the strain H129 proteins, showing the largest overall
number of amino acid differences from the wild-type reference strain 17 (y axis) versus the subset of these changes that are also different from the
wild-type strain F (x axis) and are thus unique to H129. The most extreme points are labeled to identify the proteins. (B) Because gene length
affects the total possible number of observed mutations, the same data were normalized for gene length. The scatter plot presents H129 proteins
with the largest overall percentage of amino acids differing from the reference strain 17 (y axis), versus the subset of these changes that are also
different from wild-type strain F (x axis). (C) Amino acid alignment of the ICP34.5 (RL1) protein in strains 17, F, and H129. Previously described
functional domains are boxed, while amino acid residues that differ from reference strain 17 are highlighted in red. Amino acids that match the
reference are represented by dots for clarity; dashes represent deletions in the respective strains.
VOL. 84, 2010SEQUENCE VARIATION IN HSV-1 GENOMES: F, H129, AND 175309
genes that have large numbers of amino acid changes, both
overall and with respect to gene length, are likely candidates to
explain all or part of the H129 phenotype.
ICP34.5 and other candidates that could account for the
H129 spread defect. Substantial amino acid changes may affect
protein structure and function. ICP34.5 (RL1) is a well-known
neurovirulence gene previously demonstrated to affect the
spread of HSV-1 strains in vivo (6, 82). The H129 strain has
one extra arginine in an N-terminal arginine-rich domain of
ICP34.5 (38) and two unique amino acid changes that fall on
either side of the Beclin-binding domain mediating ICP34.5’s
effect on autophagy (Fig. 4C) (50). The other H129-specific
changes in ICP34.5 are two small deletions, one of which is in
the Ala-Thr-Pro (ATP) reiteration. Although long reiterated
sequences are not determined with accuracy by de novo assem-
bly, H129 has an extremely short ATP reiteration of only 33 bp,
which we validated by PCR (data not shown). Short ATP
reiterations in ICP34.5 have been previously associated with
decreased neurovirulence (7, 38). The C terminus of ICP34.5
has a domain akin to that of the mammalian protein GADD34
(growth arrest and DNA damage), which blocks protein shutoff
by host cells and facilitates viral replication. However, this
domain is unchanged in both newly sequenced strains (25).
ICP34.5’s role in neurovirulence and these H129-specific
changes in the amino acid sequence suggest ICP34.5 as a prime
candidate for further studies of the H129 phenotype.
We also examined the coding sequence differences of a
number of other candidate proteins. The short tegument pro-
tein UL11 has a total of four amino acid changes in these
strains, two of which are specific to H129. UL11 is highly
conserved among herpesviruses and plays a role in virion en-
velopment through its interaction with the tegument protein
UL16 (4, 31, 35, 84); however, none of the observed changes lie
in the functional interaction domains of this protein. Glyco-
protein gI (US7) is another potential candidate because of its
dimerization with glycoprotein gE (US8) and its roles in im-
munoglobulin binding, axonal sorting, and virulence (43, 64).
H129 has 17 amino acid changes in gI, of which 8 are shared
with the wild-type strain F and another 7 result from a change
in length of a VNTR in gI. Although Norberg and colleagues
have shown that the amino acids encoded by this reiteration
are substrates for O-linked glycosylation, the VNTR varies in
length among many clinical isolates, making its change unlikely
to be responsible for the unique phenotype of H129 (48, 49).
The remaining two mutations in gI that are unique to H129 lie
outside its known functional domains. Another glycoprotein,
gL (UL1), has five amino acid changes unique to the H129
strain, plus an additional three shared with the F strain. Gly-
coprotein gL (UL1) is part of the HSV-1 fusion complex that
includes glycoproteins gH, gB, and gD (52). Three of the
H129-specific changes lie near a region recently suggested to
be part of a gL-gH interaction domain (21), which if disabled
could make gL an attractive candidate to explain part of the
H129 phenotype. The largest number of amino acid changes in
the H129 strain was found in the essential tegument protein
VP1/2 (UL36) (1, 16, 29, 62). This multifunctional protein is
also the largest in HSV-1, at 3,139 amino acids, a length that
dwarfs the 18 amino acid changes in the H129 strain when
these changes are normalized for length. These additional can-
didate proteins, either alone or together, may contribute to the
anterograde spread phenotype of the H129 strain and warrant
Background mutation detection: the UL13 kinase. In addi-
tion to uncovering mutations in the H129 strain, we found an
unexpected mutation in the wild-type strain F isolate: a frame-
shift in the UL13 kinase gene resulting from the deletion of
one C in a mononucleotide run of six Cs. This frameshift
changes the amino acid sequence of UL13 from amino acid
120 forward and then introduces a stop codon that truncates
the protein at residue 150 instead of the normal length of 518
amino acids. To verify this mutation, we PCR amplified this
region and directly sequenced the PCR product to assess any
variability in the stock population. All plaque-purified strain F
stocks in our lab carried this mutation, while isolates of strains
NS, RE, and several ICP34.5 mutants of strain F did not (82).
