The role of ORF66 protein in VZV replication and pathogenesis

Varicella-zoster virus (VZV) is a herpesvirus that causes chickenpox (varicella), becomes latent in the nervous system, and later reactivates to cause shingles (zoster). The pathogenesis of VZV involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin, probably via infection of T cells. This work demonstrates that VZV T cell infection is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine kinase encoded by ORF66 is essential for efficient replication of VZV in T cells, but does not appear to play a major role in infection of skin. 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 VZ virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity VZV to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFNgamma. 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, suggesting that the ORF66 kinase plays a unique role during infection of T cells.; As a protein kinase, ORF66 contains a recognizable conserved kinase domain. Mutational analysis of target residues within this domain demonstrated that disrupting the ATP anchor motif with an alanine substitution at residue G102 was associated with a marked decrease in infectious virus production in SCID-hu T cell xenografts and increased susceptibility of infected T cells to apoptosis, similar to pOka66S. In contrast, pOka66G102A grew to titers equivalent to pOka in vitro and had slightly reduced growth in skin, comparable to the reduction observed with pOka66S. These experiments indicate that the ORF66 protein supports T cell tropism of VZV by contributing to immune evasion and enhancing the survival of infected T cells, and that this activity depends upon its kinase motif.