Construction of Varicella-Zoster Virus Recombinants from Parent Oka Cosmids and Demonstration that ORF65 Protein Is Dispensable for Infection of Human Skin and T Cells in the SCID-hu Mouse Model

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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 POKA⌬10; 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 POKA⌬10 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 POKA⌬10 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.Varicella-zoster virus (VZV), the causative agent of varicella (chicken pox) and zoster (shingles), belongs to the Varicellovirus genus within the Alphaherpesvirinae subfamily of Herpesviridae (3,17,48). VZV pathogenesis is mediated by the capacity of the virus to infect circulating T cells, skin cells, and sensory ganglia. Studies of VZV have lagged behind investigations of the other human herpesviruses because its growth in vitro is highly cell associated and yields low titers of unstable, cell-free virus. The genomic organization of VZV is closely related to that of herpes simplex virus type 1 (HSV-1), which has allowed predictions about the functions of many VZV genes (12,13,29). However, the pathogenesis of VZV and HSV-1 infections differs substantially, implying divergences in viral gene functions. Cosmids derived from VZV genomic DNA have made it possible to perform functional analysis of VZV gene products by deleting open reading frames (ORFs) or introducing targeted mutations into the VZV genome (9). We have used this approach to generate VZV recombinants from the vaccine OKA strain (VOKA) and the low-passage clinical isolate, parent OKA strain (POKA) (28, 42). The purpose of these experiments was to use cosmid mutagenesis to assess the functions of ORF10 in POKA replication in vitro and in the pathogenesis of VZV infection of human skin and T cells in vivo.VZV ORF10 encodes a protein of 410 amino acids (...

“…Four overlapping POKA cosmids, pvFsp73, pvSpe14, pvPme2, and pvSpe23, were used to delete ORF10 or to make targeted mutations within ORF10 (42) (Fig. 1A).…”

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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

Che

Zerboni

Sommer

et al. 2006

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“…ROKA47S, an ORF47 null mutant, was kindly provided by Jeffrey Cohen, National Institutes of Health (12). Recombinant viruses were isolated by the transfection of human melanoma cells with the mutated cosmid and the four intact cosmids (15,31). Melanoma cells were maintained in tissue culture medium (Mediatech, Washington, D.C.) supplemented with 10% fetal calf serum (Tissue Culture Biologicals, Tulare, Calif.), nonessential amino acids, and antibiotics.…”

Section: Methodsmentioning

confidence: 99%

Differential Requirement for Cell Fusion and Virion Formation in the Pathogenesis of Varicella-Zoster Virus Infection in Skin and T Cells

Besser¹,

Ikoma

Fabel

et al. 2004

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The protein product of varicella-zoster virus (VZV) ORF47 is a serine/threonine protein kinase and tegument component. Evaluation of two recombinants of the Oka strain, rOka47⌬C, with a C-terminal truncation of ORF47, and rOka47D-N, with a point mutation in the conserved kinase motif, showed that ORF47 kinase function was necessary for optimal VZV replication in human skin xenografts in SCID mice but not in cultured cells. We now demonstrate that rOka47⌬C and rOka47D-N mutants do not infect human T-cell xenografts. Differences in the growth of kinase-defective ORF47 mutants allowed an examination of requirements for VZV pathogenesis in skin and T cells in vivo. Although virion assembly was reduced and no virion transport to cell surfaces was observed, epidermal cell fusion persisted, and VZV polykaryocytes were generated by rOka47⌬C and rOka47D-N in skin. Virion assembly was also impaired in vitro, but VZV-induced cell fusion continued to cause syncytia in cultured cells infected with rOka47⌬C or rOka47D-N. Intracellular trafficking of envelope glycoprotein E and the ORF47 and IE62 proteins, components of the tegument, was aberrant without ORF47 kinase activity. In summary, normal VZV virion assembly appears to require ORF47 kinase function. Cell fusion was induced by ORF47 mutants in skin, and cell-cell spread occurred even though virion formation was deficient. VZV-infected T cells do not undergo cell fusion, and impaired virion assembly by ORF47 mutants was associated with a complete elimination of T-cell infectivity. These observations suggest a differential requirement for cell fusion and virion formation in the pathogenesis of VZV infection in skin and T cells.Varicella-zoster virus (VZV) is a ubiquitous human herpes virus and the causative agent of varicella (chicken pox) and zoster (shingles) (1,8). Varicella is characterized by viremia and skin lesions. VZV infects T cells, which may be a mechanism for its transport from respiratory epithelial sites of inoculation to dermal and epidermal cells (1,19,20,38). Thus, T cells as well as skin are critical targets for VZV pathogenesis. VZV establishes latency in sensory ganglia and causes zoster upon reactivation. Two major advances have provided new opportunities for understanding the molecular mechanisms of VZV pathogenesis. First, VZV cosmids permit the construction of VZV recombinant viruses with targeted genetic mutations (4,15,22). Second, the SCIDhu mouse model, in which skin and T-cell xenografts are infected in vivo, makes it possible to define the effects of genetic mutations in VZV on virulence for differentiated human cells within their unique tissue microenvironments (2, 3, 13, 24-28, 31, 34, 35, 37).VZV virion production begins with the assembly of capsids, which appear to undergo initial envelopment at the nuclear membrane and de-envelopment in the cytoplasm. Final envelopment in the trans-Golgi network (TGN) (7) is followed by virion transport to cell surfaces. Syncytium formation, a hallmark of VZV infection in cultured cells, reflects cell fu...

