Characterization of the triplet repeats in the central domain of the γ
            134·5 protein of herpes simplex virus 1

The γ 134·5 protein of herpes simplex virus 1 (HSV-1) consists of an amino-terminal domain, a central domain with triplet repeats (Ala–Thr–Pro) and a carboxyl-terminal domain. The triplet repeats are a unique feature of the γ 134·5 protein encoded by HSV-1, but the number of repeats varies among different strains. Notably, the central domain containing the triplet repeats is implicated in neuroinvasion. In this report, it has been shown that partial or full deletion of triplet repeats, i.e. from ten to either three or zero, in the γ 134·5 protein has no effect on the virus response to interferon. The triplet deletion mutants replicate efficiently in CV-1 and mouse 10T1/2 cells. However, in mouse 3T6 cells, these mutants grow with delayed growth kinetics. This decrease in growth, compared with wild-type HSV-1(F), does not result from failure of the virus to suppress the RNA-dependent protein kinase response, but rather from a delay in virus release or egress. Accordingly, these mutant viruses are predominantly present within infected cells. These results indicate that deletions in the central domain of the γ 134·5 protein impair virus egress, but not virus response to interferon.

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Functional Comparison of Herpes Simplex Virus 1 (HSV-1) and HSV-2 ICP27 Homologs Reveals a Role for ICP27 in Virion Release

Journal of Virology ◽

10.1128/jvi.02994-14 ◽

2014 ◽

Vol 89 (5) ◽

pp. 2892-2905 ◽

Cited By ~ 10

Author(s):

Donglim Park ◽

Joseph Lalli ◽

Lenka Sedlackova-Slavikova ◽

Stephen A. Rice

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Infected Cell ◽

Regulatory Protein ◽

Late Gene Expression ◽

Herpes Simplex Virus 1 ◽

Amino Terminal ◽

Virion Release ◽

Simplex Virus ◽

Hsv 1

ABSTRACTNumerous studies have focused on the regulatory functions of ICP27, an immediate-early (IE) protein of herpes simplex virus 1 (HSV-1). However, its homolog in HSV-2, termed ICP27t2, has been little studied. Here, we used two different approaches to functionally compare ICP27t2 and ICP27. In transfection-based assays, ICP27t2 closely resembled ICP27 in its capacity to enhance HSV-1 late gene expression, suppress the splicing of a viral intron, and complement the growth of an HSV-1 ICP27 null mutant. To study ICP27t2 in the context of viral infection, we engineered K2F1, an HSV-1 mutant that encodes ICP27t2 in place of ICP27. In Vero cells, K2F1 replicated with wild-type (WT) kinetics and yields, expressed delayed-early and late proteins normally, and was fully capable of activating several cellular signal transduction pathways that are ICP27 dependent. Thus, we conclude that ICP27t2 and ICP27 are functionally very similar and that ICP27t2 can mediate all ICP27 activities that are required for HSV-1 replication in cell culture. Surprisingly, however, we found that K2F1 forms plaques that are morphologically different from those of WT HSV-1. Investigation of this trait demonstrated that it results from the decreased release of progeny virions into the culture medium. This appears to be due to a reduction in the detachment of K2F1 progeny from the extracellular surface of the infected cell. We identified two HSV-1 ICP27 amino-terminal deletion mutants with a similar release defect. Together, these results demonstrate that ICP27 plays a heretofore-unappreciated role in modulating the efficiency of progeny virion release.IMPORTANCEICP27 is an essential, multifunctional regulatory protein that has a number of critical roles in the HSV-1 life cycle. Although ICP27 homologs are encoded by all known members of theHerpesviridae, previous work with several of these homologs has shown that they cannot substitute for ICP27 in the context of HSV-1-infected cells. Here, we identify ICP27t2 as the first homolog that can efficiently replace ICP27 in HSV-1 infection. Unexpectedly, our results also reveal that the sequence of the ICP27 gene can affect the release of HSV-1 progeny virions from the infected cell. Thus, our comparative study has revealed a novel function for ICP27 in the regulation of virus release.

