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Author ManuscriptAuthor Manuscript Author Manuscript Author . Author manuscript; Manuscript Author available in PMC 2018 December 01. Published in final edited form as: Virology. 2017 December ; 512: 124–131. doi:10.1016/j.virol.2017.09.016.

Shared ancestry of 1 strain Patton with recent clinical isolates from Asia and with strain KOS63

Aldo Pourcheta, Richard Copinb, Matthew C. Mulveyc, Bo Shopsina,b, Ian Mohra, and Angus C. Wilsona,# aDepartment of , New York University School of Medicine, New York, New York, USA bDepartment of Medicine, New York University School of Medicine, New York, New York, USA cBeneVir Biopharm, Inc., Gaithersburg, Maryland, USA

Abstract 1 (HSV-1) is a widespread that persists for life, replicating in surface tissues and establishing latency in peripheral ganglia. Increasingly, molecular studies of latency use cultured models developed using recombinant such as HSV-1 GFP- US11, a derivative of strain Patton expressing green fluorescent protein (GFP) fused to the viral US11 protein. Visible fluorescence follows viral DNA replication, providing a real time indicator of productive and reactivation. Patton was isolated in Houston, Texas, prior to 1973, and distributed to many laboratories. Although used extensively, the genomic structure and phylogenetic relationship to other strains is poorly known. We report that wild type Patton and the GFP-US11 recombinant contain the full complement of HSV-1 genes and differ within the unique regions at only eight nucleotides, changing only two amino acids. Although isolated in , Patton is most closely related to Asian viruses, including KOS63.

Keywords Herpes simplex virus; HSV-1; viral genomic sequences; latency; Illumina sequencing; strain origins; phylogenetics; reactivation; neurovirulence; viral pathogenesis

INTRODUCTION An important and widespread human pathogen, herpes simplex virus 1 (HSV-1 or alternatively, Human alphaherpesvirus 1) is considered the archetypal member of the neurotrophic subfamily (Roizman et al., 2013). More than 70% of the human population become seropositive for HSV-1 during their lifetime, with most establishing a lifelong latent infection punctuated by reactivation events that produce new

#Address correspondence to Angus Wilson, [email protected]. A.P. and R.C. contributed equally to this work. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Pourchet et al. Page 2

infectious viruses (Nahmias et al., 1990). Recurring reactivation is responsible for many of Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author the clinical manifestations associated with HSV-1, including painful oral lesions (herpetic lesions), blindness, lasting nerve pain, cognitive impairment and life-threatening viral encephalitis. Despite decades of study there are currently no licensed vaccines against HSV-1 or antiviral drugs that can deplete the latent reservoir (Coen and Whitley, 2011). This deficit provides a strong impetus to better understand the mechanisms of pathogenesis and the control of latency and reactivation (Bloom, 2016; Wilson and Mohr, 2012).

Live-animal infection models have been invaluable in identifying viral determinants of latency and pathogenesis and in exposing the key role of host immunity (Wagner and Bloom, 1997). However, as studies of virus-host interactions become more detailed there is increased need for robust in vitro models of latency that simplify the complex biology of natural and at the same time allow for more nuanced experimental manipulation of host factors (Bloom, 2016; Wilson and Mohr, 2012). The recent development of several in vitro models has benefitted from recombinant viruses that express markers that can be visualized in live cells (Camarena et al., 2010; Cliffe et al., 2015; Kuhn et al., 2012; Pourchet et al., 2017; Thellman et al., 2017). One example is HSV-1 GFP-US11 (strain Patton), a that expresses enhanced green fluorescent protein (EGFP) as an in-frame fusion to the viral US11 protein (Benboudjema et al., 2003). The GFP-US11 fusion protein is expressed with true-late (γ2) kinetics and accumulates at levels sufficient for detection by light microscopy after the onset of viral DNA replication. This provides an easily scored indicator of acute infection and reactivation (Camarena et al., 2010). In all other measures, HSV-1 GFP-US11 replicates with the same efficiency and kinetics as the parental wild type Patton virus (Benboudjema et al., 2003). Despite extensive usage in studies of productive replication, latency, and pathogenesis (Bonneau and Jennings, 1989; Karp et al., 1997; Robey et al., 1976), the complete sequence and gene composition of HSV-1 strain Patton has not been reported.

