A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.

Two new species of antigens were detected in simian virus 40-transformed mouse cells, in addition to the large (94,000 daltons) and small (20,000 daltons) tumor antigens. These antigens were immunoprecipitated from cell extracts by using anti-T serum and not normal, nonimmune serum. One of these was a protein with a molecular weight of approximately 130,000 and was present in some but not all SV40-transformed mouse cells. The other, which we have named Tau antigen, has a molecular weight of 56,000 as estimated by electrophoresis through acrylamide gels and was found in all virus-transformed cells examined. The 13,000-daltons antigen contained about 15 methionine-tryptic peptides which were also present in the large SV40 tumor antigen as determined by ion-exchange chromatography. This strongly suggested that the protein was virus coded. The 56,000-dalton Tau antigen appeared to share only two methionine-tryptic peptides with the large species of SV40 tumor antigen, as determined by ion-exchange and paper chromatographies. Our results are compatible with a cellular origin for Tau antigen. However, our data do not exclude the possibility that this protein contains sequences specified by the virus DNA.

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Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparation of > 95% purity (specific activity z 8500 pmol . min-' . pg protease-') with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the Nor the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity ( z 5 pmol . min-l . pg protein-').The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome (AIDS) [l, 21. One of the potential targets for the inhibition of virus maturation is the virally encoded protease which is responsible for the processing of the precursor polyproteins into the necessary structural proteins and replication enzymes [3, 41. Recently, it was demonstrated that inhibitors of the HIV-1 protease arrest the maturation of HIV-I-like particles [5, 61. Structural analysis of the active HIV-1 protease has shown that it is a homodimer possessing one active site. The active site contains two sequences of AspThrGly, in common with the other known aspartic acid proteases [7, 81. Active-site mutation (Asp25) of the protease resulted in the production of non-infectious progeny virus [9].Although dimerization of the protease in the polyprotein form is clearly a prerequisite for activity [7, 81, there is no knowledge on the mechanism of activation of the protease from the Gag-Pol polyprotein and the order of the various cleavages that occur in the polyprotein. In order to study the mechanism of HIV-1 protease action in vitro, we developed a system to produce large quantities of purified HIV-1 protease. Others have expressed the protease gene (297 bp) in Escherichia coli, but with very low yield [lo]. The protease gene expressed as a fusion with the flanking truncated gag-pol region sequences resulted in the self-processing of the protease in E. coli and purification involved several successive steps resulting again in low recovery of pure protease [ll].Here we describe the expression of the HIV-1 protease gene containing a portion of the flanking pol gene in fusion with the malE gene of E. coli which codes for the maltose ...