Herpes Simplex Virus Gene Research Articles

DPV UL41 gene encoding protein induces host shutoff activity and affects viral replication

The virion host shutoff (VHS) protein, encoded by the UL41 gene of herpes simplex virus (HSV), specifically degrades mRNA and induces host shutoff. VHS and its homologs are highly conserved in the Alphaherpesvirinae subfamily. However, the role of the duck plague virus (DPV) UL41 gene is unclear. In this study, we found that the DPV UL41 gene-encoded protein (pUL41) degrades RNA polymerase (pol) II-transcribed translatable RNA and induces protein synthesis shutoff. DPV pUL41 was dispensable for viral replication, but the UL41-deleted mutant virus exhibited a significant viral growth defect and plaque size reduction in Duck embryo fibroblast (DEF) cells. Furthermore, DPV pUL41 regulated viral mRNA accumulation to affect viral DNA replication, release and cell-to-cell spread.

Splicing factor SRSF2-centric gene regulation

Serine/arginine-rich splicing factor 2 (SRSF2) is a splicing factor that is widely expressed in a variety of mammalian cell types. Increasing evidence has confirmed that SRSF2 plays vital roles in a number of biological and pathological processes. Therefore, it is important to understand how its expression is regulated, and how it regulates the expression of its target genes. Recently, we found that SRSF2 expression could be upregulated by herpes simplex virus-1 (HSV-1) infection, and that altered SRSF2 expression, in turn, epigenetically regulates the transcription of HSV-1 genes. Further studies on T cell exhaustion demonstrated that upregulated SRSF2 in exhausted T cells elevated the levels of multiple immune checkpoint molecules by associating with the acyl-transferases, P300 and CBP, and by altering histone modification near the transcription start sites of these genes, thereby influencing signal transducer and activator of transcription 3 binding to these gene promoters. These findings suggest that SRSF2 acts as an important sensor and effector during disease progression. Here, we discuss the molecules that regulate SRSF2 gene expression and their associated mechanisms, and the mechanisms via which SRSF2 regulates the expression of target genes, thus providing novel insights into the central role of SRSF2 in gene regulation.

Herpes Simplex Virus-1 infection in human primary corneal epithelial cells is blocked by a stapled peptide that targets processive DNA synthesis

PurposeAcyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus −1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. MethodsA series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. ResultsThe stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. ConclusionsHydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.

Design of an Interferon-Resistant Oncolytic HSV-1 Incorporating Redundant Safety Modalities for Improved Tolerability

Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. In vivo, this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1.

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

BackgroundFollowing 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.MethodsWe 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.ResultsHere, 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.ConclusionsThese 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.

Investigating the effect of Herpes Simplex Virus 1 Latency-Associated Non-Coding RNAs on the Human Neuronal Transcriptome

Herpes simplex virus 1 (HSV-1) is a prevalent neurotropic virus that persists for the host’s lifetime due to HSV-1 establishing latency in sensory neurons. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 1.5kb or 2.0kb major LAT intron and several microRNAs. These latency-associated non-coding RNAs (ncRNAs) have been reported to impact the establishment, maintenance and reactivation from latency. However, the molecular mechanisms of these ncRNAs are not fully characterised, especially in the context of human neurons. This study investigated how the latency-associated ncRNAs affect the human neuronal transcriptome. We developed an experimental system to deliver the latency-associated ncRNAs to human neurons, differentiated from SH-SY5Y neuroblastoma cells. The cells were infected with a replication-defective HSV-1 mutant, in1382, that establishes a quiescent infection in which LAT is strongly expressed. Alternatively, we utilised lentiviruses engineered to express the first 3.1kb of LAT, without or with mutations in splice sites that prevents splicing of the major LAT intron, or five HSV-1 microRNAs, shown to be abundant in latently infected human ganglia. Following RNA-Seq of uninfected versus infected or transduced SH-SY5Y cells, we identified 178 host genes that had significant differential expression in response to in1382 quiescent infection and lentivirus delivery of LAT or the latency-associated microRNAs. A subset of these were validated by PCR. This work provides insight into possible roles of the latency-associated ncRNAs in neuronal cell biology and latency that could aid future investigations examining how HSV-1 latency affects human neurons.

