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SUMMARY:In vesicular stomatitis virus inocula containing the transmissible interfering component (T) an exponential relation, a t low doses, between inoculum concentration and virus yield suggested that adsorption of one T particle was enough to exclude infective virus. This relation was not maintained a t high doses of inoculum, possibly because maximal interference required several hours between adsorption of T and virus particles. An assay method for T, based on the dose which gave 37% of the yield from T-free inocula, showed that the T content of inocula was usually related to passage history and yield of virus on undiluted passage. Treatment with immune serum did not appear to neutralize T, which sedimented in the centrifuge more slowly than did virus. T was much less rapidly inactivated at 56O and by U.V.irradiation than was the infectivity. Despite resemblances between the agent T, incomplete influenza virus and interferon, direct evidence is lacking that T is an incomplete form of the vesicular stomatitis virus or an interferon-like substance.In a previous paper (Cooper & Bellett, 1959) we described a phenomenon with vesicular stomatitis virus which resembled the interference due to 'incomplete "virus in influenza (von Magnus, 1951). It was concluded that the low yields from serial undiluted passage of virus were due to a transmissible interfering component (T). The present paper attempts a further definition of the properties of the agent T, and a comparison of T with incomplete influenza virus and influenza; interferon . METHODSThe preparation'of media, virus stocks and tissue cultures, and the assay of virus by plaque count were described previously (Cooper, 1957; Cooper, 1958a; Cooper & Bellett, 1959). Virus titres are expressed as plaque-forming units (pfu)/ml., dilutions being made in phosphate-buffered saline (PBS).Assay of the inteyfering agent T. Samples (0.5 ml.) of the virus stock to be assayed in twofold serial dilutions in the medium composed of Earle's saline + 5 mg. lactalbumin hydrolysatelml. + 2 mg. yeast extractlml. + 5 % (v/v) horse serum were added to washed confluent (20 hr.) chick embryo cell monolayers. After 1 hr. at 87" 5'ml. of 'conditioned' medium (fluid from 20 hr. monolayers) were added, and 20 hr. later the supernatant fluids were removed and plaque-assayed immediately, or after 2-4 days at -2 O O . The derivation of the content of T agent from these values is discussed below.

Mutants dl312, d1314, hrl, and hr3 with mutations in region ElA of adenovirus type 5 were defective for the induction of cell cycle abnormalities detectable by flow cytometry, cell DNA replication, thymidine kinase production, and chromosome aberrations and did not synthesize the viral DNA-binding protein (E2A) in rat cells. dl311, a leaky ElA mutant, induced cell cycle effects at high multiplicity in only one of three experiments, and synthesized the DNA-binding protein. hr7 (E1B) gave a wild-type response in all tests. d1313 was also positive in all tests, although it induced fewer polyploid cells than did wild-type virus, probably because of the leftward extension of the d1313 E1B deletion into ElA. sub315 and sub316, with mutations which also span the E1A-E1B border, synthesized DNAbinding protein, but caused no cell cycle alterations detectable by flow cytometry in rat or mouse cells. Although the participation of other viral early regions cannot be completely excluded, our results suggest that alteration of cell cycle progression is a direct effect of EtA unrelated to its control of other viral early regions, and may be the function of ElA in transformation.

Biochemical events were investigated in the G1 to S phase progression induced in quiescent rodent cells by human adenovirus type 5 (Ad5) and by serum. Thymidine kinase activity increased after infection of cells with Ad5 or addition of 10% serum. These stimulations were additive. An early viral gene was responsible for induction by Ad5, but the early mutants ts36, ts37, and ts125 induced thymidine kinase at the permissive and nonpermissive temperatures. Several differences were found between cells stimulated by serum compared with Ad5. Induction of thymidine kinase was delayed in Ad5-infected cells, insensitive to 0.01 microgram/ml actinomycin D and relatively resistant to reduced Ca2+ compared with induction by serum. Ornithine decarboxylase was induced by serum, but not by Ad5, alpha-Methylornithine had little effect on the induction of thymidine kinase by Ad5, but reduced the induction of thymidine kinase by serum, suggesting that Ad5-induced entry into S phase is uncoupled from polyamine biosynthesis. Methylglyoxal bis(guanylhydrazone), however, prevented the induction of thymidine kinase by both serum and Ad5. Adenovirus infection appears to induce cellular DNA synthesis and thymidine kinase in G1-arrested cells by a mechanism different from serum, and bypasses events in the normal G1 to S phase progression.

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