PubMed · 909085
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R E Gelinas, P A Myers, R J Roberts. 1977. Two sequence-specific endonucleases from Moraxella bovis.. https://doi.org/10.1016/0022-2836(77)90290-x
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The ability of five simian cell lines--BGM, LLC-MK2, MA104, Vero and BSC-1--to isolate enteroviruses and adenoviruses from faeces was assessed and compared with that of primary or secondary rhesus monkey kidney (RhMK) and HEp2 cultures. For enteroviruses, LLC-MK2 and BGM were best of the five lines, detecting 77% and 73% respectively of 88 strains isolated, but neither was as good as RhMK, which detected 88%. Both Vero and BSC-1 detected less than 50%. All cell types were good at isolating poliovirus and coxsackie B strains, although LLC-MK2 and BGM were slightly better than MA104, Vero, BSC-1 and HEp2 for the latter. Greatest variation in sensitivity occurred with coxsackie A9 and echovirus isolates, with all lines being less sensitive than RhMK. However, the combined isolation rate for BGM, LLC-MK2 and HEp2 was the same as that for RhMK and HEp2, although the strains not detected by each cell combination were different. Both Vero and BSC-1 were reasonable for detecting adenoviruses, but neither was as good as HEp2. The reasons for variations in results, and the future use for such cell lines for isolating viruses from clinical and environmental samples, are discussed.
Although maximal synthesis of HSP70 is induced early (6-12 hr) after adenovirus type 5 (Ad5) infection of HEp-2 or HeLa cells, the total amount of HSP70 appears to be increased at late times of infection (18-24 hr). Since virion structural proteins also accumulate at these times, we investigated the possible interaction between Ad5 structural proteins and HSP70 by immunoprecipitation of infected cell extracts with antibodies to either ATP-affinity-purified HSP70 or to CsCl-gradient-purified Ad5 virions. We found that HSP70 and a 62-kDa Ad-specific protein coimmunoprecipitated from infected cell extracts. Antibody which recognizes one of these two proteins does not cross-react with the other. Thus, the association between HSP70 and the 62-kDa protein appears specific. Using different antisera to specific adenovirus structural proteins, we have identified the 62-kDa protein as the Ad5 fiber protein.
The replication of adenovirus DNA requires, in addition to several host factors, three virus-encoded proteins: a DNA binding protein, the precursor of the terminal protein (pTP), and a DNA polymerase (Ad pol). Ad pol and pTP form a tight complex that is necessary for the initiation step in DNA replication. To perform mutation scanning of the adenovirus type 5 pTP and Ad pol a series of in-frame linker insertions of a 12-mer oligonucleotide d(CCCATCGATGGG) were introduced into cloned viral DNA fragments containing coding sequences of these proteins. The insertions are located at recognition sites for several blunt end-cutting restriction endonucleases. Forty different sites were mutagenized and the mutated genes were transferred to a plasmid that contains the left 42% of the adenovirus genome. They were rebuilt into the viral genome by means of in vivo recombination between plasmid DNA and digested adenovirus DNA-TP complex. The resulting viral genomes were tested for viability and rescued virus was analyzed for the presence of the inserted linker oligonucleotide. This procedure resulted in recovery of a number of viable virus mutants with insertions in the pTP or Ad pol genes, all of which are phenotypically silent. The other mutations did not allow virus production. The positions of these apparent lethal codon insertion mutations were useful to identify regions of functional importance in both proteins. It can be concluded that the precursor-specific region of pTP plays an important role in virus multiplication.