Suicide Gene Therapy Mediated with Exosomes Produced by Mesenchymal Stem/Stromal Cells Stably Transduced with HSV Thymidine Kinase.

Mesenchymal stem/stromal cells (MSCs) prepared from various human tissues were stably transduced with the suicide gene herpes simplex virus thymidine kinase (HSVTK) by means of retrovirus infection. HSVTK-transduced MSCs express the suicide gene and in prodrug ganciclovir (GCV) presence induced cell death by intracellular conversion of GCV to GCV-triphosphate. The homogenous population of HSVTK-MSCs were found to release exosomes having mRNA of the suicide gene in their cargo. The exosomes were easily internalized by the tumor cells and the presence of ganciclovir caused their death in a dose-dependent manner. Efficient tumor cell killing of glioma cell lines and primary human glioblastoma cells mediated by HSVTK-MSC exosomes is reported. Exosomes produced by suicide gene transduced MSCs represent a new class of highly selective tumor cell targeted drug acting intracellular with curative potential.

Herpes Simplex Virus 1-Specific CD8+ T Cell Priming and Latent Ganglionic Retention Are Shaped by Viral Epitope Promoter Kinetics.

Reactivation of herpes simplex virus 1 (HSV-1) from neurons in sensory ganglia such as the trigeminal ganglia (TG) is influenced by virus-specific CD8+ T cells that infiltrate the ganglia at the onset of latency and contract to a stable activated tissue-resident memory population. In C57BL/6 mice, half of HSV-specific CD8+ T cells (gB-CD8s) recognize one dominant epitope (residues 498 to 505) on glycoprotein B (gB498-505), while the remainder (non-gB-CD8s) recognize 19 subdominant epitopes from 12 viral proteins. To address how expression by HSV-1 influences the formation and ganglionic retention of CD8+ T cell populations, we developed recombinant HSV-1 with the native immunodominant gB epitope disrupted but then expressed ectopically from different viral promoters. In mice, the epitope expressed from the gB promoter restored full gB-CD8 immunodominance to 50%. Intriguingly, earlier expression from constitutive, immediate-early, and early promoters did not significantly increase immunodominance, indicating that these promoters cannot elicit more than half of the CD8 compartment. Epitope expressed from candidate viral promoters of "true late" HSV-1 genes either delayed or reduced the priming efficiency of gB-CD8s and their levels in the TG at early times. HSV expressing the epitope from the full latency-associated transcript promoter did not efficiently prime gB-CD8s; however, gB-CD8s primed by a concurrent wild-type flank infection infiltrated the TG and were retained long term, suggesting that latent epitope expression is sufficient to retain gB-CD8s. Taken together, the data indicate that viral promoters shape latent HSV-1-specific CD8+ T cell populations and should be an important consideration in future vaccine design.IMPORTANCE Latency of HSV-1 in host neurons enables long-term persistence from which reactivation may occur to cause recurrent diseases, such as blinding herpetic stromal keratitis. Latency is not antigenically silent, and viral proteins are sporadically expressed at low levels without full virion production. This protein expression is recognized by ganglion-resident HSV-1-specific CD8+ T cells that maintain a protective resident population. Since these T cells can influence lytic/latent decisions in reactivating neurons, we argue that improving their ganglionic retention and function may offer a strategy in vaccine design to reduce reactivation and recurrent disease. To understand factors driving the infiltration and retention of ganglionic CD8s, we examined several HSV recombinants that have different viral promoters driving expression of the immunodominant gB epitope. We show that the selection of epitope promoter influences CD8+ T cell population hierarchies and their function.

Comparison of HEp-2 and Vero Cell Responses Reveal Unique Proapoptotic Activities of the Herpes Simplex Virus Type 1 α0 Gene Transcript and Product.

