SPONTANEOUS OCCURRENCE OF EARLY-REGION-1A REITERATION MUTANTS OF TYPE-5-ADENOVIRUS IN PERSISTENTLY INFECTED HUMAN T-LYMPHOCYTES

Mutants of type 5 adenovirus (Ad5) with reiterated DNA sequences in th e Era region appeared in a human T-lymphocyte cell line, Molt-4, persi stently infected with H5sub304, a deletion/substitution mutant that ha s a wild-type phenotype in viral replication. Endonuclease analyses an d DNA sequencing revealed DNA reiteration in each mutant. In the four representative mutants investigated, the DNA reiterations all started within a six-base-pair consensus sequence, G(or C)CTGTG, located in th e second exon of the E1a region (at nt 1333, 1367, or 1419). There was not any DNA homology between the breakpoints in the second exon and t he inserting sequences (starting at nt 532, 710, or 792). Northern ana lyses suggested that the reiterated splicing sites of the representati ve mutants were all used in RNA splicing, and the closest donor and re cipient joints were used most frequently. These observations imply tha t during persistent infection Ad5 underwent spontaneous mutations by s equence-specific breakage and nonhomologous end-end joining recombinat ion events. These Ela reiteration mutants could be propagated in HeLa, A549, and Ks cells; they were genetically stable; and they killed CRE F cells at a strikingly high frequency. Preliminary observations tend to correlate this CREF cell killing with the accumulation of the early viral proteins and/or viral DNA in the infected cells. This degree of cell damage was not observed in Ad5wt or H5sub304 infection of CREF c ells. The observed Ela reiterations provide a model to gain insight in to understanding the evolutionary events of some, if not all, adenovir us types during many years of symbiotic, persistent relationship in hu man tonsils and adenoids and possibly other lymphoid organs. (C) 1997 Academic Press.