Cohesin promotes HSV-1 lytic transcription by facilitating the binding of RNA Pol II on viral genes.

BACKGROUND: Herpes Simplex Virus type I (HSV-1) is a large double-stranded DNA virus that enters productive infection in epithelial cells and reorganizes the host nucleus. Cohesin, a major constituent of interphase and mitotic chromosomes comprised of SMC1, SMC3, and SCC1 (Mcd1/Rad21), SCC3 (SA1/SA2), have diverse functions, including sister chromatid cohesion, DNA double-stranded breaks repair, and transcriptional control. Little is known about the role of cohesin in HSV-1 lytic infection. METHODS: We measured the effect on HSV-1 transcription, genome copy number, and viral titer by depleting cohesin components SMC1 or Rad21 using RNAi, followed by immunofluorescence, qPCR, and ChIP experiments to gain insight into cohesin's function in HSV-1 transcription and replication. RESULTS: Here, we report that cohesion subunits SMC1 and Rad21 are recruited to the lytic HSV-1 replication compartment. The knockdown results in decreased viral transcription, protein expression, and maturation of viral replication compartments. SMC1 and Rad21 knockdown leads to the reduced overall RNA pol II occupancy level but increased RNA pol II ser5 phosphorylation binding on viral genes. Consistent with this, the knockdown increased H3K27me3 modification on these genes. CONCLUSIONS: These results suggest that cohesin facilitates HSV-1 lytic transcription by promoting RNA Pol II transcription activity and preventing chromatin's silencing on the viral genome.

HSV-1 infection and pathogenesis in the tree shrew eye following corneal inoculation.

Herpes simplex virus type I (HSV-1) infection causes inflammation in the cornea known as herpes simplex virus keratitis (HSK), a common but serious corneal disease. It is not entirely clear whether the virus during recurring infection comes from the trigeminal ganglia or the eye tissue, including the retina and ciliary ganglion. Because the tree shrew is closely related to primates and tree shrew eye anatomic structures are similar to humans, we studied HSV-1 corneal infection in the tree shrew. We found that HSK symptoms closely mimic those found in human HSK showing typical punctiform and dendritic viral keratitis during the acute infection period. Following the HSV-specific lesions, complications such as stromal scarring, corneal thickening (primary infection), opacity, and neovascularization were observed. In the tree shrew model, following ocular inoculation, the cornea becomes infected, and viral protein can be detected using anti-HSV-1 antibodies in the epithelial layer and retina neuronal ganglion cells. The HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 3 days post-infection (dpi), peaking at 5 dpi. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the Latency Associated Transcripts (LATs) continue to accumulate. Interestingly, after the acute infection, we still detected abundant active HSV-1 in tree shrew eyes. Further, we found HSV-1 persistent in the ciliary ganglion and cornea. These findings are discussed in support of the tree shrew as a non-human primate HSK model, which could be useful for mechanistic studies of HSK.

Correction to: Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

An amendment to this paper has been published and can be accessed via the original article.

Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

BACKGROUND: Following acute infection, Herpes Simplex virus-1 (HSV-1) establishes lifelong latency and recurrent reactivation in the sensory neurons of trigeminal ganglia (TG). Infected tree shrew differs from mouse and show characteristics similar to human infection. A detailed transcriptomic analysis of the tree shrew model could provide mechanistic insights into HSV-1 infection in humans. METHODS: We sequenced the transcriptome of infected TGs from tree shrews and mice, and 4 human donors, then examined viral genes expression up to 58 days in infected TGs from mouse and tree shrew, and compare the latency data with that in human TGs. RESULTS: Here, we found that all HSV-1 genes could be detected in mouse TGs during acute infection, but 22 viral genes necessary for viral transcription, replication and viral maturation were not expressed in tree shrew TGs during this stage. Importantly, during latency, we found that LAT could be detected both in mouse and tree shrew, but the latter also has an ICP0 transcript signal absent in mouse but present in human samples. Importantly, we observed that infected human and tree shrew TGs have a more similar LAT region transcription peak. More importantly, we observed that HSV-1 spontaneously reactivates from latently infected tree shrews with relatively high efficiency. CONCLUSIONS: These results represent the first longitudinal transcriptomic characterization of HSV-1 infection in during acute, latency and recurrent phases, and revealed that tree shrew infection has important similar features with human infection.