Previous studies have provided evidence suggesting a role for apoptosis in the control of Herpes Simplex Virus 1 (HSV-1) latency. HSV-1 induces and then later blocks apoptosis in infected cells. The immediate early viral gene α0, which synthesizes the ICP0 protein, is necessary and sufficient for HSV-1-induced apoptosis in human epithelial (HEp-2) cells. While previous research showed that ICP0 protein synthesis is not necessary for HSV-1-induced apoptosis in infected HEp-2 cells, circumstantial evidence suggested that it might be needed in infected African green monkey kidney (Vero) cells. In this study, we determined the specific aspects of α0 needed to trigger apoptosis in these two cell types. HEp-2 cells transfected with α0 expressing plasmids that generated either full-length, truncated, or no detectable (multiple stop codons) ICP0 protein died through apoptosis. This indicates that ICP0 protein is not necessary for α0-induced apoptosis and that α0 mRNA alone has apoptotic induction properties in HEp-2 cells. We next investigated the primary structure of α0’s mRNA to better define its proapoptotic ability. Since α0 is one of the few HSV-1 genes that are spliced, we transfected cells with a plasmid expressing ICP0 from cDNA copy, pcDNAICP0. The cells transfected with pcDNAICP0 underwent apoptosis at a level equivalent to those transfected with the genomic copy of α0, which indicates that neither splicing events nor introns are required for the apoptotic function of α0 in HEp-2 cells. Next, we studied the ability of α0 to cause apoptosis in Vero cells. Since HSV-1-induced apoptosis in Vero cells requires protein synthesis early in infection, proteins synthesized with immediate early kinetics may facilitate apoptosis. Vero cells were transfected with plasmids producing either full-length ICP0 or ICP0 truncated at codon 212. Full-length ICP0, but not truncated ICP0, induced apoptosis in Vero cells. Together, these results suggest that α0 gene expression triggers apoptosis, but ICP0 protein is needed to facilitate apoptosis in Vero cells. In addition, ICP0’s facilitation activity may lie in its carboxyl-terminated domain. Thus, our results demonstrate that α0’s mRNA and protein possess proapoptotic properties. The requirement for ICP0 protein during HSV-dependent apoptosis appears to be cell type specific.

Investigating the impact of Herpes simplex virus type 1 latency-associated non-coding RNAs on apoptosis in human neuronal cells

Herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons, allowing it to persist for the lifetime of the host. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 2.0 kb major LAT intron and several microRNAs. These non-coding RNAs (ncRNAs) have been reported to influence latency, possibly through limiting apoptosis of infected cells. As these studies have used animal models or non-neuronal cell culture, we have developed a differentiated human neuroblastoma (SH-SY5Y cells) model to examine their effect in human neuronal cells. We have infected these neuronal cultures with replication-defective HSV-1, which establishes a quiescent infection and strongly expresses the latency ncRNAs. We show that quiescent HSV-1 infection reproducibly protects differentiated SH-SY5Y from etoposide-induced apoptosis. We are also further defining the contribution of different LAT ncRNAs using recombinant lentiviruses to drive expression of the LAT intron or microRNAs. Furthermore, we are also currently exploring the mechanisms of this anti-apoptosis effect, and broader virus-neuron interactions by characterising whether the human neuronal transcriptome is altered by LAT RNA expression. Improving our understanding of the molecular interactions underpinning HSV-1 latency in neurons could help develop novel therapies to target HSV-1 latency.

Temperature-sensitive origin-binding protein as a tool for investigations of herpes simplex virus activities in vivo.

While it is fairly clear that herpes simplex virus (HSV) DNA replication requires at least seven virus-encoded proteins in concert with various host cell factors, the mode of this process in infected cells is still poorly understood. Using HSV-1 mutants bearing temperature-sensitive (ts) lesions in the UL9 gene, we previously found that the origin-binding protein (OBP), a product of the UL9 gene, is only needed in the first 6 hours post-infection. As this finding was just a simple support for the hypothesis of a biphasic replication mode, we became convinced through these earlier studies that the mutants tsR and tsS might represent suitable tools for more accurate investigations in vivo. However, prior to engaging in highly sophisticated research projects, knowledge of the biochemical features of the mutated versions of OBP appeared to be essential. The results of our present study demonstrate that (i) tsR is most appropriate for cell biological studies, where only immediate early and early HSV gene products are being expressed without the concomital viral DNA replication, and (ii) tsS is a prime candidate for the analysis of HSV DNA replication processes because of its reversibly thermosensitive OBP-ATPase, which allows one to switch on the initiation of DNA synthesis precisely.