The importance of HSV-1 strain differences is well recognized, especially in animal pathogenesis models, and may reflect differences in their capacity to cause in humans (Dix et al., 1983; Sedarati and Stevens, 1987). For a number of years, the only complete HSV-1 genome sequence was for strain 17 (Brown et al., 1973; McGeoch et al., 1988), but with recent advances in high-throughput sequencing technology, the complete or near-complete sequences of many laboratory strains and clinical isolates have been determined (Bowden et al., 2006; Colgrove et al., 2015; Macdonald et al., 2012a; 2012b; Pfaff et al., 2016; Szpara et al., 2014; Watson et al., 2012). This very substantial increase in sequence information has revealed extensive genotypic variation between isolates, mostly involving single nucleotide polymorphisms (SNPs) along with small insertions and deletions. Some viruses exhibit larger deletions and frame shifts that may be deleterious for growth in vivo, however, for the most part individual strains appear relatively stable even after repeated passage in culture (Colgrove et al., 2015). Using sequence variation, individual HSV-1 strains can be grouped into three major clades or phylogroups of African, Asian and European/North American origin (Bowden et al., 2006; Parsons et al., 2015; Szpara et al., 2014; 2010). This diversity is thought to reflect the extended co-evolution of HSV-1 with humans and for the most part the distribution of genotypes mirrors the migration of our species across the globe (Bowden et al., 2006; Sakaoka et al., 1994).

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Strain Patton was isolated from a recurrent oral lesion and distributed to other researchers by Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author William E. Rawls M.D., a member of the faculty in the Department of Virology and Epidemiology at Baylor College of Medicine in Houston, Texas (Duff and Rapp, 1973). Unfortunately, the ethnicity, travel history and health status of the donor was not published. Over successive years the genome of the virus has been characterized by restriction endonuclease mapping (Denniston et al., 1981; Enquist et al., 1979; Graham et al., 1978) and small portions have been sequenced (Umene et al., 1984; Watson et al., 1982). However, the exact composition and arrangement of viral genes, especially those involved in important traits such as neurovirulence, remain unknown. Likewise, there is no information on the phylogenetic relationship of Patton to other HSV-1 strains.

The approximately 152-kb double-stranded DNA genome of sequenced HSV-1 strains share a common structure and relatively high (68%) G/C content (McGeoch et al., 1988; Roizman et al., 2013). There are 80 or so single copy open reading frames (ORFs) and non-coding RNA genes distributed across the unique long (UL, 106.5 kb) and unique short (US, 13.5 kb) regions, which are flanked by terminal and internal long inverted (TRL/IRL, 9.2 kb) and short inverted (TRS/IRS, 6.6 kb) repeats. There are also small microsatellite repeats (less than 100 bp each) and short reiterated sequences (less than 50 bp each) that together account for much of the sequence variation between HSV-1 strains.

Here we have used Illumina sequencing of viral DNA to derive complete genome sequences for both the parental HSV-1 strain Patton and recombinant derivative GFP-US11, the goals being to determine the gene repertoire and sequence of each virus, and to establish the evolutionary relationship to other laboratory strains. We identified the full complement of HSV-1 open reading frames (ORFs) and non-coding RNA genes in each viral genome. Using strain 17 as reference, no large deletions or rearrangements in gene order were identified. Despite extensive engineering and passage within the laboratory, only eight single nucleotide polymorphisms (SNPs) were found in non-repetitive regions of the recombinant virus compared to the parental sequence and of these, only two SNPs are predicted to change the corresponding protein sequence. Unexpectedly, lineage analysis showed that strain Patton is related to strain KOS (referred to here as KOS63, (Bowen et al., 2016)) another widely used laboratory strain first isolated at Baylor College of Medicine, Texas in the early 1960’s (Smith, 1964). Numerous differences in the genomic sequences as well as previously reported differences in biological properties such as neurovirulence confirm that KOS63 and Patton are distinct viruses. We conclude that wild type strain Patton and the versatile GFP-US11 reporter virus are functional viruses that encode the full complement of known HSV-1 genes and belong to a clade of viruses originating in Asia.

