scholarly journals Essential Functions of the Unique N-Terminal Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain in Viral Replication and in the Pathogenesis of Skin Infection

2006 ◽  
Vol 80 (19) ◽  
pp. 9481-9496 ◽  
Author(s):  
Barbara Berarducci ◽  
Minako Ikoma ◽  
Shaye Stamatis ◽  
Marvin Sommer ◽  
Charles Grose ◽  
...  
Keyword(s):  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Mutational Analysis ◽  
Terminal Region ◽  
Functional Region ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Cell Spread ◽  
Cell Cell

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.

scholarly journals 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

2004 ◽  
Vol 78 (22) ◽  
pp. 12406-12415 ◽  
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.

scholarly journals Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry

2008 ◽  
Vol 83 (1) ◽  
pp. 228-240 ◽  
Author(s):  
Barbara Berarducci ◽  
Jaya Rajamani ◽  
Mike Reichelt ◽  
Marvin Sommer ◽  
Leigh Zerboni ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Viral Entry ◽  
Rich Region ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Viral Particles ◽  
Infected Cells ◽  
Cell Spread ◽  
Cell Cell

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, in contrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-ΔCys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin.

scholarly journals Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

Virology ◽  
2016 ◽  
Vol 492 ◽  
pp. 82-91 ◽  
Author(s):  
Mohamed I. Khalil ◽  
Xibing Che ◽  
Phillip Sung ◽  
Marvin H. Sommer ◽  
John Hay ◽  
...  
Keyword(s):  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Mutational Analysis ◽  
Immediate Early ◽  
Varicella Zoster ◽  
Early Protein

scholarly journals Glycoprotein I of Varicella-Zoster Virus Is Required for Viral Replication in Skin and T Cells

2002 ◽  
Vol 76 (16) ◽  
pp. 8468-8471 ◽  
Author(s):  
Jennifer Moffat ◽  
Hideki Ito ◽  
Marvin Sommer ◽  
Shannon Taylor ◽  
Ann M. Arvin
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Human Skin ◽  
Deletion Mutant ◽  
Varicella Zoster ◽  
Glycoprotein I

ABSTRACT Varicella-zoster virus (VZV) glycoprotein I (gI) is dispensable in cell culture; the SCIDhu model of VZV pathogenesis was used to determine whether gI is necessary in vivo. The parental and repaired viruses grew in human skin and thymus/liver implants, but the gI deletion mutant was not infectious. Thus, gI is essential for VZV infectivity in skin and T cells.

scholarly journals The Requirement of Varicella Zoster Virus Glycoprotein E (gE) for Viral Replication and Effects of Glycoprotein I on gE in Melanoma Cells

Virology ◽  
2002 ◽  
Vol 304 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Chengjun Mo ◽  
Jay Lee ◽  
Marvin Sommer ◽  
Charles Grose ◽  
Ann M. Arvin
Keyword(s):  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Melanoma Cells ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Virus Glycoprotein ◽  
Glycoprotein I

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

2006 ◽  
Vol 80 (6) ◽  
pp. 3116-3121 ◽  
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.

scholarly journals 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

2003 ◽  
Vol 77 (10) ◽  
pp. 5607-5620 ◽  
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.

scholarly journals Phosphorylation by the Varicella-Zoster Virus ORF47 Protein Serine Kinase Determines whether Endocytosed Viral gE Traffics to the trans-Golgi Network or Recycles to the Cell Membrane

2002 ◽  
Vol 76 (21) ◽  
pp. 10980-10993 ◽  
Author(s):  
T. K. Kenyon ◽  
Jeffrey I. Cohen ◽  
Charles Grose
Keyword(s):  
Varicella Zoster Virus ◽  
Cytoplasmic Tail ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Varicella Zoster ◽  
Virion Production ◽  
Golgi Network ◽  
Cell Spread ◽  
Cell Cell

ABSTRACT Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the trans-Golgi network (TGN), while in cell-cell spread, surface glycoproteins fuse cells into syncytia. In this report, we delineate a potential role for serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoprotein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA 83:8967-8971, 1986), although respective roles of viral and cellular protein kinases have never been delineated. VZV ORF47 is a viral serine protein kinase that recognized a consensus sequence similar to that of casein kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as “team players” in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.

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