Selection and Preliminary Characterization of Temperature-Sensitive Mutants of Type 5 Adenovirus

Abstract

Eight temperature-sensitive (ts) mutants that replicate normally at 32 C but poorly, if at all, at 39.5 C have been isolated from mutagenized stocks of a wildtype strain of type 5 adenovirus. Three mutagens were employed: nitrous acid, hydroxylamine, and nitrosoguanidine. Ts mutants were isolated from mutagenized viral stocks with frequencies between 0.01 and 0.1. All eight mutants had reversion frequencies of 10-5 or less. Complementation experiments in doubly infected cultures at the nonpermissive temperature separated the mutants into three nonoverlapping complementation groups. Complementation yields ranged from a 2.3to a 3,000fold increase over the sums of the yields from the two singly infected controls. Genetic recombination was also demonstrated; approximate recombination frequencies ranged from 0.1 to 15%. Preliminary biochemical and immunological characterization of the mutants indicated that: (i) the single mutant in complementation group I did not replicate its deoxyribonucleic acid (DNA) or synthesize late proteins at the nonpermissive temperature but did inhibit host DNA synthesis to 25 % of an uninfected control; (ii) the four group II mutants replicated viral DNA, shut off host DNA synthesis, synthesized penton base and fiber, but did not synthesize immunologically detectable hexon; the three mutants in complementation group III synthesized viral DNA, shut off host DNA synthesis, and made immunologically reactive capsid proteins (hexon, penton base, and fiber).

Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

“…represented the DNA-binding protein encoded by early region E2A (Levine et al, 1975;Lewis et al, 1976;Grodzicker et al, 1977) cross-linked to viral DNA. This conclusion has recently been confirmed (P.K.Chatterjee, unpublished observations) using the H5ts 125 mutant (Ensinger and Ginsberg, 1972), which induces the production of only small quantities of non-functional DNA-binding protein at non-permissive temperatures (Levine et al, 1975). Additional cross-linked species, exhibiting apparent mol.…”

“…The productive cycle of subgroup C human adenoviruses is conventionally considered to comprise the early and late phases, demarcated by the onset of viral DNA synthesis (see Tooze, 1982). Entry into the late phase, which is characterized by the synthesis of large quantities of virion components and inhibition of cellular gene expression (Flint, 1984), depends upon viral DNA replication (Feldman and Rapp, 1966;Ginsberg et al, 1967;Ensinger and Ginsberg, 1972;Wilkie et al, 1973). Nevertheless, inhibition of viral DNA synthesis subsequent to its initiation does not halt late RNA production (Carter and Ginsberg, 1976).…”

Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5

“…We have also determined the site of mutation in H5ts125. This mutant, isolated originally by Ensinger and Ginsberg (29),carries a lesion in the structural gene of DBP, which affects viral DNA replication (1,30). DBP extracted from H5tsl25-infected cells is thermolabile for binding to single-stranded DNA (31).…”

Conditional repression of the E2 transcription unit in E1-E3-deleted adenovirus vectors is correlated with a strong reduction in viral DNA replication and late gene expression in vitro

“…The E2B region provides the precursor terminal protein (80 kDa) and the viral DNA polymerase (140 kDa); the gene product of E2A is the abundant nuclear DNA-binding protein (DBP [72 kDa]) (1,19). Studies of DBP functions have been supported by temperature-sensitive mutants, such as Ad5Hts.125, that are unable to replicate viral DNA at a nonpermissive temperature (39ЊC) (10). Besides its role during viral DNA replication, there have been regulatory functions ascribed to DBP, such as repression of transcription from the E4 region (25) and transcriptional activation of the major late promoter (3).…”