Human Asymptomatic Epitope Peptide/CXCL10-Based Prime/Pull Vaccine Induces Herpes Simplex Virus-Specific Gamma Interferon-Positive CD107+ CD8+ T Cells That Infiltrate the Corneas and Trigeminal Ganglia of Humanized HLA Transgenic Rabbits and Protect against Ocular Herpes Challenge.

Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that infects the cornea, causing potentially blinding herpetic disease. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull vaccine was tested in a human leukocyte antigen (HLA) transgenic rabbit model of ocular herpes (HLA Tg rabbits). Three peptide epitopes were selected, from the HSV-1 membrane glycoprotein C (UL44400-408), the DNA replication binding helicase (UL9196-204), and the tegument protein (UL25572-580), all preferentially recognized by CD8+ T cells from "naturally protected" HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were immunized with a mixture of these three ASYMP CD8+ T cell peptide epitopes (UL44400-408, UL9196-204, and UL25572-580), which were delivered subcutaneously with CpG2007 adjuvant (prime). Fifteen days later, half of the rabbits received a topical ocular treatment with a recombinant neurotropic adeno-associated virus type 8 (AAV8) vector expressing the T cell-attracting CXCL10 chemokine (pull). The frequency and function of HSV-specific CD8+ T cells induced by the prime/pull vaccine were assessed in the peripheral blood, cornea, and trigeminal ganglion (TG). Compared to the cells generated in response to peptide immunization alone, the peptide/CXCL10 prime/pull vaccine generated frequent polyfunctional gamma interferon-positive (IFN-γ+) CD107+ CD8+ T cells that infiltrated both the cornea and TG. CD8+ T cell mobilization into the cornea and TG of prime/pull-vaccinated rabbits was associated with a significant reduction in corneal herpesvirus infection and disease following an ocular HSV-1 (strain McKrae) challenge. These findings draw attention to the novel prime/pull vaccine strategy for mobilizing antiviral CD8+ T cells into tissues to protect against herpesvirus infection and disease.IMPORTANCE There is an urgent need for a vaccine against widespread herpes simplex virus infections. The present study demonstrates that immunization of HLA transgenic rabbits with a peptide/CXCL10 prime/pull vaccine triggered mobilization of HSV-specific CD8+ T cells locally into the cornea and TG, the sites of acute and latent herpesvirus infections, respectively. Mobilization of antiviral CD8+ T cells into the cornea and TG of rabbits that received the prime/pull vaccine was associated with protection against ocular herpesvirus infection and disease following an ocular HSV-1 challenge. These results highlight the importance of the prime/pull vaccine strategy to bolster the number and function of protective CD8+ T cells within infected tissues.

Herpes Simplex Virus 1 Infection of Tree Shrews Differs from That of Mice in the Severity of Acute Infection and Viral Transcription in the Peripheral Nervous System.

UNLABELLED: Studies of herpes simplex virus (HSV) infections of humans are limited by the use of rodent models such as mice, rabbits, and guinea pigs. Tree shrews (Tupaia belangeri chinensis) are small mammals indigenous to southwest Asia. At behavioral, anatomical, genomic, and evolutionary levels, tree shrews are much closer to primates than rodents are, and tree shrews are susceptible to HSV infection. Thus, we have studied herpes simplex virus 1 (HSV-1) infection in the tree shrew trigeminal ganglion (TG) following ocular inoculation. In situ hybridization, PCR, and quantitative reverse transcription-PCR (qRT-PCR) analyses confirm that HSV-1 latently infects neurons of the TG. When explant cocultivation of trigeminal ganglia was performed, the virus was recovered after 5 days of cocultivation with high efficiency. Swabbing the corneas of latently infected tree shrews revealed that tree shrews shed virus spontaneously at low frequencies. However, tree shrews differ significantly from mice in the expression of key HSV-1 genes, including ICP0, ICP4, and latency-associated transcript (LAT). In acutely infected tree shrew TGs, no level of ICP4 was observed, suggesting the absence of infection or a very weak, acute infection compared to that of the mouse. Immunofluorescence staining with ICP4 monoclonal antibody, and immunohistochemistry detection by HSV-1 polyclonal antibodies, showed a lack of viral proteins in tree shrew TGs during both acute and latent phases of infection. Cultivation of supernatant from homogenized, acutely infected TGs with RS1 cells also exhibited an absence of infectious HSV-1 from tree shrew TGs. We conclude that the tree shrew has an undetectable, or a much weaker, acute infection in the TGs. Interestingly, compared to mice, tree shrew TGs express high levels of ICP0 transcript in addition to LAT during latency. However, the ICP0 transcript remained nuclear, and no ICP0 protein could be seen during the course of mouse and tree shrew TG infections. Taken together, these observations suggest that the tree shrew TG infection differs significantly from the existing rodent models. IMPORTANCE: Herpes simplex viruses (HSVs) establish lifelong infection in more than 80% of the human population, and their reactivation leads to oral and genital herpes. Currently, rodent models are the preferred models for latency studies. Rodents are distant from primates and may not fully represent human latency. The tree shrew is a small mammal, a prosimian primate, indigenous to southwest Asia. In an attempt to further develop the tree shrew as a useful model to study herpesvirus infection, we studied the establishment of latency and reactivation of HSV-1 in tree shrews following ocular inoculation. We found that the latent virus, which resides in the sensory neurons of the trigeminal ganglion, could be stress reactivated to produce infectious virus, following explant cocultivation and that spontaneous reactivation could be detected by cell culture of tears. Interestingly, the tree shrew model is quite different from the mouse model of HSV infection, in that the virus exhibited only a mild acute infection following inoculation with no detectable infectious virus from the sensory neurons. The mild infection may be more similar to human infection in that the sensory neurons continue to function after herpes reactivation and the affected skin tissue does not lose sensation. Our findings suggest that the tree shrew is a viable model to study HSV latency.

