Genomic location dictates lytic promoter activity during herpes simplex virus latency

Herpes simplex virus 1 (HSV-1) is a significant pathogen that establishes life-long latent infections with intermittent episodes of resumed disease. In mouse models of HSV infection, persistent low-level lytic gene expression has been detected during latency in the absence of spontaneous reactivation events leading to new virus production. This viral activity during latency has been reported using a sensitive Cre-marking model for several lytic gene promoters placed in one location in the HSV-1 genome. Here we extend these findings in the same model by examining first, the activity of an ectopic lytic gene promoter in other places in the genome and second, whether native promoter activity might be detectable. We found that both for ectopic and native lytic gene promoters, Cre expression during latency was detected in our model, but only when the promoter was located near the ends of the unique long genome segment. This location is significant because it is in close proximity to the region from which latency associated transcripts (LAT) are derived. These results show for the first time that native HSV-1 lytic gene promoters can produce protein products during latency, but that this activity is only detectable when they are located close to the LAT locus. Author summary HSV is a significant human pathogen and the best studied model of mammalian virus latency. Traditionally the active (lytic) and inactive (latent) phases of infection were considered to be distinct, but the notion of latency being entirely quiescent is evolving due to the detection of some lytic gene expression during latency. Here we add to this literature by finding that activity can be found for native lytic gene promotors as well as for constructs placed ectopically in the HSV genome. However, this activity was only detectable when these promoters were located close by a region known to be transcriptionally active during latency. These data have implications for our understanding of HSV gene regulation during latency and the extent to which transcriptionally active regions are insulated from adjacent parts of the viral genome.


