Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication, and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo

ABSTRACT Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Δ491RSRR494 and point mutation 491GSGG494 in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Δ491RSRR494 or 491GSGG494 mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBΔ491RSRR494 or pOka-gB491GSGG494 virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.

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Functions of the ORF9-to-ORF12 Gene Cluster in Varicella-Zoster Virus Replication and in the Pathogenesis of Skin Infection

Journal of Virology ◽

10.1128/jvi.00303-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5825-5834 ◽

Cited By ~ 51

Author(s):

Xibing Che ◽

Mike Reichelt ◽

Marvin H. Sommer ◽

Jaya Rajamani ◽

Leigh Zerboni ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Gene Cluster ◽

Skin Infection ◽

Cultured Cells ◽

Human Tonsil ◽

Reading Frame ◽

Varicella Zoster ◽

The Individual

ABSTRACT The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKAΔ10/11), ORF11 and ORF12 (POKAΔ11/12), or ORF10, ORF11 and ORF12 (POKAΔ10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKAΔ11, POKAΔ10/11, and POKAΔ11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKAΔ10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.

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Functions of the C-Terminal Domain of Varicella-Zoster Virus Glycoprotein E in Viral Replication In Vitro and Skin and T-Cell Tropism In Vivo

Journal of Virology ◽

10.1128/jvi.78.22.12406-12415.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12406-12415 ◽

Cited By ~ 34

Author(s):

Jennifer Moffat ◽

Chengjun Mo ◽

Jason J. Cheng ◽

Marvin Sommer ◽

Leigh Zerboni ◽

...

Keyword(s):

T Cell ◽

Viral Replication ◽

Varicella Zoster Virus ◽

Point Mutations ◽

Glycoprotein E ◽

Varicella Zoster ◽

Terminal Domain ◽

C Terminus

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication. To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an “acid cluster” comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.

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Recombination of Globally Circulating Varicella-Zoster Virus

Journal of Virology ◽

10.1128/jvi.00437-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7133-7146 ◽

Cited By ~ 49

Author(s):

Peter Norberg ◽

Daniel P. Depledge ◽

Samit Kundu ◽

Claire Atkinson ◽

Julianne Brown ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Genetic Recombination ◽

Human Herpesvirus ◽

Infectious Laryngotracheitis Virus ◽

Live Attenuated Vaccine ◽

Herpes Simplex Viruses ◽

Varicella Zoster ◽

Laryngotracheitis Virus

ABSTRACTVaricella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombinein vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, eitherin vivoorin vitroduring passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade.IMPORTANCEAlthough genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

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The Immediate-Early 63 Protein of Varicella-Zoster Virus: Analysis of Functional Domains Required for Replication In Vitro and for T-Cell and Skin Tropism in the SCIDhu Model In Vivo

Journal of Virology ◽

10.1128/jvi.78.3.1181-1194.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1181-1194 ◽

Cited By ~ 40

Author(s):

Armin Baiker ◽

Christoph Bagowski ◽

Hideki Ito ◽

Marvin Sommer ◽

Leigh Zerboni ◽

...

Keyword(s):

T Cell ◽

Varicella Zoster Virus ◽

Nuclear Localization ◽

Regulatory Protein ◽

Immediate Early ◽

Functional Domains ◽

Reading Frame ◽

Varicella Zoster

ABSTRACT The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 US1.5 protein, which is expressed colinearly with ICP22 (US1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.

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Anti-Glycoprotein H Antibody Impairs the Pathogenicity of Varicella-Zoster Virus in Skin Xenografts in the SCID Mouse Model

Journal of Virology ◽

10.1128/jvi.01338-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 141-152 ◽

Cited By ~ 15

Author(s):

Susan E. Vleck ◽

Stefan L. Oliver ◽

Mike Reichelt ◽

Jaya Rajamani ◽

Leigh Zerboni ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Neutralizing Antibodies ◽

Plasma Membranes ◽

Virus Entry ◽

Varicella Zoster ◽

Virus Particles ◽

Early Endosomes ◽

Glycoprotein H

ABSTRACT Varicella-zoster virus (VZV) infection is usually mild in healthy individuals but can cause severe disease in immunocompromised patients. Prophylaxis with varicella-zoster immunoglobulin can reduce the severity of VZV if given shortly after exposure. Glycoprotein H (gH) is a highly conserved herpesvirus protein with functions in virus entry and cell-cell spread and is a target of neutralizing antibodies. The anti-gH monoclonal antibody (MAb) 206 neutralizes VZV in vitro. To determine the requirement for gH in VZV pathogenesis in vivo, MAb 206 was administered to SCID mice with human skin xenografts inoculated with VZV. Anti-gH antibody given at 6 h postinfection significantly reduced the frequency of skin xenograft infection by 42%. Virus titers, genome copies, and lesion size were decreased in xenografts that became infected. In contrast, administering anti-gH antibody at 4 days postinfection suppressed VZV replication but did not reduce the frequency of infection. The neutralizing anti-gH MAb 206 blocked virus entry, cell fusion, or both in skin in vivo. In vitro, MAb 206 bound to plasma membranes and to surface virus particles. Antibody was internalized into vacuoles within infected cells, associated with intracellular virus particles, and colocalized with markers for early endosomes and multivesicular bodies but not the trans-Golgi network. MAb 206 blocked spread, altered intracellular trafficking of gH, and bound to surface VZV particles, which might facilitate their uptake and targeting for degradation. As a consequence, antibody interference with gH function would likely prevent or significantly reduce VZV replication in skin during primary or recurrent infection.

