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N W Blake

Publications and source records attributed to N W Blake.

14 recordsLinked to original sources
PubMed2000

Novel intertypic recombinants of epstein-barr virus in the chinese population.

Among 34 Epstein-Barr virus isolates from nonimmunocompromised Chinese donors, we identified three intertypic recombinants with type 1 sequences at the EBNA2 locus and type 2 sequences at some or all of the EBNA3A, -3B, and -3C loci. These appear to have arisen from independent, evolutionarily recent recombination events; such events may be commoner in nonimmunocompromised populations than hitherto imagined.

Base Sequence↗
PubMed1999

Inhibition of antigen presentation by the glycine/alanine repeat domain is not conserved in simian homologues of Epstein-Barr virus nuclear antigen 1.

Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.

Alanine↗
PubMed1998

Antigen presenting phenotype of Hodgkin Reed-Sternberg cells: analysis of the HLA class I processing pathway and the effects of interleukin-10 on epstein-barr virus-specific cytotoxic T-cell recognition.

Approximately 40% of Hodgkin's disease (HD) cases in Western countries carry Epstein-Barr virus (EBV) in the malignant Hodgkin-Reed-Sternberg (H-RS) cells. HLA class I-restricted cytotoxic T lymphocytes (CTLs) with specificity for viral antigens expressed in H-RS cells therefore have therapeutic potential. However, a prerequisite for CTL therapy is that the tumor target be capable of processing and presenting endogenously expressed antigens via the transporter associated with antigen processing (TAP)-dependent HLA class I pathway. We have assessed the antigen-presenting phenotype of H-RS cells in two ways. First, immunohistochemical analysis of 38 HD biopsies showed that H-RS cells were uniformly TAP1/TAP2-positive and expressed HLA class I in the majority (18 of 24, 75%) of EBV-positive cases compared with only 4 of 14 (29%) of EBV-negative cases. Second, using a panel of 5 H-RS cell lines, we showed that 4 of 5 could process and present EBV proteins to HLA class I-restricted EBV-specific CTL clones. Others have reported that human interleukin-10 (IL-10), which is expressed by H-RS cells in the majority of EBV-positive HD cases, can abrogate CTL recognition in some circumstances. However, IL-10 pretreatment of the H-RS lines or of the EBV-specific CTLs had no such effect in this system. These results support the possibility that EBV-specific CTLs may be used to treat virus-positive HD.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Transporter (TAP)-independent processing of a multiple membrane-spanning protein, the Epstein-Barr virus latent membrane protein 2.

Antigen presentation to CD8+ cytotoxic T lymphocytes (CTL) usually involves proteolytic cleavage of antigen in the cytosol and the delivery of epitope peptides onto major histocompatibility complex class I molecules in the endoplasmic reticulum (ER) via the heterodimeric peptide transporter TAP1/TAP2. In the few exceptional cases where TAP-independent presentation of an endogenously expressed protein has been observed, the epitope-containing domain of the protein either has naturally accessed or has been directed into the ER lumen where it is thought to become susceptible to ER proteases. Here, we describe a novel example of TAP-independent processing involving the Epstein-Barr virus (EBV) latent membrane protein LMP2, a multiple membrane-spanning protein with minimal projection into the ER. Expression of LMP2 in the TAP-T2 cell line, whether from the resident EBV genome or from a recombinant vaccinia virus vector vacc-LMP2, rendered the cells sensitive to recognition by CTL clones specific for two HLA-A2.1-restricted peptide epitopes, LMP2 329-337 or 426-434. Vacc-LMP2-mediated sensitization to lysis required expression of the antigen de novo in T2 cells and was blocked by brefeldin A. In the same experiments, two other EBV-specific CTL epitopes, one derived from LMP2 but restricted through a different HLA allele (A11), the other restricted through A2.1 but derived from a different viral protein (BMLF1), did not display TAP-independent processing. The results are discussed in relation to the unusual topology of LMP2 in the membrane and the position of the epitope peptides within that structure.

ATP-Binding Cassette Transporters↗
PubMed1996

Similarity in genome organization between Molluscum contagiosum virus (MCV) and vaccinia virus (VV): identification of MCV homologues of the VV genes for protein kinase 2, structural protein VP8, RNA polymerase 35 kDa subunit and 3beta-hydroxysteroid dehydrogenase.