Introduction
HSV-1 infects around 67% of world population and generally causes mild disease in the form of cold sores (Looker et al. 2015).For most individuals HSV-1 disease is infrequent and often asymptomatic, however that is not always the case and the virus can cause severe diseases such as neonatal herpes, and herpes keratitis and encephalitis.Initial HSV-1 infection usually begins in the skin or mucosal epithelium, during which expression of viral genes occurs in a sequential cascade, categorised as immediate-early (IE), early (E) and late (L), ultimately resulting in the production of new virus particles (Honess andRoizman 1974, Barklie Clements, Watson andWilkie 1977).The late genes are further divided into two subclasses as leaky-late ( 1 ) or truelate ( 2 ), where the expression of latter strictly occurs following DNA replication (Holland et al. 1980, Conley et al. 1981).The virus quickly spreads to the cell bodies of innervating neurons within the sensory or autonomous nervous system, where latency is established allowing viral persistence.Virus replication may occur in neurons during productive infection, but it is not necessary for the establishment of latency (Speck andSimmons 1991, Speck andSimmons 1992).During latency, the HSV-1 genome persists in a non-productive state within the latently-infected neurons, while retaining the potential for reactivation.
The HSV-1 genome as it is packaged in the virion has two segments of unique sequence, termed unique long (U L ) and unique short (U S ), each of which is bracketed by repeats at each end (R L and R S ).By convention, the genome is considered to start with the long segment and genes are numbered from left to right in U L and U S (Fig 1A).
To complete this nomenclature, the repeats that are at the ends of the genome are referred to as being terminal (TR L and TR S ) and those that join the long and short segments are internal (IR L and IR S ).Once inside a host cell, the genome becomes a circle and the long and short segments are able to flip with respect to each other, so there is no biological distinction between internal and terminal repeats.These repeated regions are large enough to encode some genes, meaning they are present in two copies, with each copy adjacent to different genes from U L or U S .
HSV-1 latency at the level of whole organism is largely quiescent as spontaneous reactivation events are exceptionally rare (Laycock et al. 1991, Feldman et al. 2002, Gebhardt and Halford 2005).However, this perspective is challenged when viral activity is examined at the cellular level.High level gene expression during latency is limited to non-coding RNAs known as latency-associated transcripts (LATs) and certain micro-RNAs that originate from the LAT region.The expression of LATs begins early during infection, which aligns with the simultaneous establishment of latency and productive infection at the cellular level (Speck and Simmons 1992) (Spivack and Fraser 1988).Studies employing careful PCR and in situ hybridisation analysis have revealed that high levels of LATs are found in only 5 to 30% of latently-infected neurons at a given time in latency (Mehta et al. 1995, Maggioncalda et al. 1996, Ellison et al. 2000, Chen et al. 2002b, Wang et al. 2005a).Several reports over the years have provided evidence supporting the presence of lytic transcripts in the latentlyinfected trigeminal ganglia of mice, challenging the traditional view of strict viral latency (Green, Courtney and Dunkel 1981, Kramer and Coen 1995, Tal-Singer et al. 1997, Kramer et al. 1998, Chen et al. 2002a, Feldman et al. 2002, Pesola et al. 2005, Maillet et al. 2006, Ma et al. 2014).One notable report showed that lytic genes belonging to one of the classes were expressed in almost two-thirds of infected neurons (Ma et al. 2014), a far higher frequency than neurons exhibiting spontaneous reactivation (Margolis et al. 2007).This suggests that lytic gene transcription is a common phenomenon during latency in the absence of overt reactivation and is likely to be biologically relevant as an increase in viral activity were correlated with a progressive response from the host (Ma et al. 2014).
There is also evidence that some lytic transcripts may generate protein during latency, which is from two main sources.First, immune infiltrates consisting primarily of virusspecific and activated CD8 + T cells have been found in latently-infected sensory ganglia (Halford, Gebhardt and Carr 1996, Khanna et al. 2003, van Lint et al. 2005, van Velzen et al. 2013, Feldman et al. 2002, Sawtell 2003, Margolis et al. 2007).
Second, the use of Cre reporter mice, such as ROSA26R (Soriano 1999), infected with recombinant viruses that express Cre-from lytic gene promoters suggests these promoters are active during latency.In the ROSA26R model, any Cre expression leads to the indelible marking of neurons and acts as a record of historic gene expression (Proenca et al. 2008, Wakim et al. 2008).In these experiments, accumulation of marked neurons was observed during latency indicating that lytic gene promoters were able to drive Cre expression beyond the acute infection.
Promoters for infected cell protein (ICP)47 (U S 12), ICP6 (U L 39), and glycoprotein B (gB) (U L 27) were able to drive Cre expression during latency, but not ICP0 (Russell and Tscharke 2016).Thus, the expression of lytic gene expression during latency could stem from unsuccessful abortive reactivation events by the virus, incomplete repression of the genome, or more speculatively, a latency-associated gene expression program (Singh and Tscharke 2020).
It is important to acknowledge a significant limitation of the study conducted by Russell and Tscharke, which is that the promoters were not examined in their natural location (Russell and Tscharke 2016).A recent study investigating the ICP47 promoter activity in latency using the same model found no evidence of Cre expression from either its native location or from the intergenic region between U L 26 and U L 27 (U L 26/U L 27).
Importantly however, this study recapitulated previous data showing expression from the ICP47 promoter in latency when placed between U L 3 and U L 4 (U L 3/U L 4), using the original and several newly made viruses (Russell andTscharke 2016, Velusamy et al. 2023).These data led to the speculation that the placement of this promoter close to the LAT promoter, which is active during latency might be required for expression to be detected during latency.
Here we have systematically investigated whether the expression of lytic genes during latency is influenced by the location of genes within HSV-1 genome, using an extended set of ectopic and native lytic gene promoters.This was accomplished by utilizing expression constructs inserted into multiple sites within the genome and then examining native promoter activity at sites found to be permissive for ectopic expression constructs.The results show the first evidence of gene expression from native HSV lytic gene promoters during latency and confirm that the level of this activity is influenced by promoter location in the HSV-1 genome.

