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

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 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 (...

The Ubiquitous Cellular Transcriptional Factor USF Targets the Varicella-Zoster Virus Open Reading Frame 10 Promoter and Determines Virulence in Human Skin Xenografts in SCIDhu Mice In Vivo

“…The resulting PCR products were inserted by triple ligation into the pT7Blue-3 SacI-EcoRI vector, which had been digested with the enzymes SacI and EcoRI, to obtain the pT7Blue-3 SacI-EcoRI plasmid with the ORF10 regulatory element Ϫ87 to Ϫ97 deleted. This fragment was cloned back into the pvFsp73 cosmid as described previously (6). Similar strategies were used to delete ϩ39 to ϩ63, Ϫ179 to ϩ63, and a six-nucleotide substitution in the USF binding site within the ORF10 regulatory element.…”

“…VZV recombinants were made as described previously (6). Briefly, two-or three-step PCRs with the pT7Blue-3 SacI-EcoRI plasmid (6) as a template were used to delete a fragment or mutate nucleotides within the ORF10 intergenic region.…”

“…Recombinant viruses, designated POKA10-pro⌬Ϫ87/Ϫ97, POKA10-⌬pro, POKA10-pro⌬ϩ39/ϩ63, and POKA10-pro⌬USF, were isolated by transfection of human MeWo cells with the mutated pvFsp73 cosmid and three intact cosmids, pvSpe14, pvPme2, and pvSpe23 (6). MeWo cells were maintained in minimum essential medium (Mediatech, Washington, DC) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA), nonessential amino acids, and antibiotics.…”

“…However, in contrast to VP16, ORF10 protein is dispensable for VZV replication in vitro and in SCIDhu T-cell xenografts in vivo, but it is critical for VZV growth in human skin in vivo. An ORF10-null mutant, POKA⌬10, exhibited deficiencies in virion formation and cellcell spread in infected skin (6), but deleting ORF10 had no consequences for VZV replication in T-cell xenografts in SCIDhu mice compared to results with wild-type POKA. In addition to equal infectious virus yields, POKA⌬10-infected T-cell xenografts showed the same pattern of T-cell depletion observed with fully virulent VZV at late time points after infection of T-cell xenografts (6).…”

“…An ORF10-null mutant, POKA⌬10, exhibited deficiencies in virion formation and cellcell spread in infected skin (6), but deleting ORF10 had no consequences for VZV replication in T-cell xenografts in SCIDhu mice compared to results with wild-type POKA. In addition to equal infectious virus yields, POKA⌬10-infected T-cell xenografts showed the same pattern of T-cell depletion observed with fully virulent VZV at late time points after infection of T-cell xenografts (6). Given this evidence of the specific importance of ORF10 for the pathogenesis of VZV infection of skin and the lack of information about the functional architecture of its promoter region and the regulation of ORF10 expression in the context of the VZV genome during replication, we undertook a detailed analysis of the ORF10 promoter and its responsiveness to VZV and cellular transactivators both in vitro and in SCIDhu skin xenografts in vivo.…”