Herpes Simplex Virus Entry by a Nonconventional Endocytic Pathway

ABSTRACT Herpes simplex virus 1 (HSV-1) causes significant morbidity and mortality in humans worldwide. HSV-1 enters epithelial cells via an endocytosis mechanism that is low-pH dependent. However, the precise intracellular pathway has not been identified, including the compartment where fusion occurs. In this study, we utilized a combination of molecular and pharmacological approaches to better characterize HSV entry by endocytosis. HSV-1 entry was unaltered in both cells treated with small interfering RNA (siRNA) to Rab5 or Rab7 and cells expressing dominant negative forms of these GTPases, suggesting entry is independent of the conventional endo-lysosomal network. The fungal metabolite brefeldin A (BFA) and the quinoline compound Golgicide A (GCA) inhibited HSV-1 entry via beta-galactosidase reporter assay and impaired incoming virus transport to the nuclear periphery, suggesting a role for trans-Golgi network (TGN) functions and retrograde transport in HSV entry. Silencing of Rab9 or Rab11 GTPases, which are involved in the retrograde transport pathway, resulted in only a slight reduction in HSV infection. Together, these results suggest that HSV enters host cells by an intracellular route independent of the lysosome-terminal endocytic pathway. IMPORTANCE Herpes simplex virus 1 (HSV-1), the prototype alphaherpesvirus, is ubiquitous in the human population and causes lifelong infection that can be fatal in neonatal and immunocompromised individuals. HSV enters many cell types by endocytosis, including epithelial cells, the site of primary infection in the host. The intracellular itinerary for HSV entry remains unclear. We probed the potential involvement of several Rab GTPases in HSV-1 entry and suggest that endocytic entry of HSV-1 is independent of the canonical lysosome-terminal pathway. A nontraditional endocytic route may be employed, such as one that intersects with the trans-Golgi network (TGN). These results may lead to novel targets for intervention.

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Herpes simplex virus entry by a non-conventional endocytic pathway

10.1101/2020.09.28.317867 ◽

2020 ◽

Author(s):

Giulia Tebaldi ◽

Suzanne M. Pritchard ◽

Anthony V. Nicola

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Epithelial Cells ◽

Retrograde Transport ◽

Endocytic Pathway ◽

Host Cells ◽

Rab Gtpases ◽

Transport Pathway ◽

Simplex Virus ◽

Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) causes significant morbidity and mortality in humans worldwide. HSV-1 enters epithelial cells via an endocytosis mechanism that is low pH-dependent. However, the precise intracellular pathway has not been identified, including the compartment where fusion occurs. In this study, we utilized a combination of molecular and pharmacological approaches to better characterize HSV entry by endocytosis. HSV-1 entry was unaltered in both cells treated with siRNA to Rab5 or Rab7 and cells expressing dominant-negative forms of these GTPases, suggesting entry is independent of the conventional endo-lysosomal network. The fungal metabolite brefeldin A (BFA) and the quinoline compound Golgicide A (GCA) inhibited HSV-1 entry via beta-galactosidase reporter assay and impaired incoming virus transport to the nuclear periphery, suggesting a role for trans Golgi network (TGN) functions and retrograde transport in HSV entry. Silencing of Rab9 or Rab11 GTPases, which are involved in the retrograde transport pathway, resulted in only a slight reduction in HSV infection. Together these results suggest that HSV enters host cells by an intracellular route independent of the lysosome-terminal endocytic pathway.IMPORTANCEHSV-1, the prototype alphaherpesvirus, is ubiquitous in the human population and causes lifelong infection that can be fatal in neonatal and immunocompromised individuals. HSV enters many cell types by endocytosis, including epithelial cells, the site of primary infection in the host. The intracellular itinerary for HSV entry remains unclear. We probed the potential involvement of several Rab GTPases in HSV-1 entry, and suggest that endocytic entry of HSV-1 is independent of the canonical lysosome-terminal pathway. A non-traditional endocytic route may be employed, such as one that intersects with the TGN. These results may lead to novel targets for intervention.