RESULTS Comparison of the parental wild type and GFP-US11 Patton genomes Total DNA was isolated from free preparations of wild type and GFP-US11 HSV-1 Patton viral particles, produced by infection of Vero (African green monkey kidney) cells and used to construct paired libraries for high-throughput Illumina sequencing. Sequencing of GFP-US11 was performed by NYU Langone Medical Center Genome Technology Center (GTC), whereas the wild type virus was sequenced by Aperiomics (Ashburn, VA). Reads

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(38,696,673 for wild type and 3,789,912 for GFP-US11) were compared against the rhesus Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author macaque genome to eliminate those derived from contaminating host DNA and low-quality sequence reads were also removed. The remaining high-quality reads (Fig. 1A) were assembled into continuous genome sequences guided by the HSV-1 strain 17 syn+ reference sequence (GenBank Accession Number JN555585). For the Patton GFP-US11 virus trimming and genome assembly was performed using the VirGA (VirAmp) online viral genome assembly pipeline, whereas proprietary software was used to assemble the Patton wild virus (see methods).

With the exception of the enhanced green fluorescent protein (EGFP) cassette inserted immediately upstream of the US11 initiation codon in the GFP-US11 virus (Benboudjema et al., 2003), the sequences of the wild type and GFP-US11 viruses showed a remarkable degree of sequence identity. After excluding low confidence polymorphisms in regions of low sequence complexity, we identified eight single nucleotide differences of high confidence between the two genomes (listed in Fig. 1B). Of these, only two polymorphisms were predicted to alter sequences: Ala-114 changed to Val in the minor protein UL17 involved in DNA packaging and retention (Thurlow et al., 2006; Toropova et al., 2011), and Ala-211 changed to Val in the large tegument protein and ubiquitin-specific protease UL36 (Newcomb and Brown, 2010). The small number of differences, resulting in either no change to the amino acid sequence or conservative substitutions (two cases of Ala to Val), reveals an unexpectedly high degree of genomic stability over numerous passages and manipulations in the laboratory.

Conservation of coding and non-coding sequences When compared strain 17, we identified more than 700 single nucleotide polymorphisms (SNPs) and 18 small insertion/deletion events (indels) in the wild type Patton sequence. All 74 single copy ORFs were present in both of the Patton sequences, together with the coding sequence for enhanced green fluorescent protein (EGFP) inserted N-terminal to the initiation codon of US11 in the GFP-US11 virus (Benboudjema et al., 2003). The predicted fusion protein included 14 additional residues derived from the shuttle used in the construction. Only 18 ORFs (listed in Fig. 2A) were identical at the amino acid level between the Patton viruses and strain 17. The remaining ORFs contained at least one amino acid difference, resulting in 228 substitutions out of a total of 38,169 codons (0.6% divergence). As might be predicted, the large tegument protein encoded by ORF36, the largest ORF in the HSV-1 genome, contained the largest number of differences, totaling 26 out of 3,164 codons (0.8% divergence).

