- Volume 71, Issue 2, 1990

The E7 open reading frame of human papillomavirus type 16 (HPV-16) encodes a protein that can immortalize primary rat cells, cooperate with the ras oncoprotein to transform low passage rat cells and transform established rodent cells to anchorage independence. The immortalizing and cooperation functions have been investigated using a series of point mutations that introduce single amino acid changes into the E7 protein in two distinct regions. Certain mutations altering amino acids conserved between the E7 protein of genital HPV types, the adenovirus E1 a protein and simian virus 40 large T antigen abolished the ability of the E7 protein to immortalize or cooperate with ras in a focus forming assay. Mutations in a consensus sequence for a casein kinase II recognition site, which is also shared by E1a and large T, reduced immortalizing activity, but did not affect the ability to cooperate with ras. Single mutations disrupting cysteine motifs, which form putative zinc-binding sites in the second region, reduced the activity of the E7 protein, whereas double mutants, in which neither of the cysteine motifs remained intact, showed no or very low activity. The activity of the mutants in immortalization and cooperation assays was essentially the same as their transforming activities in NIH 3T3 cells. This indicates that these three functions of E7 map to overlapping domains which cannot be separated by these mutations in the region of E1a/large T homology or the cysteine motifs.

Chemical cross-linking using ethylene glycol succinimi- dyl succinate (EGS) and dithiobispropionimidate (DTBP) was performed to determine the association of herpes simplex virus type 1 glycoprotein C (HSV-1 gC) with its nearest neighbours. Human embryonic lung (HEL) cells were infected with HSV-1 strain KOS, treated with EGS, lysed with Nonidet P40, immuno- precipitated with monoclonal antibodies specific for gC, and analysed by SDS-PAGE. These analyses demonstrated the presence of cross-linked complexes that migrated with an apparent M r in the range 150000 to 260000. Two-dimensional SDS-PAGE (non-reduced and then reduced) analyses of HSV-1-infected HEL cells treated with the cleavable cross-linker DTBP demonstrated that molecules that comigrated with gC were the only components of these high M r complexes. Immunoelectroblot (Western blot) analyses using polyclonal rabbit antiserum specific for gC verified that the high M r complexes contained gC. These results indicated that gC molecules may be localized in the infected cell membrane as dimers.

Human fibroblast cells infected with varicella-zoster virus (VZV) showed a slight increase in lipoprotein synthesis, with the production of two major viral lipoproteins, as detected by radioimmunoprecipitation (RIP). Three bands of M r 73000, 90000 and 97000 were identified as forms of the VZV gpl glycoprotein. All three incorporated both palmitic and myristic acid, and were shown by thin-layer chromatography to contain myristic, palmitic and stearic acids. A very strong band corresponding to 7000 M r, which may represent the product of VZV gene 49, was detected after RIP and in VZV-infected cells, and was shown to contain almost entirely myristic acid. Several minor bands were also detected. The possible functions of the lipoproteins are discussed.

The DNA of bovine adenovirus (BAV) serotype 4, a member of the subgroup 2 BA Vs, has been cloned and mapped with 11 restriction enzymes. Southern blot hybridizations probed by a clone containing about 50% of the BAV-4 genome revealed a very strong and extended DNA sequence homology amongst the members of subgroup 2, but no homology was detectable to the subgroup 1 bovine, or any of those human (HAV) and porcine adenovirus serotypes examined. These findings were strengthened by reciprocal hybridizations. When using the cloned hexon gene region of BAV-3 (subgroup 1) or the total genome of HAV-2 as probes, again no homology could be shown to the bovine subgroup 2 serotypes. The extent of DNA homology detectable between the members of bovine subgroup 1, the porcine and the human serotypes was variable, but in general less expressed than that observed within the bovine subgroup 2.

A cDNA clone prepared from hepatitis delta virus (HDV) RNA extracted from human serum was subcloned in the bacterial expression vector pPL31 to produce a fusion protein consisting of the first 98 amino acids of MS2 polymerase and of 64 amino acids from near the N-terminal region of hepatitis delta antigen (HDAg). The fusion protein was shown to be related to HDAg by a commercial sandwich immunoassay (Abbott) and immunoblotting with human anti- HDAg serum. Antiserum against the fusion protein was raised in rabbits and used to identify HDAg extracted from the serum and liver of an HDV-infected woodchuck and chimpanzee and from the serum of an HDV-infected human, by immunoblotting and immunohistology. A single, major polypeptide of 24K was detected in both serum and liver extracts, with a minor polypeptide of 26K sometimes present. Liver extracts also contained lower M r polypeptides thought to be degradation products, the major species being 22·5K. The same pattern of staining was obtained with human anti-HDAg serum. Absorption experiments with the expressed protein and cross-competition experiments with the rabbit antiserum suggest that a major immunodominant region of HDAg is present near the N-terminal end of the antigen, between positions 1561 and 1368 on the genome. Both the expressed protein and rabbit antiserum were shown to be good diagnostic reagents.