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The Solute Carrier Transporter SLC15A3 Participates in Antiviral Innate Immune Responses against Herpes Simplex Virus-1

Journal of Immunology Research ◽

10.1155/2018/5214187 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Longzhen He ◽

Baocheng Wang ◽

Yuanyuan Li ◽

Leqing Zhu ◽

Peiling Li ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Immune Responses ◽

Innate Immune ◽

Host Cells ◽

Type I ◽

Innate Immune Responses ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

The innate immune response is the first line defense against viral infections. Novel genes involved in this system are continuing to emerge. SLC15A3, a proton-coupled histidine and di-tripeptide transporter that was previously found in lysosomes, has been reported to inhibit chikungunya viral replication in host cells. In this study, we found that SLC15A3 was significantly induced by DNA virus herpes simplex virus-1(HSV-1) in monocytes from human peripheral blood mononuclear cells. Aside from monocytes, it can also be induced by HSV-1 in 293T, HeLa cells, and HaCaT cells. Overexpression of SLC15A3 in 293T cells inhibits HSV-1 replication and enhances type I and type III interferon (IFN) responses, while silencing SLC15A3 leads to enhanced HSV-1 replication with reduced IFN production. Moreover, we found that SLC15A3 interacted with MAVS and STING and potentiated MAVS- and STING-mediated IFN production. These results demonstrate that SLC15A3 participates in anti-HSV-1 innate immune responses by regulating MAVS- and STING-mediated signaling pathways.

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A novel role for phagocytosis-like uptake in herpes simplex virus entry

The Journal of Cell Biology ◽

10.1083/jcb.200509155 ◽

2006 ◽

Vol 174 (7) ◽

pp. 1009-1021 ◽

Cited By ~ 154

Author(s):

Christian Clement ◽

Vaibhav Tiwari ◽

Perry M. Scanlan ◽

Tibor Valyi-Nagy ◽

Beatrice Y.J.T. Yue ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dominant Negative ◽

Endocytic Pathway ◽

Herpes Simplex Virus 1 ◽

Mode Of Entry ◽

Virion Envelope ◽

Membrane Protrusions ◽

Simplex Virus ◽

Hsv 1

It is becoming increasingly clear that herpesviruses can exploit the endocytic pathway to infect cells, yet several important features of this process remain poorly defined. Using herpes simplex virus-1 (HSV-1) as a model, we demonstrate that endocytosis of the virions mimic many features of phagocytosis. During entry, HSV-1 virions associated with plasma membrane protrusions followed by a phagocytosis-like uptake involving rearrangement of actin cytoskeleton and trafficking of the virions in large phagosome-like vesicles. RhoA GTPase was activated during this process and the mode of entry was cell type–specific. Clathrin-coated vesicles had no detectable role in virion trafficking as Eps15 dominant-negative mutants failed to affect HSV-1 uptake. Binding and fusion of the virion envelope with the phagosomal membrane is likely facilitated by clustering of nectin-1 (or HVEM) in phagosomes, which was observed in infected cells. Collectively, our data suggests a novel mode of uptake by which the virus can infect both professional and nonprofessional phagocytes.

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The XPO6 Exportin Mediates Herpes Simplex Virus 1 gM Nuclear Release Late in Infection

Journal of Virology ◽

10.1128/jvi.00753-20 ◽

2020 ◽

Vol 94 (21) ◽

Author(s):