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Role of the arginine cluster in the disordered domain of Herpes Simplex Virus 1 UL34 for the recruitment of ESCRT-III for viral primary envelopment

Journal of Virology ◽

10.1128/jvi.01704-21 ◽

2021 ◽

Author(s):

Jun Arii ◽

Kosuke Takeshima ◽

Yuhei Maruzuru ◽

Naoto Koyanagi ◽

Yoshitaka Nakayama ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Nuclear Export ◽

Herpes Simplex Virus 1 ◽

Amino Terminal ◽

Arginine Residues ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

During the nuclear export of nascent nucleocapsids of herpesviruses, the nucleocapsids bud through the inner nuclear membrane (INM) by acquiring the INM as a primary envelope (primary envelopment). We recently reported that herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), which consists of UL34 and UL31, interacts with an ESCRT-III adaptor ALIX and recruits ESCRT-III machinery to the INM for efficient primary envelopment. In this study, we identified a cluster of six arginine residues in the disordered domain of UL34 as a minimal region required for the interaction with ALIX as well as the recruitment of ALIX and an ESCRT-III protein CHMP4B to the INM in HSV-1-infected cells. Mutations in the arginine cluster exhibited phenotypes similar to those with ESCRT-III inhibition reported previously, including the mis-localization of NEC, induction of membranous invagination structures containing enveloped virions, aberrant accumulation of enveloped virions in the invaginations and perinuclear space, and reduction of viral replication. We also showed that the effect of the arginine cluster in UL34 on HSV-1 replication was dependent primarily on ALIX. These results indicated that the arginine cluster in the disordered domain of UL34 was required for the interaction with ALIX and the recruitment of ESCRT-III machinery to the INM to promote primary envelopment. IMPORTANCE Herpesvirus UL34 homologs contain conserved amino-terminal domains that mediate vesicle formation through interactions with UL31 homologs during primary envelopment. UL34 homologs also comprise other domains adjacent to their membrane-anchoring regions, which differ in length, are variable in herpesviruses and do not form distinguished secondary structures. However, the role of these disordered domains in infected cells remains to be elucidated. In this study, we present data suggesting that the arginine cluster in the disordered domain of HSV-1 UL34 mediates the interaction with ALIX, thereby leading to the recruitment of ESCRT-III machinery to the INM for efficient primary envelopment. This is the first study to report the role of the disordered domain of a UL34 homolog in herpesvirus infections.

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Herpes Simplex Virus 1 γ134.5 Protein Inhibits STING Activation That Restricts Viral Replication

Journal of Virology ◽

10.1128/jvi.01015-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 20

Author(s):

Shuang Pan ◽

Xing Liu ◽

Yijie Ma ◽

Youjia Cao ◽

Bin He

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virulence Factor ◽

Dna Sensing ◽

Viral Inhibition ◽

Herpes Simplex Virus 1 ◽

Amino Terminal ◽

Viral Virulence ◽

Simplex Virus ◽

Hsv 1

ABSTRACTThe γ134.5 gene of herpes simplex virus 1 (HSV-1) encodes a virulence factor that promotes viral pathogenesis. Although it perturbs TANK-binding kinase 1 (TBK1) in the complex network of innate immune pathways, the underlying mechanism is obscure. Here we report that HSV-1 γ134.5 targets stimulator of interferon genes (STING) in the intracellular DNA recognition pathway that regulates TBK1 activation. In virus-infected cells the γ134.5 protein associates with and inactivates STING, which leads to downregulation of interferon regulatory factor 3 (IRF3) and IFN responses. Importantly, HSV-1 γ134.5 disrupts translocation of STING from the endoplasmic reticulum to Golgi apparatus, a process necessary to prime cellular immunity. Deletion of γ134.5 or its amino-terminal domain from HSV-1 abolishes the observed inhibitory activities. Consistently, an HSV mutant that lacks functional γ134.5 replicated less efficiently in STING+/+than in STING−/−mouse embryonic fibroblasts. Moreover, reconstituted expression of human STING in the STING−/−cells activated IRF3 and reduced viral growth. These results suggest that control of the DNA sensing pathway by γ134.5 is advantageous to HSV infection.IMPORTANCEViral inhibition of innate immunity contributes to herpes simplex virus pathogenesis. Although this complex process involves multiple factors, the underlying events remain unclear. We demonstrate that an HSV virulence factor γ134.5 precludes the activation of STING, a central adaptor in the intracellular DNA sensing pathway. Upon HSV infection, this viral protein engages with and inactivates STING. Consequently, it compromises host immunity and facilitates HSV replication. These observations uncover an HSV mechanism that is likely to mediate viral virulence.

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Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria

Journal of Virology ◽

10.1128/jvi.01995-16 ◽

2016 ◽

Vol 91 (2) ◽

Cited By ~ 5

Author(s):

Pooja Chadha ◽

Akua Sarfo ◽

Dan Zhang ◽

Thomas Abraham ◽

Jillian Carmichael ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Tegument Protein ◽

Herpes Simplex Virus 1 ◽

Transfected Cells ◽

Terminal Domain ◽

Domain Interactions ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined. IMPORTANCE The HSV-1-encoded tegument protein UL16 is involved in multiple events of the virus replication cycle, ranging from virus assembly to cell-cell spread of the virus, and hence it can serve as an important drug target. Unfortunately, a lack of both structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV-infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just UL16 of HSV-1 but also its homologs in other herpesviruses.