Ectopic gB promoter activity in HSV-1 latency depends on genomic location
Previous studies have demonstrated that the infection of ROSA26R mice with HSV-1 recombinants expressing Cre under the control of gB and ICP47 promoters from U L 3/U L 4, led to promoter activation and efficient marking of neurons during latency.By contrast, the ICP47 promoter could not drive Cre expression that was detectable in ROSA26R mice during latency either from its native location (at the border of R S and U S ) or from U L 26/U L 27. Together these results suggest a location-dependent effect on ICP47 promoter activity during latency (Russell andTscharke 2016, Velusamy et al. 2023).Notably, U L 3/U L 4 is relatively close to the LAT promoter in TR L , which is transcriptionally active in latency.To test whether proximity to the LAT promoter might be a determinant of expression during latency for lytic gene promoters more generally, we made two new viruses: Both had an eGFP/Cre fusion gene under the control of the HSV-1 gB promoter (gB_eGC) but in the first, this was located in U L 26/U L 27 and in the second it was placed between U L 55 and U L 56 (U L 55/U L 56) (Fig 1A).These viruses were named gB_eGC_UL26/27 and gB_eGC_UL55/56, respectively.U L 26/U L 27 is roughly in the middle of the U L segment, far from either copy of the LAT promoter, whereas U L 55/U L 56 is at the far end of U L , close to the LAT promoter in IR L .
The use of U L 26/U L 27 as an insertion site has been shown not to compromise replication or pathogenesis (Russell, Stefanovic and Tscharke 2015), but U L 55/U L 56 has not been evaluated.Therefore both new viruses were characterised carefully.We confirmed the insertion of gB_eGC in the correct region for these new viruses by a whole genome digest (S2A,B Fig) .Then, we checked if the eGFP/Cre fusion protein was expressed in Vero cells infected with these viruses by western blotting.
Unmodified HSV-1 KOS and pC_eGC (described previously in (Russell and Tscharke 2016)) were used as negative and positive controls respectively, while GAPDH was used as a loading control (Fig 1B ,C).We could detect eGFP/Cre protein, but noted that there were low molecular weight fragments at lower levels that were likely cleaved or degraded products of the protein (Fig 1B ,C).Next, we validated that Cre was functional when expressed by the new viruses using a Cre-reporter cell line Vero SUA, again using HSV-1 KOS and pC_eGC as controls (Rinaldi, Marshall and Preston 1999) (S3A-S3F Fig).Finally, we showed that replication of these viruses was the same as for the parent HSV-1 KOS in Vero cells (Fig 1D ,E) and in mice infected by our flank tattoo model (Fig 1F) (Russell et al. 2015).
After successfully validating these viruses we used them to investigate the activity of their ectopic gB promoters.ROSA26R mice were tattoo-infected on the flank with HSV-1 gB_eGC_UL26/27 or gB_eGC_UL55/56, their dorsal root ganglia (DRG) from thoracic 5 to lumbar 1 dermatomes were isolated, stained for β-gal activity and the number of β-gal-expressing neurons were counted (S4A,B Fig) .In the case of HSV-1 gB_eGC_UL26/27, we saw a decrease in the number of β-gal + cells from day 5 to 10, which remained stable thereafter (Fig 1G).However, the number of DRG with β-gal + cells, which represents the spread of the virus, was similar between days 5 and 10, reduced on days 20 and 40, and remained stable thereafter in latency (Fig 1H).By contrast, HSV-1 gB_eGC_UL55/56 the β-gal + cell number was similar for all times up to day 40, but was increased at day 100 compared with day 20 and earlier (Fig 1I).
The number of DRG with at least one β-gal + cell was also increased at day 100 compared with earlier days (Fig 1J).Thus the result for HSV-1 gB_eGC_UL55/56 is reminiscent of what was seen when the same expression cassette was placed in U L 3/U L 4, at a similar distance to the LAT promoter, but at the other end of U L. (Russell and Tscharke 2016).However, there was no evidence that the gB promoter was active during latency when driving Cre from U L 26/U L 27, similar to what was found for the ICP47 promoter (Velusamy et al. 2023).Together, these results demonstrate that close proximity to the LAT promoter allows lytic gene activity to be detected during HSV-1 latency in the Cre/ROSA26R marking model.

