Investigations in the SCID-hu mouse model of VZV pathogenesis support the concept that primary VZV infection involves entry of infected T cells into the circulation during an initial viremic phase and that these migrating T cells carry infectious virus from sites of respiratory mucosal inoculation to the skin. Tonsil CD4+
T cells, especially activated, memory subpopulations that are programmed for tissue immune surveillance, are highly permissive for VZV infection in vitro and can transfer virus from the circulation to human skin xenografts in the SCID-hu mouse model (28
). VZV must then overcome innate immune barriers, mediated by epidermal cell production of IFN-α, in order to create the characteristic vesicular cutaneous lesions (27
). In the current experiments, we have characterized the pattern of VZV virion formation within T cells in vivo and, using comparisons of pOka and pOka66S, a pOka-derived ORF66 stop codon mutant, we have identified the importance of the ORF66 protein for VZV virion production, interference with apoptosis, and inhibition of the IFN pathway in VZV-infected T cells.
Like all herpesviruses, VZV virion production must begin with the packaging of viral DNA in precursor procapsids in the nucleus of the infected cell (46
). Our EM experiments showed that formation of VZV nucleocapsids and complete VZV virions was extensive in primary differentiated human T cells infected with pOka in vivo. Our EM analyses of skin xenografts have also shown production of authentic VZV virions in vivo, in contrast to the predominance of aberrant virions in cultured cells (6
). These observations are consistent with a role for release of intact VZV virions from T cells into skin during VZV pathogenesis and support our evidence that VZV must be released from T cells in order to infect other T cells in vivo (6
Some pOka-infected T cells also had striking intranuclear inclusions with multiple membranous layers surrounding dense collections of capsids, which were not observed in epidermal cells. Similar membrane-bound nuclear structures have been found in T cells infected in vitro with human herpesvirus 6, which is a T-lymphotropic betaherpesvirus (45
). The human herpesvirus 6 inclusions were termed “tegusomes” because the presence of partially tegumented capsids suggested that tegument acquisition might occur at these sites. However, pOka intranuclear virions did not appear to be tegumented, and many pOka-infected T cells contained virions without evidence of these structures, indicating that formation of intranuclear vesicles is not required for VZV nucleocapsid assembly in T cells infected within the intact tissue microenvironment in vivo.
Preventing ORF66 translation was associated with a dramatic reduction in VZV virulence in T-cell xenografts. The results with pOka66S were comparable to those obtained with vOka-derived ROKA66S, indicating that ORF66 protein is a critical determinant of infection of T cells by a low-passage clinical isolate as well as a tissue culture-passaged VZV strain (37
). EM experiments showed that the difference between pOka and pOka66S replication reflected a marked decrease in the intranuclear assembly of nucleocapsids in pOka66S-infected T cells. These observations indicate that ORF66 protein is required for early events in VZV replication in T cells in vivo, in addition to its role in the nuclear exclusion of IE62 late in infection (23
). The impact of preventing ORF66 translation on virion production was specific for T cells infected in vivo and was not observed in melanoma cells infected with pOka66S in vitro. When the ORF66 homologue US
3 was deleted from pseudorabies virus, virions accumulated in the perinuclear space, suggesting a defect in de-envelopment (24
). This phenomenon was not observed in T cells infected with pOka66S, indicating that the ORF66 protein was not required for nuclear egress of VZV virions, to the extent that virions were made in the absence of the ORF66 protein. This difference may be due to the absence of a corresponding phosphorylation site for ORF66 kinase in the VZV homologue of UL
34, a target of HSV US
), or because ORF66 protein is not an absolutely essential structural component in primary VZV virions.
VZV spread in skin, as well as in cultured cells, is characterized by the formation of polykaryocytes and extensive syncytia resulting from cell-cell fusion. Syncytium formation can occur even when virion assembly is severely disrupted, as shown in studies of rOka47ΔC and rOka47D-N, two VZV mutants defective in ORF47 protein kinase activity (6
). In contrast, virion assembly and release of infectious virus particles appears to be necessary for T-cell tropism, because VZV-infected T cells do not undergo fusion with adjacent cells. The defective virion assembly of rOka47ΔC and rOka47D-N mutants was associated with a complete block in replication in T cells in vivo (6
). Some VZV virion production was observed in T cells infected with pOka66S in vivo, consistent with detectable, albeit limited viral replication. Although ORF47 protein is a major VZV kinase/tegument protein, the analyses of pOka66S and vOka-derived ROKA66S demonstrate that ORF47 protein does not compensate for the absence of ORF66 protein functions in T cells. The deficiency in T-cell tropism associated with eliminating ORF66 protein appears to result from a defect in virion production specific for these target cells. In contrast to the ORF47 mutants, virion assembly was not disrupted in pOka66S-infected melanoma cells and pOka66S infection was decreased only slightly in skin xenografts. This phenotype was not detected with ROka66S, probably because vOka is already attenuated for growth in skin (37
). Thus, ORF66 protein appeared to provide a minor growth advantage in skin, perhaps because ORF47 protein function is sufficient or because an epidermal cell protein, not present in T cells, compensates for the lack of ORF66 protein.
