Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors.

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.     

Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus

Precursor polyproteins containing translational products of the gag gene of Moloney murine leukemia virus were purified by gel electrophoresis and cleaved into large fragments by hydroxylamine, mild acid hydrolysis, or cyanogen bromide. The hydroxylamine cleavage method (specific for asparagine-glycine bonds) was adapted especially for this study. The electrophoretic mobility and antigenicity of the fragments and, in some cases, the presence or absence of [35S]methionine revealed detailed information on the structure of Pr65gag, gPr80gag, and Pr75gag (the unglycosylated variant of gPr80gag formed in vivo in the presence of tunicamycin or in vitro in a reticulocyte cell-free system). When compared with Pr65gag, gPr80gag contains 7,000 daltons of additional amino acids, presumably as, or as part of, a leader sequence at or very close to its N terminus. We present evidence that this leader may have replaced part of the p15 sequence. Furthermore, gPr80gag contains three separate carbohydrate groups. One is attached to the presumed leader sequence or to the p15 domain, and two are attached to the p30 domain. Each of the Moloney murine leukemia virus gag precursor proteins Pr65gag, gPr80gag, and Pr75gag corresponds with a read-through product into the pol gene. We designated these products Pr180gag-pol, gPr200gag-pol, and Pr190gag-pol (the unglycosylated variant of gPr200gag-pol), respectively. gPr200gag-pol contains all of the extra amino acids and carbohydrate groups present in gPr80gag and at least one carbohydrate group in its pol sequences.