Mice were infected with influenza A virus by aerosol. Bronchoalveolar washings obtained from infected mice contained interleukin 1 (IL-1) and tumour necrosis factor (TNF) activities. IL-1 was present at day 4 postinfection but not at day 7. TNF activity was present at day 4 and day 7 post-infection. The presence of both these monokines was coincident with increased cell populations in the lungs. In vitro studies demonstrated that macrophages from non-infected mice produce IL- 1 and TNF activities in response to live influenza A virus stimulation. These results suggest that a direct interaction between virus and alveolar macrophages leads to IL-I and TNF production during the course of infection and could account for both the immune responses and the pathology that occur during influenza A virus infection.

The sequences of the P mRNA and protein of strain 18537 of antigenic subgroup B of human respiratory syncytial virus were determined by sequencing cloned cDNAs of intracellular mRNA. Comparison with the corresponding sequences of the A2 strain of subgroup A showed that there was extensive sequence identity at both the nucleotide (80% identity) and amino acid (90% identity) levels. The P proteins contained a single divergent region (52% amino acid identity) flanked by highly conserved domains (96% identity). The previously observed differences in electrophoretic mobilities between the P proteins of subgroup A strains and certain subgroup B strains could not be attributed to differences in the polypeptide Mr of the primary translation product.

The gene encoding the spike glycoprotein (S) of bovine enteric coronavirus (BECV) was cloned and its complete sequence of 4092 nucleotides was determined. This sequence contained a single long open reading frame with a coding capacity of 1363 amino acids (Mr 150747). The predicted protein had 19 N-glycosylation sites. A signal sequence comprising 17 amino acids was observed starting from the first methionine residue. A potential peptidase cleavage site was located between amino acids 763 and 767. These cleavages explain the maturation of the primary product of the S gene to SI (Mr 104692) and S2 (Mr 84175) spike structural proteins. Two amphipathic α-helices (amino acids 1007 to 1077 and 1269 to 1294) which may constitute the 12 nm stalk of the viral spike were also observed; another a-helix (amino acids 1305 to 1335) may be involved in the anchorage of the spike in the viral membrane. Comparison of this protein sequence to the described homologous mouse hepatitis (MHV) strain A59 and MHV-JHM S protein sequences led us to suggest that MHV-A59 and MHV-JHM S genes could be derived from a deletion of the BECV S gene.

A single gene is known to have a predominant influence on scrapie incubation period in mice. In crosses between strains that give a short incubation period, such as NZW mice, and those which give a long incubation period, such as I/LnJ mice, long incubation period was dominant using a Chandler scrapie agent isolate. Recently a close linkage was found between the incubation period gene and the prion protein (PrP) structural gene in I/LnJ mice crossed to NZW mice. Because this linkage suggested an important role for PrP in the pathogenesis of scrapie we sought to verify the linkage between these genes and extended the analysis to three additional mouse strains. All four of the mouse strains that we evaluated, I/LnJ, P/J, MA/MyJ, and RIIIS/J, had incubation periods longer than those of the NZW mice to which they were crossed. In addition, all four strains shared an XbaI restriction enzyme polymorphism, which suggested that all four strains might also exhibit linkage between the incubation period and the PrP structural gene. Very strong linkage between PrP and incubation period was found in I/LnJ and P/J mice crossed to NZW mice, whereas less obvious linkage was demonstrated for MA/MyJ mice crossed to NZW mice. In MA/MyJ mice genes other than PrP also had an obvious influence on incubation period. In RIIIS/J mice no linkage was shown. Although linkage between PrP and incubation period was very significant in I/LnJ and P/J mice, a few animals were identified in both crosses that represented potential recombinants in which PrP and incubation period did not segregate together. Therefore, although these phenotypes are certainly linked in I/LnJ and P/J mice, it is possible that PrP and incubation period are controlled by separate genes.

The nucleotide sequence of a naturally occurring satellite RNA (satRNA) of cucumber mosaic virus strain F (CMV-F) isolated from Petasites japonicum Miq. has been determined. F-satRNA is 338 nucleotides long and possesses a single open reading frame (ORF; residues 11 to 91) in its 5′-terminal region. Analysis of in vitro translation products of the RNA revealed that the major peptide synthesized had an M t of 2800, corresponding to the value (M r 2874) calculated from the amino acid sequence predicted from the ORF. Furthermore, a ribosome-binding fragment of the RNA in the initiation step of an in vitro translation system was found to contain the first AUG codon (residues 11 to 13). These results suggest that of the F-satRNA sequence, the coding region for the peptide can be assigned to the region from AUG to GGU (residues 11 to 91), accompanied by a termination codon UGA.

Several dsRNA segments, ranging in size from 0·8 to 19·5 kbp, have been isolated from citrus plants infected with the VT and ST strains of citrus tristeza virus (CTV) and translated, after denaturation, in a reticulocyte lysate cell-free system. Only two small dsRNA segments of 0·8 and 1·7 kbp were efficiently translated and showed a consistent pattern of products. The major translation product of the 0·8 kbp dsRNA was a 27K polypeptide, immunoprecipitated by antiserum to the CTV coat protein. The major translation product of the 1·7 kbp dsRNA segment was a 21·5K peptide. The possibility that the CTV coat protein may be translated from a subgenomic viral messenger RNA is discussed.