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Assembly of VP26 in herpes simplex virus-1 capsid implicated by 3-D structure of recombinant capsid

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140579 ◽

1995 ◽

Vol 53 ◽

pp. 840-841

Author(s):

Z. Hong Zhou ◽

Jing He ◽

Joanita Jakana ◽

J. D. Tatman ◽

Frazer J. Rixon ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

3D Structure ◽

Structure Study ◽

Herpes Simplex Virus 1 ◽

Shell Proteins ◽

Life Threatening ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus-1 (HSV-1) is a ubiquitous virus which is implicated in diseases ranging from self-curing cold sores to life-threatening infections. The 2500 Å diameter herpes virion is composed of a glycoprotein spike containing, lipid envelope, enclosing a protein layer (the tegument) in which is embedded the capsid (which contains the dsDNA genome). The B-, and A- and C-capsids, representing different morphogenetic stages in HSV-1 infected cells, are composed of 7, and 5 structural proteins respectively. The three capsid types are organized in similar T=16 icosahedral shells with 12 pentons, 150 hexons, and 320 connecting triplexes. Our previous 3D structure study at 26 Å revealed domain features of all these structural components and suggested probable locations for the outer shell proteins, VP5, VP26, VP19c and VP23. VP5 makes up most of both pentons and hexons. VP26 appeared to bind to the VP5 subunit in hexon but not to that in penton.

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Herpes simplex virus 1 targets IRF7 via ICP0 to limit type I IFN induction

Scientific Reports ◽

10.1038/s41598-020-77725-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

David Shahnazaryan ◽

Rana Khalil ◽

Claire Wynne ◽

Caroline A. Jefferies ◽

Joan Ní Gabhann-Dromgoole ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Immune Responses ◽

Tear Film ◽

Cell Protein ◽

Type I ◽

Type I Ifn ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

AbstractHerpes simplex keratitis (HSK), caused by herpes simplex virus type 1 (HSV-1) infection, is the commonest cause of infectious blindness in the developed world. Following infection the virus is initially suspended in the tear film, where it encounters a multi-pronged immune response comprising enzymes, complement, immunoglobulins and crucially, a range of anti-viral and pro-inflammatory cytokines. However, given that HSV-1 can overcome innate immune responses to establish lifelong latency throughout a susceptible individual’s lifetime, there is significant interest in understanding the mechanisms employed by HSV-1 to downregulate the anti-viral type I interferon (IFN) mediated immune responses. This study aimed to investigate the interactions between infected cell protein (ICP)0 and key elements of the IFN pathway to identify possible novel targets that contribute to viral immune evasion. Reporter gene assays demonstrated the ability of ICP0 to inhibit type I IFN activity downstream of pathogen recognition receptors (PRRs) which are known to be involved in host antiviral defences. Further experiments identified interferon regulatory factor (IRF)7, a driver of type I IFN, as a potential target for ICP0. These findings increase our understanding of the pathogenesis of HSK and suggest IRF7 as a potential therapeutic target.

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Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

Viruses ◽

10.3390/v13020196 ◽

2021 ◽

Vol 13 (2) ◽

pp. 196

Author(s):

Sara Artusi ◽

Emanuela Ruggiero ◽

Matteo Nadai ◽

Beatrice Tosoni ◽

Rosalba Perrone ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Antiviral Activity ◽

Herpes Simplex Virus 1 ◽

Tem Analysis ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.

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Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells

Journal of Virology ◽

10.1128/jvi.00271-17 ◽

2017 ◽

Vol 91 (12) ◽

Cited By ~ 10

Author(s):