The original stock of strain F in our laboratory displayed a
mixed population of mutant and wild-type sequence, demon-
strating the likely source of the frameshift found in the plaque-
purified stock used for sequencing. The sequence of UL13 in
all other strains matches that of the original strain 17 reference
at this position, indicating that our sequenced strain is indeed
a UL13 mutant. All amino acid comparisons between strains in
this paper were done with a corrected version of UL13. The
strain F genome sequence submitted to NCBI has been cor-
rected to the parental version, with a notation of the location
of the frameshift in the sequenced isolate.
Genome sequencing of clinical and lab isolates of HSV-1
provides rich data on interstrain variations at both the DNA
and amino acid levels. It presents the opportunity to map
simple and complex phenotypes of interest to specific genes, as
we have done with the unique strain H129, whose anterograde
spread phenotype is of crucial interest to the field of neural
circuit tracing. Defining the full genetic spectrum of any virus
stock also allows one to find previously undetected mutations,
as demonstrated by the unexpected UL13 kinase mutation
found in our otherwise-wild-type strain F. Further analysis of
these data, including complementation testing with the candi-
date mutations of the H129 strain, will allow us to determine
causality in these genotype-phenotype connections.
Future advances in genome sequencing of herpesviruses.
One sequencing run provided extensive coverage (?1,000-
fold) of the genome (Fig. 1), far beyond the depth used for
most genome sequencing projects (12, 26, 27, 39, 40, 61). Fu-
ture sequencing will be done by multiplexing four or more
strains per run, providing more power for interstrain compar-
isons. To handle the enormous sequence output of these
projects, improvements in de novo assembly will be required
for facile analysis. We used a combination of de novo assembly
followed by alignment to position large blocks of sequence
along the reference genome, but this method cannot fully ad-
dress the possibility of transpositions or other rearrangements.
Standard restriction fragment length polymorphism methods
can be used to address these issues, and deep sequencing
technologies using longer reads or paired-end sequencing may
also assist in assembly. We cannot overemphasize the impor-
tance of the source of DNA used for future sequencing
projects. Our data demonstrate that plaque-purified viral DNA
5310SZPARA ET AL.J. VIROL.
may fix variations from the original stock into sequence arti-
facts, as demonstrated by the UL13 kinase mutation. Single
genomes that are cloned into BACs reflect cloning of a single
genome from a diverse population, and they will likely have
similar issues of genetic bottlenecks and unintentionally se-
Limitations of VNTR sequencing. The HSV-1 genome con-
tains 24 documented VNTRs or reiterations (41, 42). In both
HSV and the related alphaherpesvirus varicella-zoster virus,
VNTR lengths vary between strains and also during multiple
passages of the same strain (37, 46, 53, 72, 74–77). Precision in
defining the length of reiterated sequences is impossible for
most sequencing technologies, and even paired-end reads do
not offer precise length determinations because of variations in
the insert size of the sequencing libraries. Thus, many pub-
lished genome studies across a wide range of species either do
not report data for reiterated sequences or exclude data map-
ping to repetitive regions from any further analyses (26, 27, 39,
40, 61). While the approach used here yielded sequence reads
covering all HSV-1 reiterations, currently available assembly
methods precluded accurate length determinations for about
half of the HSV-1 reiterations (22). According to current ge-
nome finishing standards recommended for all species from
viral to eukaryotic, these HSV-1 genomes would be considered
noncontiguous finished because of the imprecision of VNTR
length (9). Future efforts to determine VNTR length by tradi-
tional sequencing methods may allow for further understand-
ing of variations in these regions.
Background mutation detection. The loss of a functional
UL13 kinase protein in our plaque-purified isolate of HSV-1
strain F provides a cautionary note to our confidence in pur-
portedly wild-type laboratory strains and also to the genetic
background of strains used for directed mutagenesis. It is com-
mon to assume that DNA genomes are inherently stable and
exhibit almost no variation in sequence during laboratory pas-
sage. However, until now, we have been unable to comprehen-
sively analyze the entire genome complement, or background,
of any given strain, and thus our knowledge of genetic drift in
culture has been limited at best. The UL13 kinase, like many
HSV-1 proteins, is not required for growth in vitro and only
marginally affects virus fitness in cell culture (11, 51, 71), al-
lowing its mutation to pass unnoticed. Surprisingly, the homol-
ogous UL13 kinase of MDV is also frequently deleted during
laboratory passage, suggesting that mutation of UL13 may
provide some as-yet-unknown adaptive advantage to growth in
cultured cells (5, 28, 68). There are at least two other examples
of nonessential genes found to be truncated in passaged HSV
lab strains: a terminal truncation of gI (Us7) in the KOS321
strain (48), and a truncated vhs protein (UL41) in the HSV-2
HG52 strain (20). As high-throughput sequencing technologies
become more facile and widespread, it may be feasible to
routinely sequence lab isolates and mutagenized strains, in
order to screen for unexpected, unnoticed mutations. In this
regard, a powerful use of whole genome sequencing will be the
analysis of suppressor mutations, which is a useful method to
detect genetic interactions.
