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...

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes two clinically different diseases: varicella (or chickenpox), during the primary infection, and zoster (or shingles), during virus reactivation from latency (3). The VZV genome is about 125 kb, the smallest among the human herpesviruses, and encodes at least 70 unique open reading frames (ORFs) (10,17,22). Although VZV and herpes simplex virus (HSV) types 1 and 2, the other two human alphaherpesviruses, show similarities in genome organization and share a number of homologous genes, VZV has unique mechanisms of pathogenesis that must be explained by its genetic differences from HSV. These mechanisms include T-cell tropism, which results in cell-associated viremia during primary VZV infection, and the characteristic formation of large polykaryocytes due to cell-cell fusion during skin infection (29).The VZV genome encodes nine putative glycoproteins that are known or presumed to be involved in different steps during the viral replication cycle: attachment and entry into the target cell, envelopment of the viral particles, cell-cell spread, and egress. The glycoprotein E (gE) is a 623-amino-acid (aa) typical type I membrane glycoprotein encoded by ORF68. gE is the most abundant glycoprotein expressed on the plasma membrane and in the cytoplasm of infected cells, and it is present on the virion envelope (10,20). gE is a multifunctional protein that has been shown to be involved in cell fusion and to localize to the trans-Golgi network (TGN), where the virus undergoes secondary envelopment (13,33,34,51).VZV gE moves from the endoplasmic reticulum to the Golgi membrane for its complete maturation (20). gE is recycled from the plasma membrane and targeted to the TGN through signal sequences contained in the C terminus (1, 63), which have been identified as the endocytosis motif YAGL (aa 582 to 585), and the TGN localizat...

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication.To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an "acid cluster" comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.Varicella-zoster virus (VZV) is an alphaherpesvirus with a genome of ϳ125,000 bp, encoding at least 70 unique open reading frames (ORFs) (3,8,10). Primary VZV infection causes varicella, which is characterized by cell-associated viremia and the formation of vesicular skin lesions that contain high concentrations of cell-free virions (3,19,35). It is believed that VZV is transmitted by aerosolized virions and infected cell material shed from skin and respiratory epithelium, although this process has not been tested in animal models. VZV preferentially infects memory T cells that have skin homing markers, as a mechanism for its transfer from respiratory epithelial sites of inoculation to dermal and epidermal cells (19). VZV establishes latency in sensory ganglia and causes herpes zoster upon reactivation. Thus, VZV pathogenesis requires infection of circulating lymphocytes, skin, and neural cells. The highly cell-associated nature of VZV replication in vitro and its very restricted infectivity in nonhuman species have been obstacles to understanding how viral gene products contribute to VZV replication and to the infection of specific target cells that are important for the viral life cycle in the host. Two advances have provided new opportunities to analyze the molecular mechanisms of VZV infectivity in vitro and in vivo. First, the use of VZV cosmids permits the identification of VZV genes, or regions within the coding sequence for particular viral prot...

Varicella-zoster virus (VZV) is the only human herpes virus for which a vaccine has been licensed. A clinical VZV isolate, designated the parent Oka (pOka) strain was passed in human and non-human fibroblasts to produce vaccine Oka (vOka). The pOka and vOka viruses exhibit similar infectivity in cultured cells but healthy susceptible individuals given vaccines derived from vOka rarely develop the cutaneous vesicular lesions characteristic of varicella. Inoculation of skin xenografts in the SCIDhu mouse model of VZV pathogenesis demonstrated that vOka had a reduced capacity to replicate in differentiated human epidermal cells in vivo (Moffat, J.F., Zerboni, L., Kinchington, P.R., Grose, C., Kaneshima, H., Arvin A.M., 1998a. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol. 72:965-74). In order to investigate the attenuation of vOka in skin, we made chimeric pOka and vOka recombinant viruses from VZV cosmids. Six chimeric pOka/vOka viruses were generated using cosmid sets that incorporate linear overlapping fragments of VZV DNA from cells infected with pOka or vOka. The cosmid sets consist of pOka and vOka DNA segments that have identical restriction sites. As expected, the growth kinetics and plaque morphologies of the six chimeric pOka/vOka viruses were indistinguishable in vitro. However, the chimeric viruses exhibited varying capacities to replicate when evaluated in skin xenografts in vivo. The presence of ORFs 30-55 from the pOka genome was sufficient to maintain wild-type infectivity in skin. Chimeric viruses containing different vOka components retained the attenuation phenotype, suggesting that vOka attenuation is multi-factorial and can be produced by genes from different regions of the vOka genome.

These findings suggest that children may plateau in CD4+ T-cell responses to influenza antigens with repeated exposures and that the number of exposures may play a large role in building a memory CD4+ T-cell response to influenza A, perhaps independently from age.