Reactivation of HSV-1 following explant of tree shrew brain.

Herpes Simplex Virus type I (HSV-1) latently infects peripheral nervous system (PNS) sensory neurons, and its reactivation leads to recurring cold sores. The reactivated HSV-1 can travel retrograde from the PNS into the central nervous system (CNS) and is known to be causative of Herpes Simplex viral encephalitis. HSV-1 infection in the PNS is well documented, but little is known on the fate of HSV-1 once it enters the CNS. In the murine model, HSV-1 genome persists in the CNS once infected through an ocular route. To gain more details of HSV-1 infection in the CNS, we characterized HSV-1 infection of the tree shrew (Tupaia belangeri chinensis) brain following ocular inoculation. Here, we report that HSV-1 enters the tree shrew brain following ocular inoculation and HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 5 days post-infection (p.i.), peaking at 10 days p.i. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the LAT intron region continues to be expressed. Live virus could be recovered from the olfactory bulb and brain stem tissue. Viral proteins could be detected using anti-HSV-1 antibodies and anti-ICP4 antibody, during the acute stage but not beyond. In situ hybridization could detect LAT during acute infection in most brain regions and in olfactory bulb and brain stem tissue well beyond the acute stage. Using a homogenate from these tissues' post-acute infection, we did not recover live HSV-1 virus, supporting a latent infection, but using a modified explant cocultivation technique, we were able to recover reactivated virus from these tissues, suggesting that the HSV-1 virus latently infects the tree shrew CNS. Compared to mouse, the CNS acute infection of the tree shrew is delayed and the olfactory bulb contains most latent virus. During the acute stage, a portion of the infected tree shrews exhibit symptoms similar to human viral encephalitis. These findings, together with the fact that tree shrews are closely related to primates, provided a valuable alternative model to study HSV-1 infection and pathogenesis in the CNS.

Early nucleosome deposition on, and replication of, HSV DNA requires cell factor PCNA.

Herpes simplex virus (HSV) is a double-stranded DNA virus that can cause lytic infections in epithelial cells of the skin and latent infections in neuronal cells of the peripheral nervous system. After virion attachment to the cell membrane, the capsid enters the cytoplasm and is transported to the nucleus. Following docking at the nuclear pore, the HSV DNA, and contents of the virion, are injected into the nucleus. The viral DNA that enters the nucleus is devoid of histones, but begins to be covered with them soon after entry. The covering of histones, in the form of nucleosomes, reaches a maximum during the early stages of infection and drops off during late infection (after DNA replication). However, during latency, the genome is saturated with nucleosomes. In this study, we examine the role of proliferating cell nuclear antigen (PCNA), a cellular DNA polymerase accessory protein (processivity factor), and cell DNA polymerases in histone deposition during the early stages of HSV infection. Using SiRNA knockdown, and a cytosine arabinoside (araC) chemical inhibitor, we conclude that PCNA is important for viral replication and histone deposition. However, cell DNA polymerases that bind PCNA do not appear to be required for these processes and PCNA does not appear to bind to the viral DNA polymerase (which has its own viral processivity factor).