Native gB promoter does not make protein in latency
The next aim was to check if the native promoter of gB makes protein in latency.This was achieved by combining the use of a 2A peptide with HSV-1 Cre/ROSA26R system.A 2A sequence induces steric hinderance during translation such that two proteins can be made at an equimolar ratio from a single mRNA (Donnelly et al. 2001, Atkins et al. 2007, Ahier and Jarriault 2014, Daniels et al. 2014, Tulloch, Luke and Ryan 2017, Szymczak et al. 2004).We made a recombinant virus in which the stop codon at the end of U L 27 was replaced with sequences encoding the highly efficient T2A (Liu et al. 2017)  .This suggests that HSV-1 UL27-T2A-Cre may have a defect in transport to, or growth in neurons.When ROSA26 mice were infected to determine native gB promoter activity, we observed few marked neurons in four out of 11 mice on day 5, and none on days 10, 20 and 40 (Fig 2E).We decided to extend the experiment to days 140 and 240, instead of day 100 as done previously, and found that majority of the mice did not have any marked neurons on these days (Fig 2E).The lack in activity from the native gB promoter in latency is consistent with what was observed for ectopic promoter placed in the same location, but the marking at day 5 was surprisingly low.This low marking might be due to less virus in neurons, but also it is possible that the T2A sequence is not behaving as expected in vivo.

T2A sequence can be used to study native promoter activity
To validate the use of T2A sequences in our HSV-1 Cre-marking model, we generated a recombinant virus with a T2A sequence between eGFP and Cre, under the control of gB promoter inserted in the U L 3/U L 4 region (Fig 3A).This new virus was called gB_eGTC_UL3/4 and was designed to match the previously published HSV-1 gB_eGC (Russell and Tscharke 2016), allowing a direct comparison of marking ability between these two viruses.
The expression of Cre as an independent protein by the new virus was confirmed by western blotting (Fig 3B) and this virus showed similar growth kinetics to the parent both in vitro and in vivo (Figs 3C,D).To investigate the efficiency of neuronal marking when a T2A is used, groups of ROSA26 mice were infected with HSV-1 gB_eGTC_UL3/4 or HSV-1 gB_eGC.DRG were stained to count the number of β-galexpressing neurons at days 30, 100 and 200 after infection.We found that neither the number of β-gal + cells nor the number of DRG with any β-gal + cells were significantly different between the viruses at any day (Fig 3E, F).Further, in accordance with previous findings there was accumulation of β-gal + cells in latency regardless of presence of a T2A sequence (Fig 3E) (Russell and Tscharke 2016).Therefore, we conclude that insertion of T2A sequence does not affect neuronal marking in the ROSA26/Cre reporter system.

Protein expression from native promoters in latency
Finally, we investigated the activity of native promoters towards the ends of U L where expression of Cre from ectopic cassettes was able to be detected during latency.We chose U L 3 and U L 56, replacing their stop codons with a T2A, followed by Cre to make UL3-T2A-Cre and UL56-T2A-Cre, respectively (Fig 4A).Both viruses expressed Cre as an independent protein (Figs 4B,C) and had growth similar to the parent virus in vitro and in vivo (Figs 4D-G).Finally, we used these viruses to examine the activity of native promoters in the Cre marking system.When Cre was driven from the native U L 3 promoter, we found that β-gal + neurons were either non-existent or very rare, limited to one or two neurons per mouse, and to single DRG on days 5 and 10 (Fig 4H).This low level of marking was seen on days 20, 40 and 140, but there was a trend towards a higher mean and a greater fraction of mice having any marking with each later time (Fig 4H,I).At 300 days after infection, the average number of β-gal + neurons and the number of DRG with marked neurons were significantly increased compared with all times up to day 40 ( Figs 4H,I).Marking of neurons in ROSA26R mice by HSV-1 UL56-T2A-Cre was similar to that seen with UL3-T2A-Cre, except that an early peak was seen on day 5, before the number fell to very low levels on day 10.Thereafter there was a trend of increased means that became statistically significant compared with days 10, 20 and 40 at day 300 (Fig 4J).Similarly, number of DRG with at least one βgal + cell increased on day 300 compared with days 10 and 20 (Fig 4K).These data slow that the native U L 3 and U L 56 promoters can drive detectable Cre expression during HSV-1 latency.

