Herpes Simplex Virus Replication Research Articles

The propagation of HSV-1 in high autophagic activity

Autophagy is an intracellular process involving double-membrane vacuoles that ultimately merge with the lysosomes and play a key role in the inhibition of herpessimplex virus 1 (HSV-1) proliferation. This virus is an agent of some lethal neuronal diseases like encephalitis. HSV-1 requires the expression of its latent proteins, such as ICP34.5, to promote cell infection, which disrupts the autophagy process. In this study, we aimed to evaluate the effect of autophagy induction on HSV-1 replication in host cells at the early and late stages of its replication. Furthermore, we explored the consequences of autophagy induction before and after cell infection.Cells were transfected through Beclin-1-expressing plasmids. Autophagy induction was performed with microtubule-associated protein 1 light chain 3 (LC3-II) as an autophagosome formation marker by using flow cytometry. In the first stage, HSV-1 was inoculated into transfected cells 18 hours post-transfection. Next, viral DNA was extracted 18 and 48 hours post-infection, and eventually viral copies per milliliter were calculated through real-time polymerase chain reaction (PCR). For the second stage, the plasmid containing Beclin-1 was transfected to the cells following virus inoculation to examine the influence of autophagy induction after cell infection.Study results have shown that in neuroblastoma cells autophagy activation reduces virus yield from 4×10 5 copies/ml (control sample) to 9×10 4 copies/ml at 24 h postinfection and viral load after 48 h declines up to 1×10 6 copies/ml, which is less than that of the control sample about 5 logs. However, in HeLa cells, we observed a significant reduction in autophagy induction with reducing HSV-1 propagation. Despite these results, HSV-1 proliferation in both cell types increased and these viruses were able to maintain their ability to propagate even in high autophagic activity. Hyperactivation of autophagy can only slow the rate of virus replication. This study may provide new insight into the effect of autophagy on HSV-1 replication.

HSV-1 and Zika Virus but Not SARS-CoV-2 Replicate in the Human Cornea and Are Restricted by Corneal Type III Interferon

Here, we report our studies of immune-mediated regulation of Zika virus (ZIKV), herpes simplex virus 1 (HSV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the human cornea. We find that ZIKV can be transmitted via corneal transplantation in mice. However, in human corneal explants, we report that ZIKV does not replicate efficiently and that SARS-CoV-2 does not replicate at all. Additionally, we demonstrate that type III interferon (IFN-λ) and its receptor (IFNλR1) are expressed in the corneal epithelium. Treatment of human corneal explants with IFN-λ, and treatment of mice with IFN-λ eye drops, upregulates antiviral interferon-stimulated genes. In human corneal explants, blockade of IFNλR1 enhances replication of ZIKV and HSV-1 but not SARS-CoV-2. In addition to an antiviral role for IFNλR1 in the cornea, our results suggest that the human cornea does not support SARS-CoV-2 infection despite expression of ACE2, a SARS-CoV-2 receptor, in the human corneal epithelium.

THYMIDINE KINASE AS A CAUSATIVE FACTOR FOR TYPE 1 HERPES SIMPLEX VIRUS RESISTANCE AGAINST ACYCLOVIR

ABSTRACTBackground: Herpes Simplex Virus (HSV) infection demonstrates a high prevalence in the world. Acyclovir, one of guanine synthetic analogues, is commonly used to treat infections caused by HSV. HSV resistance against acyclovir may occur, especially in immunocompromised and immunocompetent patients, as the consequence of viral mutations. Thymidine kinase (TK) is an HSV tegument protein which plays an important role in HSV-1 resistance against acyclovir. Purpose: The purpose of this article is to review the mechanisms of TK mutation that cause HSV-1 resistance against acyclovir. Review: Acyclovir involves three stages of viral thymidine kinase phosphorylation to form acyclovir triphosphate. It prevents HSV replication by acting as a competitive inhibitor of viral DNA polymerase and a chain terminator in viral DNA synthesis. Resistance is associated with viral TK mutation that is encoded by UL23 gene. Long-term use of acyclovir may promote thymidine kinase mutation in immunocompromised and immunocompetent patients via three mechanisms, namely absolute insufficiency in TK activity (TK-negative), depletion in TK synthesis, and inability in TK phosphorylation which consequently hinders the phosphorylation of acyclovir. Herpes TK gene contains a series of cytosine and guanosine, that are important for the function and the mutation of HSV by producing incomplete or fewer enzymes as the result of nucleotide addition or elimination in homopolymer process. Conclusion: HSV-1 resistance against acyclovir is evolved from TK mutations, in the form of TK-negative, TK low-producing, and TK altered mutants, that are unable to phosphorylate TK and accordingly disrupt acyclovir phosphorylation to convert acyclovir triphosphate. Keyword : Acyclovir, Herpes simplex virus, Resistance, Thymidine kinase

