- Volume 84, Issue 11, 2003

The full complement of genes encoded by Orf virus (ORFV) is not yet known. A cDNA library was constructed using mRNA isolated 5 h post-infection from cells infected with ORFV in vitro and grown in the presence of cytosine arabinoside. Using 12 non-overlapping probes representing the entire genome of the Orf-11 strain of the virus, cDNA clones representing individual genes expressed early in infection were isolated. Thirty-eight early genes were identified, either via isolation of their cDNA from the library or via Northern blotting. Twenty-nine of the isolated cDNAs represented orthologues of other poxvirus genes or had been identified previously as genes of ORFV, whilst seven appeared unrelated to any known poxvirus gene or indeed to any known gene in the DNA databases. The sequences described in this paper constitute approximately 30 kb of the ORFV genome and contain the complete or partial sequence of 47 genes.

Ascoviruses (family Ascoviridae) are large, enveloped, double-stranded (ds)DNA viruses that attack lepidopteran larvae and pupae, and are unusual in that they are transmitted by parasitic wasps during oviposition. Previous comparisons of DNA polymerase sequences from vertebrate and invertebrate viruses suggested that ascoviruses are closely related to iridoviruses. This relationship was unexpected because these viruses differ markedly in virion symmetry, genome configuration and cellular pathology. Here we present evidence based on sequence comparisons and phylogenetic analyses of a greater range of ascovirus proteins and their homologues in other large dsDNA viruses that ascoviruses evolved from iridoviruses. Consensus trees for the major capsid protein, DNA polymerase, thymidine kinase and ATPase III from representative ascoviruses, algal viruses (family Phycodnaviridae), vertebrate and invertebrate iridoviruses (family Iridoviridae) and African swine fever virus (ASFV; family Asfarviridae) showed that ascovirus proteins clustered most closely with those of the lepidopteran iridovirus Chilo iridescent virus (CIV) (Invertebrate iridescent virus 6). Moreover, analysis of the presence or absence of homologues of an additional 50 proteins encoded in the genome of Spodoptera frugiperda ascovirus (SfAV-1a) showed that about 40 % occurred in CIV, with lower percentages encoded by the genomes of, respectively, vertebrate iridoviruses, phycodnaviruses and ASFV. The occurrence of three of these genes in SfAV-1a but not CIV was indicative of the evolutionary differentiation of ascoviruses from invertebrate iridoviruses.

The early protein P35 from the baculovirus Autographa californica nucleopolyhedrovirus is a direct inhibitor of caspases and can block apoptosis in a wide variety of systems. In addition, it has been linked to the regulation of viral gene expression, shut-down of protein synthesis in infected insect cells and malignant transformation of mouse fibroblasts. By yeast-two-hybrid screening we identified the RPB11a subunit of human RNA polymerase II as an interaction partner of P35. Specificity of the interaction was confirmed by affinity blotting. By immunocytology, P35 was in part found in the nucleus of transfected cells. Homology searches further revealed that P35 has structural similarity with RPB3, the subunit of RNA polymerase II that has been demonstrated to interact directly with RPB11a. When transfected into human colon carcinoma cells, P35 was able to enhance the activity of E-cadherin and β-actin promoters by about a factor of two as measured by luciferase reporter assay. P35 and hRPB11a together enhanced the E-cadherin activity about three- to fourfold. These data suggest an additional role for P35 in the regulation of cellular transcription.

Open reading frame 94 (Ha94) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is 1086 bp long and a homologue of Autographa californica multiple NPV ORF109. The gene is conserved among all baculoviruses whose genomes have been completely sequenced so far and is thus considered a baculovirus core gene. Ha94 transcripts were detected from 24 to 96 h post-infection (p.i.) of HzAM1 cells with HaSNPV. Polyclonal antiserum raised to a GST–HA94 fusion protein recognized a 43 kDa protein, HA94, in infected cell lysates from 36 to 96 h p.i., suggesting that Ha94 is a late gene. Western blot analysis of proteins present in budded virus and occlusion-derived virus (ODV) showed that Ha94 encodes a structural component of ODV. When ODVs were fractionated further into nucleocapsid and envelope components, Western blot analysis indicated that the encoded protein was associated with both the nucleocapsid and the envelope. In summary, data available indicated that Ha94 encodes a novel ODV-specific protein of HaSNPV, designated ODV-EC43.

Autographa californica nucleopolyhedrovirus (AcMNPV), the type member of the virus family Baculoviridae, infects pest insects and has been the subject of many studies for its development as a biopesticide. It is also the virus upon which most of the commercial baculovirus protein expression systems are based. AcMNPV infection of cultured host Spodoptera frugiperda (Sf9) cells can induce a number of alterations of host cell properties including altering the cellular cytoskeleton, an arrest of the cell cycle in G2/M, and the global shutoff of host protein translation. Additionally, several cellular transcripts have been shown to be down-regulated following AcMNPV infection. In this study, we take a differential display approach to address whether a global down-regulation of Sf9 host transcripts occurs at late times of infection. Additionally, we also use this approach to search for host mRNAs which are up-regulated at early times of infection, and may be important for facilitating baculovirus infection. From these experiments we can confirm a global down-regulation of Sf9 mRNA levels at late times of infection. We also found that up-regulation of individual host gene RNA levels at early times of infection did not occur frequently. One host transcript which was found to be transiently up-regulated as a result of AcMNPV infection was an Sf9 Hsc70 gene. Hsc70 proteins have been shown to play a vital role in the life-cycle of other large DNA viruses, which suggests that this protein is also important for baculovirus infection.

Experiments were conducted to determine if the 37 kDa protein (37K) of Soil-borne wheat mosaic virus (SBWMV) is a virus movement protein. First, evidence was obtained that indicated that 37K has the ability to move from cell to cell, similar to other virus movement proteins (MPs). Plasmids containing the GFP gene fused to the SBWMV 37K, the coat protein (CP) or the CP readthrough domain (RT) ORFs were delivered by biolistic bombardment to wheat and tobacco leaves. In wheat leaves, cell-to-cell movement of GFP–37K was observed, while GFP, GFP–CP and GFP–RT accumulated primarily in single cells. All fusion proteins accumulated in single cells in tobacco leaves. Thus, cell-to-cell movement is a specific property of 37K that occurs in SBWMV host plants. Subcellular accumulation of 37K was studied using SBWMV-infected and 37K-expressing transgenic wheat. In infected and transgenic wheat leaves, 37K accumulated in the cell wall, similar to other virus MPs, and in aggregates in the cytoplasm. Phylogenetic studies were conducted to compare the furovirus 37K proteins with members of the 30K superfamily of virus MPs. Amino acid sequences of the furovirus 37K proteins were aligned with the MPs from 43 representative viruses. The furovirus 37K proteins were found to reside in a clade that also contained the dianthovirus MPs. Combined, these data suggest that SBWMV 37K is probably a virus MP.

Sodium hydroxide (NaOH) solutions are widely used for the purification of contaminated equipment, as they are known to inactivate a variety of pathogens. However, information about their effect on agents causing transmissible spongiform encephalopathy (TSE) is sparse and contradictory. Scrapie hamster brain homogenate, containing the disease-associated form of the prion protein (PrPSc), was exposed to NaOH. Kinetics studies showed that treatment of brain homogenate with millimolar concentrations of NaOH rapidly abolished the proteinase K-resistant form of the prion protein (PrPres). NaOH treatment converted PrPSc into a protease-sensitive form, either in solution or when adsorbed to a metallic surface. If infectivity of TSEs is linked with PrPres, the results imply that inactivation of TSE occurs more efficiently than currently assumed.