Oncolytic herpes simplex virus and immunotherapy

BackgroundOncolytic viruses have been proposed to be employed as a potential treatment of cancer. Well targeted, they will serve the purpose of cracking tumor cells without causing damage to normal cells. In this category of oncolytic viral drugs human pathogens herpes simplex virus (HSV) is especially suitable for the cause. Although most viral infection causes antiviral reaction in the host, HSV has multiple mechanisms to evade those responses. Powerful anti-tumor effect can thus be achieved via genetic manipulation of the HSV genes involved in this evading mechanism, namely deletions or mutations that adapt its function towards a tumor microenvironment. Currently, oncolytic HSV (oHSV) is widely use in clinical; moreover, there’s hope that its curative effect will be further enhanced through the combination of oHSV with both traditional and emerging therapeutics.ResultsIn this review, we provide a summary of the HSV host antiviral response evasion mechanism, HSV expresses immune evasion genes such as ICP34.5, ICP0, Us3, which are involved in inducing and activating host responses, so that the virus can evade the immune system and establish effective long-term latent infection; we outlined details of the oHSV strains generated by removing genes critical to viral replication such as ICP34.5, ICP0, and inserting therapeutic genes such as LacZ, granulocyte macrophage colony-stimulating factor (GM-CSF); security and limitation of some oHSV such G207, 1716, OncoVEX, NV1020, HF10, G47 in clinical application; and the achievements of oHSV combined with immunotherapy and chemotherapy.ConclusionWe reviewed the immunotherapy mechanism of the oHSV and provided a series of cases. We also pointed out that an in-depth study of the application of oHSV in cancer treatment will potentially benefits cancer patients more.

Linking a cell-division gene and a suicide gene to define and improve cell therapy safety.

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety, however, is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1, but none has quantitatively defined the safety level of transplant therapies. Here, using genome-engineering strategies, we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore, we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here, we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic.

In Vivo Knockdown of the Herpes Simplex Virus 1 Latency-Associated Transcript Reduces Reactivation from Latency.

During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Mechanisms of Host IFI16, PML, and Daxx Protein Restriction of Herpes Simplex Virus 1 Replication.

The initial events after DNA virus infection involve a race between epigenetic silencing of the incoming viral DNA by host cell factors and expression of viral genes. Several host gene products, including the nuclear domain 10 (ND10) components PML (promyelocytic leukemia) and Daxx (death domain-associated protein 6), as well as IFI16 (interferon-inducible protein 16), have been shown to restrict herpes simplex virus 1 (HSV-1) replication. Whether IFI16 and ND10 components work together or separately to restrict HSV-1 replication is not known. To determine the combinatorial effects of IFI16 and ND10 proteins on viral infection, we depleted Daxx or PML in primary human foreskin fibroblasts (HFFs) in the presence or absence of IFI16. Daxx or IFI16 depletion resulted in higher ICP0 mutant viral yields, and the effects were additive. Surprisingly, small interfering RNA (siRNA) depletion of PML in the HFF cells led to decreased ICP0-null virus replication, while short hairpin RNA (shRNA) depletion led to increased ICP0-null virus replication, arguing that different PML isoforms or PML-related proteins may have restrictive or proviral functions. In normal human cells, viral DNA replication increases expression of all classes of HSV-1 genes. We observed that IFI16 repressed transcription from both parental and progeny DNA genomes. Taken together, our results show that the mechanisms of action of IFI16 and ND10 proteins are independent, at least in part, and that IFI16 exerts restrictive effects on both input and replicated viral genomes. These results raise the potential for distinct mechanisms of action of IFI16 on parental and progeny viral DNA molecules.IMPORTANCE Many human DNA viruses transcribe their genomes and replicate in the nucleus of a host cell, where they exploit the host cell nuclear machinery for their own replication. Host factors attempt to restrict viral replication by blocking such events, and viruses have evolved mechanisms to neutralize the host restriction factors. In this study, we provide information about the mechanisms of action of three host cell factors that restrict replication of herpes simplex virus (HSV). We found that these factors function independently and that one acts to restrict viral transcription from parental and progeny viral DNA genomes. These results provide new information about how cells counter DNA virus replication in the nucleus and provide possible approaches to enhance the ability of human cells to resist HSV infection.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification—exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK). Its expression results in specific vulnerability of genetically modified cells to prodrug—ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating “emergency exit” switch allowing eradication of transplanted cells in case of their malfunction.

