One approach to identifying viral entry receptors is based on identifying cell lines that are resistant to viral entry. These resistant cell lines can be used for screening of cDNA libraries containing candidate viral receptor genes, as was done successfully for the identification of HSV-1 entry receptors (40
). In these studies, a reporting system consisting of a lac
Z gene driven by the HSV-1 immediate early ICP4 promoter (ICP4/β-Gal) was employed so that HSV-1 entry could be measured by β-Gal production. We wished to test the feasibility of this approach for identifying VZV receptors by focusing our studies on cultured nonprimate cells, most of which do not support productive VZV infection. Restriction of productive VZV infection in the rodent cell lines studied here was found to occur downstream of virus entry, similar to previous observations made with cultured rat neurons and mouse neuroblastomas (4
). Inhibition of infectivity in CHO-K1 cells was observed in the presence of PAA, and both ORF68 and gE transcripts were detected in infected CHO-K1 cells. While these observations support the conclusions that replication of the VZV genome can take place in CHO-K1 cells and that a block to productive infection may occur late in the viral life cycle, we do not presently know if the levels of genome replication in CHO-K1 cells and fully permissive cells are equivalent. Thus, it is possible that a block to productive VZV infection occurs at the level of genome replication.
In addition to the two species (mouse and hamster) used for the experiments reported here, we have observed that cultured chicken embryo fibroblasts also form blue foci after infection with ROka-lacZ (data not shown). Thus, VZV may be able to utilize an entry receptor that is conserved across many species. In this respect, it is worth noting that the M6P-binding domains of CI-MPR, which are implicated to be important for VZV entry (7
), are conserved across many species (10
). The ability of CF VZV to enter CHO-K1 cells is a significant distinction from HSV-1 and implies that VZV is able to utilize an entry receptor that is unique from those used by HSV-1. The existence of novel alphaherpesvirus entry receptors on CHO-K1 cells has also been postulated for equine herpesvirus 1 (EHV-1) (14
) and pseudorabies virus (PRV) (45
Our findings are consistent with a pathway of CF VZV entry into CHO-K1 cells that involves cell surface interactions with heparan sulfate and CI-MPR and that can proceed by a low-pH-dependent endocytic pathway. Receptors for gD do not appear to play a role in CF VZV entry. This result must be interpreted with caution because the numbers of surface-localized gD receptors in the CHO-K1-based cell lines utilized for our analyses are known to be low and to vary between lines (31
). Nevertheless, our results are consistent with the notion that unique receptors are required for CF VZV entry. It will be pertinent to determine whether VZV, like HSV-1, utilizes multiple entry receptors and/or multiple entry mechanisms to gain entry to the different cell types encountered during infection of the host. The entry of free virions into host cells is relevant for at least three steps in the most recently proposed model of primary VZV pathogenesis (32
). Infection of a new host is initiated primarily by airborne free virions released from cutaneous lesions, which invade epithelial cells in the upper respiratory tract. The virus subsequently infects T cells within tonsillar lymphoid tissues; infected T cells then enter the circulation and transport virus to the skin. Infected T cells do not undergo fusion with adjacent cells (39
), and infected T cells can produce abundant amounts of complete, enveloped virions (55
). Consequently, the initial infection of T cells and the spread of virus from T cells to other target cells are both thought to be mediated by free virions.
We have demonstrated that the ICP4/β-Gal reporter gene, combined with a sensitive chemiluminescence-based β-Gal detection assay, can monitor VZV entry into small numbers of target cells at a low MOI. The ICP4/β-Gal reporter system should therefore provide a useful tool for further investigations into VZV entry, including both entry mediated by CF virus and that mediated by CA virus. Understanding the fundamentals of CF VZV entry into relevant target cell types, such as human respiratory epithelial cells and T cells, and how this process is distinct from entry mediated by CA virus should provide insight into the pathogenesis of VZV. Entry studies may also uncover new means for blocking VZV infection of target cells and for improving the infectivity of live attenuated varicella-zoster vaccines.
This study indicates that restriction of productive VZV infection in CHO-K1 cells occurs after the initiation of virus gene expression, likely late in the viral life cycle. Even though at least one late structural protein, gE, is produced in VZV-infected CHO-K1 cells, no evidence of progeny virion production was detected. For CHO-K1 cells infected with PRV, a 10,000-fold reduction in progeny virion production was noted in comparison to that in fully permissive RK13 cells (45
). This defect could not be corrected by expression of nectin-1, prompting speculation that the restriction of PRV in CHO-K1 cells occurs during entry as well as at a step(s) downstream. The postentry block to PRV infection is believed to occur after early gene expression but has not been characterized further. In contrast, more modest decreases in progeny virion production were noted in CHO-K1 cells infected with EHV-1 (14
) and in HVEM-expressing CHO-K1 cells infected with HSV-1 (40
). Thus, CHO-K1-derived cells are generally considered to be fully permissive for both EHV-1 and HSV-1.
One striking characteristic of VZV-infected CHO-K1 cells is the strictly nuclear localization of IE62. IE62 staining in NIH 3T3 cells also appeared to be nuclear (Fig. ). Phosphorylation of IE62 by a viral protein kinase encoded by ORF66 is required in order for IE62 to relocate from the nucleus to the cytoplasm (13
). Relocation of phosphorylated IE62 to the cytoplasm enables its incorporation into the tegument of progeny virions. The nuclear confinement of IE62 and the lack of virion production that we observed in infected CHO-K1 cells bear resemblance to observations made with a POka mutant in which ORF66 production was prevented by stop codon insertion (POka66S). IE62 was observed to be strictly nuclear in POka66S-infected MeWo cells, and severe defects in progeny virion formation were also observed in POka66S-infected T cells (55
). The failure of IE62 to reach the cytoplasm may be one plausible explanation for the failure of progeny virion production in VZV-infected CHO-K1 cells. While we do not yet know whether VZV virion formation in CHO-K1 cells is directly dependent on proper IE62 localization, we speculate that restriction of VZV in CHO-K1 cells could arise from a defect in the production of ORF66 and/or its ability to phosphorylate IE62. This defect would prevent IE62 from escaping the nucleus, which in turn could impair virion assembly. In contrast to our observations with infected CHO-K1 cells, IE62 staining in rat neurons infected in vivo was observed only in the cytoplasm, not in the nucleus (18
). This pattern of IE62 staining is consistent with that observed in latently infected human neurons (22
). The dissimilarity in IE62 staining patterns observed for rodent cell lines infected in vitro versus rat neurons infected in vivo lends credence to the notion that infections established in the rat model of VZV latency are not simply abortive infections. A comprehensive study of viral gene expression and viral protein localization in a variety of cultured rodent cells may help to clarify whether rodent models of VZV latency represent truly latent infections capable of reactivation or abortive infections. Further studies to elucidate the mechanism of postentry restriction of VZV in nonprimate cells may uncover new cellular targets for antiviral intervention and lead to the development of suitable nonprimate models for studying VZV pathogenesis and latency.