Discussion
In this study, we used a Cre/ROSA26R mouse model to investigate the influence of location of a lytic promoter in the HSV-1 genome on its activity during latency, as well as the ability of native lytic gene promoters to drive protein expression in latency.To achieve this, the gB promoter was studied in ectopic constructs from three locations: U L 3/U L 4, U L 26/U L 27 and U L 55/U L 56.In addition, the native promoters of gB, U L 3 and U L 56 were analysed for their activity by using a T2A sequence to separate the HSV protein from Cre (Karimnia et al. 2021, Nasamu et al. 2021).The results of neuronal marking by all the recombinant viruses used here have been summarised in Fig 5 , showing each as percent of maximum number of neurons marked to show the differences in kinetics clearly (Fig 5).These show clearly that both for non-native and for native promoters, activity in latency was detectable from U L 3/U L 4 and U L 55/U L 56, but not from U L 26/U L 27.
Prior to this study, only two promoters have been studied using ROSA26R/Cre marking models when placed in two different locations.First, there was no accumulation of β-gal+ cells in latency when Cre was driven by the ICP0 promoter, regardless of whether it was placed in the U L 3/U L 4 or within the U S 5 gene (Proenca et al. 2008, Russell andTscharke 2016).This finding suggested that while a set of other promoters was active from U L 3/U L 4, placement in this location alone was not adequate to guarantee activity during latency (Russell and Tscharke 2016).Second, while the promoter for ICP47 was active from U L 3/U L 4 during latency, this activity was not detectable from either U L 26/U L 27 region or U S 12, its native location (Velusamy et al. 2023).Taken together with our new data shown here, it would seem that a location near the LAT promoter is necessary, if not sufficient to endow a lytic promoter with enough activity during latency that it can be detected in the ROSA26R/Cre model.
LATs are known to play a significant role in the maintenance of a latent state as well as reactivation (Bloom, Giordani andKwiatkowski 2010, Phelan, Barrozo andBloom 2017).Specifically during latency, the LAT region is characterised by the presence of euchromatic marks (Kubat et al. 2004b, Kubat et al. 2004a, Neumann et al. 2007, Wang et al. 2005b), suggesting that it may exert a permissive effect on neighbouring genes.However, considering the entire region from which RNA transcripts are regularly produced in latent neurons, it is proximity to the promoter that seems most important in leading to detectable activity during latency.The native promoter for ICP47 is within R S , which is within 5 kb from where the 3' ends of the L/ST transcripts and the precursor for miR-H17 are transcribed, and yet no activity was able to be detected for this promoter during latency (Phelan et al. 2017).This is by contrast to a set of ICP47 promoter variants, all of which were active in latency when driving eGFP/Cre from U L 3/U L 4 (Velusamy et al. 2023).Considering our new data, it is interesting to note that the average number of marked neurons at day 100 was lower for gB_eGC compared with gB_eGC_UL55/56 (29 and 70, respectively).This order of marking for the gB promoter from these two locations corresponds to their distance from the LAT promoter: The gB promoter in U L 55/U L 56 is 2.6 kb from the start of LAT transcription whereas it is 4.1 kb for viruses that use U L 3/U L 4. Arguing against a simple correlation between distance from LAT and permissiveness for expression during latency, is that the fold increase in β-gal + cells from the start of latency to day 100 was higher for gB_eGC