HSV-1 expresses a number of noncoding RNAs (ncRNA), including several viral microRNAs and the latency-associated transcripts. These ncRNAs are the only viral products consistently detected in latently infected ganglia (Umbach et al., 2008; 2009). Compared to strain 17, we found that seven of the 18 stem-loop structures (pre-miRNAs) reported in miRBase (Release 21) contained single nucleotide polymorphisms (SNPs). In two instances the SNPs lie within the sequence predicted to form the functional microRNA (hsv1-miR-H3–5p and hsv1-miR-H5-3p), and thus might potentially alter their functions. However, both of these RNAs are present at very low levels and their validity as bona fide

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microRNAs has been questioned (Flores et al., 2013). Thus it seems that the microRNA Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author repertoire of the Patton viruses is essentially equivalent to strain 17 (Jurak et al., 2014).

Analysis of strain phylogeny To understand the phylogenetic relationship of Patton to other commonly used laboratory strains and a growing inventory of clinical isolates, we performed lineage analysis comparing sequence polymorphisms in the non-repetitive portion of the genomes to determine likely evolutionary relationships. As shown in Fig. 3A, Patton clustered with KOS63 and clinical isolates from India (HSV1_India HSV-1/0116209/India/2011), China (CR38/1980–1988, RDH193), South Korea (R11/1980–1988, R62/1980–1988), and Japan (S23/1980–1983, S25/1980–1983) (Bondre et al., 2016; Sakaoka et al., 1994; 1985). Consistent with other studies, this analysis aligned strain 17 with McKrae and F in a separate European/North American clade (Pfaff et al., 2016; Szpara et al., 2014). Thus it appears that Patton belongs to a clade of viruses that originate in Asia, some of which are associated with viral encephalitis (Bondre et al., 2016).

The similarity of Patton to Asian genotypes was unexpected given that the virus is thought to have been isolated in the United States. There is a similar geographic disconnect for KOS63, which was also isolated at Baylor, albeit a number of years earlier, but also clusters with genotypes of Asian rather than North American/European origin (Bowen et al., 2016; Smith, 1964). Numerous sequence differences clearly indicate that Patton and KOS63 are distinct viruses and in fact only 15 of the 74 predicted protein products are identical at the amino acid level (Fig. 2B). Sequence differences are also evident in the partial sequence of the US2 gene, for which Patton and KOS63 differ at multiple positions (Fig. 3B). Nucleotide sequence polymorphisms in glycoprotein G (gG, US4) and glycoprotein I (gI, US7) have been used to define three major genotypes termed A, B and C, that correlate to the three major geographic clades (Norberg, 2010; Norberg et al., 2004). A simpler assay to distinguished these genotypes based on digestion of an amplified segment of the US2 gene by the endonuclease EcoO109I has been proposed (Glück et al., 2015). However, this is uninformative when applied to the Patton genome, which in this limited region is identical to the sequences of North American/European (genotype B) strains H129 and F. Thus simple genotyping tests may not accurately assign all viruses and a combination of tests or better still, genome sequencing should be used.

Despite the phylogenetic proximity of Patton to KOS63 and India/2011, there are many SNPs that are predicted to alter the protein coding potential of the corresponding ORFs. Compared to strain 17 the largest number of non-synonymous changes are in UL36, UL43, US5, US7, and UL4 (Fig. 3A). Not surprisingly, the very large UL36 sequence also contains the greatest number of differences between Patton and stains KOS (Fig. 3B) and India/2011 (Fig. 3C). No other gene showed a comparable number of changes.

Determinants of neurovirulence Popular laboratory strain KOS63 exhibits reduced neurovirulence relative to 17 in outbred Swiss Webster mice following footpad inoculation (Thompson et al., 1986). This important phenotypic difference has been attributed to polymorphisms impacting the expression and

