The 55-kDa product of early region lB (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation.

The specific contributions of human adenovirus type 5 early region 1B (E1B) proteins were examined using mutants which synthesize these products individually. In cooperation with E1A, transformation of primary baby rat kidney cells was achieved with either the 176R protein or 496R protein alone, albeit at an efficiency considerably less than that observed when both were present. These results indicate that transformation mediated by either E1B product can proceed independently, but that the processes involved are additive.

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In addition to major proteins of 19K and 55K (176 and 496 residues, 176R and 496R, respectively), early region 1B (E1B) of human adenovirus type 5 (Ad5) is predicted to encode at least three other polypeptides of 156R, 93R and 84R that share 79 amino-terminal residues with 496R. We have used a series of specific antipeptide sera to identify and partially characterize these proteins. 84R was produced in large amounts, 156R somewhat less, and 93R at very low levels. Synthesis of 176R, 496R, as well as the E2A 72K DNA-binding protein commenced shortly after that of E1A proteins in Ad5-infected KB cells. Production of 156R, 93R and 84R began somewhat later, but prior to the synthesis of the late structural protein IX and hexon. 156R, which is composed of the 79 amino-terminal and 77 carboxy-terminal amino acids of 496R, migrated on SDS-PAGE as two species which appeared to differ by their degree of phosphorylation. 156R and 496R yielded identical tryptic phosphopeptides that contained both phosphoserine and phosphothreonine, and one of these was immunoprecipitated by a serum specific for the carboxy terminus. These results suggested that Ser-490 and/or Ser-491 as well as Thr-495 are major sites of phosphorylation in these proteins.

SUMMARYCo-infection of cells with vesicular stomatitis viruses of the Indiana and New Jersey serotypes results in interference. Using specifically-labelled immunofluorescent antibodies, it was demonstrated that within any one co-infected cell, one virus serotype replicated to the relative exclusion of the other serotype. This result was further substantiated by an examination of the virus serotypes released by infectious centres co-infected with both viruses. Dominance of one serotype over the other was shown to be a function of the relative multiplicity of the two viruses. Superinfection by the second serotype at a higher multiplicity resulted in dominance by the second virus during the early period (up to I'5 h) post-infection. After this time, the minority virus was able to overcome this dominance. Dominance of the majority virus was also abolished by u.v. inactivation.Cell protein synthesis appeared to be less affected in cells infected with both serotypes than when infection was with a single serotype. INTRODUCTIONInfection of a cell culture with one virus type can, in certain circumstances, reduce the yield of a second virus type from those same cells. Examples of this heterologous interference have been studied by Marcus & Carver (I967) as well as a number of other workers (Bratt & Rubin, I968; Rott et al. I972). In contrast to the interference seen between the widely different virus groups described by these workers, interference mediated by a particular group of defective particles of vesicular stomatitis virus (VSV) is effective only against the homotypic virus strain (Huang & Wagner, I966; Crick & Brown, I973). We have previously reported that a second class of defective particles (LT particle) of the Indiana serotype of VSV can interfere not only with virus of the Indiana and closely related Cocal serotypes but can equally well interfere with the heterotypic New Jersey strain of VSV (Prevec & Kang, I97O; Prevec, t974). Heterotypic interference as mediated by LT particles does not appear to be simply a case of heterologous interference since LT particles do not interfere with two other rhabdoviruses, Piry and Chandipura (Prevec, I974).In an effort to gain some insight into the possible mechanism of heterotypic interference we re-examined the effect of co-infection of cells with non-defective (infectious) B particles of the Indiana and New Jersey serotypes. A previous study by Cooper (I958) has shown that * To whom reprint requests should be sent. Antisera and fluorescence reagents. The production and titre of the rabbit anti-NJ antiserum has been previously described (Kang & Prevec, 197o). This antiserum was conjugated with fluorescein according to the method described by Sternberger (1974). Guinea pig anti-Ind antiserum was raised in guinea pigs by 3 weekly subcutaneous injections of I ml of an equal volume of Freund's adjuvant and a suspension of ~ x ~o 9 Ind virus/ml in PBS. Two weeks after the last injection the animals were bled, and the antiserum was tested and found to be specific for Ind viru...