scholarly journals 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

2004 ◽  
Vol 78 (3) ◽  
pp. 1181-1194 ◽  
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.

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

2008 ◽  
Vol 82 (12) ◽  
pp. 5825-5834 ◽  
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.

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 Phosphorylation of the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein IE62 by the VZV Open Reading Frame 66 Protein Kinase

2006 ◽  
Vol 80 (4) ◽  
pp. 1710-1723 ◽  
Author(s):  
Amie J. Eisfeld ◽  
Stephanie E. Turse ◽  
Sara A. Jackson ◽  
Edwina C. Lerner ◽  
Paul R. Kinchington
Keyword(s):  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Nuclear Import ◽  
Regulatory Protein ◽  
Recombinant Baculovirus ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Transcriptional Regulatory

ABSTRACT IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

scholarly journals Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency

2005 ◽  
Vol 79 (8) ◽  
pp. 5069-5077 ◽  
Author(s):  
Jeffrey I. Cohen ◽  
Tammy Krogmann ◽  
Sebastien Bontems ◽  
Catherine Sadzot-Delvaux ◽  
Lesley Pesnicak
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Localization Signal ◽  
Carboxy Terminal

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.

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

2006 ◽  
Vol 80 (7) ◽  
pp. 3238-3248 ◽  
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.

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 Promoter Sequences of Varicella-Zoster Virus Glycoprotein I Targeted by Cellular Transactivating Factors Sp1 and USF Determine Virulence in Skin and T Cells in SCIDhu Mice In Vivo

2003 ◽  
Vol 77 (1) ◽  
pp. 489-498 ◽  
Author(s):  
Hideki Ito ◽  
Marvin H. Sommer ◽  
Leigh Zerboni ◽  
Hongying He ◽  
Dwayne Boucaud ◽  
...  
Keyword(s):  
T Cells ◽  
Varicella Zoster Virus ◽  
Host Cells ◽  
Upstream Sequence ◽  
Upstream Stimulatory Factor ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Glycoprotein I

ABSTRACT Varicella-zoster virus (VZV) glycoprotein I is dispensable in cell culture but necessary for infection of human skin and T cells in SCIDhu mice in vivo. The gI promoter contains an activating upstream sequence that binds the cellular transactivators specificity factor 1 (Sp1) and upstream stimulatory factor (USF) and an open reading frame 29 (ORF29)-responsive element (29RE), which mediates enhancement by ORF29 DNA binding protein of immediate-early 62 (IE62)-induced transcription. Recombinants, rOKAgI-Sp1 and rOKAgI-USF, with two base pair substitutions in Sp1 or USF sites, replicated like rOKA in vitro, but infectivity of rOKAgI-Sp1 was significantly impaired in skin and T cells in vivo. A double mutant, rOKAgI-Sp1/USF, did not replicate in skin but yielded low titers of infectious virus in T cells. The repaired protein, rOKAgI:rep-Sp1/USF, was as infectious as rOKA. Thus, disrupting gI promoter sites for cellular transactivators altered VZV virulence in vivo, with variable consequences related to the cellular factor and the host cell type. Mutations in the 29RE of the gI promoter were made by substituting each of four 10-bp blocks in this region with a 10-bp sequence, GATAACTACA, that was predicted to interfere with enhancer effects of the ORF29 protein. One of these mutants, which was designated rOKAgI-29RE-3, had diminished replication in skin and T cells, indicating that ORF29 protein-mediated enhancement of gI expression contributes to VZV virulence. Mutations within promoters of viral genes that are nonessential in vitro should allow construction of recombinant herpesviruses that have altered virulence in specific host cells in vivo and may be useful for designing herpesviral gene therapy vectors and attenuated viral vaccines.

scholarly journals Varicella-Zoster Virus (VZV) ORF17 Protein Induces RNA Cleavage and Is Critical for Replication of VZV at 37oC but Not 33oC

2002 ◽  
Vol 76 (21) ◽  
pp. 11012-11023 ◽  
Author(s):  
Hitoshi Sato ◽  
Lawrence D. Callanan ◽  
Lesley Pesnicak ◽  
Tammy Krogmann ◽  
Jeffrey I. Cohen
Keyword(s):  
Varicella Zoster Virus ◽  
Deletion Mutant ◽  
Host Protein ◽  
Parental Virus ◽  
Rna Cleavage ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Cotton Rats ◽  
Simplex Virus

ABSTRACT Varicella-zoster virus (VZV) open reading frame 17 (ORF17) is homologous to herpes simplex virus (HSV) UL41, which encodes the viral host shutoff protein (vhs). HSV vhs induces degradation of mRNA and rapid shutoff of host protein synthesis. An antibody to ORF17 protein detected a 46-kDa protein in VZV-infected cells. While HSV vhs is located in virions, VZV ORF17 protein was not detectable in virions. ORF17 protein induced RNA cleavage, but to a substantially lesser extent than HSV-1 vhs. Expression of ORF17 protein did not inhibit expression from a β-galactosidase reporter plasmid, while HSV type 1 vhs abolished reporter expression. Two VZV ORF17 deletion mutants were constructed to examine the role of ORF17 in virus replication. While the ORF17 VZV mutants grew to peak titers that were similar to those of the parental virus at 33°C, the ORF17 mutants grew to 20- to 35-fold-lower titers than parental virus at 37°C. ORF62 protein was distributed in a different pattern in the nuclei and cytoplasm of cells infected with an ORF17 deletion mutant at 37°C compared to 33°C. Inoculation of cotton rats with the ORF17 deletion mutant resulted in a level of latent infection similar to that produced by inoculation with the parental virus. The importance of ORF17 protein for viral replication at 37°C but not at 33°C suggests that this protein may facilitate the growth of virus in certain tissues in vivo.

scholarly journals 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

2009 ◽  
Vol 83 (15) ◽  
pp. 7495-7506 ◽  
Author(s):  
Stefan L. Oliver ◽  
Marvin Sommer ◽  
Leigh Zerboni ◽  
Jaya Rajamani ◽  
Charles Grose ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Protein Localization ◽  
Glycoprotein B ◽  
Primary Loop ◽  
Varicella Zoster ◽  
Artificial Chromosomes ◽  
Recognition Motif ◽  
Fusion Loop

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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