Ovine herpesvirus 1 (OVHV-1) thymidine kinase locus sequence analysis: evidence that OVHV-1 belongs to the Macavirus genus of the Gammaherpesvirinae subfamily.

The HindIII-HincII fragment of the 5.5 kbp H11 HindIII clone of ovine herpesvirus 1 (OvHV-1) was cloned and its primary structure was determined by preparation of nested deletion subclones and their sequencing. Sequence analysis of the overlapping clones revealed that 3239 bp OvHV-1 fragment contains complete thymidine kinase (TK) gene, a partial open reading frame of ORF20 and that encoding glycoprotein H (gH). The conserved OvHV-1 TK displayed the highest similarity to homologous TK proteins encoded by members of the Macavirus genus of the Gammaherpesvirinae subfamily. These data including our previous analysis of the partial sequence of VP23 homologue might serve as further evidence that OvHV-1 should be categorized within the genus Macavirus of the Herpesviridae family.

Nucleotide sequence of the gene encoding the major capsid protein of herpesvirus of turkeys.

The gene encoding the major capsid protein (MCP) VP5 of herpesvirus of turkeys (HVT) was identified and sequenced. It has a single open reading frame of 4236 nucleotides encoding 1412 aa protein. The gene is flanked by VP23 and UL20 sequences and is localized in the unique long region (UL) within the BamHI-B fragment. Comparison of amino acid homology has shown its clear position among the alpha-herpesviruses rather than beta- or gamma-herpesviruses. The VP5 is expressed from polycistronic mRNA together with the UL20 and the VP23 genes. The 7,2 kb RNA transcript is lacking any promoter elements or polyA signal in intergenomic regions between VP5 and UL20 or VP5 and VP23 genes, respectively. Multiple alignment of known major capsid protein sequences of all herpesvirus groups revealed presence of seven highly homologous clusters suggesting-that the corresponding protein domains might play an important role in folding of MCP and assembly of herpesvirus capsid.

Characterization of RKZ isolate of ovine herpesvirus 1.

Cytopathic effect (CPE) characterized mainly by foci of rounded cells was observed in cultures of primary plexus choroideus cells from healthy lamb following cryopreservation. It was possible to transmit the infectious agent to other primary cells of ovine origin by co-cultivation with infected cells. By indirect immunofluorescence microscopy it was found that high percentage of sheep (65-80% in 3 different herds from Slovakia) are infected with this infectious agent. Electron microscopy of cells with CPE revealed the presence of herpesvirus particles. Viral DNA was isolated from infected cells using pulse-field gel electrophoresis and further used as probe in Southern blot analysis. The probe reacted specifically only with DNA from cells infected with Ovine herpesvirus 1 (OvHV-1) but not with DNA of other ruminant herpesviruses. Some of the HindIII restriction fragments of DNA of the obtained OvHV-1 isolate denominated RKZ were cloned. Part of the H9 clone was sequenced identifying a gene that encoded a polypeptide homologous to conserved herpesvirus VP23 structural protein. From comparison of the sequence of this clone with VP23 sequences of other herpesviruses it was deduced that OvHV-1 might be classified within the Rhadinovirus genus of the Gammaherpesvirinae subfamily. The sequencing of the H9 clone of DNA of RKZ isolate enabled establishment of sensitive and highly specific polymerase chain reaction (PCR) assay for detection of OvHV-1.

Herpesvirus of turkeys homologue of HSV VP16 is structurally related to varicella zoster virus trans-inducing protein encoded by ORF 10.

Expression of the immediate-early genes of alpha-herpesviruses is stimulated by a family of trans-inducing factors represented by VP16 of HSV-1 and ORF10 gene product of VZV. We have identified and determined the nucleotide sequence of the UL48 gene encoding the herpesvirus of turkeys (HVT) homologue of HSV VP16. The gene maps to the BamHI-J fragment and appears to be expressed in a form of bicistronic transcript together with UL49. The deduced amino acid sequence of the protein encoded by HVT UL48 gene shows 55% identity with MDV UL48 gene product. Like the majority of related proteins in other alpha-herpesviruses, the protein encoded by HVT UL48 gene lacks the acidic C-terminal tail, known to possess the transactivation capacity of HSV VP16. Hydrophobic cluster analysis has revealed that its predicted domain composition is closely related to the transactivator protein encoded by ORF10 of VZV. However, the putative amino-terminal activation domain of the HVT homologue of HSV VP16 does not contain a typical horseshoe-like hydrophobic cluster found in other alpha-herpesvirus homologues, suggesting either that it acts as a transactivator via a different activation domain or that its transactivation potential is diminished.

Identification and characterization of a cDNA clone derived from the Marek's disease tumour cell line RPL1 encoding a homologue of alpha-transinducing factor (VP16) of HSV-1.

We have identified and sequenced a 2.3 kb cDNA clone RPL(N.S) 6 derived from the Marek's disease virus (MDV)-transformed cell line RPL1, which contained open reading frames (ORFs) homologous to UL49 (VP22) and UL48 (VP16) of herpes simplex virus. Northern blot hybridization identified a 2.5 kb transcript corresponding to this cDNA clone in the total RNA from MSB1 lymphoblastoid cells, but not in RNA from the original RPL1 cells, most probably due to the very low level of its transcription. In vitro translation demonstrated that both MDV UL49 and UL48 can be expressed from a single mRNA.

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.