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Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System

Journal of Virology ◽

10.1128/jvi.02666-06 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9024-9033 ◽

Cited By ~ 60

Author(s):

Zhen Zhang ◽

Jenny Rowe ◽

Weijia Wang ◽

Marvin Sommer ◽

Ann Arvin ◽

...

Keyword(s):

Bacterial Artificial Chromosome ◽

Varicella Zoster Virus ◽

Artificial Chromosome ◽

Wild Type Virus ◽

Growth Curve Analysis ◽

Type Virus ◽

Wild Type ◽

Varicella Zoster

ABSTRACT To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZVBAC) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZVLuc). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZVLuc genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.

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Varicella-Zoster Virus Open Reading Frame 10 Is a Virulence Determinant in Skin Cells but Not in T Cells In Vivo

Journal of Virology ◽

10.1128/jvi.80.7.3238-3248.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3238-3248 ◽

Cited By ~ 25

Author(s):

Xibing Che ◽

Leigh Zerboni ◽

Marvin H. Sommer ◽

Ann M. Arvin

Keyword(s):

Varicella Zoster Virus ◽

Open Reading Frame ◽

Early Gene ◽

Pathogenic Potential ◽

Reading Frame ◽

Virion Assembly ◽

Varicella Zoster ◽

Skin Cells

ABSTRACT The open reading frame 10 (ORF10) of varicella-zoster virus (VZV) encodes a tegument protein that enhances transactivation of VZV genes and has homology to herpes simplex virus type 1 (HSV-1) VP16. While VP16 is essential for HSV replication, ORF10 is dispensable for vaccine OKA (VOKA) growth in vitro. We used parent OKA (POKA) cosmids to delete ORF10, producing POKAΔ10; point mutations that disrupted the acidic activation domain and the putative motif for binding human cellular factor 1 (HCF-1) in ORF10 protein yielded POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF viruses. Deleting ORF10 or mutating these two functional domains had no effect on VZV replication, immediate-early gene transcription, or virion assembly in vitro. However, deleting ORF10 reduced viral titers and the extent of cutaneous lesions significantly in SCIDhu skin xenografts in vivo compared to POKA. Epidermal cells infected with POKAΔ10 had significantly fewer DNA-containing nucleocapsids and complete virions compared to POKA; extensive aggregates of intracytoplasmic viral particles were also observed. Altering the activation or the putative HCF-1 domains of ORF10 protein had no consequences for VZV replication in vivo. Thus, the decreased pathogenic potential of POKAΔ10 in skin could not be attributed to absence of these ORF10 protein functions. In contrast to skin cells, deleting ORF10 did not impair VZV T-cell tropism in vivo, as assessed by infectious virus yields. We conclude that ORF10 protein is required for efficient VZV virion assembly and is a specific determinant of VZV virulence in epidermal and dermal cells in vivo.

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Mutational Analysis of the Varicella-Zoster Virus ORF62/63 Intergenic Region

Journal of Virology ◽

10.1128/jvi.80.6.3116-3121.2006 ◽

2006 ◽

Vol 80 (6) ◽

pp. 3116-3121 ◽

Cited By ~ 17

Author(s):

Jeremy O. Jones ◽

Marvin Sommer ◽

Shaye Stamatis ◽

Ann M. Arvin

Keyword(s):