in U L 3/U L 4 than U L 55/U L 56 region.This can be seen as a steeper slope for the grey compared to the purple line in Fig 5B .Some caution is required here because these comments rely on comparisons across experiments, but we note that the slope for gB_eGC is similar here as in a previous publication (Russell and Tscharke 2016) and also for the independently made gB_eGTC_UL3/4, suggesting the observations are likely to be robust.There also remains an open question as to when during infection the expression of these promoters is being affected by proximity to the LAT promoter.LATs can be expressed in some neurons from the earliest times after infection, presumably having a role in the establishment of latency and any expression at this time sets a baseline of marked neurons in the ROSA26R/Cre model.
We chose to study the gB promoter in part because activated, gB-specific T cells are found in latently-infected DRG, which strongly suggests that gB antigen is expressed during latency (Mackay et al. 2012b, Mackay et al. 2012a).However, we were not able to detect Cre expression from this promoter, either when linked to the native gB with a T2A, or from an additional copy of this promoter from the same part of the genome.Moreover, the average number of marked neurons that demonstrate survival of neurons after expression of the gB promoter at any time after infection was very low.
The apparent inconsistency between the immunological studies of gB expression and ours could be attributed to the comparatively low sensitivity of our reporter system compared to the ability of T cells to detect extremely low levels of antigen.Where T cells may be able to detect as few as a handful of antigenic peptides, significantly higher levels of Cre are likely to be required to ensure migration to the nucleus, access to the target site and recombination of the loxP sites (Dause and Kirby 2020, Irvine et al. 2002, Purbhoo et al. 2004).
Evidence for immunological detection of lytic antigens goes beyond gB and the phenotype of resident T cells to include persistent cytokine expression by infiltrating immune cells (Chen et al. 2000).Notably, the continual cytokine expression is present in the ganglia even after infection with a thymidine kinase deficient virus that is unable to replicate and reactivate (Chen et al. 2000), suggesting that full engagement of lytic cascade is not required for the activation of immune cells.Our observation that native late gene promoters, including a true-late gene (U L 3), can drive protein expression in latency aligns with the idea that lytic gene expression during this phase of infection is de-coupled from the ordered cascade of productive infection.Whether this activity is a component of animation phase of reactivation which never rendered complete (Kim et al. 2012, Cliffe et al. 2015, Cuddy et al. 2020, Dochnal et al. 2022), or a part of latency program of gene expression involved in maintenance of a latent state (Singh and Tscharke 2020), remains to be elucidated.
Finally, we conclude that detection of native lytic gene promoter expression during latency using ROSA26R/Cre models can be interpreted simply as showing that these promoters can be active in latent infection.It also suggests that activity is highest from promoters close to the LAT promoter and that areas of the genome at the ends of U L are not entirely insulated from the de-repression of the LAT region during latency.
However, the failure to detect promoter activity in latency from across the genome requires more caution in interpretation and is better considered to be falling below the limit of sensitivity of the model than to be entirely absent.

