Open Reading Frame S/L of Varicella-Zoster Virus Encodes a Cytoplasmic Protein Expressed in Infected Cells

Varicella-zoster virus (VZV) encodes six genes that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 2 (ORF2), was expressed as a 31-kDa phosphoprotein in the membranes of infected cells. Unlike equine and bovine herpesvirus type 1 ORF2 homologs that are associated with virions, VZV virions contained no detectable ORF2 protein. The ORF2 deletion mutant established a latent infection in cotton rats at a frequency and with a number of VZV genomes similar to that of the parental virus. ORF63 transcripts, a hallmark of latent infection, were present in ganglia latently infected with both the ORF2 deletion mutant and parental VZV. Thus, ORF2 is the first VZV gene shown to be dispensable for establishment of latent infection in an animal model. Varicella-zoster virus (VZV) is a member of the alphaherpesvirus subfamily. This subfamily includes the genera Simplexvirus, such as herpes simplex virus (HSV), and Varicellovirus. The varicelloviruses include VZV, equine herpesvirus type 1 (EHV-1), bovine herpesvirus type 1 (BHV-1), and simian varicella virus (SVV). VZV encodes at least 70 unique genes, most of which are conserved in HSV. However, open reading frames (ORFs) 1, 2, 13, 32, 57, and S/L do not have homologs in HSV (6). Five of these genes, ORFs 1, 13, 32, 57, and S/L have been shown to be dispensable for replication of VZV in vitro (4,5,9,17,26).VZV ORF2 is predicted to encode a 26-kDa protein containing 238 amino acids (10). Although VZV ORF2 does not have an HSV homolog, it does share positional and limited sequence homology with several animal alphaherpesvirus genes. These genes include the BHV-1 circ gene, EHV-1 gene 3 (UL1), EHV-4 gene 3, and Marek's disease virus ORF71 (12,16,29,30). Interestingly, ORF2 is the only gene in VZV that does not have a homolog in SVV (13).VZV and HSV establish lifelong latent infections in sensory ganglia in humans. Unlike latency in HSV infection, in which only the latency-associated transcript RNA is expressed, multiple viral transcripts have been detected during VZV latency (7,8,18,20,24). While many studies have evaluated which HSV genes are dispensable for latency, it is unknown whether specific VZV genes are required for the virus to establish a latent infection. Here we show that VZV ORF2 is dispensable for viral replication in cell culture and for establishment of latent infection in an animal model. VZV ORF2 encodes a 31-kDa phosphoprotein. RNA prepared from VZV-infected cells revealed a 900-base transcript in infected cells using a probe complementary to ORF2 (data not shown). To verify that the ORF2 protein is expressed in VZV-infected cells, rabbit antibody to a maltose binding protein-ORF2 fusion protein containing amino acids 28 to 238 of ORF2 was produced. Immunoprecipitation of [35 S]methionine-labeled cells using antiserum to ORF2 showed a 31-kDa protein in VZV-infected cells (Fig. 1A).While ORF2 is predicted to encode a 26-kDa protein on the basis of its amino acid sequence, the larger size of the protein in virus-infected...

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confidence: 99%

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confidence: 99%

Varicella-Zoster Virus Open Reading Frame 2 Encodes a Membrane Phosphoprotein That Is Dispensable for Viral Replication and for Establishment of Latency

Sato

Pesnicak

Cohen

2002

“…The genome consists of two main coding regions, the unique long (U L ) and unique short (U S ) regions, each of which is flanked by internal repeat (IR) and terminal repeat (TR) sequences. Three ORFs, including ORF63/70 as well as ORF62/71 and ORF64/69, are located within the repeat sequences and are therefore duplicated within the VZV genome (7).The development of VZV cosmid systems has permitted the functional analysis of VZV gene products by deleting ORFs or introducing targeted mutations into the viral genome (6,15,29). In previous experiments, we showed that IE63 protein was essential for generating infectious virus from our VZV cosmids (42).…”

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confidence: 99%

“…The development of VZV cosmid systems has permitted the functional analysis of VZV gene products by deleting ORFs or introducing targeted mutations into the viral genome (6,15,29). In previous experiments, we showed that IE63 protein was essential for generating infectious virus from our VZV cosmids (42).…”

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confidence: 99%

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

Baiker¹,

Bagowski²,

Ito³

et al. 2004

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 U S 1.5 protein, which is expressed colinearly with ICP22 (U S 1). 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.Varicella-zoster virus (VZV), a member of the alphaherpesvirus subfamily of the Herpesviridae, causes varicella during primary infection and herpes zoster when it reactivates from latency in sensory ganglia. The VZV genome is a doublestranded DNA molecule of approximately 125 kb with open reading frames (ORFs) that are known or predicted to encode at least 70 distinct gene products (1). The genome consists of two main coding regions, the unique long (U L ) and unique short (U S ) regions, each of which is flanked by internal repeat (IR) and terminal repeat (TR) sequences. Three ORFs, including ORF63/70 as well as ORF62/71 and ORF64/69, are located within the repeat sequences and are therefore duplicated within the VZV genome (7).The development of VZV cosmid systems has permitted the functional analysis of VZV gene products by deleting ORFs or i...

“…Even more surprising, we noted that VZV Ellen had exactly the same mutation in ORF0 as the vaccine strain, while lacking other shared polymorphisms with vOka (17,20). This ORF had been overlooked in the original 1986 publication of the Dumas sequence and has not been considered a prime candidate for the attenuation phenotype.…”

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confidence: 99%

The Attenuated Genotype of Varicella-Zoster Virus Includes an ORF0 Transitional Stop Codon Mutation

Peters

Tyler

Carpenter

et al. 2012

Varicella-zoster virus (VZV) is the first of the human herpesviruses to be attenuated and subsequently approved as a live vaccine to prevent varicella and herpes zoster. Both the attenuated VZV vaccine, called vaccine Oka or vOka, and the parental strain pOka have been completely sequenced. Yet the specific determinants of attenuation are uncertain. The open reading frame (ORF) with the most single nucleotide polymorphisms (SNPs), ORF62, encodes the regulatory protein IE62, but IE62 studies have failed to define a specific SNP associated with attenuation. We have completed next-generation sequencing of the VZV Ellen genome, a strain known to be highly attenuated by its very limited replication in human skin xenografts in the SCID mouse model of VZV pathogenesis. A comparative analysis of the Ellen sequence with all other complete VZV sequences was extremely informative. In particular, an unexpected finding was a stop codon mutation in Ellen ORF0 (herpes simplex virus UL56 homolog) identical to one found in vOka, combined with the absence of polymorphisms in most Ellen ORFs that were known to be mutated in vOka. The mutated ORF0 protein was also imaged in both two dimensions and three dimensions by confocal microscopy. The probability of two VZV strains not connected by a recent common ancestor having an identical ORF0 SNP by chance would be 1 × 10 −8 , in other words, extremely unlikely. Taken together, these bioinformatics analyses strongly suggest that the stop codon ORF0 SNP is one of the determinants of the attenuation genotype of live VZV vaccines.