Antagonism of interferon signaling by fibroblast growth factors promotes viral replication

Fibroblast growth factors (FGFs) play key roles in the pathogenesis of different human diseases, but the cross‐talk between FGFs and other cytokines remains largely unexplored. We identified an unexpected antagonistic effect of FGFs on the interferon (IFN) signaling pathway. Genetic or pharmacological inhibition of FGF receptor signaling in keratinocytes promoted the expression of interferon‐stimulated genes (ISG) and proteins in vitro and in vivo. Conversely, FGF7 or FGF10 treatment of keratinocytes suppressed ISG expression under homeostatic conditions and in response to IFN or poly(I:C) treatment. FGF‐mediated ISG suppression was independent of IFN receptors, occurred at the transcriptional level, and required FGF receptor kinase and proteasomal activity. It is not restricted to keratinocytes and functionally relevant, since FGFs promoted the replication of herpes simplex virus I (HSV‐1), lymphocytic choriomeningitis virus, and Zika virus. Most importantly, inhibition of FGFR signaling blocked HSV‐1 replication in cultured human keratinocytes and in mice. These results suggest the use of FGFR kinase inhibitors for the treatment of viral infections.

Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

Single AAV-Mediated CRISPR-SaCas9 Inhibits HSV-1 Replication by Editing ICP4 in Trigeminal Ganglion Neurons

Herpes simplex keratitis (HSK) is the most common cause of corneal blindness in developed nations, caused by primary or recurrent herpes simplex virus 1 (HSV-1) infection of the cornea. Latent infection of HSV-1, especially in the trigeminal ganglion (TG), causes recurrence of HSV-1 infection. As antiviral treatment is not effective on latent HSV-1, to test the possibility of inhibiting HSV-1 by SpCas9 (Streptococcus pyogenes Cas9) or SaCas9 (Staphylococcus aureus Cas9), ICP0 and ICP4, two important genes required for HSV-1 replication and reactivation, were chosen as targets. In Vero cells, SpCas9 and SaCas9 targeting ICP0 or ICP4 can effectively inhibit the proliferation of HSV-1 without affecting cell viability. No significant guide RNA (gRNA)-dependent off-targets were observed in the human genome by digenome sequencing and deep sequencing verification. Adeno-associated virus 1 (AAV1)-mediated delivery of SaCas9 inhibits HSV-1 replication by targeting ICP4 in mouse primary TG neuronal cells. SpCas9 and SaCas9 are able to inhibit HSV-1 infection in Vero cells and mouse TG neuronal cultures with high efficiency and good biosafety. AAV1-mediated delivery of SaCas9 shows great potential in treating HSK and inhibiting HSV-1 in TG neurons. Further investigations may be needed to test the inhibition of latent infections, which may result in the development of novel methods for treating viral diseases.

MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.

TRIM21 Aggravates Herpes Simplex Virus Epithelial Keratitis by Attenuating STING-IRF3-Mediated Type I Interferon Signaling.

Herpes simplex virus-1 (HSV-1) is the leading cause of infectious blindness in the developed world. HSV-1 infection can occur anywhere in the eye, and the most common presentation is epithelial keratitis. In the HSV epithelial keratitis mice model, we detected the expression of TRIM21 and then investigated the clinical relationship between TRIM21 and HSV epithelial keratitis by silencing TRIM21. Through the clinical scores and histopathology examination, we found that TRIM21 can effectively reduce the severity of HSV epithelial keratitis. Furthermore, silencing TRIM21 significantly controlled the virus particle release at 1, 3, and 5 days post-HSV-1 infection. Notably, the production of IFN-β was enhanced, and the secretion of pro-inflammatory cytokines (IL-6 and TNF-a) was inhibited. Next, human corneal epithelial cells were pretreated with lentivirus or siRNA, respectively, so that TRIM21 expression was overexpressed or silenced. We focused on the regulation of STING-IRF3 and type I interferon signaling after infected with HSV-1. In conclusion, our results have identified that TRIM21 is abnormally high expressed in HSV epithelial keratitis. TRIM21 enhances the replication of HSV-1 in corneal epithelial cells via suppressing the production of type I IFN by inhibiting STING/IRF3 signaling. It also promotes the secretion of IL-6 and TNF-a, thereby aggravating the severity of HSV epithelial keratitis.