Molluscum contagiosum virus (MCV) and vaccinia virus (VV) are serologically unrelated poxviruses with a disparate genome composition (MCV, 66% G+C; VV, 33% G+C). Molecular studies of MCV have been hindered by the inability to propagate the virus in cells cultured in vitro. We sequenced 7765 bp of MCV DNA cloned from four widely spaced regions throughout the MCV genome and identified a total of 11 potential open reading frames (ORF), designated CX1-11. These include MCV homologues of the VV genes encoding protein kinase 2, structural protein VP8, RNA polymerase 35 kDa subunit and 3beta-hydroxysteroid dehydrogenase. The position and orientation of the MCV ORFs was collinear to the VV genome, with the exception of the region around ORF CX11 which is inverted in the MCV genome.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Vaccinia virus serpins B13R (SPI-2) and B22R (SPI-1) encode M(r) 38.5 and 40K, intracellular polypeptides that do not affect virus virulence in a murine intranasal model.

A characterization of genes B13R (SPI-2) and B22R (SPI-1) from vaccinia virus strain Western Reserve (WR) is presented. These genes are transcribed early during infection and the predicted encoded proteins show similarity to the superfamily of serine protease inhibitors (serpins). The 5' transcriptional initiation site of each gene was mapped by primer extension experiments to 71-72 and 31 nucleotides upstream of the B13R and B22R open reading frames (ORFs), respectively. Each ORF was expressed in Escherichia coli and specific antisera were raised against the protein produced. These antisera were used to identify the B13R- and B22R-encoded proteins in vaccinia virus-infected cells as stable, intracellular, nonglycosylated proteins of M(r) 38.5K and M(r) 40K, respectively. The B22R gene product was detected in all orthopoxviruses tested including cowpox, rabbitpox, and vaccinia strains WR, Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W. In contrast, the B13R gene product had a more limited distribution and was not detected in Copenhagen, Tashkent, Lister, and Tian Tan. Viable virus deletion mutants that lacked only B13R or B22R coding sequences (delta B13R and delta B22R) and revertant viruses in which the deleted gene was restored were constructed by transient dominant selection. The growth of the deletion mutants in cell culture was indistinguishable from that of wild-type virus. Additionally the virulence of each deletion mutant was indistinguishable from wild-type and revertant viruses in a murine intranasal model.

Administration, Intranasal↗
PubMed1995

A single amino acid substitution in the capsid protein VP1 of coxsackievirus B3 (CVB3) alters plaque phenotype in Vero cells but not cardiovirulence in a mouse model.

We previously described a large plaque attenuant (p14V-1) derived from a cardiovirulent Coxsackievirus B3 (CVB3) and showed that there were no major determinants of either attenuation or plaque phenotype in the 5' nontranslated region (5'NTR). Part of the region encoding the last 124 amino acids of VP3 and the first 106 amino acids of VP1 of the attenuant was then sequenced and compared to the wild-type. Three nucleotide changes were found in the VP1 coding region: a silent single base change at nucleotide position 2467 (C to U) and a double-base change at position 2690-1 (AA to GT), which leads to a change from lysine to serine at amino acid position 80. This mutation maps to the begining of B-C loop of the three-dimensional structure of VP1 of CVB3, where a distinct surface projection is formed. Two infectious chimeric cDNA clones were constructed, based on a cardiovirulent cDNA construct. In one construct, the 5'NTR and the VP3-VP1 region were from p14V-1 and in the other, only the VP3-VP1 region was from this attenuant. Both chimeric viruses produced large plaques on Vero cell monolayers, similar to p14V-1 but larger than the prototypic cardiovirulent virus. In vivo experiments showed that both chimeric viruses induced myocarditis in a murine model, similar to wild-type virus. We conclude that mutation serine-80 in capsid protein VP1 of p14V-1 is a determinant of the large plaque phenotype but is not responsible for attenuation.

Amino Acid Sequence↗
PubMed1995

Vaccinia virus serpins B13R and B22R do not inhibit antigen presentation to class I-restricted cytotoxic T lymphocytes.