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amino acid sequences of US9 and US8A, which are thought to be required for invasion of Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author the central nervous system (CNS) following inoculation at a peripheral site (Negatsch et al., 2011). The predicted US9 and US8A sequences in Patton encode proteins of 90 and 159 amino acids respectively, equivalent to strain 17 and other neurovirulent viruses. Another important determinant of neurovirulence is the ICP34.5 protein encoded by the RL1 gene located in the long repeat region (Chou et al., 1990). ICP34.5 contributes to viral neuropathogenicity in murine and guinea pig infection models by promoting and spread in the CNS (Valyi-Nagy et al., 1994; Whitley et al., 1993). This is achieved through interactions with multiple host regulatory proteins including beclin 1, TANK- binding kinase (TBK1), phosphatase PP1α and eIF2α (Wilcox and Longnecker, 2016). Several HSV-1 strains carry polymorphisms in the RL1 coding sequence that impact neurovirulence (Bower et al., 1999; Mao and Rosenthal, 2003). The predicted ICP34.5 protein encoded by Patton is 236 amino acids in length, the shortest version of the protein reported to date (Fig. 5), and this reflects a deletion of three Asp residues within the N- terminal beclin-binding domain and a contraction of the repetitive Ala-Thr-Pro (ATP triplet) near the center of the polypeptide.

DISCUSSION The goal of this study was to derive the complete genomic sequences for the parental (wild type) HSV-1 strain Patton virus and the GFP-US11 recombinant. Patton has been used extensively in laboratory studies, including, but not limited to, early studies of mRNA splicing (Watson et al., 1981), identification of proteins involved in viral DNA replication (Graham et al., 1978), drug resistance (Furman et al., 1981), pathogenesis (Bonneau and Jennings, 1989; Mohr et al., 2001; Morahan et al., 1981; Ward et al., 2003), and oncolytic virus development (Pourchet et al., 2016; Taneja et al., 2001). The GFP-US11 recombinant has served as a versatile reporter simplifying the development of in vitro latency models (Camarena et al., 2010; Kuhn et al., 2012; Pourchet et al., 2017; Roehm et al., 2011), and has been used in more detailed studies of reactivation (Kim et al., 2012), maintenance of latency by nerve growth factor signaling (Camarena et al., 2010; Kobayashi et al., 2012), and the suppression of reactivation by (Linderman et al., 2017). Understanding the genomic structure of Patton and its evolutionary relationship to other strains and clinical isolates is therefore important.

Consistent with the very similar replication and virulence properties of the GFP-US11 recombinant relative to the parental strain, we found no major differences in gene composition and organization, which mirrored those of the archetype, strain 17. Thirteen of the predicted protein sequences were identical between Patton and 17 (Fig. 2A), comparable to the conservation between 17 and KOS63 (twelve identical ORFs) (Macdonald et al., 2012b). By comparison nineteen proteins are predicted to be identical between KOS63 and McKrae (Macdonald et al., 2012a). The invariant amino acid sequence of replication origin binding protein UL9 is noteworthy because of its large size (851 amino acids). Of significance, Patton encodes the full-length versions of envelope glycoprotein I (US7) and the UL13 kinase, both of which are truncated in other laboratory strains (Norberg et al., 2004; Szpara et al., 2010). The intact coding potential of Patton clearly supports its use as an experimental virus.

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The high degree of sequence conservation between the parental and recombinant Patton Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author viruses is unexpected given the extensive manipulations required to construct the GFP-US11 reporter virus (Benboudjema et al., 2003). First US11 was replaced with the coding sequence for EGFP in the context of a Δ34.5 recombinant to produce the virus Δ34.5ΔUS11. Next, the ICP34.5 (RL1) gene was reintroduced to create GFPΔUS11 and finally, the US11 ORF was re-introduced into GFPΔUS11 to produce the GFP-US11 virus. Each step involved several rounds of plaque purification and two of the steps also included selection on human U251 glioblastoma cells to obtain recombinants that had restored the ICP34.5 or US11 functions. The absence of significant deletions and other rearrangements implies a high degree of genomic stability, comparable to that observed for long term propagation of KOS63 (Colgrove et al., 2015).