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons.

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.

Timing Is Everything.

ABSTRACTN. Drayman et al. in their recent article (mBio 8:e01612-17, 2017, https://doi.org/10.1128/mBio.01612-17) have used dynamic proteomics and machine learning to show that the cell cycle state of any individual cell affects the outcome of a productive herpes simplex virus 1 (HSV-1) infection. Cells infected from early G1 through S were most permissive for expression of genes from the HSV-1 genome, whereas cells infected in late G2 to mitosis were much less so. Most of the infected cells that underwent mitosis became permanently nonpermissive for HSV-1 gene expression afterward. The cell cycle stage accounted for 60% of the success of infection, and cell density and motility accounted for most of the rest. To successfully reactivate, HSV-1 must express its genes in neurons and cells of the spinosum and granulosum epidermis strata. These cells are permanently in the cell cycle stages most permissive for HSV-1 gene expression, and none reenters mitosis, thus maximizing the efficiency of a successful HSV-1 reactivation before the adaptive immunity can control it.

Comparative Analysis of Gene Therapy Systems Expressing Two Oncotherapeutic Genes under Control of a Single Promotor

To improve the safety profile and specificity in cancer gene therapy applications, а set of singleplasmid vectors for expression of suicide thymidine kinase gene of the herpes simplex virus (HSVtk) and mouse OX40L gene (m0X40L) involved in regulation of immune response was constructed. Therapeutic transgene expression was directed by a strong constitutive promoter of human cytomegalovirus (CMV), while translation occurred on an RNA template due to an encephalomyocarditis virus internal ribosome entry site (construct CMV-HSVtk-IRES-mOX40L) or 2A-peptide of Porcine Teschovirus-1 (construct CMV-HSVtk-2AmOX40L) inserted between them. The constructs obtained were functionally validated by transient transfection of C26 cells (mouse colon carcinoma). Both bicystronic vectors demonstrated strong cytotoxic activity and produced cytotoxic protein HSVtk at the similar level. To analyze efficiency of mOX40L expression, C26 transfected cells were immunofluorescence-labelled with anti-OX40L-antibodies conjugated with phycoerythrin following flow cytometric analysis. Production of mOX40L was higher in the case of CMV-HSVtk- 2A-mOX40L. Better performance of this expression vector was correlated with our data. Previously, we have demonstrated that a higher level of transgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence. Thus, transfection with the vector based on the 2A peptide sequence is optimal for achieving high efficiency of expression of both oncotherapeutic genes.

Oncolytic Herpes Simplex Virus ICP47 Deletion Reverses Tumor Immune Evasion

Herpes simplex virus (HSV) is an enveloped, double-stranded DNA virus that has been used with modification as oncolytic viruses (OVs) against a number of tumor types. OVs represent a new class of therapeutic agents that promote anti-tumour responses through a dual mechanism of action that is dependent on selective tumor cell killing and the induction of systemic anti-tumour immunity. Among OVs, HSVs preferentially replicate in and lyse cancer cells, leading to in situ autovaccination, adaptive anti-virus and anti-tumor immunity. Suppression of antitumor immunity after OV therapy has been observed and the molecular and cellular mechanisms of action are recently reported. ICP47, a small protein produced by the herpes simplex virus, is considered as an important factor in the evasion of cellular immune responses in HSV-infected cells. Therefore, reviewing the research status of ICP47 is certainly helpful to improve the anti-tumor effect of oncolytic HSVs (oHSVs). Here, this review will focus on the following contents: 1) Anti-tumor mechanism of OVs; 2) Functions of early HSV genes; 3) The mechanism of immune escape of ICP47; 4) Recombinant HSV against cancer; 5) The functional verification of ICP47 deletion. This review highlights the current understanding of recombinant HSVs against cancers.