Completely conserved proteins: potential therapeutic tar-
gets. The complete conservation of 10 coding sequences across
all three strains suggests that this group includes proteins vital
to viral function in vivo and in vitro and less tolerant of se-
quence variation. In comparing these 10 genes to other se-
quences available in GenBank and to the published genome of
the mutant HF10 strain, only four of these, UL31, UL35,
UL45, and gJ (US5), were still invariant (78). As more genome
sequences become available, it will be important to see if these
proteins remain unchanged. Further examination on a protein-
by-protein level may also reveal that some genes with only one
or two coding changes are in fact also highly conserved, with
minor changes that do not affect their functional domains. The
preservation of a coding sequence unit across a large number
of divergent HSV strains indicates a promising target for an-
Defining the H129 mutant genotype and phenotype. The full
genotype of the previously uncharacterized strain H129 is of
significant interest to the neuroanatomical circuit tracing com-
munity, because it is the only strain known whose spread is
limited to the forward, or anterograde, direction (3, 17, 24, 59,
69, 86). This phenotype complements the opposing retrograde-
only spread of the related alphaherpesvirus PRV strain Bartha,
as well the rabies virus-derived tracers (19, 73). Finding all of
the sequence differences in the H129 strain is the first step
toward defining the causative mutation(s). Because there is as
yet no in vitro assay for the H129 directional spread phenotype,
testing of complementation and sufficiency will require either
the development of such an assay or the use of rodent models.
Given the mutations observed in several candidate genes, such
as ICP34.5 (RL1), gL (UL1), and UL36, the phenotype of
H129 may well be polygenic, adding complexity to future stud-
ies. However, the ability of this unique strain to provide insight
to neuronal biology and viral infection makes it a worthy goal.
Since the original source of the H129 clinical isolate was an
encephalitic patient (17), an interesting question arises: was
the unique biology of H129 involved in the disease? It also is
possible that the H129 phenotype had nothing to do with the
disease. The patient may have had other genetic differences
that led to viral encephalitis, and these provided an opportu-
nity for the H129 mutant to thrive. Unfortunately, the lack of
patient samples from that time and the inability for further
testing in humans preclude our ability to answer these ques-
tions. The best insight may come from future studies, where if
a case of herpetic encephalitis is observed, both the patient
genome and the viral genome can be assayed simultaneously.
By correlation, we may then be able to predict whether HSV-
induced encephalitis usually results from patient genetics, viral
genetics, or a combination of both.
Use of comparative virology and genome sequencing to map
complex phenotypes. These data provided the complete se-
quence of two new genomes of HSV-1 and demonstrated the
large degree of coding sequence variability in a DNA virus of
high replication fidelity. The abundance of protein-level vari-
ation provides an impetus to continue sequencing projects
aimed at discovering the sequence variabilities in clinical iso-
lates of HSV-1. Clearly, these methods provide rich data for
comparisons across strains, but they also directly suggest
straightforward experiments to map specific genotype differ-
ences to known phenotypic differences.
In the case of hard-to-study clinical phenotypes, such as
latency, reactivation, and tissue tropism, high-throughput ge-
nome sequencing of divergent virus strains will now enable
unbiased and comprehensive association of phenotypes to dif-
VOL. 84, 2010 SEQUENCE VARIATION IN HSV-1 GENOMES: F, H129, AND 17 5311
ferences at multiple genetic loci. In our proof-of-principle ex-
ample, we used the new sequence of strain F, in combination
with the previously published strain 17, to help identify the
likely causative mutations in the mutant strain H129. Similarly,
genome sequencing could be used to map complex traits, such
as tendency to latency or reactivation frequency, where candi-
date loci could be found by comparing variations across the
genomes of multiple genetically divergent strains that share
these phenotypes. Whole-genome assay techniques will pro-
vide data and a means to map viral genotype differences to
phenotypes previously defined in human patients, particularly
those that are difficult to accurately replicate or study in animal
and cell culture models.
We thank J. Buckles, C. Chiriac, Y. Tafuri, and the Lewis-Sigler
Institute for Integrative Genomics Microarray Facility for technical
support. We thank M. Llina ´s, M. Lyman, O. Kobiler, members of the
Enquist lab, and anonymous reviewers for feedback on these data and
We acknowledge funding from a Center Grant (NIH/NIGMS P50
GM071508), the New Jersey Commission on Spinal Cord Research
(M.L.S.), NIH P40 RR 018604 (L.W.E. and M.L.S.), and a supplement
to NIH R01 AI 033063 (M.L.S.).
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