Szpara and colleagues highlighted ten proteins (UL15, UL16, UL20, UL31, UL35, UL45, gK/UL53, UL55, ICP22/US1 and gJ/US5) that are invariant between strains 17, F, and H129 raising the interesting notion that these could be used as strain-independent antiviral targets (Szpara et al., 2010). Only five of the ten were unchanged in Patton: capsid protein VP26 (UL35), membrane proteins UL20 and UL45, envelope glycoprotein K (UL53), and the tegument protein UL55. Interestingly UL20, UL45, UL53 and UL55 are considered dispensable for growth in culture and it remains a mystery why these genes have remained unchanged. Likewise, the close relationship of strain Patton, a laboratory strain isolated more than 40 years ago, to recent clinical isolates from South Korea, Japan, China and India is striking given the large number of HSV-1 genes considered dispensable for growth in cell culture. This unanticipated stability calls into question the claim that only recent clinical isolates are suitable strains for the development of oncolytic HSV-1 derivatives (Liu et al., 2003).

One of the rationales for obtaining genome sequences for different HSV-1 strains is the identification of pathogenic determinants, especially any that might contribute to the severest disease manifestations such as viral encephalitis. Polymorphisms in the regulatory and coding regions of US8a, US9 and ICP34.5 (RL1) have each been correlated with differences in the replication and spread of virus in of the CNS (Chou et al., 1990; Negatsch et al., 2011; Valyi-Nagy et al., 1994; Whitley et al., 1993). We found that the predicted amino acids sequences of US9 and US8a of the Patton viruses are identical to those of strain 17 and other isolates. However, the ICP34.5 protein encoded by Patton is the shortest example known to date due to the deletion of three aspartate residues within the beclin-binding domain and to fewer copies of a reiterated Ala-Thr-Pro (ATP) triplet that is already known to vary in length between strains (Jing and He, 2005; Szpara et al., 2010). Despite the observed variability in the ATP repetitive segment, the neurovirulence of the Patton strain is comparable to other highly neurovirulent strains following intracranial administration and the virus can invade the CNS from the periphery (Chou et al., 1990; Mohr et al., 2001). In addition, the Patton strain can access and replicate within the murine trigeminal ganglia following corneal infection (Ward et al., 2003). Thus far, the impact of variations within the ICP34.5 ATP repetitive segment have not had any detectable impact on ICP34.5 function or on the spread of virus within the nervous system in small animal models. In addition, recent clinical isolates from East Asian and North American lineages contain similarly shortened ATP repeats with only one additional copy of the motif compared to Patton strain. The

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forces governing the expansion and contraction of repetitive motifs in HSV1 genomes are Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author incompletely understood, but could conceivably involve recombination between small homologous segments during replication (Umene, 1986).

The most surprising finding of this study is the unexpected relationship of Patton to strain KOS63, a virus with clear affinities to East Asian rather than North American isolates (Bowen et al., 2016). Although the published details are limited, the indications are that the Patton virus was isolated at Baylor University College of Medicine in Houston, Texas prior to 1973 (Duff and Rapp, 1973). This is intriguing because some years earlier, KOS63 virus, another virus with an Asian genotype, was cultured from a recurrent oral lesion by Kendall Owen Smith, an influential virologist on the faculty at Baylor (Smith, 1964). Although isolated at the same institution, it is clear that KOS63 and Patton are distinct viruses. From the onset it was known that the viruses differ in their ability to transform hamster embryo fibroblasts (Duff and Rapp, 1973) and as shown in this study, comparisons of the genome sequences reveal numerous SNPs, some of which result in non-synonymous changes in protein sequence. Smith had served with the US Army in Korea (1950 to 1953) and it has been suggested that he may have been infected by the virus that gave rise to KOS63 during this military service in Asia (Grose, 2014). In the absence of fresh information we cannot exclude the possibility that both KOS63 and Patton were isolated by Smith but were named and propagated separately. Interestingly, strain KOS79 was also isolated from Smith almost two decades after the isolation of KOS63 but this virus is clearly distinct from both KOS63 and Patton, aligning more closely to European and North American strains (Bowen et al., 2016; Dix et al., 1983).

