Characterization of an early temperature-sensitive and cytocidal double mutant of adenovirus type 2.
Human adenovirus type 2 mutant, H2 ts 111, presented a double phenotype: temperature-sensitive (ts) for initiation and elongation of DNA synthesis, and cytocidal (cyt) by its large-plaque formation and the nucleolytic cleavage of both viral and cellular DNAs. Both characters were recessive since they were efficiently complemented by wild-type or other DNA-negative ts mutants. H2 ts 111 DNA--terminal protein complex formed at 33 degrees C and chased at 39.5 degrees C showed a decreased affinity for glass fibre filters, concurrently with the loss of protein-linked DNA ends. H2 ts 111 DNA breakdown occurring upon shift-up to 39.5 degrees C therefore appeared to start in close proximity to the genome extremities. Marker rescue experiments showed that the ts character was abolished by co-infection with plasmid recombinants containing whole or part of the E2A region, encoding for the 72K DNA-binding protein. The N-terminal domain of this 72K protein has been assigned between 0 and 200 amino acids, and is supposed to have a function in late transcription control. Since H2 ts 111 mapped between 0 and 300 residues (63.6 to 65.9 map units), its mutation was most likely located between 200 and 300 amino acids, namely in the C-terminal domain of the protein, which is involved in DNA replication. Recombination between H2 ts 111 and H5 dl 313 mutant revealed that the cyt function was localized in the E1B zone, between 3.8 and 11.3 map units. The nucleolytic and cytocidal effects were complemented by HEK 293 cells, an H5-transformed cell line expressing the left-most 12.5% of the viral genome. H2 ts 111 appeared, therefore, as a double ts-cyt mutant. The gene product rendered temperature-sensitive by the H2 ts 111 mutation was found to act stoichiometrically, and not catalytically, a result compatible with a lesion in the 72K protein. Although inactive at the non-permissive temperature, the early 72K protein was normally synthesized and stable in H2 ts 111-infected cells at 39.5 degrees C. Assymetric complementation obtained with the DNA-defective H5 ts 36 implied a certain degree of type specificity in the DNA-binding protein function.
D000260:Adenoviruses, Human / Q000235:genetics*; D003588:Cytopathogenic Effect, Viral; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005816:Genetic Complementation Test; D009154:Mutation; D011995:Recombination, Genetic; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics
D004268:DNA-Binding Proteins; D014764:Viral Proteins