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An Adenovirus Cytocidal Function Related to the Control of a Cellular pH 4 Endonuclease Activity

Laboratoire de Virologie Moléculaire, INSERM U. 233, 2, Place de Verdun, 59045 Lille Cédex, France

An adenovirus (Ad) interserotypic recombinant (H2cyt141) between temperature-sensitive mutant H2ts111 of Ad2 and deletion mutant H5dl313 of Ad5 was isolated and characterized. It was phenotypically ts⁺, dl⁺, hr⁺ and formed large plaques (or cytocidal: cyt). It contained the right 89% of Ad5 DNA and the leftmost 11% of Ad2 DNA. Genetic recombination data suggested the cytocidal mutation lay in the transforming region E1B, confirming sequence analysis. The cytocidal effect resulted in part from the breakdown of cellular DNA. Host cell and virus DNA breakdown induced by H2cyt141 appeared cell-dependent: it occurred in HeLa, KB or BHK-21 cells, but not in CV1 or 293 cells. In human cells the cyt effect was recessive and adenovirus DNA degradation was prevented by co-infection with adenovirus wild-type (H2WT), other adenovirus serotypes or simian virus 40 (SV40). In simian cells, H2cyt141 did not inhibit SV40 DNA replication, unlike H2WT. The amount of H2cyt141 DNA integrated in human cell DNA at early stages of the lytic cycle was found to be significantly lower than for H2WT. Novobiocin inhibited viral DNA breakdown in human cells. Cellular DNA extracted from H2cyt141-infected cells exhibited a repeat band pattern in gel electrophoresis reminiscent of the nuclease digestion pattern of chromatin, with monosome-size fragments as the digestion limit. The H2cyt141-induced nucleolytic effect would therefore occur in the linker regions of cell DNA and might result from the observed stimulation (by a factor of greater than 100) of an acidic (optimum pH 4.0) endonuclease activity. The nucleolytic effect also appeared to be recessive in vitro and absent in mixed samples containing extracts from H2cyt141-infected cells plus extracts from H2WT- or mock-infected cells. The virus gene product responsible for the enhancement of the acidic endonuclease was found to function stoichiometrically and not catalytically. The cytocidal and nucleolytic effects of the viral E1B region 19K protein may be mediated by a cellular inhibitor of acidic endonuclease.

Keywords: adenovirus, E1B, cytocidal effect, endonuclease

Received 6 December 1984; accepted 14 May 1985.