If T-cell infection mediates VZV transfer from respiratory sites of inoculation to skin, modifying the induction of apoptosis in T cells may be useful during VZV pathogenesis. We found that T cells infected with pOka were protected from activation of caspase 3, a marker of apoptosis, compared to T cells infected with pOka66S. These data suggest that ORF66 protein may function to inhibit apoptosis in VZV-infected T cells. A number of studies have shown that deletion of HSV-1 US
3, the ORF66 homologue, results in apoptosis of HSV-infected cells (10
3 has also been shown to prevent the cleavage activation of procaspase 3 and resulting caspase cascade in response to stress-inducing stimuli, likely through a mechanism involving interactions with Bcl-2 family proteins (4
). Previous observations about HSV-1 US
3, combined with our new information about the effect of ORF66 on apoptosis in T cells, suggest that US
3-related gene products such as the ORF66 protein interfere with apoptosis. Additionally, microarray analysis of the transcriptional profiles of VZV-infected primary human T cells and fibroblasts showed that the transcription of caspase 8 was decreased in infected T cells but not in HFFs or skin (18
). Other evidence that may signify a tissue-specific antiapoptosis mechanism comes from the discovery by Hood et al. that VZV-infected human sensory neurons are resistant to apoptosis while HFFs are not (15
Like other herpesviruses, VZV has evolved mechanisms for the transient evasion of host control by innate and adaptive immune mechanisms. Previous experiments showed that ORF66 contributes to the down-regulation of MHC class I cell surface expression on VZV-infected T cells (1
). Our current experiments indicate that ORF66 protein may also help the virus to survive in infected T cells by inhibiting the signaling of the IFN pathway by IFN-γ. Such a mechanism could be important for VZV pathogenesis, since IFN-γ production by natural killer cells is an important innate immune response. VZV infection of fibroblasts inhibits IFN-γ signal transduction via the Jak/Stat pathway by causing a 10-fold reduction in Stat1α and a 90-fold reduction in Jak2 and an associated block in transcription of IFN regulatory factor 1 and class II transactivator in fibroblasts (2
). IFN-γ-induced phosphorylation of Stat1 was reduced in pOka-infected T cells, but not in T cells infected with pOka66S. Thus, ORF66 protein may function to modulate the effects of IFN-γ on VZV infection of T cells as well as contributing to MHC class I down-regulation (1
Although preventing ORF66 translation by inserting a stop codon into the pOka ORF66 gene was compatible with replication, as it was in vOka, these mutagenesis experiments demonstrated that complete or partial deletions of ORF66 did not allow recovery of infectious virus in our cosmid system. The observations with the complete deletion of ORF66, which overlaps with the gI (ORF67) promoter region, were consistent with our analyses of the gI (ORF67) promoter in the context of the VZV genome (17
). The VZV gI protein is dispensable in vitro (30
). Nevertheless, altering one of the ORF29 response elements of the gI promoter, designated 29RE4, did not allow VZV replication (13
). Of interest, neither the complete ORF66 deletion nor the 29RE4 substitution could be rescued, whereas the partial deletion of ORF66, which did not disrupt the 29RE4 region, was rescued by inserting the complete ORF66 sequence at the AvrII cloning site. These observations suggest that this region of the VZV genome has functions that are independent of expression of the ORF66 or gI proteins. In contrast, conservative substitutions in putative phosphorylation sites of the ORF66 protein at residues 48 and 331 had no effect on pOka replication in vitro or in T cells or skin in vivo.
In summary, VZV infection of T cells was associated with robust virion production and modulation of the apoptosis and IFN pathways in human T cells. Preventing ORF66 protein expression impaired the growth of the low-passage pOka virus in SCID-hu T-cell xenografts in vivo, increased the susceptibility of infected T cells to apoptosis, and reduced the capacity of the virus to interfere with induction of the interferon pathway by exposure to IFN-γ. Our working model is that VZV pathogenesis depends on the assembly and release of virions from T cells and that infected T cells must survive long enough to transport VZV from respiratory sites of inoculation to the skin (27
). ORF66 protein appears to have a unique role in virion production that is specific for T cells and also supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.