The precise origins of strain Patton will probably remain a mystery. Unfortunately, key persons involved with the original isolation and propagation of Patton are deceased and our efforts to uncover additional information on the source of the isolate have been unsuccessful. This leaves us to speculate that the ancestor of strain Patton was either carried to Texas by a returning veteran of the wars in Korea or Vietnam, as has been suggested for KOS63 (Grose, 2014), or alternatively, was brought to Texas by immigrants from the Indian Subcontinent or East Asia where similar viruses circulate.

METHODS Next generation sequencing Wild type and GFP-US11 viruses were propagated from stocks maintained in our laboratories by multi-cycle infection of Vero (ATCC® CCL-81™) cells. Genomic DNA was extracted as described previously (Pourchet et al., 2016) without further fragmentation and subjected to Illumina sequencing. Sequencing of GFP-US11 was performed by NYU Langone Medical Center Genome Technology Center (GTC), whereas the wild type virus was sequenced by Aperiomics (Ashburn, VA). For Patton GFP-US11, the sequence was assembled using VirGA (version 1.0), a galaxy-based viral genome assembly workflow (Parsons et al., 2015; Wan et al., 2015) provided on-line and free of charge by Moriah Szpara and colleagues (Pennsylvania State University). In brief, after trimming to remove redundant and repetitive sequences, the remaining short reads were assembled into longer contigs that could be orientated and joined into a draft genome using a reference-guided

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assembler and a scaffold based contig extender tool. Scaffolding and gene annotation were Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author performed using strain 17 (GenBank Accession JN555585) as the reference. The Patton wild type, the sequence was assembled at Aperiomics using proprietary software after automated coverage reduction to minimize false SNP calling.

For SNP calling analysis, we used Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2009) to map reads from each genome sequences onto the HSV strain 17 reference sequence. BWA outputs were analyzed and annotated using SAMtools (Li et al., 2009), GATK (McKenna et al., 2010) and ANNOVAR (Wang et al., 2010). To minimize false positives, SNPs in repetitive regions were removed from the analysis.

Phylogenetic analysis Phylogenetic analysis was based on 3031 high-confidence variable positions identified between Patton and 14 additional HSV-1 genomes, specifying HSV-2 as the outgroup. SNPs were retrieved after pair-wise genome alignment using MAUVE (Darling et al., 2010). Maximum likelihood phylogenies were obtained using PhyML (Guindon et al., 2005), and HKY model. Branch robustness was assessed through bootstrapping (1000 pseudo- replicates).

Additional analysis Alignments for individual protein sequence using MegAlign Pro software (DNAStar Inc.) and online version of the basic local alignment search tool (BLAST) provided by the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Nucleotide sequence accession number All genomic data are available at BioProject NCBI, accession PRJNA406924.

Acknowledgments

Illumina sequencing of GFP-US11 was performed by the NYU Langone Medical Center Genome Technology Center (GTC), which receives support from the National Institutes of Health (NIH) by grants from the National Center for Advancing Translational Sciences (NCATS UL1 TR00038), and by a Center Support Grant (P30CA016087) to the Laura and Isaac Perlmutter Cancer Center. We thank Moriah Szpara and Daniel Renner for valuable assistance with VirGA genome assembly workflow. Lynn Enquist (Princeton University), Charles Grose (University of Iowa) and Janet Butel (Baylor College of Medicine) provided valuable input on the history of Patton and contemporaneous HSV-1 strains. This study was supported by grants from the National Institutes of Health to A.C.W. (AI103933), I.M. (AI073898 and GM056927) and B.S. (AI103268 and N272201400019C).