The nuclear localization signal-mediated nuclear targeting of herpes simplex virus 1 early protein UL2 is important for efficient viral production.

Herpes simplex virus 1 (HSV-1) is a representative alphaherpesvirus that can provoke a series of severe diseases to human being, but its exact pathogenesis is not perfectly understood. UL2, a uracil-DNA glycosylase involved in the process of HSV-1 DNA replication, has been shown to be predominantly targeted to the nuclei in our previous study, yet little is established regarding the subcellular localization signal or its related function of UL2 during HSV-1 propagation. Here, by creating a number of UL2 variants merged with enhanced yellow fluorescent protein, an authentic nuclear localization signal (NLS) of UL2 was, for the first time, identified and profiled to amino acids (aa) 1 to 17 (MKRACSRSPSPRRRPSS), and 12RRR14 was indispensable for its nuclear accumulation. Besides, the predicted nuclear export signal (aa 225 to 240) of UL2 was determined to be nonfunctional. Based on the HSV-1 bacterial artificial chromosome and homologous recombination technique, three recombinant viruses with mutations of the identified NLS, deletion and revertant of UL2 were constructed to assess the effect of UL2 nuclear targeting on HSV-1 replication. Compared to the wild type HSV-1, UL2 deletion remarkably restrained viral production, and mutation of NLS targeting UL2 to cytoplasm (pan-cellular distribution) in recombinant virus-infected cells showed a certain degree of deficiency in HSV-1 proliferation. Moreover, recombinant virus with UL2 deletion exhibited serious damages of viral DNA synthesis and mRNA expression, and these processes were partially disrupted in the recombinant virus with UL2 NLS mutation. Collectively, we had established a functional NLS in UL2 and showed that the NLS-mediated nuclear translocation of UL2 was important for efficient production of HSV-1. These data were of significance for further clarifying the biological function of UL2 during HSV-1 infection.

Guanidine modifications enhance the anti‐herpes simplex virus activity of (E,E)‐4,6‐bis(styryl)‐pyrimidine derivatives in vitro and in vivo

The worldwide prevalence of herpes simplex virus (HSV) and shortage of efficient therapeutic strategies to counteract it are global concerns. In terms of treatment, the widely utilized anti-HSV drugs such as acyclovir have serious limitations, for example, drug resistance and side effects. Here, we have identified the guanidine-modified (E,E)-4,6-bis(styryl)-pyrimidine (BS-pyrimidine) derivative compound 5d as an inhibitor of HSV and further elucidated the anti-HSV mechanisms of compound 5d both in vitro and in vivo. Cytopathic effect inhibition assay, plaque assay, and immunofluorescence assay were used to evaluate the anti-HSV effects of compound 5d in vitro. Membrane fusion assays, immunofluorescence assays, Western blotting assays, and pull-down assays were used to explore the anti-HSV mechanisms of compound 5d. HSV-1-infected mice, combined with haematoxylin-eosin staining and quantitative RT-PCR, were used to study the anti-HSV effects of compound 5d in vivo. The guanidine-modified compound 5d rather than the un-modified compound 3a effectively inhibited both HSV-1 and HSV-2 multiplication in different cell lines, more effectively than acyclovir. Compound 5d may block virus binding and post-binding processes such as membrane fusion, by targeting virus gB protein. In addition, compound 5d may also down-regulate the cellular PI3K/Akt signalling pathway to interfere with HSV replication. Treatment with compound 5d also markedly improved survival and decreased viral titres in HSV-infected mice. Thus, the guanidine-modified BS-pyrimidine derivatives have the potential to be developed into novel anti-HSV agents targeting both virus gB protein and cellular PI3K/Akt signalling pathways.