Hugo Boruchowicz ◽

Josiane Hawkins ◽

Kendra Cruz-Palomar ◽

Roger Lippé

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Nuclear Membrane ◽

Viral Protein ◽

Herpes Simplex Virus 1 ◽

Nuclear Membranes ◽

Proteomics Approach ◽

Simplex Virus ◽

Golgi Network ◽

Hsv 1

ABSTRACT The glycoprotein M of herpes simplex virus 1 (HSV-1) is dynamically relocated from nuclear membranes to the trans-Golgi network (TGN) during infection, but molecular partners that promote this relocalization are unknown. Furthermore, while the presence of the virus is essential for this phenomenon, it is not clear if this is facilitated by viral or host proteins. Past attempts to characterize glycoprotein M (gM) interacting partners identified the viral protein gN by coimmunoprecipitation and the host protein E-Syt1 through a proteomics approach. Interestingly, both proteins modulate the activity of gM on the viral fusion machinery. However, neither protein is targeted to the nuclear membrane and consequently unlikely explains the dynamic regulation of gM nuclear localization. We thus reasoned that gM may transiently interact with other molecules. To resolve this issue, we opted for a proximity-dependent biotin identification (BioID) proteomics approach by tagging gM with a BirA* biotinylation enzyme and purifying BirA substrates on a streptavidin column followed by mass spectrometry analysis. The data identified gM and 170 other proteins that specifically and reproducibly were labeled by tagged gM at 4 or 12 h postinfection. Surprisingly, 35% of these cellular proteins are implicated in protein transport. Upon testing select candidate proteins, we discovered that XPO6, an exportin, is required for gM to be released from the nucleus toward the TGN. This is the first indication of a host or viral protein that modulates the presence of HSV-1 gM on nuclear membranes. IMPORTANCE The mechanisms that enable integral proteins to be targeted to the inner nuclear membrane are poorly understood. Herpes simplex virus 1 (HSV-1) glycoprotein M (gM) is an interesting candidate, as it is dynamically relocalized from nuclear envelopes to the trans-Golgi network (TGN) in a virus- and time-dependent fashion. However, it was, until now, unclear how gM was directed to the nucleus or evaded that compartment later on. Through a proteomic study relying on a proximity-ligation assay, we identified several novel gM interacting partners, many of which are involved in vesicular transport. Analysis of select proteins revealed that XPO6 is required for gM to leave the nuclear membranes late in the infection. This was unexpected, as XPO6 is an exportin specifically associated with actin/profilin nuclear export. This raises some very interesting questions about the interaction of HSV-1 with the exportin machinery and the cargo specificity of XPO6.

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Characterization of a Proteolytically Stable D-Peptide That Suppresses Herpes Simplex Virus 1 Infection: Implications for the Development of Entry-Based Antiviral Therapy

Journal of Virology ◽

10.1128/jvi.02979-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1932-1938 ◽

Cited By ~ 18

Author(s):

Dinesh Jaishankar ◽

Abraam M. Yakoub ◽

Anita Bogdanov ◽

Tibor Valyi-Nagy ◽

Deepak Shukla

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Host Cells ◽

Therapeutic Application ◽

Herpes Simplex Virus 1 ◽

Corneal Blindness ◽

Simplex Virus ◽

Hsv 1

Uncontrolled herpes simplex virus 1 (HSV-1) infection can advance to serious conditions, including corneal blindness or fatal encephalitis. Here, we describe a highly potent anti-HSV-1 peptide (DG2) that inhibits HSV-1 entry into host cells and blocks all aspects of infection. Importantly, DG2 is highly resistant to proteases and shows minimal toxicity, paving the way for prophylactic or therapeutic application of the peptidein vivo.

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HCFC1R1 Deficiency Blocks Herpes Simplex Virus-1 Infection by Inhibiting Nuclear Translocation of HCFC1 and VP16

10.1101/2020.03.13.991679 ◽

2020 ◽

Author(s):

