Superinfection Exclusion of Alphaherpesviruses Interferes with Virion Trafficking

Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection interferes with secondary viral infections within that same cell. Although SIE has been observed across many viruses, it has remained relatively understudied. A recently characterized glycoprotein D (gD) -independent SIE of alphaherpesviruses presents a novel mechanism of co-infection restriction for Herpes Simplex Virus Type 1 (HSV-1) and Pseudorabies virus (PRV). In this study, we evaluated the role of multiplicity of infection (MOI), receptor expression, and trafficking of virions to gain greater insight into potential mechanisms of alphaherpesvirus SIE. We observed that high MOI secondary viral infections were able to overcome SIE in a manner that was independent of receptor availability. Utilizing recombinant viruses expressing fluorescent protein fusions, we assessed virion localization during SIE through live fluorescent microscopy of dual-labeled virions and localization of capsid assemblies. Analysis of these assemblies confirmed changes in the distribution of capsids during SIE. These results indicate that SIE during PRV infection inhibits viral entry or fusion while HSV-1 SIE inhibits infection through a post-entry mechanism. Although the timing and phenotype of SIE is similar between alphaherpesviruses, the related viruses implement different mechanisms to restrict coinfection.

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gD-Independent Superinfection Exclusion of Alphaherpesviruses

Journal of Virology ◽

10.1128/jvi.00089-16 ◽

2016 ◽

Vol 90 (8) ◽

pp. 4049-4058 ◽

Cited By ~ 20

Author(s):

A. Criddle ◽

T. Thornburg ◽

I. Kochetkova ◽

M. DePartee ◽

M. P. Taylor

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Viral Infections ◽

Herpes Simplex Virus 1 ◽

Viral Genomes ◽

Superinfection Exclusion ◽

Development Of Resistance ◽

Simplex Virus ◽

Spread Of Infection ◽

Hsv 1

ABSTRACTMany viruses have the capacity to prevent a cell from being infected by a second virus, often termed superinfection exclusion. Alphaherpesviruses, including the human pathogen herpes simplex virus 1 (HSV-1) and the animal herpesvirus pseudorabies virus (PRV), encode a membrane-bound glycoprotein, gD, that can interfere with subsequent virion entry. We sought to characterize the timing and mechanism of superinfection exclusion during HSV-1 and PRV infection. To this end, we utilized recombinant viruses expressing fluorescent protein (FP) markers of infection that allowed the visualization of viral infections by microscopy and flow cytometry as well as the differentiation of viral progeny. Our results demonstrated the majority of HSV-1- and PRV-infected cells establish superinfection exclusion by 2 h postinfection. The modification of viral infections by virion inactivation and phosphonoacetic acid, cycloheximide, and actinomycin D treatments indicated new protein synthesis is needed to establish superinfection exclusion. Primary infection with gene deletion PRV recombinants identified that new gD expression is not required to establish superinfection exclusion of a secondary viral inoculum. We also identified the timing of coinfection events during axon-to-cell spread, with most occurring within a 2-h window, suggesting a role for cellular superinfection exclusion during neuroinvasive spread of infection. In summary, we have characterized a gD-independent mechanism of superinfection exclusion established by two members of the alphaherpesvirus family and identified a potential role of exclusion during the pathogenic spread of infection.IMPORTANCESuperinfection exclusion is a widely observed phenomenon initiated by a primary viral infection to prevent further viruses from infecting the same cell. The capacity for alphaherpesviruses to infect the same cell impacts rates of interviral recombination and disease. Interviral recombination allows genome diversification, facilitating the development of resistance to antiviral therapeutics and evasion of vaccine-mediated immune responses. Our results demonstrate superinfection exclusion occurs early, through a gD-independent process, and is important in the directed spread of infection. Identifying when and where in an infected host viral genomes are more likely to coinfect the same cell and generate viral recombinants will enhance the development of effective antiviral therapies and interventions.

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Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

Journal of Virology ◽

10.1128/jvi.00598-09 ◽

2009 ◽

Vol 83 (14) ◽

pp. 6978-6986 ◽

Cited By ~ 20

Author(s):

M. G. Lyman ◽

C. D. Kemp ◽

M. P. Taylor ◽

L. W. Enquist

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Membrane Topology ◽

Human Pathogens ◽

Herpesvirus Type ◽

Varicella Zoster ◽

Green Fluorescent ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.