Chromatin assembly on herpes simplex virus 1 DNA early during a lytic infection is Asf1a dependent.

Herpes simplex virus (HSV) is a large DNA virus which is characterized by its ability to form latent infections in neurons of the peripheral nervous system. Although histones are found in the capsids of small DNA viruses (papovaviruses), none are found in the capsids of large HSV. However, after entry into the infected cell nucleus, the HSV genome begins to associate with nucleosomes during the earliest stages of infection. In contrast, late during infection, newly replicated viral DNA does not appear to associate with nucleosomes, suggesting that histones are deposited specifically on input viral DNA. The mechanisms of deposition and removing histones from the viral genome are unclear. Recently, histone chaperones, involved in the assembly and disassembly of nucleosomes, have been identified. Human antisilencing factor 1 (Asf1) is one such factor which is involved in both the assembly and disassembly of nucleosomes in cellular systems. In this study, we have examined the effect of small interfering RNA (siRNA) knockdown of Asf1a on HSV infections in HeLa cells. Both viral replication and growth were found to be decreased. Also, viral DNA was significantly less protected from micrococcal nuclease (MNase) digestion up to 6 h postinfection (hpi). However, transcription of the immediate early (IE) genes ICP0 and ICP4 was significantly upregulated at 3 h postinfection. Also, these genes were found to be less protected from MNase digestion and, therefore, less associated with nucleosomes. These results suggest that Asf1a plays a role in regulating IE genes by assembling chromatin onto histone-free viral DNA by 3 h postinfection.

DNA damage promotes herpes simplex virus-1 protein expression in a neuroblastoma cell line.

Although the induction of the cellular DNA damage response by herpes simplex virus-1 (HSV-1) infection of epithelial cells in tissue culture promotes productive infection, there has been no experimental observation of the effect of the cellular DNA damage response on HSV-1 infection in vivo or in neuronal derived cell lines in tissue culture. Thus, it has been speculated that the lack of cellular DNA damage induction during infection of neurons may promote latency in these cells. This work examines the profile of HSV-1 promoter induction and protein expression, in the absence or presence of infection; using cellular DNA damage inducing topoisomerase inhibitors (Camptothecin and Etoposide) on a neuroblastoma cell line (C1300) in which HSV-1 infection fails to induce the DNA damage response. In the absence of infection, a plasmid expressing the immediate early ICP0 promoter was the most induced by the DNA damage drug treatments compared to the early (RR) and late (VP16) gene promoters. Similarly, drug treatment of C1300 cells infected with HSV-1 virus showed enhanced protein expression for ICP0, but not ICP4 and VP16 proteins. However, when the cells were infected with a HSV-1 virus defective in the immediate early gene trans-activator VP16 (in814) and treated with the DNA damaging drugs, there was enhanced expression of immediate early and late HSV-1 proteins. Although, viral infection of the neuroblastoma cell alone did not induce DNA damage, cellular DNA damage induced by drug treatments facilitated viral promoter induction and viral protein expression. This implicates a mechanism by which HSV-1 viral genes in a quiescent or latent state may become induced by cellular DNA damage in neuronal cells to facilitate productive infection.

The half-life of the HSV-1 1.5-kb LAT intron is similar to the half-life of the 2.0-kb LAT intron.

Herpes simplex virus type 1 establishes a latent infection in the sensory neurons of the peripheral nervous system of humans. Although about 80 genes are expressed during the lytic cycle of the virus infection, essentially only one gene is expressed during the latent cycle. This gene is known as the latency-associated transcript (LAT), and it appears to play a role in the latency cycle through an anti-apoptotic function in the 5' end of the gene and miRNA encoded along the length of the transcript which downregulate some of the viral immediate-early gene products. The LAT gene is about 8.3 kb long and consists of two exons separated by an unusual intron. The intron between the exons consists of two nested introns. This arrangement of introns has been called a twintron. Furthermore, the larger (2 kb) intron has been shown to be very stable. In this study, we measure the stability of the shorter 1.5-kb nested intron and find its half-life is similar to the longer intron. This was achieved by deleting the 0.5-kb overlapping intron from a plasmid construct designed to express the LAT transcript from a tet-inducible promoter and measuring the half-life of the 1.5-kb intron in tissue culture cells. This finding supports the hypothesis that it is the common branch-point region of these nested introns that is responsible for their stability.

Persistent inflammation and neuronal loss in the mouse brain induced by a modified form of attenuated herpes simplex virus type I.