Varicella Zoster Virus ◽

Binding Site ◽

Intergenic Region ◽

Binding Sites ◽

Mutational Analysis ◽

Heat Shock Factor 1 ◽

Cultured Fibroblasts ◽

Varicella Zoster

ABSTRACT The varicella-zoster virus (VZV) ORF62/63 intergenic region was cloned between the Renilla and firefly luciferase genes, which acted as reporters of ORF62 and ORF63 transcription, and recombinant viruses were generated that carried these reporter cassettes along with the intact native sequences in the repeat regions of the VZV genome. In order to investigate the potential contributions of cellular transregulatory proteins to ORF62 and ORF63 transcription, recombinant reporter viruses with mutations of consensus binding sites for six proteins within the intergenic region were also created. The reporter viruses were used to evaluate ORF62 and ORF63 transcription during VZV replication in cultured fibroblasts and in skin xenografts in SCIDhu mice in vivo. Mutations in putative binding sites for heat shock factor 1 (HSF-1), nuclear factor 1 (NF-1), and one of two cyclic AMP-responsive elements (CRE) reduced ORF62 reporter transcription in fibroblasts, while mutations in binding sites for HSF-1, NF-1, and octamer binding proteins (Oct-1) increased ORF62 reporter transcription in skin. Mutations in one CRE and the NF-1 site altered ORF63 transcription in fibroblasts, while mutation of the Oct-1 binding site increased ORF63 reporter transcription in skin. The effect of each of these mutations implies that the intact binding site sequence regulates native ORF62 and ORF63 transcription. Mutation of the only NF-κB/Rel binding site had no effect on ORF62 or ORF63 transcription in vitro or in vivo. The segment of the ORF62/63 intergenic region proximal to ORF63 was most important for ORF63 transcription, but mutagenesis also altered ORF62 transcription, indicating that this region functions as a bidirectional promoter. This first analysis of the ORF62/63 intergenic region in the context of VZV replication indicates that it is a dual promoter and that cellular transregulatory factors affect the transcription of these key VZV regulatory genes.

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Mutational Analysis of Open Reading Frames 62 and 71, Encoding the Varicella-Zoster Virus Immediate-Early Transactivating Protein, IE62, and Effects on Replication In Vitro and in Skin Xenografts in the SCID-hu Mouse In Vivo

Journal of Virology ◽

10.1128/jvi.77.10.5607-5620.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5607-5620 ◽

Cited By ~ 43

Author(s):

Bunji Sato ◽

Hideki Ito ◽

Stewart Hinchliffe ◽

Marvin H. Sommer ◽

Leigh Zerboni ◽

...

Keyword(s):

Cell Culture ◽

Varicella Zoster Virus ◽

Infectious Virus ◽

Mutational Analysis ◽

Single Copy ◽

Open Reading Frames ◽

Immediate Early ◽

Varicella Zoster

ABSTRACT The varicella-zoster virus (VZV) genome has unique long (UL) and unique short (US) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. The immediate-early 62 (IE62) protein, encoded by open reading frame 62 (ORF62) and ORF71 in these repeats, is the major VZV transactivating protein. Mutational analyses were done with VZV cosmids generated from parent Oka (pOka), a low-passage clinical isolate, and repair experiments were done with ORF62 from pOka and vaccine Oka (vOka), which is derived from pOka. Transfections using VZV cosmids from which ORF62, ORF71, or the ORF62/71 gene pair was deleted showed that VZV replication required at least one copy of ORF62. The insertion of ORF62 from pOka or vOka into a nonnative site in US allowed VZV replication in cell culture in vitro, although the plaque size and yields of infectious virus were decreased. Targeted mutations in binding sites reported to affect interaction with IE4 protein and a putative ORF9 protein binding site were not lethal. Single deletions of ORF62 or ORF71 from cosmids permitted recovery of infectious virus, but recombination events repaired the defective repeat region in some progeny viruses, as verified by PCR and Southern hybridization. VZV infectivity in skin xenografts in the SCID-hu model required ORF62 expression; mixtures of single-copy recombinant OkaΔ62 (rOkaΔ62) or rOkaΔ71 and repaired rOka generated by recombination of the single-copy deletion mutants were detected in some skin implants. Although insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was necessary for cell-cell spread in differentiated human skin tissues in vivo.

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Molecular and therapeutic aspects of varicella–zoster virus infection

Expert Reviews in Molecular Medicine ◽

10.1017/s146239940500966x ◽

2005 ◽

Vol 7 (15) ◽

pp. 1-24 ◽

Cited By ~ 6

Author(s):

Mark Quinlivan ◽

Judith Breuer

Keyword(s):

Varicella Zoster Virus ◽

Mononuclear Cells ◽

Small Animal ◽

Varicella Zoster Virus Infection ◽

Peripheral Blood Mononuclear ◽

Live Attenuated Vaccine ◽

Narrow Host Range ◽

Varicella Zoster

Varicella–zoster virus (VZV) is a highly species-specific member of the Herpesviridae family. The virus exhibits multiple cell tropisms, infecting peripheral blood mononuclear cells and skin cells before establishing latency in sensory neurons. Such tropisms are essential both for primary infection, which manifests itself as chickenpox (varicella), and subsequent reactivation to cause herpes zoster (shingles). The highly cell-associated nature of the virus, coupled with its narrow host range, has resulted in the lack of an animal model that mimics its diseases in humans, thereby greatly hindering the study of events in VZV pathogenesis. Despite this, extensive studies both in vitro and in vivo in small-animal models have provided a fascinating insight into molecular events that govern VZV diseases. In addition, VZV has become the first human herpes virus for which a live attenuated vaccine has been developed.

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