Impact of acyclovir use on survival of patients with ventilator-associated pneumonia and high load herpes simplex virus replication

BackgroundHerpes simplex virus (HSV) replication can be detected in the respiratory secretions of a high proportion of ventilated intensive care unit (ICU) patients. However, the clinical significance remains poorly defined. We investigated whether patients with ventilator-associated pneumonia not responding to antibiotics and in whom high levels of HSV could be detected in respiratory secretions benefit from acyclovir treatment.MethodsRespiratory secretions (bronchoalveolar lavage fluid or tracheal aspirates) were tested for HSV replication by quantitative real-time PCR. ICU survival times, clinical parameters, and radiographic findings were retrospectively compared between untreated and acyclovir treated patients with high (> 105 HSV copies/mL) and low (103–105 HSV copies/mL) viral load.ResultsFifty-seven low and 69 high viral load patients were identified. Fewer patients with high viral load responded to antibiotic treatment (12% compared to 40% of low load patients, p = 0.001). Acyclovir improved median ICU survival (8 vs 22 days, p = 0.014) and was associated with a significantly reduced hazard ratio for ICU death (HR = 0.31, 95% CI 0.11–0.92, p = 0.035) in high load patients only. Moreover, circulatory and pulmonary oxygenation function of high load patients improved significantly over the course of acyclovir treatment: mean norepinephrine doses decreased from 0.05 to 0.02 μg/kg body weight/min between days 0 and 6 of treatment (p = 0.049), and median PaO2/FiO2 ratio increased from 187 to 241 between day 3 and day 7 of treatment (p = 0.02). Chest radiographic findings also improved significantly (p < 0.001).ConclusionsIn patients with ventilator-associated pneumonia, antibiotic treatment failure, and high levels of HSV replication, acyclovir treatment was associated with a significantly longer time to death in the ICU and improved circulatory and pulmonary function. This suggests a causative role for HSV in this highly selected group of patients.

Mangiferin: A promising natural xanthone from Mangifera indica for the control of acyclovir – resistant herpes simplex virus 1 infection

Mangiferin is found in many plant species as the mango tree (Mangifera indica) with ethnopharmacological applications and scientific evidence. The emergence of resistant herpes simplex virus (HSV) strains to Acyclovir (ACV) has encouraged the search for new drugs. We investigated the in vitro and in vivo activity of mangiferin obtained from M. indica against ACV-resistant HSV-1 (AR-29) and sensitive (KOS) strains. The in vitro activity was performed under varying treatment protocols. The substance showed a CC50 > 500 μg/mL and IC50 of 2.9 μg/mL and 3.5 μg/mL, respectively, for the AR-29 and KOS strains. The in vivo activity was performed in Balb/c mice treated with 0.7% topical mangiferin formulation. This formulation inhibited most effectively the AR-29 strain, attenuated the lesions, postponed their appearance or enhanced healing, in comparison to control group. We demonstrated the potentiality of mangiferin from M. indica to control HSV replication with emphasis to ACV-resistant infection.

Interferon Kappa Is Important for Keratinocyte Host Defense against Herpes Simplex Virus-1.

Type I interferon kappa (IFNκ) is selectively expressed in human keratinocytes. Herpes simplex virus-1 (HSV-1) is a human pathogen that infects keratinocytes and causes lytic skin lesions. Whether IFNκ plays a role in keratinocyte host defense against HSV-1 has not been investigated. In this study, we found that IFNκ mRNA expression was induced by addition of recombinant IFNκ and poly (I:C); and its expression level was significantly greater than IFNa2, IFNb1, and IFNL1 in both undifferentiated and differentiated normal human epidermal keratinocytes (NHEKs) under resting and stimulation conditions. Although IFNe was expressed at a relatively higher level than other IFNs in resting undifferentiated NHEK, its expression level did not change after stimulation with recombinant IFNκ and poly (I:C). HSV-1 infection inhibited gene expression of IFNκ and IFNe in NHEK. Silencing IFNκ in NHEK led to significantly enhanced HSV-1 replication in both undifferentiated and differentiated NHEK compared to scrambled siRNA-transfected cells, while the addition of recombinant IFNκ significantly reduced HSV-1 replication in NHEK. In addition, we found that IFNκ did not regulate protein expression of NHEK differentiation markers. Our results demonstrate that IFNκ is the dominant type of IFNs in keratinocytes and it has an important function for keratinocytes to combat HSV-1 infection.