Herpes simplex virus-1 (HSV-1) is a widespread neurotropic virus that can reach the brain and cause a rare but acute herpes simplex encephalitis (HSE) with a high mortality rate. Most patients present with changes in neurological and behavioral status, and survivors suffer long-term neurological sequelae. To date, the pathogenesis leading to brain damage is still not well understood. HSV-1 induced encephalitis in the central nervous system (CNS) in animals are usually very diffuse and progressing rapidly, and mostly fatal, making the analysis difficult. Here, we established a mouse model of HSE via intracerebral inoculation of modified version of neural-attenuated strains of HSV-1 (deletion of ICP34.5 and inserting a strong promoter into the latency-associated transcript region), in which the LMR-αΔpA strain initiated moderate productive infection, leading to strong host immune and inflammatory response characterized by persistent microglia activation. This viral replication activity and prolonged inflammatory response activated signaling pathways in neuronal damage, amyloidosis, Alzheimer's disease, and neurodegeneration, eventually leading to neuronal loss and behavioral changes characterized by hypokinesia. Our study reveals detailed pathogenic processes and persistent inflammatory responses in the CNS and provides a controlled, mild and non-lethal HSE model for studying long-term neuronal injury and increased risk of neurodegenerative diseases due to HSV-1 infection.

The herpes simplex virus type 1 latency-associated transcript can protect neuron-derived C1300 and Neuro2A cells from granzyme B-induced apoptosis and CD8 T-cell killing.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LAT's antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LAT's antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LAT's ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LAT's antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo.

Direct evidence that HSV DNA damaged by ultraviolet (UV) irradiation can be repaired in a cell type-dependent manner.

Infection of permissive cells, in tissue culture, with herpes simplex virus (HSV) has been reported to induce host DNA damage repair responses that are necessary for efficient viral replication. However, direct repair of the damaged viral DNA has not, to our knowledge, been shown. In this report, we detect and determine the amount of damaged HSV-1 DNA, following introduction of experimentally damaged HSV genomes into tissue cultures of permissive Vero, NGF differentiated PC12 cells and primary rat neurons, using a method of detection introduced here. The results show that HSV-1 strain 17 DNA containing UV-induced DNA damage is efficiently repaired, in Vero, but not NGF differentiated PC12 cells. The primary rat neuronal cultures were capable of repairing the damaged viral DNA, but with much less efficiency than did the permissive Vero cells. Moreover, by conducting the experiments with either an inhibitor of the HSV polymerase (phosphonoacetic acid [PAA]) or with a replication defective DNA polymerase mutant virus, HP66, the results suggest that repair can occur in the absence of a functional viral polymerase, although polymerase function seems to enhance the efficiency of the repair, in a replication independent manner. The possible significance of varying cell type mediated repair of viral DNA to viral pathogenesis is discussed.

The histone variant H3.3 regulates gene expression during lytic infection with herpes simplex virus type 1.

It has been proposed that incorporation of the histone variant H3.3 within actively transcribed regions of a genome helps to facilitate transcription. In this report we use lytic infection by herpes simplex virus type 1 (HSV-1) as a model to examine the temporal profile of histone H3 incorporation and to determine whether the variant histone H3.3 has a direct effect on transcription. We find that canonical H3.1 and variant H3.3 exhibit distinct temporal associations with the genome in cell lines expressing equal amounts of epitope-tagged H3 variants. At the earliest times examined after infection, the HSV-1 genome is incorporated into chromatin that predominantly contains the variant H3.3, whereas incorporation of canonical H3.1 occurs later in infection and is dependent on replication of the HSV-1 genome. Further, inhibition of H3.3 association, via reduced expression of the H3.3 chaperone HIRA, significantly reduces the levels of HSV-1 mRNA. These findings show that incorporation of H3.3 facilitates transcription, and they provide new evidence for a regulatory role of chromatin composition during HSV-1 acute infection.

Evidence that herpes simplex virus DNA derived from quiescently infected cells in vitro, and latently infected cells in vivo, is physically damaged.