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Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author HIGHLIGHTS

• Next generation sequencing of parental strain Patton and GFP-US11 recombinant viruses

• Remarkable conservation of genome sequence despite extensive manipulations

• Both viruses contain the full complement of viral genes • Strain Patton is closely related to KOS63 and recent isolates from Asia

• Identify deletion of three residues within the beclin-binding domain of ICP34.5 protein

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Fig. 1. Sequencing of parental HSV-1 strain Patton and the GPF-US11 recombinant (A) Summary of sequencing coverage for the wild type and recombinant viruses. Pre- processed sequence reads were aligned to the new draft genomes to assess the coverage depth of each assembly. This is plotted (y axis) against the length of the HSV-1 genome (x axis). (B) List of high confidence single nucleotide polymorphisms (SNPs) between parental Patton virus and its derivative GFP-US11. Additional polymorphisms in regions of low sequence complexity have been excluded. Two SNPs are predicted to change the amino acid sequence of the UL17 capsid protein and UL36 large tegument protein.

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Fig 2. Listings of the most highly conserved viral proteins (A) List of thirteen ORFs in Patton that encode polypeptides identical to counterparts in strain 17. (B) Listing of fifteen polypeptides that are identical in strain KOS63. Only four of the predicted polypeptides (UL18, UL33, UL35, and UL55) are identical between all three viruses.

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Fig. 3. Phylogenetic relationship of HSV-1 GFP-US11 (Patton) to selected Asian, European and North American strains (A) Lineage analysis was performed using genome sequences of the following strains and clinical isolates: 17 (GenBank Accession JN555585); McKrae (JQ730035); HF10 (DQ889502); KT425109/KOS79 (KT425109); F (GU734771); H129 (GU734772); Patton (this study); KOS63 (KT425110); India/2011 (KJ847330); CR38 (HM585508); RDH193 (KT425108); R11 (HM585514); R62 (HM585515); S23 (HM585512) and S25 (HM585513). The more distantly related sequence of HSV-2 strain HG52 (GenBank Accession Z86099) was used as an outlier. Affiliation to each of the three major HSV-1 genotypes is indicating using a colored box, designated as genotype A (mauve), genotype B

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(green) and genotype C (ochre). Patton is indicated in blue. (B) In silico application of the Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author US2 genotyping method (Glück et al., 2015), which is based on the presence or absence of two EcoO109I endonuclease cleavage sites (boxed) within the US2 coding region (nucleotides 134,151 to 134,256 of strain 17). Nucleotides that differ from the Patton sequence are indicated in white font and genotype designation follows the color scheme used in panel A.

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Fig. 4. Top five divergent polypeptides encoded by Patton relative to strains 17, KOS63 and India/2011 Divergence refers to SNPs that give rise to changes in the protein coding potential of Patton compared to other strains. The five open reading frames carrying the largest number of non- synonymous codon changes are listed in order of greatest divergence based on comparisons of strain Patton to (A) reference strain 17 (JN555585), (B) KOS63 (KT425110), and (C) India/2011 (KJ847330). For each polypeptide, the predicted polypeptide length as defined by strain 17 is given, along with the predicted number of changes in Patton. Where known the principal function of the protein is stated.

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Fig. 5. Sequence variation in the neurovirulence determinant, ICP34.5 (A) Schematic showing the principal domains and sequence features of the HSV-1 ICP34.5 protein encoded by the RL1 gene. The C-terminus shares sequence homology with the host GADD34 (growth arrest and DNA damage-inducible gene 34) protein. Numbering corresponds to the sequence of reference strain 17. (B, C) Partial sequences centering on the N-terminal beclin-binding domain and the repeated ATP triplet derived from the following HSV-1 genomes: Patton (this study); 17 (YP_009137073); India/2011 (KJ847330); S25 (ADM23673); F [Szpara] (ADD60013); F [Roizman] (P08353); RDH193 (ALM22702); 66S (AKG59690); McKrae (AFP86362); KOS63 (AFE62826), and H129 (ADD60090).

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