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The Capsid and Tegument of the Alphaherpesviruses Are Linked by an Interaction between the UL25 and VP1/2 Proteins

Journal of Virology ◽

10.1128/jvi.01113-07 ◽

2007 ◽

Vol 81 (21) ◽

pp. 11790-11797 ◽

Cited By ~ 105

Author(s):

Kelly Elizabeth Coller ◽

Joy I-Hsuan Lee ◽

Aki Ueda ◽

Gregory Allan Smith

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Protein Product ◽

Tegument Protein ◽

Viral Propagation ◽

Tegument Proteins ◽

Green Fluorescent ◽

Simplex Virus ◽

Carboxy Terminal ◽

Hsv 1

ABSTRACT How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.

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Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

10.1101/392555 ◽

2018 ◽

Author(s):

Chao Qin ◽

Rui Zhang ◽

Yue Lang ◽

Anwen Shao ◽

Aotian Xu ◽

...

Keyword(s):

Gene Transcription ◽

Pseudorabies Virus ◽

Viral Infections ◽

Type I Interferon ◽

Interferon Response ◽

Interferon Α ◽

Type I ◽

Viral Inhibition ◽

Simplex Virus ◽

Hsv 1

AbstractType I interferon response plays a prominent role against viral infection, which is frequently disrupted by viruses. Here, we report Bcl-2 associated transcription factor 1 (Bclaf1) is degraded during the alphaherpesvirus Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) infections through the viral protein US3. We further reveal that Bclaf1 functions critically in type I interferon signaling. Knockdown or knockout of Bclaf1 in cells significantly impairs interferon-α (IFNα)-mediated gene transcription and viral inhibition against US3 deficient PRV and HSV-1. Mechanistically, Bclaf1 maintains a mechanism allowing STAT1 and STAT2 to be efficiently phosphorylated in response to IFNα, and more importantly, facilitates IFN-stimulated gene factor 3 (ISGF3) binding with IFN-stimulated response elements (ISRE) for efficient gene transcription by directly interacting with ISRE and STAT2. Our studies establish the importance of Bclaf1 in IFNα-induced antiviral immunity and in the control of viral infections.

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TRPC1 participates in the HSV-1 infection process by facilitating viral entry

Science Advances ◽

10.1126/sciadv.aaz3367 ◽

2020 ◽

Vol 6 (12) ◽

pp. eaaz3367 ◽

Cited By ~ 1

Author(s):

DongXu He ◽

AiQin Mao ◽

YouRan Li ◽

SiuCheung Tam ◽

YongTang Zheng ◽

...

Keyword(s):

Viral Entry ◽

Transient Receptor Potential ◽

Trp Channels ◽

Critical Role ◽

Receptor Potential ◽

Infection Process ◽

Ocular Abnormality ◽

Simplex Virus ◽

Hsv 1

Mammalian transient receptor potential (TRP) channels are major components of Ca2+ signaling pathways and control a diversity of physiological functions. Here, we report a specific role for TRPC1 in the entry of herpes simplex virus type 1 (HSV-1) into cells. HSV-1–induced Ca2+ release and entry were dependent on Orai1, STIM1, and TRPC1. Inhibition of Ca2+ entry or knockdown of these proteins attenuated viral entry and infection. HSV-1 glycoprotein D interacted with the third ectodomain of TRPC1, and this interaction facilitated viral entry. Knockout of TRPC1 attenuated HSV-1–induced ocular abnormality and morbidity in vivo in TRPC1−/− mice. There was a strong correlation between HSV-1 infection and plasma membrane localization of TRPC1 in epithelial cells within oral lesions in buccal biopsies from HSV-1–infected patients. Together, our findings demonstrate a critical role for TRPC1 in HSV-1 infection and suggest the channel as a potential target for anti-HSV therapy.

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Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry

Journal of Virology ◽

10.1128/jvi.02123-16 ◽

2016 ◽

Vol 91 (5) ◽

Cited By ~ 17

Author(s):

Darin J. Weed ◽

Suzanne M. Pritchard ◽

Floricel Gonzalez ◽

Hector C. Aguilar ◽

Anthony V. Nicola

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Viral Entry ◽

Low Ph ◽

Fusion Activity ◽

Ph Inactivation ◽

Target Membrane ◽

Fusion Domain ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions. IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane.

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Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection

Journal of Virology ◽

10.1128/jvi.73.5.4110-4119.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4110-4119 ◽

Cited By ~ 135

Author(s):

Gillian Elliott ◽

Peter O’Hare

Keyword(s):

Herpes Simplex Virus ◽

Green Fluorescent Protein ◽

Herpes Simplex ◽

Fluorescent Protein ◽

Time Lapse ◽

Live Cell ◽

Cell Periphery ◽

Green Fluorescent ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.