Using polymerase chain reaction (PCR) and alkaline gel electrophoresis, the authors show that, compared with DNA derived from virions used to establish infection, herpes simplex virus DNA derived from quiescently infected rat pheochromocytoma (PC12) cells in culture accumulates alkaline-labile lesions. That is, compared with equivalent amounts of virion DNA, viral DNA from nerve growth factor-differentiated long-term infected cells in culture is consistently 3 to 10 times more refractory to amplification by PCR. Despite using equal mole amounts of DNA isolated from quiescently infected cells (determined by quantitative Southern blots), DNA from quiescently infected cells could not be detected by PCR under conditions in which the virion-derived DNA was easily detected. Refractoriness to PCR was confirmed by analysis with a ligation-mediated PCR technique. The refractoriness was not the result of genomic circularization. The refractoriness was, however, related to the time that the quiescently infected cells had been maintained in culture. The refractoriness to PCR was taken as an indication that the viral DNA was damaged. This hypothesis was confirmed by showing that viral DNA from quiescently infected PC12 cells accumulated alkaline-labile DNA lesions, as determined by alkaline gel electrophoresis. The phenomenon was not limited to tissue culture, because viral DNA derived from the ganglia of latently infected mice is also 3 to 10 times more refractory to amplification than are equivalent amounts of virion-derived genomes. Taken together, these results represent the first evidence that herpes simplex virus DNA is physically damaged as a function of long-term infection. Implications for viral reactivation and pathogenesis are discussed.

Temporal association of the herpes simplex virus genome with histone proteins during a lytic infection.

Previous work has determined that there are nucleosomes on the herpes simplex virus (HSV) genome during a lytic infection but that they are not arranged in an equally spaced array like in cellular DNA. However, like in cellular DNA, the promoter regions of several viral genes have been shown to be associated with nucleosomes containing modified histone proteins that are generally found associated with actively transcribed genes. Furthermore, it has been found that the association of modified histones with the HSV genome can be detected at the earliest times postinfection (1 h postinfection) and increases up to 3 h postinfection. However from 3 h to 6 h postinfection (the late phase of the replication cycle), the association decreases. In this study we have examined histone association with promoter regions of all kinetic classes of genes. This was done over the time course of an infection in Sy5y cells using sucrose gradient sedimentation, bromodeoxyuridine labeling, chromatin immunoprecipitation assays, Western blot analysis, trypsin and DNase digestion, and quantitative real-time PCR. Because no histones were detected inside HSV type 1 capsids, the viral genome probably starts to associate with histones after being transported from infecting virions into the host nucleus. Promoter regions of all gene classes (immediate early, early, and late) bind with histone proteins at the start of viral gene expression. However, after viral DNA replication initiates, histones appear not to associate with newly synthesized viral genomes.

Transduction of brain by herpes simplex virus vectors.

An imposing obstacle to gene therapy is the inability to transduce all of the necessary cells in a target organ. This certainly applies to gene transfer to the brain, especially when one considers the challenges involved in scaling up transduction from animal models to use in the clinic. Non-neurotropic viral gene transfer vectors (e.g., adenovirus, adeno-associated virus, and lentivirus) do not spread very far in the nervous system, and consequently these vectors transduce brain regions mostly near the injection site in adult animals. This indicates that numerous, well-spaced injections would be required to achieve widespread transduction in a large brain with these vectors. In contrast, herpes simplex virus type 1 (HSV-1) is a promising vector for widespread gene transfer to the brain owing to the innate ability of the virus to spread through the nervous system and form latent infections in neurons that last for the lifetime of the infected individual. In this review, we summarize the published literature of the transduction patterns produced by attenuated HSV-1 vectors in small animals as a function of the injection site, and discuss the implications of the distribution for widespread gene transfer to the large animal brain.

Attenuated Phenotype and Immunogenic Characteristics of a Mutated Herpes Simplex Virus 1 Strain in the Rhesus Macaque.

Herpes simplex virus type 1(HSV-1) presents a conundrum to public health worldwide because of its specific pathogenicity and clinical features. Some experimental vaccines, such as the recombinant viral glycoproteins, exhibit the viral immunogenicity of a host-specific immune response, but none of these has achieved a valid epidemiological protective efficacy in the human population. In the present study, we constructed an attenuated HSV-1 strain M3 through the partial deletion of UL7, UL41, and the latency-associated transcript (LAT) using the CRISPR/Cas9 system. The mutant strain exhibited lowered infectivity and virulence in macaques. Neutralization testing and ELISpot detection of the specific T-cell responses confirmed the specific immunity induced by M3 immunization and this immunity defended against the challenges of the wild-type strain and restricted the entry of the wild-type strain into the trigeminal ganglion. These results in rhesus macaques demonstrated the potential of the attenuated vaccine for the prevention of HSV-1 in humans.