Cell lines and viruses
Vero cells were obtained from American Type Culture Collection (ATCC, CCL-81).Cre reporter assays were performed in Vero SUA cells (gift from Prof Stacey Efstathiou, (Rinaldi et al. 1999)).Both cell lines were cultured in minimum essential medium (MEM; ThermoFisher Scientific) supplemented with 2 or 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 4 mM L-glutamine (ThermoFisher Scientific), 5 mM HEPES buffer (ThermoFisher Scientific) and 55 µM β-mercaptoethanol To construct pgB_eGTC_UL3/4, the flanking U L 3 and U L 4 arms, gB promoter, eGFP and Cre-BGH polyA sequences were amplified from pT gB_eGC (Russell and Tscharke 2016).T2A sequence was synthesised as two complementary dioxynucleotides (Liu et al. 2017).These fragments were cloned into the BamHI site of pCR bluntII vector (Invitrogen, Life Technologies) maintaining the original configuration as pT gB_eGC (Russell and Tscharke 2016) except that T2A was inserted in frame between eGFP and Cre.

Recombinant virus generation and in vitro growth analysis
All the recombinant viruses were constructed using a transfection/infection method described previously (Velusamy, Gowripalan andTscharke 2020, Russell et al. 2015).

Ethics statement
All mice were sourced from Australian Phenomics Facility (APF), Canberra, Australia.
This study was conducted according to ethical requirements and approval from Australian National University Animal Experimentation Ethics Committee under protocols A2017/39 and A2020/42.The research was undertaken in accordance with Australian Capital Territory Animal Welfare Act 1992 and Australian code for the care and use of animals for scientific purposes, 8th edition 2013.

Mice and infections
At least eight weeks old, female, specific pathogen free C57BL/6 or B6.129S4-Gt(ROSA)26Sortm1So/J (ROSA26R) (Soriano 1999) were used for experiments.The mice were housed and bred at APF. ROSA26R mice were a gift from Dr. Francis R.
Carbone (University of Melbourne, Australia).Mice were anaesthetised by intraperitoneal injection with Avertin (2,2,2-tribromoethanol in 2-methyl-butanol) or Ketamine/Xylazine mix before infection with 1  10 8 PFU/ml of virus on the shaved flank.The procedure was followed as described previously except that the needle was charged for 20 s in the virus suspension and tattooed for a period of 20 s (Russell et al. 2015).The virus dose and infection route were same for all the experiments.

Virus titration from skin and DRG
Mice were euthanised with a rising concentration of CO 2 , the infected area of the skin (2.5 cm vertically  0.8 cm horizontally) and the DRG (T5-L1) on the ipsilateral side were excised, and collected in 500 µl MEM (without serum) 5 days after infection.The tissue samples were snap frozen, 5 mm diameter stainless steel bead (Qiagen) was added to all the tubes and the tissue was homogenised in TissueLyser II (Qiagen) at an oscillation frequency of 30 Hz for 90 s twice.Homogenates were subjected to three freeze/thaw cycles and the amount of infectious virus was quantified by a standard plaque assay on Vero cells.

Detection of β-gal expression
To check expression of β-gal in vitro, confluent Vero SUA cells were left untreated or infected with appropriate virus at an MOI of 0.05 PFU/cell and incubated at 37 °C with 5% CO 2 for 1 hr.The unabsorbed virus was removed, replaced with fresh medium (MEM with 2% serum) and cells were further incubated for 36 hrs.Following incubation, the cells were washed with PBS (Sigma-Aldrich), fixed with 2% paraformaldehyde (Electron Microscopy Sciences)/ 0.5% glutaraldehyde (Sigma-Aldrich) (in PBS) for 4 hrs at 4 °C, washed with PBS again and incubated in the permeabilization solution (2 mM magnesium chloride (Ajax FineChem), 0.01% (w/v) sodium deoxycholate (Sigma-Aldrich), 0.02% (v/v) IGEPAL CA-630 (Sigma-Aldrich), 5 mM potassium ferrocyanide (Sigma-Aldrich) and 5 mM potassium ferricyanide (Sigma-Aldrich) in PBS) containing 1 mg/ml X-gal (Sigma-Aldrich; prepared fresh as a 40 mg/ml stock in N,N-Dimethylformamide (Sigma-Aldrich)) overnight at 4 °C.Cells were washed with PBS, overlayed with 50% glycerol (Sigma-Aldrich; in PBS), and then visualised and imaged using Olympus CKX41 microscope fitted with Olympus DP22 digital camera.
To enumerate β-gal-expressing cells in vivo, mice were euthanised with a rising concentration of CO 2 , and the DRG (T5-L1) on the ipsilateral side were collected individually in fixative as above.The DRG were incubated on ice for 1 hr and then washed twice with PBS before adding permeabilization buffer as before.Following further incubation at 4 °C for 30 mins, the solution was replaced with permeabilization buffer containing 1 mg/ml X-gal (prepared as above) and DRG were incubated at 4 °C for 12-16 hrs.DRG were washed with PBS again and incubated in 50% glycerol (in PBS) overnight.The DRG were visualised and imaged using Olympus CKX41 microscope equipped with Olympus DP22 digital camera.The β-gal + cells were either counted manually or with the aid of ImageJ software (Schneider, Rasband and Eliceiri 2012).