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Soluble V Domain of Nectin-1/HveC Enables Entry of Herpes Simplex Virus Type 1 (HSV-1) into HSV-Resistant Cells by Binding to Viral Glycoprotein D

Journal of Virology ◽

10.1128/jvi.80.1.138-148.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 138-148 ◽

Cited By ~ 33

Author(s):

Heechung Kwon ◽

Qing Bai ◽

Hyun-Jung Baek ◽

Kelly Felmet ◽

Edward A. Burton ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cell Surface ◽

Viral Entry ◽

Glycoprotein D ◽

Viral Glycoprotein ◽

Virus Attachment ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1123), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1123, approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1123 did not associate with the cell surface. sNec1123-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.

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HIV-1 Replication in HIV-Infected Individuals Is Significantly Reduced When Peripheral Blood Mononuclear Cells Are Superinfected with HSV-1

The Scientific World JOURNAL ◽

10.1100/2012/102843 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6

Author(s):

Taneth Yamsuwan ◽

Chintana Chirathaworn ◽

Pokrath Hansasuta ◽

Parvapan Bhattarakosol

Keyword(s):

T Lymphocytes ◽

Viral Entry ◽

Mononuclear Cells ◽

Multiplicity Of Infection ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Immunodeficiency Virus ◽

Simplex Virus ◽

Hiv 1 ◽

Hsv 1

Herpes simplex virus (HSV) can cause generalized infection in human immunodeficiency virus- (HIV-) infected patients leading to death. This study investigated HSV-1 replication in PBMCs from 25 HIV-infected individuals and 15 healthy donors and the effects of HSV-1 superinfection on HIV-1 production. Herpes viral entry mediator (HVEM) receptor on T lymphocytes was also evaluated. Our results confirmed that the number of activated (CD3+ and CD38+) T lymphocytes in HIV-infected individuals (46.51±17.54%) was significantly higher than in healthy donors (27.54±14.12%,Pvalue = 0.001) without any significant differences in HVEM expression. Even though the percentages of HSV-1 infected T lymphocytes between HIV-infected individuals (79.25±14.63%) and healthy donors (80.76±7.13%) were not different (Pvalue = 0.922), yet HSV-1 production in HIV-infected individuals (47.34±11.14×103 PFU/ml) was significantly greater than that of healthy donors (34.17±8.48×103 PFU/ml,Pvalue = 0.001). Moreover, HSV-1 virions were released extracellularly rather than being associated with the cells, and superinfection of HSV-1 at a multiplicity of infection (MOI) of 5 significantly decreased HIV production (Pvalue < 0.001).

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Multiple Peptides Homologous to Herpes Simplex Virus Type 1 Glycoprotein B Inhibit Viral Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00793-08 ◽

2008 ◽

Vol 53 (3) ◽

pp. 987-996 ◽

Cited By ~ 33

Author(s):

Radeekorn Akkarawongsa ◽

Nina E. Pocaro ◽

Gary Case ◽

Aaron W. Kolb ◽

Curtis R. Brandt

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Viral Entry ◽

Herpes Simplex Virus Type ◽

Glycoprotein B ◽

Induced Mutations ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The 773-residue ectodomain of the herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) has been resistant to the use of mutagenic strategies because the majority of the induced mutations result in defective proteins. As an alternative strategy for the identification of functionally important regions and novel inhibitors of infection, we prepared a library of overlapping peptides homologous to the ectodomain of gB and screened for the ability of the peptides to block infection. Seven of 138 15-mer peptides inhibited infection by more than 50% at a concentration of 100 μM. Three peptides (gB94, gB122, and gB131) with 50% effective concentrations (EC50s) below 20 μM were selected for further studies. The gB131 peptide (residues 681 to 695 in HSV-1 gB [gB-1]) was a specific entry inhibitor (EC50, ∼12 μM). The gB122 peptide (residues 636 to 650 in gB-1) blocked viral entry (EC50, ∼18 μM), protected cells from infection (EC50, ∼72 μM), and inactivated virions in solution (EC50, ∼138 μM). We were unable to discern the step or steps inhibited by the gB94 peptide, which is homologous to residues 496 to 510 in gB-1. Substitution of a tyrosine in the gB122 peptide (Y640 in full-length gB-1) reduced the antiviral activity eightfold, suggesting that this residue is critical for inhibition. This peptide-based strategy could lead to the identification of functionally important regions of gB or other membrane proteins and identify novel inhibitors of HSV-1 entry.

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