Lipidomic profiling of virus infection identifies mediators that resolve herpes simplex virus-induced corneal inflammatory lesions.

Lipid mediators (LMs) play a pivotal role in the induction and resolution of inflammation. To identify and elucidate their involvement during virus infection, multiple reaction monitoring (MRM) based liquid chromatography-tandem mass spectrometry lipidomic profiling of 62 lipid species was performed in this study. Results show that RAW264.7 macrophages differentially produce specific LMs signals depending on difference in virus pathogenicity. Integration of large-scale lipidomics with targeted gene expression data revealed mediators, such as RVD3, 18-HEPE, 11(12)-EET etc. correlated with the pathogenic phase of the infection. The herpes simplex virus (HSV)-induced keratitis model demonstrates that 11(12)-EET treatment represents a novel alternative for treating viral infection.

Inhibition of ULK1 and Beclin1 by an α-herpesvirus Akt-like Ser/Thr kinase limits autophagy to stimulate virus replication

Autophagy is a powerful host defense that restricts herpes simplex virus-1 (HSV-1) pathogenesis in neurons. As a countermeasure, the viral ICP34.5 polypeptide, which is exclusively encoded by HSV, antagonizes autophagy in part through binding Beclin1. However, whether autophagy is a cell-type-specific antiviral defense or broadly restricts HSV-1 reproduction in nonneuronal cells is unknown. Here, we establish that autophagy limits HSV-1 productive growth in nonneuronal cells and is repressed by the Us3 gene product. Phosphorylation of the autophagy regulators ULK1 and Beclin1 in virus-infected cells was dependent upon the HSV-1 Us3 Ser/Thr kinase. Furthermore, Beclin1 was unexpectedly identified as a direct Us3 kinase substrate. Although disabling autophagy did not impact replication of an ICP34.5-deficient virus in primary human fibroblasts, depleting Beclin1 and ULK1 partially rescued Us3-deficient HSV-1 replication. This shows that autophagy restricts HSV-1 reproduction in a cell-intrinsic manner in nonneuronal cells and is suppressed by multiple, independent viral functions targeting Beclin1 and ULK1. Moreover, it defines a surprising role regulating autophagy for the Us3 kinase, which unlike ICP34.5 is widely encoded by alpha-herpesvirus subfamily members.

Eczema Herpeticum: Clinical and Pathophysiological Aspects.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in the world. AD is a complex pathology mainly characterized by an impaired skin barrier, immune response dysfunction, and unbalanced skin microbiota. Moreover, AD patients exhibit an increased risk of developing bacterial and viral infections. One of the most current, and potentially life-threatening, viral infection is caused by herpes simplex virus (HSV), which occurs in about 3% of AD patients under the name of eczema herpeticum (EH). Following a first part dedicated to the clinical features, virological diagnosis, and current treatments of EH, this review will focus on the description of the pathophysiology and, more particularly, the presently known predisposing factors to herpetic complications in AD patients. These factors include those related to impairment of the skin barrier such as deficit in filaggrin and anomalies in tight and adherens junctions. In addition, low production of the antimicrobial peptides cathelicidin LL-37 and human β-defensins; overexpression of cytokines such as interleukin (IL)-4, IL-13, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP); or downregulation of type I to III interferons as well as defect in functions of immune cells such as dendritic, natural killer, and regulatory T cells have been involved. Otherwise, genetic polymorphisms and AD topical calcineurin inhibitor treatments have been associated with an increased risk of EH. Finally, dysbiosis of skin microbiota characterized in AD patients by Staphylococcus aureus colonization and toxin secretion, such as α-toxin, has been described as promoting HSV replication and could therefore contribute to EH.

The Interaction Mechanism Between Herpes Simplex Virus 1 Glycoprotein D and Host Antiviral Protein Viperin.

