Varicella-zoster virus (VZV) ORF65 virion protein is dispensable for replication in cell culture and is phosphorylated by casein kinase II, but not by the VZV protein kinases.

The unique short region of varicella zoster virus (VZV) encodes four genes. One of these, ORF65, is predicted to encode an 11-kDa protein. Antibody to ORF65 protein immunoprecipitated a 16-kDa protein from the membrane fraction of VZV-infected cells. ORF65 protein was shown to be phosphorylated by casein kinase II. The VZV ORF47 or ORF66 protein kinases were not required for phosphorylation of ORF65. VZV with a large deletion in ORF65 was constructed and was shown to be dispensable for replication of virus in cell culture. The herpes simplex virus homolog of VZV ORF65 has been reported to be located in the nucleus of infected cells and in virions as a tegument protein, whereas the pseudorabies virus homolog is located in the Golgi apparatus of infected cells and in virions as a type II membrane protein. The ORF65 protein localized to the Golgi apparatus in virus-infected cells and was located in virions, most likely as a type II membrane protein. Thus, VZV ORF65 more closely resembles its pseudorabies virus homolog in its localization in infected cells and virions.

Nucleotide sequences that distinguish Oka vaccine from parental Oka and other varicella-zoster virus isolates.

The sequences of approximately 34 kb from the 3' end of the varicella-zoster virus (VZV) Oka vaccine strain and the previously sequenced Dumas strain were compared. Sequence differences were noted in the coding sequences of several VZV open reading frames (ORFs), including ORFs 48, 51, 52, 55, 56, 58, 59, 60, 62, 64, and 68. Tests based on differences in the ORF62 gene and in the ORF64 poly-A region successfully distinguished the Oka vaccine strain from its wild-type parent and from other Japanese and US clinical isolates. These changes remained stable after passage of the virus in humans.

Epstein-Barr virus BARF1 protein is dispensable for B-cell transformation and inhibits alpha interferon secretion from mononuclear cells.

The Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus.

The peptide recognized by HLA-A68.2-restricted, squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes is derived from a mutated elongation factor 2 gene.

The identification of naturally processed tumor peptides that can stimulate a tumor-specific, CTL response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One type of cancer in which a tumor-specific, CTL response has been observed is squamous cell carcinoma of the lung. In the system investigated here, the tumor-specific CTLs are HLA-A68.2 restricted. Immunoaffinity chromatography was used to isolate the HLA-A68.2 molecules from the tumor cell line, and peptide was eluted with acid from the HLA-A68.2 molecules and subjected to three rounds of separation by reversed phase-high performance liquid chromatography (RP-HPLC). To determine which fractions contained the peptide recognized by the tumor-specific CTLs, an aliquot of each RP-HPLC fraction was added to the autologous, B-lymphoblastoid cell line, and the cells were then tested as targets for tumor-specific CTLs. After the third round of RP-HPLC, mass spectrometry was used to sequence individual peptide candidates, and a peptide with a m/z of 497 was identified as the active peptide. Collision-activated dissociation of m/z 497 allowed identification of the peptide sequence as ETVSEQSNV. With the exception of a single amino acid difference (glutamic acid versus glutamine as the sixth position in the peptide), this peptide is identical to residues 581 to 589 of elongation factor 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the tumor cells that accounts for the difference between the two peptide sequences. Although a similar analysis did not reveal the presence of the mutation in three additional lung cell carcinomas, this does not rule out the possibility that a survey of a larger population of tumor cells would reveal the presence of the mutation at a low frequency. These results demonstrate the utility of this approach for identifying tumor-specific antigens that are the targets of a CTL response.