Molecular basis for a geographic variation of varicella-zoster virus recognized by a peptide antibody.

A live attenuated varicella vaccine, derived from a Japanese isolate, is currently being widely used to modulate disease caused by varicella-zoster virus. Differentiation of the vaccine from wild-type isolates has been and will continue to be critical in the assessment of the vaccine in the United States. This has largely relied upon identification of characteristic DNA polymorphisms in the vaccine strain in the United States. This has largely relied upon identification of characteristic DNA polymorphisms in the vaccine strain. In this report, we describe the identification of a new sequence polymorphism, located in the N-terminal coding sequence of open reading frame (ORF) 10. This variation results in the synthesis of an ORF 10 protein that is differentially recognized by antibodies to an ORF 10 synthetic peptide. The variation appears to be completely restricted to Japanese strains, including that used for the live attenuated varicella vaccine. As such, this polymorphism and the antibodies that differentially recognize it could prove highly useful in the assessment of the Japanese vaccine in the United States.

Transcriptional mapping of varicella-zoster virus regulatory proteins.

Varicella-zoster virus (VZV) expresses four proteins that influence viral transcriptional events and that also are homologous to herpes simplex virus type 1 (HSV-1) immediate-early proteins. However, their transcription and the mechanisms by which it is regulated are not yet resolved. To identify the promoter regions, a precise knowledge of the initiation and termination of the encoded RNAs is first required. In this report, we summarize the complete and precise mapping of the RNA transcripts of two of these genes--those from open reading frames 4 and 63. In addition, several elements of their promoter regulatory regions have been identified and predicted. Structural and functional studies of the regulatory sequences suggest that these two VZV genes may be regulated in a fashion different from that of their HSV-1 counterparts.

Use of polymerase chain amplification reaction for the detection of adenoviruses in ocular swab specimens.

PURPOSE: To evaluate the application of polymerase chain reaction (PCR) methodology as a potential diagnostic tool for the detection of adenovirus DNA in ocular swab samples. METHODS: Oligonucleotides derived from the adenovirus hexon gene were used to amplify a 306-base pair (bp) product by PCR. Radiolabeled oligonucleotides derived from sequences within the amplified product were used as specific probes. Specificity was determined against DNA of 13 adenovirus serotypes (types 1 to 11, inclusive, and types 19 and 37) and from nonadenoviral DNAs. Limits of detection were determined by PCR amplification of known amounts of purified adenovirus serotype 2 DNA. The assay was tested on 107 ocular swab samples and correlated to results obtained from tissue culture and a commercial immunoassay (Adenoclone). RESULTS: The 306-bp PCR product was amplified from all adenovirus serotypes tested, but not from negative control DNAs. As little as 15 fg of adenovirus type 2 DNA could be detected by PCR and ethidium bromide stain. Using a simplified sample preparation procedure, 46 of 58 adenovirus culture-positive but Adenoclone-negative swabs were positive by PCR (79% sensitivity). All (11 of 11) Adenoclone-positive clinical eye swabs tested were positive by PCR (100% sensitivity). Only 1 of 38 nonadenoviral ocular swab samples was positive by PCR (97% specificity). CONCLUSIONS: PCR appeared to be highly suitable for the diagnosis of adenovirus in ocular swabs, offering important improvements in speed over tissue culture isolation and in sensitivity over immunoassay.

Regulated nuclear localization of the varicella-zoster virus major regulatory protein, IE62.

The major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), IE62, accumulates within the nucleus of transfected and VZV-infected cells. Data are presented to show that nuclear localization of IE62 is dependent upon charged amino acids mapping to residues 677-685 of the 1310 residue protein. Furthermore, coexpression of VZV open-reading frame (ORF) 66 with IE62 results in the accumulation of cytoplasmic forms of IE62, suggesting that the ORF 66 protein can override the IE62 nuclear localization signal. Consistent with this, cytoplasmic forms of IE62 were detected in VZV-infected cells at late but not immediate early times after infection. The ORF 66 protein, a putative protein kinase, did not affect nuclear localization of other proteins, and IE62 nuclear localization was not affected by coexpression with VZV ORF 47, another putative kinase. These results suggest that IE62 nuclear functions may be specifically regulated by the VZV ORF 66 protein.

Nuclear accumulation of IE62, the varicella-zoster virus (VZV) major transcriptional regulatory protein, is inhibited by phosphorylation mediated by the VZV open reading frame 66 protein kinase.

IE62, the major transcriptional activator protein encoded by varicella-zoster virus (VZV), locates to the nucleus when expressed in transfected cells. We show here that cytoplasmic forms of IE62 accumulate in transfected and VZV-infected cells as the result of the protein kinase activity associated with VZV open reading frame 66 (ORF66). Expression of the ORF66 protein kinase but not the VZV ORF47 protein kinase impaired the ability of coexpressed IE62 to transactivate promoter-reporter constructs. IE62 that was coexpressed with the ORF66 protein accumulated predominantly in the cytoplasm, whereas the normal nuclear localization of other proteins was not affected by the ORF66 protein. In cells infected with VZV, IE62 accumulated in the cytoplasm at late times of infection, whereas in cells infected with a VZV recombinant unable to express ORF66 protein (ROka66S), IE62 was completely nuclear. Point mutations introduced into the predicted serine/threonine catalytic domain and ATP binding domain of ORF66 abrogated its ability to influence IE62 nuclear localization, indicating that the protein kinase activity was required. The region of IE62 that was targeted by ORF66 was mapped to amino acids 602 to 733. IE62 peptides containing this region were specifically phosphorylated in cells coexpressing the ORF66 protein kinase and in cells infected with wild-type VZV but were not phosphorylated in cells infected with ROka66S. We conclude that the ORF66 protein kinase phosphorylates IE62 to induce its cytoplasmic accumulation, most likely by inhibiting IE62 nuclear import.

