T-Cell Tropism and the Role of ORF66 Protein in Pathogenesis of Varicella-Zoster Virus Infection

Abstract

The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-␥. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells. Varicella-zoster virus (VZV)is an alphaherpesvirus that causes chicken pox, or varicella, establishes lifelong latency in the sensory ganglia, and later reactivates to cause shingles, or herpes zoster. The pathogenesis of primary VZV infection is characterized by inoculation of respiratory mucosa, followed by a cell-associated viremia and a subsequent vesicular rash that develops 10 to 21 days after exposure (3). T cells appear to be a major target cell for VZV viremia. The virus infects primary human T cells in vitro and exhibits tropism for T cells in thymus/liver xenografts in the severe combined immunodeficiency (SCID)-hu mouse model in vivo (28,35,49). VZV alters cellular gene expression in T cells, as shown by downregulation of major histocompatibility (MHC) class I protein expression and microarray analysis of gene transcription (1, 18). Importantly, T cells have the capacity to transport infectious VZV through the circulation, resulting in the formation of typical cutaneous lesions in SCID-hu skin xenografts (29). The goal of these experiments was to further investigate the T-cell tropism of VZV by examining virion formation, effects on apoptotic and interferon (IFN) pathways in human T cells, and the contributions of the ORF66 protein to these processes, based on previous evidence that preventing ORF66 protein expression decreased VZV virulence in T-cell xenografts in vivo (37).The putative early gene ORF66 encodes a 47-kDa protein that localizes to both nuclei and cytoplasm of infected cells in vitro and is present in the VZV virion (22,50). Sequence analysis has revealed that ORF66 is highly homologous to serine/threonine protein kinases in other...

Histone Deacetylases 1 and 2 Are Phosphorylated at Novel Sites during Varicella-Zoster Virus Infection

“…2). The HSV-1 U S 3 kinases are required for phosphorylation of HDAC1 and HDAC2 (39)(40)(41), and VZV encodes ORF66p, an ortholog of these proteins (36,37,42,47). We demonstrate that a VZV mutant lacking ORF66p kinase activity is unable to induce hyperphosphorylation of HDAC1 and HDAC2 (Fig.…”

“…Deletion of ORF66 or abolishment of its kinase activity results in a modest impairment of growth in several cell types (11,42,43,46). We noted that the cytopathic effect and plaque size of wild-type virus were greater than those of VZV-ORF66KD.…”

“…6A, lane 17). We conclude that HDAC2 is not a directly phosphorylated target of ORF66p (11,42,43,46). The functional roles of ORF66p that are necessary to direct efficient replication in these various cell types remain undefined.…”

“…Based on their homology, it is suspected that ORF66p and U S 3 phosphorylate similar viral and cellular targets, although there are few data to substantiate this hypothesis. Both are known to share some functions, including the ability to block apoptosis of infected cells in a cell-typedependent manner (36,37,42). At present, it is unknown if ORF66p phosphorylates HDAC1 and/or HDAC2 or if it has any role in blocking histone deacetylation to enhance VZV replication.…”

Pseudorabies virus US3-mediated inhibition of apoptosis does not affect infectious virus production

“…For Alphaherpesvirinae, the largest subfamily of the family Herpesviridae, the conserved US3 serine/threonine kinase has been shown to possess antiapoptotic properties in herpes simplex virus type 1 (HSV-1) (Benetti et al, 2003;Cartier et al, 2003a, b;Jerome et al, 1999;Leopardi et al, 1997;Munger et al, 2001;Munger & Roizman, 2001;Ogg et al, 2004), HSV-2 (Asano et al, 1999(Asano et al, , 2000Hata et al, 1999;Murata et al, 2002), varicellazoster virus (Schaap et al, 2005), Marek's disease virus (Schumacher et al, 2008) and pseudorabies virus (PRV) (Deruelle et al, 2007;Geenen et al, 2005), but not in bovine herpesvirus type 1 (Takashima et al, 1999). Both PRV and HSV US3 render cells less susceptible to apoptosis induced by infection (Asano et al, 1999(Asano et al, , 2000Geenen et al, 2005;Leopardi et al, 1997), overexpression of proapoptotic proteins of the Bcl-2 family (Ogg et al, 2004), or several exogenous apoptotic stimuli (Cartier et al, 2003b;Geenen et al, 2005;Hata et al, 1999;Murata et al, 2002).…”