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

Abstract

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...

A full UL13 open reading frame in Marek's disease virus (MDV) is dispensable for tumor formation and feather follicle tropism and cannot restore horizontal virus transmission of rRB-1B in vivo

“…In terms of cellular tropism, this result also shows that the rRB-1B devoid of a full UL13 ORF is capable of spreading to T cells in vivo. This result was surprising since the VZV ORF47 is required for efficient replication in T lymphocytes in vitro and for growth in human fetal lymphocytes implanted in mice with severe combined immunodeficiency (SCID) [3,28]. This obvious discrepancy suggests that there is a difference between VZV and MDV with respect to the viral determinants involved in the T-cell tropism of the two viruses in their target species.…”

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

“…VZV plaque formation can be fully preserved in cultured cells infected with VZV mutants even when virion assembly is severely defective, as we demonstrated with our VZV ORF47 kinase-null mutants (6). However, the EM analysis of melanoma cells infected with POKA and POKA⌬10 revealed no consequences of ORF10 deletion on VZV capsid formation or subsequent steps in virion maturation in vitro.…”

“…The SCIDhu mouse model, in which skin and T-cell xenografts are infected in vivo, provides a unique opportunity to evaluate VZV gene functions in human tissue microenvironments through comparative studies of intact VZV and VZV mutants (25,32). Experiments in the SCIDhu mouse model are important for determining how VZV gene products contribute to pathogenesis because VZV proteins, such as glycoprotein I, the ORF47 and ORF66 protein kinases, or defined functional domains within these proteins may be dispensable in cultured cells but remain essential for VZV replication in skin, T-cell xenografts, or both in vivo (5,6,31,33,34,49).…”

Downregulation of Varicella-Zoster Virus (VZV) Immediate-Early ORF62 Transcription by VZV ORF63 Correlates with Virus Replication In Vitro and with Latency

“…Since ORF63 encodes a tegument protein (28), its localization in the virus may be required for efficient assembly of virions. Two VZV ORF47 protein kinase mutants demonstrate similar phenotypes, with growth kinetics similar to that of parental virus in cultured cells but with reduced growth in human skin xenografts (2). Both mutants produce fewer and slightly smaller viral particles than parental virus in both melanoma cells and human skin xenografts.…”