Yangkun Shen ◽

Zhoujie Ye ◽

Xiangqian Zhao ◽

Zhihua Feng ◽

Jinfeng Chen ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Host Cell ◽

Host Cells ◽

The Novel ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Novel Target ◽

Simplex Virus ◽

Hsv 1

ABSTRACTUpon HSV-1 infection, viral protein 16 (VP16), supported by Host Cell Factor C1 (HCFC1), is rapidly transported into the nucleus, and help to express a series of HSV-1 immediate-early proteins to begin its lytic replication. However, no direct evidence has shown if the HCFC1 deficiency can affect the proliferation of HSV-1 so far. Here, we showed that the HCFC1 deficiency led to a strong resistance to HSV-1 infection. Moreover, we identified Host Cell Factor C1 Regulator 1 (HCFC1R1) as a new host factor acting early in HSV infection for the transport of the HSV-1 capsid to the nucleus. The HCFC1R1 deficiency also led to a strong resistance to HSV-1 infection. The HCFC1R1 deficiency did not affect the attachment of HSV-1 to host cells but act early in HSV-1 infection by perturbing the formation of HCFC1/VP16 complex. Remarkably, in addition to wild-type HSV-1 infection, the host cells in the absence of either HCFC1 or HCFC1R1 showed strong resistant to the infection of TK-deficient HSV-1, which strain can course severe symptoms and tolerate to the current anti-HSV drug Acyclovir. Our data suggest that HCFC1 or HCFC1R1 may be used as the novel target for developing anti-HSV-1 therapies.IMPORTANCEHerpes simplex virus-1 (HSV-1) is widely spread in the human population and can cause a variety of herpetic diseases. Acyclovir, a guanosine analogue that targets the TK protein of HSV-1, is the first specific and selective anti-HSV-1 drug. However, the rapid emergence of resistant HSV-1 strains is occurring worldwide, endangering the efficacy of Acyclovir. Alternatively, targeting host factors is another strategy to stop HSV-1 infection. Unfortunately, although the HSV-1’s receptor, Nectin-1, was discovered in 1998, no effective antiviral drug to date has been developed by targeting Nectin-1. Targeting multiple pathways is the ultimate choice to prevent HSV-1 infection. Here we demonstrated that the deletion of HCFC1 or HCFC1R1 exhibits a strong inhibitory effect on both wild-type and TK-deficient HSV-1. Overall, we present evidence that HCFC1 or HCFC1R1 may be used as the novel target for developing anti-HSV-1 therapies with a defined mechanism of action.

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Herpes Simplex Virus 1 Has Multiple Mechanisms for Blocking Virus-Induced Interferon Production

Journal of Virology ◽

10.1128/jvi.78.16.8411-8420.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8411-8420 ◽

Cited By ~ 139

Author(s):

Gregory T. Melroe ◽

Neal A. DeLuca ◽

David M. Knipe

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Posttranslational Modifications ◽

Sendai Virus ◽

Host Cells ◽

Nuclear Accumulation ◽

Herpes Simplex Virus 1 ◽

Early Protein ◽

Simplex Virus ◽

Hsv 1

ABSTRACT In response to viral infection, host cells elicit a number of responses, including the expression of alpha/beta interferon (IFN-α/β). In these cells, IFN regulatory factor-3 (IRF-3) undergoes a sequence of posttranslational modifications that allow it to act as a potent transcriptional coactivator of specific IFN genes, including IFN-β. We investigated the mechanisms by which herpes simplex virus 1 (HSV-1) inhibits the production of IFN-β mediated by the IRF-3 signaling pathway. Here, we show that HSV-1 infection can block the accumulation of IFN-β triggered by Sendai virus (SeV) infection. Our results indicate that HSV-1 infection blocks the nuclear accumulation of activated IRF-3 but does not block the initial virus-induced phosphorylation of IRF-3. The former effect was at least partly mediated by increased turnover of IRF-3 in HSV-1-infected cells. Using mutant viruses, we determined that the immediate-early protein ICP0 was necessary for the inhibition of IRF-3 nuclear accumulation. Expression of ICP0 also had the ability to reduce IFN-β production induced by SeV infection. ICP0 has been shown previously to play a role in HSV-1 sensitivity to IFN and in the inhibition of antiviral gene production. However, we observed that an ICP0 mutant virus still retained the ability to inhibit the production of IFN-β. These results argue that HSV-1 has multiple mechanisms to inhibit the production of IFN-β, providing additional ways in which HSV-1 can block the IFN-mediated host response.