Restriction fragment length polymorphism
For extracting viral DNA, 80% confluent Vero cells were infected with appropriate virus in a 175 cm 2 area flask at an MOI of 0.05 PFU/cell for 1 hr at 37 °C with 5% CO 2 .
Fresh medium was added, and cells were further incubated for 48 hrs.The cells and supernatant were centrifuged to remove cell debris.Supernatant was further centrifuged at 17,684 g for 90 mins at 4 °C and the pellet obtained was resuspended in TE-SDS (10 mM Trizma base (pH 8.0) (Sigma-Aldrich), 1 mM EDTA (ThermoFisher Scientific) and 0.5% (w/v) SDS (Sigma-Aldrich) in water).DNA was extracted from the lysate using a standard phenol and chloroform extraction method (Sambrook and Russell 2006), digested with an appropriate restriction enzyme and electrophoresed on an agarose gel (S2 Fig) .

Statistical analysis
Statistical comparisons of means were done using a one-way or two-way analysis of variance (ANOVA) with an appropriate post-hoc test in GraphPad Prism (Version 10.0.1).A p value less than 0.05 was considered significant.

Figure legends
followed by Cre (called UL27-T2A-Cre) (Fig 2A).The expression of gB and Cre as independent proteins was verified by western blotting and this virus grew at a similar rate to parent in vitro (Figs 2B,C).Moreover, Cre was functional in the Vero SUA reporter cells (S3 Fig).Next, we assessed the growth potential of this virus in vivo, in skin and DRG of C57BL/6 mice, and we found similar viral loads in the skin, however the titre of the recombinant virus was reduced in the DRG, compared with the parent virus (Fig 2D) this plasmid was used to generate HSV-1 UL27-T2A-Cre (S1B Fig).To construct pUL3-T2A-Cre, T2A and Cre sequences were obtained as earlier, U L 3 (11184 -11610) and U L 4 (11611 -12049) containing fragments were amplified from HSV-1 KOS, and inserted into the BamHI site of pCR bluntII vector and this plasmid was used to generate HSV-1 UL3-T2A-Cre (S1C Fig).Plasmid UL56-T2A-Cre was constructed using U L 55 (115644 -116144) and U L 56 (116145 -116660) fragments amplified from HSV-1 KOS, and T2A-Cre fragment from pUL27-T2A-Cre, all cloned into BamHI site of pCR bluntII vector and this plasmid was used to generate HSV-1 UL56-T2A-Cre (S1D Fig).
The screening of desired recombinant was carried out based on fluorescence where possible or PCR.Each virus was purified further via three rounds of plaque purification and the absence of parent virus in the final round was confirmed by PCR.The introduced modification in the recombinant was verified by PCR, Sanger sequencing and restriction fragment length polymorphism (S2 Fig).The expression of Cre was verified by western blotting and the function was validated by Vero SUA reporter assays (S3 Fig).

Figure 1 :
Figure 1: Activity of ectopic gB promoter from two independent locations.

Figure 2 :
Figure 2: Evaluation of native gB promoter activity.

Figure 4 :
Figure 4: Native lytic gene promoters can express protein during HSV latency

Figure 5 :
Figure 5: Summary of lytic promoter activity in HSV-1 latency and was purchased from Addgene (plasmid 42230).The sequences coding for appropriate guide RNA were synthesised as two complementary dioxynucleotides, annealed to generate double stranded DNA fragments and inserted into the BbsI site of pX330.
Confluent Vero cells were infected with appropriate virus at an MOI of 10 PFU/cell and incubated for 20 hrs at 37 °C with 5% CO 2 .Cells were washed once with PBS and resuspended in RIPA buffer (200 mM Trizma base (Sigma-Aldrich), 150 mM Sodium