P. Lomonte

Genetic relationships between bovine herpesvirus 4 and the gammaherpesviruses epstein-Barr virus and herpesvirus saimiri

The overall arrangement of genes in the unique central part of the bovine herpesvirus type 4 (BHV-4) genome has been deduced by analysis of short DNA sequences. Twenty-three genes conserved in at least one of the completely sequenced herpesviruses have been identified and localized. All of these genes encoded amino acid sequences with higher similarity to proteins of the gammaherpesviruses Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simplex virus type 1 or the betaherpesvirus human cytomegalovirus. The genome organization of BHV-4 had also an overall colinearity with that of the gammaherpesviruses EBV and HVS. Furthermore, the BHV-4 genes content and arrangement were more similar to those of HVS than to those of EBV, suggesting that BHV-4 and HVS are evolutionarily more closely related to each other than either are to EBV. BHV-4 DNA sequences were generally deficient in CpG dinucleotide. This CpG deficiency is characteristic of gammaherpesvirus genomes and suggests that the BHV-4 latent genome is extensively methylated. Despite several biological features similar to those of betaherpesviruses, BHV-4 displays the molecular characteristics of the representative members of the gammaherpesvirinae subfamily.

Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination

Promyelocytic leukemia (PML) bodies (also called ND10) are dynamic nuclear structures implicated in a wide variety of cellular processes. ALT-associated PML bodies (APBs) are specialized PML bodies found exclusively in telomerase-negative tumors in which telomeres are maintained by recombination-based alternative (ALT) mechanisms. Although it has been suggested that APBs are directly implicated in telomere metabolism of ALT cells, their precise role and structure have remained elusive. Here we show that PML bodies in ALT cells associate with chromosome ends forming small, spatially well-defined clusters, containing on average 2–5 telomeres. Using an innovative approach that gently enlarges PML bodies in living cells while retaining their overall organization, we show that this physical enlargement of APBs spatially resolves the single telomeres in the cluster, but does not perturb the potential of the APB to recruit chromosome extremities. We show that telomere clustering in PML bodies is cell-cycle regulated and that unique telomeres within a cluster associate with recombination proteins. Enlargement of APBs induced the accumulation of telomere-telomere recombination intermediates visible on metaphase spreads and connecting heterologous chromosomes. The strand composition of these recombination intermediates indicated that this recombination is constrained to a narrow time window in the cell cycle following replication. These data provide strong evidence that PML bodies are not only a marker for ALT cells but play a direct role in telomere recombination, both by bringing together chromosome ends and by promoting telomere-telomere interactions between heterologous chromosomes.

Molecular biology of bovine herpesvirus type 4

Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.

Analysis of Bovine Herpesvirus 4 Genomic Regions Located Outside the Conserved Gammaherpesvirus Gene Blocks

Bovine herpesvirus 4 (BHV-4) DNA sequences located outside the gene blocks conserved among the gammaherpesviruses BHV-4, herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV) were analysed. Twelve potential open reading frames (ORFs) were found. Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or betaherpesviruses. Nevertheless, six of the ORFs were homologous in amino acid sequences to proteins encoded by HVS but apparently not to those encoded by EBV. Furthermore, the location and orientation of these six ORFs in the BHV-4 genome were similar to the corresponding ORFs in the HVS genome. No genes homologous to known cellular genes were found in the BHV-4 genome; this feature is the major difference between the BHV-4 and HVS genomes with regards to the overall gene content.

Bovine herpesvirus 4: genomic organization and relationship with two other gammaherpesviruses, Epstein-Barr virus and herpesvirus saimiri

Bovine herpesvirus 4 (BHV-4) belongs to the gammaherpesvirinae subfamily. Although the whole sequence of BHV-4 genome is not known it was possible, based on random sequencing, to assume that its genomic organization consists of genes clustered in blocks whose orientation and location in the genome are conserved within a herpesvirus subfamily. Between these blocks lie genes which are specific to either a particular virus or a virus subfamily. BHV-4 genome consists of 5 gene blocks conserved among the gammaherpesviruses and particularly within the Epstein-Barr virus (EBV) and the herpesvirus saimiri (HVS) genomes. Analysis of the regions located outside the gene blocks showed the presence of 12 open reading frames (ORFs). Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or beta-herpesviruses. Nevertheless, 5 ORFs were homologous in amino acid sequences to proteins encoded by HVS and one was similar to a protein encoded by both HVS and EBV. On the basis of the molecular data BHV-4 is more closely related to HVS than to EBV. Genes homologous to cellular genes have been described in both HVS and EBV genomes. No genes homologous to presently sequenced cellular genes were found among those found in the BHV-4 genome to date.

Location and characterization of the bovine herpesvirus type 4 thymidine kinase gene; comparison with thymidine kinase genes of other herpesviruses

SummaryThe location and nucleotide sequence of the bovine herpesvirus type 4 (BHV-4) thymidine kinase (TK) gene was determined. The coding region of the TK gene is 1335 nucleotides long and corresponds to a polypeptide of 445 amino acids. Comparison of TK amino acid sequences of BHV-4 and 16 herpesvirus TKs reveals a greater homology to those of the gammaherpesviruses EBV and specially HVS, than to those of alphaherpesviruses. The open reading frames detected in the vicinity of TK gene were homologous to the corresponding ones in other herpesviruses.

Analysis of the biochemical properties of, and complex formation between, glycoproteins H and L of the gamma2 herpesvirus bovine herpesvirus-4.

Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gamma-herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-beta-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31-35 kDa and diffusely migrating protein species ranging from 45-65 kDa. Tunicamycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31-35 kDa and the 45-65 kDa proteins were glycosylated, gp31-35 being a precursor of the 45-65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31-35 kDa (gp31-35) and 45-55 kDa (gp45-55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31-35 and gp45-55. gp110 and gp45-55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.