Viperin is an interferon-inducible protein that responsible for a variety of antiviral responses to different viruses. Our previous study has shown that the ribonuclease UL41 of herpes simplex virus 1 (HSV-1) can degrade the mRNA of viperin to promote HSV-1 replication. However, it is not clear whether other HSV-1 encoded proteins can regulate the function of viperin. Here, one novel viperin associated protein, glycoprotein D (gD), was identified. To verify the interaction between gD and viperin, gD and viperin expression plasmids were firstly co-transfected into COS-7 cells, and fluorescence microscope showed they co-localized at the perinuclear region, then this potential interaction was confirmed by co-immunoprecipitation (Co-IP) assays. Moreover, confocal microscopy demonstrated that gD and viperin co-localized at the Golgi body and lipid droplets. Furthermore, dual-luciferase reporter and Co-IP assays showed gD and viperin interaction leaded to the increase of IRF7-mediated IFN-β expression through promoting viperin and IRAK1 interaction and facilitating K63-linked IRAK1 polyubiquitination. Nevertheless, gD inhibited TRAF6-induced NF-κB activity by decreasing the interaction of viperin and TRAF6. In addition, gD restrained viperin-mediated interaction between IRAK1 and TRAF6. Eventually, gD and viperin interaction was corroborated to significantly inhibit the proliferation of HSV-1. Taken together, this study would open up new avenues toward delineating the function and physiological significance of gD and viperin during HSV-1 replication cycle.

Herpes Simplex Virus 1 MicroRNA miR-H28 Exported to Uninfected Cells in Exosomes Restricts Cell-to-Cell Virus Spread by Inducing Gamma Interferon mRNA.

An earlier report showed that herpes simplex virus 1 (HSV-1) expresses two microRNAs (miRNAs), miR-H28 and miR-H29, late in the infectious cycle. The miRNAs are packed in exosomes and, in recipient cells, restrict the transmission of virus from infected cells to uninfected cells. We now report that (i) miR-H28 induced the synthesis of gamma interferon (IFN-γ) in both infected cells and cells transfected with miR-H28, (ii) IFN-γ accumulated concurrently with viral proteins in infected cells, (iii) IFN-γ was produced in HEp-2 cells derived from cancer tissue and in HEK293T cells derived from normal tissue, and (iv) HSV-1 replication was affected by exposure to IFN-γ before infection but not during or after infection. The results presented in this report support the growing body of evidence indicating that HSV-1 encodes functions designed to reduce the spread of infection from infected cells to uninfected cells, possibly in order to maximize the transmission of virus from infected individuals to uninfected individuals.IMPORTANCE In this report, we show that IFN-γ is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN-γ before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN-γ to curtail its spread from infected cells to uninfected cells. In essence, this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.

Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication.

During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) UL34 and UL31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins in vitro Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein UL25 but not to the other capsid proteins tested (i.e., VP5, VP23, and UL17) and that HSV-1 NEC binding of nucleocapsids was mediated by the interaction of NEC with UL25. UL31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and UL25. We also showed that alanine substitution of UL31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles that were similar in size and morphology to primary envelopes in the perinuclear space. These results suggested that NEC binding via UL31 R281 and D282 to nucleocapsids, and probably to UL25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment.IMPORTANCE Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis have not been reported thus far. In this study, we have present data showing that two amino acids in the membrane-distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.

A unique class of lignin derivatives displays broad anti-HIV activity by interacting with the viral envelope

In Gordts et al. (2015), we have shown that lignosulfonic acid, a commercially available lignin derivative, possesses broad antiviral activity against human immunodeficiency virus (HIV) and Herpes simplex virus (HSV) by preventing viral entry into susceptible target cells. Because of the interesting safety profile as potential microbicide, we now determined the antiviral activity of a series of lignosulfonates in order to understand better which molecular features can contribute to their antiviral activity. Here, 24 structurally different lignosulfonates were evaluated for their capacity to inhibit HIV and HSV transmission and replication in various cellular assays. These derivatives differ in origin (hardwood or softwood), counter-ion used during sulphite processing (Na+, Ca2+, or NH4+), sulphur content, carboxylic acid percentage, and molecular weight fraction, which allowed to determine structure-activity relationships. We demonstrate that the broad antiviral activity of lignosulfonates is mainly dependent on their molecular weight and that their mechanism of action is based on interactions with the viral envelope glycoproteins. This makes the lignosulfonates a potential low-cost microbicide that protects women from sexual HIV and HSV transmission and thus prevents life-long infection.