Fumio Maeda ◽

Jun Arii ◽

Yoshitaka Hirohata ◽

Yuhei Maruzuru ◽

Naoto Koyanagi ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Null Mutation ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Nuclear Egress ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Upon herpes simplex virus 1 (HSV-1) infection, the CD98 heavy chain (CD98hc) is redistributed around the nuclear membrane (NM), where it promotes viral de-envelopment during the nuclear egress of nucleocapsids. In this study, we attempted to identify the factor(s) involved in CD98hc accumulation and demonstrated the following: (i) the null mutation of HSV-1 UL34 caused specific dispersion throughout the cytoplasm of CD98hc and the HSV-1 de-envelopment regulators, glycoproteins B and H (gB and gH); (ii) as observed with CD98hc, gB, and gH, wild-type HSV-1 infection caused redistribution of the endoplasmic reticulum (ER) markers calnexin and ERp57 around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of these markers; (iii) the ER markers colocalized efficiently with CD98hc, gB, and gH in the presence and absence of UL34 in HSV-1-infected cells; (iv) at the ultrastructural level, wild-type HSV-1 infection caused ER compression around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of the ER; and (v) the UL34-null mutation significantly decreased the colocalization efficiency of lamin protein markers of the NM with CD98hc and gB. Collectively, these results indicate that HSV-1 infection causes redistribution of the ER around the NM, with resulting accumulation of ER-associated CD98hc, gB, and gH around the NM and that UL34 is required for ER redistribution, as well as for efficient recruitment to the NM of the ER-associated de-envelopment factors. Our study suggests that HSV-1 induces remodeling of the global ER architecture for recruitment of regulators mediating viral nuclear egress to the NM. IMPORTANCE The ER is an important cellular organelle that exists as a complex network extending throughout the cytoplasm. Although viruses often remodel the ER to facilitate viral replication, information on the effects of herpesvirus infections on ER morphological integrity is limited. Here, we showed that HSV-1 infection led to compression of the global ER architecture around the NM, resulting in accumulation of ER-associated regulators associated with nuclear egress of HSV-1 nucleocapsids. We also identified HSV-1 UL34 as a viral factor that mediated ER remodeling. Furthermore, we demonstrated that UL34 was required for efficient targeting of these regulators to the NM. To our knowledge, this is the first report showing that a herpesvirus remodels ER global architecture. Our study also provides insight into the mechanism by which the regulators for HSV-1 nuclear egress are recruited to the NM, where this viral event occurs.

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Herpes Simplex Virus UL12.5 Targets Mitochondria through a Mitochondrial Localization Sequence Proximal to the N Terminus

Journal of Virology ◽

10.1128/jvi.02087-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2601-2610 ◽

Cited By ~ 33

Author(s):

Jennifer A. Corcoran ◽

Holly A. Saffran ◽

Brett A. Duguay ◽

James R. Smiley

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Mitochondrial Genome ◽

Nuclear Localization ◽

Full Length ◽

Mitochondrial Localization ◽

Amino Terminal ◽

The Matrix ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) gene UL12 encodes a conserved alkaline DNase with orthologues in all herpesviruses. The HSV-1 UL12 gene gives rise to two separately promoted 3′ coterminal mRNAs which encode distinct but related proteins: full-length UL12 and UL12.5, an amino-terminally truncated form that initiates at UL12 codon 127. Full-length UL12 localizes to the nucleus where it promotes the generation of mature viral genomes from larger precursors. In contrast, UL12.5 is predominantly mitochondrial and acts to trigger degradation of the mitochondrial genome early during infection. We examined the basis for these very different subcellular localization patterns. We confirmed an earlier report that the amino-terminal region of full-length UL12 is required for nuclear localization and provide evidence that multiple nuclear localization determinants are present in this region. In addition, we demonstrate that mitochondrial localization of UL12.5 relies largely on sequences located between UL12 residues 185 and 245 (UL12.5 residues 59 to 119). This region contains a sequence that resembles a typical mitochondrial matrix localization signal, and mutations that reduce the positive charge of this element severely impaired mitochondrial localization. Consistent with matrix localization, UL12.5 displayed a detergent extraction profile indistinguishable from that of the matrix protein cyclophilin D. Mitochondrial DNA depletion required the exonuclease activity of UL12.5, consistent with the idea that UL12.5 located within the matrix acts directly to destroy the mitochondrial genome. These results clarify how two highly related viral proteins are targeted to different subcellular locations with distinct functional consequences.

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Structural Variability of the Herpes Simplex Virus 1 GenomeIn VitroandIn Vivo

Journal of Virology ◽

10.1128/jvi.00223-12 ◽

2012 ◽

Vol 86 (16) ◽

pp. 8592-8601 ◽

Cited By ~ 9

Author(s):

Charlotte Mahiet ◽

Ayla Ergani ◽

Nicolas Huot ◽

Nicolas Alende ◽

Ahmed Azough ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Considerable Proportion ◽

Inverted Repeats ◽

Herpes Simplex Virus 1 ◽

Cell Extracts ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation.In vivo, for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the onein vitrowas observed, along with a considerable proportion of noncanonical assortment.

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