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Disassembly of the TRIM23-TBK1 Complex by the Us11 Protein of Herpes Simplex Virus 1 Impairs Autophagy

Journal of Virology ◽

10.1128/jvi.00497-19 ◽

2019 ◽

Vol 93 (17) ◽

Cited By ~ 10

Author(s):

Xing Liu ◽

Rachel Matrenec ◽

Michaela U. Gack ◽

Bin He

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Molecular Mechanisms ◽

Host Cells ◽

Viral Escape ◽

Wild Type Virus ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Us11 Protein ◽

Hsv 1

ABSTRACTThe Us11 protein encoded by herpes simplex virus 1 (HSV-1) functions to impair autophagy; however, the molecular mechanisms of this inhibition remain to be fully established. Here, we report that the Us11 protein targetstripartite motifprotein 23 (TRIM23), which is a key regulator of autophagy-mediated antiviral defense mediated by TANK-binding kinase 1 (TBK1). In virus-infected cells, the Us11 protein drastically reduces the formation of autophagosomes mediated by TRIM23 or TBK1. This autophagy-inhibitory effect is attributable to the binding of the Us11 protein to the ARF domain in TRIM23. Furthermore, such interaction spatially excludes TBK1 from the TRIM23 complex that also contains heat shock protein 90 (Hsp90). When stably expressed alone in host cells, the Us11 protein recapitulates the observed phenotypes seen in cells infected with the US11-expressing or wild-type virus. Consistent with this, expression of the Us11 protein promotes HSV-1 growth, while expression of TRIM23 restricts HSV-1 replication in the absence of US11. Together, these results suggest that disruption of the TRIM23-TBK1 complex by the Us11 protein inhibits autophagy-mediated restriction of HSV-1 infection.IMPORTANCEAutophagy is an evolutionarily conserved process that restricts certain intracellular pathogens, including HSV-1. Although HSV-1 is well known to inhibit autophagy, little is known about the precise molecular mechanisms of autophagy inhibition. We demonstrate that the Us11 protein of HSV-1 spatially disrupts the TRIM23-TBK1 complex, which subsequently suppresses autophagy and autophagy-mediated virus restriction. Thus, expression of the Us11 protein facilitates HSV-1 replication. These data unveil new insight into viral escape from autophagy-mediated host restriction mechanisms.

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Herpes simplex virus 1 infection induces ubiquitination of UBE1a

Biochemical Journal ◽

10.1042/bcj20200885 ◽

2020 ◽

Author(s):

Marina Ikeda ◽

Tadashi Watanabe ◽

Akihiro Ito ◽

Masahiro Fujimuro

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Protein Trafficking ◽

Protein Modification ◽

Host Cells ◽

Herpes Simplex Virus 1 ◽

Cellular Events ◽

Protein Ubiquitination ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Further, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.

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In Vitro Analysis of the Antioxidant and Antiviral Activity of Embelin against Herpes Simplex Virus-1

Microorganisms ◽

10.3390/microorganisms9020434 ◽

2021 ◽

Vol 9 (2) ◽

pp. 434

Author(s):

Tony Elias ◽

Lee H. Lee ◽

Miriam Rossi ◽

Francesco Caruso ◽

Sandra D. Adams

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Time Course ◽

Early Stage ◽

Vero Cells ◽

Host Cells ◽

Herpes Simplex Virus 1 ◽

Wide Range ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus-1 (HSV-1) causes a wide range of infections from mild to life-threatening in the human population. There are effective treatments for HSV-1 infections that are limited due HSV-1 latency and development of resistance to current therapeutics. The goal of this study was to investigate the antioxidant and antiviral effects of embelin on HSV-1 in cultured Vero cells. Oxidative stress was verified by an extensive production of a reactive oxygen species (ROS) H2O2. Vero cells were infected with a recombinant strain of HSV-1 and antiviral assays, time course attachment, penetration, and post penetration assays, confocal microscopy, qPCR, and antioxidant assays were conducted. Our results lead to the conclusion that embelin is noncytotoxic at concentrations tested ranging from 20 to 70 µM. Treatment of HSV-1 virions with embelin resulted in 98.7–100% inhibition and affected the early stage of HSV-1 infection of Vero cells, by inhibiting the attachment and penetration of HSV-1 virions to host cells. Treatment of virions with concentrations of embelin ranging from 35 to 60 µM significantly reduced the production of H2O2. In conclusion, embelin reduces oxidative damage caused by HSV-1 infection and is an effective antiviral to reduce the infection of HSV-1 in cultured Vero cells. Further studies are needed to explore the possibility of embelin as a medicinal agent.

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