Vaccinia virus (VV) inhibits the presentation of certain epitopes from influenza virus nucleoprotein (NP), haemagglutinin (HA) and non-structural 1 (NS1) proteins to CD8+ cytotoxic T lymphocytes (CTL) by an unknown mechanism. We have investigated whether VV genes B13R and B22R, which encode proteins with amino acid similarity to serine protease inhibitors (serpins), are involved in this process. Recombinant VVs were constructed which express influenza virus proteins HA, NP or NS1 and which lack serpin gene B13R or both B13R and B22R. The lysis of cells infected with these viruses by influenza virus-specific CD8+ CTL was compared to the lysis of cells infected with viruses expressing both the influenza proteins and the serpin genes. Cytotoxicity assays showed that deletion of the VV serpin genes B13R and B22R and other genes between B13R and B24R did not increase the level of lysis, indicating that these genes are not involved in inhibition of antigen presentation of the epitopes tested.

Animals↗
PubMed1993

Attenuation of a reactivated cardiovirulent coxsackievirus B3: The 5'-nontranslated region does not contain major attenuation determinants.

To investigate the molecular basis of pathogenicity of Coxsackieviruses, a virus was reactivated by transfection from a full-length cDNA clone derived from cardiovirulent Coxsackievirus B3 (CVB3). The reactivated virus, rCVB3, was passaged serially in human dermatofibroblasts (HDF). No cytopathic effect was observed up to 12 days after inoculation with rCVB3 or early-passage virus, although disintegration of the monolayers was observed with late-passage virus (10th to 14th passages). Approximately 10% of HDF inoculated with rCVB3 were positive for viral antigens by immunofluorescence using enterovirus- or CVB3-specific monoclonal antibodies. These observations, together with the low infectivity titre of rCVB3 in HDF, suggests that HDF initially support only carrier state infection. After the 14th passage, the cardiovirulence of passaged virus (p14V) in mice was attenuated by a factor of > 10(4). Phenotypic changes of plaque size were also noticed in p14V: An attenuated variant (p14V-1) that produced larger plaques than rCVB3 in Vero cells has been plaque purified. The 5'-terminus of the genome of attenuant p14V-1 was amplified by polymerase chain reaction (PCR) and its sequence determined. Only one point mutation was found within the 5'-nontranslated region (5'NTR) at position 690 (A to U) compared to the viral RNA sequence obtained for rCVB3. An intertypic chimeric virus was reactivated from a cDNA clone after replacing the 5'-terminal 891 nucleotides of the wild-type genome with the corresponding region of the attenuant p14V-1. This chimeric virus, CB3/p14V-1/1, produced wild-type plaques in Vero cells and showed cardiovirulence similar to that of rCVB3 in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Characterization of a molluscum contagiosum virus homolog of the vaccinia virus p37K major envelope antigen.

We present the first nucleotide sequence data for molluscum contagiosum virus (MCV), an unclassified poxvirus. A 2,276-bp XhoI fragment from a near left-terminal fragment of MCV subtype I (MCVI) and a 1,920-bp XhoI fragment from the corresponding locus of MCV subtype II (MCVII) were sequenced and analyzed for open reading frames (ORFs). A large, complete ORF of 1,167 bp was present in both fragments. The putative polypeptide has a calculated molecular mass of 43 kDa (p43K protein) and was shown to have a high degree of homology to the vaccinia virus p37K major envelope antigen (40% amino acid identity and 22% conservative changes). The nucleotide content of the MCV fragments sequenced was 66% G or C. The codon usage within the gene for p43K reflected this high G + C content, with position 3 of codons being predominantly G or C (82 and 87% for MCVI and MCVII, respectively). The MCV p43K-encoding gene has motifs immediately upstream which are similar to those required for vaccinia virus late gene expression. The location and direction of transcription of the MCV p43K-encoding gene were equivalent to those of the vaccinia virus p37K gene, revealing similarity in genetic organization between MCV and vaccinia virus. Another, incomplete ORF was identified downstream of the p43K-encoding gene in both MCVI and MCVII. The sequence immediately upstream of this ORF overlapped the termination codon of the p43K-encoding gene and contained a motif which had homology to the derived consensus sequence for vaccinia virus early gene promoters.

Amino Acid Sequence↗
PubMed1989

Molluscum contagiosum virus types in genital and non-genital lesions.

The endonuclease digest patterns of viral DNA from 48 genital and 45 non-genital molluscum contagiosum virus (MCV) lesions were examined. The overall ratio of MCVI to MCVII was 3.23:1. There was no predominance of either MCV type in genital lesions. No obvious morphological differences were seen between MCVI and MCVII lesions. MCVII was not found in any patient under 15 years old.

Adolescent↗