“…Effect of ORF4 deletion on the replication of VZV in melanoma cells. To delete ORF4 from the viral genome, we used a new VZV cosmid system derived from the parent Oka (pOka) virus (10). The complete genome of pOka is contained in four overlapping SuperCos 1 cosmid vectors (Stratagene, Inc.), designated pvFsp73(1-33128), pvSpe14(21796-61868), pvPme2 (53756-96035), and pvSpe23(94055-125123) (Fig.…”

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confidence: 99%

Requirement of Varicella-Zoster Virus Immediate-Early 4 Protein for Viral Replication

Sato

Sommer

Ito

et al. 2003

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Varicella-zoster virus (VZV) is an alphaherpesvirus that causes two diseases, chickenpox and zoster. VZVopen reading frame 4 (ORF4) encodes the immediate-early 4 (IE4) protein, which is conserved among alphaherpesvirus and has transactivation activity in transient transfections. To determine whether the ORF4 gene product is essential for viral replication, we used VZV cosmids to remove ORF4 from the VZV genome. Deleting ORF4 was incompatible with recovery of infectious virus, whereas transfections done by using repaired cosmids with ORF4 inserted at a nonnative site yielded virus. To analyze the functional domain of IE4, we introduced a mutation altering the C-terminal amino acids, KYFKC (K443S), which was designed to disrupt the dimerization of IE4 protein. Transfections with these mutant cosmids yielded no virus, indicating that this KYFKC motif was essential for IE4 function.Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus which causes varicella (chickenpox) during primary infection and herpes zoster (shingles) after its reactivation from sites of latency in sensory ganglia (1, 3). Both varicella and zoster continue to be serious public health problems (1, 11). The expression of VZV genes is regulated in a temporal fashion known as the lytic cascade. The product of open reading frame 4 (ORF4) has been shown to have immediate-early regulatory functions, like immediate-early 62 (IE62) and IE63 (4). The IE4 tegument protein is a 51-kDa phosphoprotein that transactivates genes of all three kinetic classes in transient expression systems and enhances IE62 transactivation (4). In turn, IE62, along with the cellular transcription factor USF, activates the ORF4 promoter (7). IE4 protein resembles its herpes simplex virus (HSV) homolog, ICP27, in its C-terminal residues (38% in amino acids 235 to 449) and complements ICP27 functions in this region, but it does not repair full deletions of the ICP27 gene (8, 9). The equine herpesvirus homolog is UL3 (12). Baudoux et al. have recently mapped IE4 domains in transient expression systems (2), demonstrating that the IE4 transactivating function depends on dimerization mediated by a KYFKC peptide in the C-terminal cysteine-rich region, amino acids 443 to 447. Other mutations in an arginine-rich region of the amino terminus, designated Rb, also reduced transactivation independently of dimerization and may be required for IE4 interactions with cellular proteins, including TATA-binding protein, transcription factor IIB, and the p50 and p65 subunits of NF-B (5). IE4 also has KH-like motifs that are involved in RNA binding which are found in conserved forms in alphaherpesvirus homologs and are required for HSV replication (13).The aim of this report was to examine the role of IE4 protein in viral replication by deleting or mutating ORF4 in the context of the viral genome. We showed that ORF4 is an essential gene and that the sequence encoding the C-terminal KYFKC motif in IE4 protein, which mediates dimerization, is required for VZV replication. Effect of ORF4 ...