The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarboninduced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12-dimethylbenz [a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz [c,h] an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas-namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells. treatment with the noncarcinogenic phorbol ester tumor promoter phorbol 12-myristate 13-acetate. In both models, tumor development can be easily visualized, tissue is readily accessible, and development of carcinomas is preceded by the appearance of benign papillomas. In the latter model, the initiation and promotion steps are distinct stages in the carcinogenic process and the ratio of papillomas to carcinomas is much higher than in the complete carcinogenesis model. The papillomas and carcinomas produced in both models appear similar by standard pathological criteria.The presence of a transforming Ha-ras oncogene in mouse skin papillomas and carcinomas induced by a single treatment with the polycyclic hydrocarbon DMBA and repetitive treatments with phorbol 12-myristate 13-acetate has been reported by Balmain and coworkers (6,7). However, the mechanism of activation was not determined. The critical change may not involve overexpression of the cellular protooncogene since Toftgard et al. (8) found no increase in RNA levels of Ha-ras or seven other protooncogenes in polycyclic hydrocarbon-induced skin tumors. Our initial studies, which included both RNA analysis and protein analysis using monoclonal antibodies to detect the ras gene product (to be published elsewhere), are consistent with these findings. Thus, we looked for qualitative rather than quantitative changes in ras and used the NIH 3T3 transfection system to facilitate these analyses.

The nucleotide sequence of the regions flanking the long terminal repeat of Rous-associated virus-2 has been determined. The region analyzed spans the ends of the viral genome and includes the terminus of the enm' gene, the 3' noncoding region. the 5' noncoding region. and the beginning of the gag gene. These data have been compared with sequences available from other avian retroviruses. The comparisons reveal sections which are highly conserved and others which are quite variable. Sequence homologies within the conserved regions suggest details concerning the mode of origin of the si-c-transducing viruses. Included in the variable section is a region (XSR) found only in certain strains of Rous-derived virus. Its absence from other oncogenic viruses indicates that these sequences are not required to elicit disease.

Assembly and maturation of retroviral particles requires the aggregation and controlled proteolytic cleavage of polyprotein core precursors by a precursor-encoded protease (PR). Active, mature retroviral PR is a dimer, and the accumulation of precursors at sites of assembly may facilitate subunit interaction and subsequent activation of this enzyme. In addition, it has been suggested that cellular cytoplasmic components act as inhibitors of PR activity, so that processing is delayed until the nascent virions leave this compartment and separate from the surface of host cells. To investigate the mechanisms that control PR activity during virus assembly, we studied the in vivo processing of retroviral gag precursors that contain tandemly linked PR subunits in which dimerization is concentration independent. Sequences encoding four different linked protease dimers were independently joined to the end of the Rous sarcoma virus (RSV) gag gene in a simian virus 40-based plasmid vector which expresses a myristoylated gag precursor upon transfection of COS-1 cells. Three of these plasmids produced gag precursors that were incorporated into viruslike particles and proteolytically cleaved by the dimers to mature core proteins that were indistinguishable from the processed products of wild-type gag. The amount of viral gag protein that was assembled and packaged in these transfections was inversely related to the relative proteolytic activities of the linked PR dimers. The fourth gag precursor, which contained the most active linked PR dimer, underwent rapid intracellular processing and did not form viruslike particles. In the absence of the plasma membrane targeting signal, processing of all four linked PR dimer-containing gag precursors was completed entirely within the cell. From these results, we conclude that the delay in polyprotein core precursor processing that occurs during normal virion assembly does not depend on a cytoplasmic inhibitor of PR activity. We suggest that dimer formation is not only necessary but may be sufficient for the initiation of PR-directed maturation of gag and gag-pol precursors.

The avian sarcoma and leukosis viruses (ASLV) encode a protease (PR) at the C terminus of gag which in vivo catalyzes the processing of both gag and gag-pol precursors. The studies reported here were undertaken to determine whether PR is able to cleave these polyproteins while it is still part of thegag precursor or whether the release of its N terminus to form free PR is necessary for full proteolytic activity. To address this question, we created a mutation that disrupts the PR cleavage site between the NC and PR coding regions of thegag gene. This mutation was introduced into a eukaryotic vector that expresses only the gag precursor and into an otherwise infectious clone of ASLV that carries the neo gene as a selectable marker. These constructs were * Corresponding author.

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