The transcriptional regulatory proteins encoded by varicella-zoster virus open reading frames (ORFs) 4 and 63, but not ORF 61, are associated with purified virus particles.

Of the five varicella-zoster virus (VZV) open reading frames (ORFs) known to encode proteins which influence viral transcriptional events, two (ORFs 10 and 62) encode proteins associated with the tegument of virus particles, where they may function during the immediate-early events of infection. In this study, antibodies which recognize the products of the three additional VZV ORFs, ORFs 4, 61, and 63, were made and used to characterize their association with virus particles. ORF 4 encoded a 52-kDa polypeptide, and antibodies to ORF 63 reacted with polypeptides of 47 and 28 kDa. Antibodies to ORF 61 recognized heterogeneous polypeptides of 62 to 66 kDa in cells infected with a vaccinia virus recombinant expressing ORF 61 and in VZV-infected melanoma cells but reacted very weakly with polypeptides of VZV-infected human foreskin fibroblasts, suggesting that cell-specific factors were involved in ORF 61 protein accumulation. Analysis of virus particles purified from melanoma cells indicated that a 52-kDa polypeptide from ORF 4 and the 47-kDa polypeptide from ORF 63, but not any from ORF 61, were associated with virus particles. The virion proteins were likely components of the tegument, as they were not solubilized by treatment of virus with mild detergents and were completely resistant to trypsin digestion unless prior envelope solubilization was performed. The products of ORFs 4 and 63 were not found in purified VZV nucleocapsids. These results suggest that forms of the ORF 4- and ORF 63-encoded transcriptional regulatory proteins are also structural and may also have roles in the immediate-early events of infection.

Virion association of IE62, the varicella-zoster virus (VZV) major transcriptional regulatory protein, requires expression of the VZV open reading frame 66 protein kinase.

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is associated with the tegument of gradient-purified virions. Here, we show that most, if not all, of the association requires the expression of open reading frame 66 (ORF66), a protein kinase. The association of IE62 with wild-type VZV virions was confirmed using immunoelectron microscopy with IE62-specific antibodies, which reacted with virions in ultrathin sections of VZV-infected cells. Fractionated purified virions from cells infected with recombinant VZV ROka contained substantial levels of the 175-kDa virion IE62 protein and also contained the ORF66 protein. However, virions from cells infected with recombinant VZV ROka66S, in which ORF66 is disrupted, lacked not only the ORF66 protein but also most of the virion 175-kDa IE62 polypeptide. The virion-associated protein kinase activity was still present in ROka66S virions, although the 175-kDa protein substrate for the virion kinase was absent, implying that the virion protein kinase is encoded by genes other than ORF66. The very low levels of IE62 in ROka66S virions indicate that ORF66 protein mediates the redistribution of IE62 to sites of tegument assembly. IE62 was resolved into several species from VZV-infected cells which showed mobility differences between ROka and ROka66S, and a specific form of IE62 was detected in ROka virions. These results are consistent with a role for the ORF66-mediated phosphorylation of IE62 that results in cytoplasmic distribution of the regulatory protein for tegument inclusion. They support a model in which VZV tegument acquisition occurs in the cytoplasm. As such, two unusual features of VZV IE62, namely, its virion inclusion and its phosphorylation and nuclear exclusion by the ORF66 protein kinase, are functionally linked.

Transcriptional mapping of the varicella-zoster virus regulatory genes encoding open reading frames 4 and 63.

Four of the 68 varicella-zoster virus (VZV) unique open reading frames (ORFs), i.e., ORFs 4, 61, 62, and 63, encode proteins that influence viral transcription and are considered to be positional homologs of herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. In order to identify the elements that regulate transcription of VZV ORFs 4 and 63, the encoded mRNAs were mapped in detail. For ORF 4, a major 1.8-kb and a minor 3.0-kb polyadenylated [poly(A)+] RNA were identified, whereas ORF 63-specific probes recognized 1.3- and 1.9-kb poly(A)+ RNAs. Probes specific for sequences adjacent to the ORFs and mapping of the RNA 3' ends indicated that the ORF 4 RNAs were 3' coterminal, whereas the RNAs for ORF 63 represented two different termination sites. S1 nuclease mapping and primer extension analyses indicated a single transcription initiation site for ORF 4 at 38 bp upstream of the ORF start codon. For ORF 63, multiple transcriptional start sites at 87 to 95, 151 to 153, and (tentatively) 238 to 243 bp upstream of the ORF start codon were identified. TATA box motifs at good positional locations were found upstream of all mapped transcription initiation sites. However, no sequences resembling the TAATGARAT motif, which confers IE regulation upon HSV-1 IE genes, were found. The finding of the absence of this motif was supported through analyses of the regulatory sequences of ORFs 4 and 63 in transient transfection assays alongside those of ORFs 61 and 62. Sequences representing the promoters for ORFs 4, 61, and 63 were all stimulated by VZV infection but failed to be stimulated by coexpression with the HSV-1 transactivator Vmw65. In contrast, the promoter for ORF 62, which contains TAATGARAT motifs, was activated by VZV infection and coexpression with Vmw65. These results extend the transcriptional knowledge for VZV and suggest that ORFs 4 and 63 contain regulatory signals different from those of the ORF 62 and HSV-1 IE genes.