Approximately 80 percent of all human sera that react with antigens of HTLV-III, the etiologic agent of the acquired immune deficiency syndrome (AIDS), recognize protein bands at 66 and 51 kilodaltons. A mouse hybridoma was produced that was specific to these proteins. Repeated cloning of the hybridoma did not separate the two reactivities. The p66/p51 was purified from HTLV-III lysates by immunoaffinity chromatography and subjected to NH2-terminal Edman degradation. Single amino acid residues were obtained in 17 successive degradation cycles. The sequence determined was a perfect translation of the nucleotide sequence of a portion of the HTLV-III pol gene. The purified p66/51 had reverse transcriptase activity and the monoclonal immunoglobulin G specifically removed the enzyme activity from crude viral extract as well as purified enzyme.

Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon (retroviral ABSTRACTWe have purified from Moloney murine leukemia virus (Mo-MuLV) a protease that has the capacity of accurately cleaving the polyprotein precursor Pr65m into the mature viral structural proteins. Both the NH2-and COOHterminal amino acid sequences have been determined and aligned with the amino acid sequence deduced from the DNA sequence of Mo-MuLV by other workers. The results show that: (1) the protease is located at the 5' end of thepol gene, and the rst four amino acids are overlapped with the 3' end of the

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.

The envelope (env) and structural (gag) gene products of human T-cell leukemia (lymphotropic) virus type III were identified by immunoaffinity chromatography, immunoprecipitation, and two-dimensional oligopeptide mapping methods. The env gene specifies a glycosylated polypeptide with a molecular weight of 160,000 (gp160) that is processed to gp120 and smaller gene products. The gag gene specifies two polypeptides of 70,000 and 55,000 molecular weight (p70 and p55), both of which contain p24, the major structural protein of the mature virion. The techniques in this study can be used to define the extent of variability of the env gene product among different virus isolates and may identify the nature and patterns of the humoral immune response that lead to an immunologically protected state.

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Radiolabeled amino acid sequencing was used to characterize gp41, an antigen of HTLV-III/LAV, the virus believed to be the etiological agent of the acquired immune deficiency syndrome. This antigen is the one most commonly detected in immunoblot assays by sera of patients with AIDS or AIDS-related complex (ARC) and other individuals infected with HTLV-III/LAV. A mouse monoclonal antibody that was reactive with gp41 precipitated a 160-kilodalton protein (gp160) in addition to gp41, but did not precipitate a 120-kilodalton protein (gp120) from extracts of metabolically labeled cells producing HTLV-III. Extracts of infected cells that had been labeled with tritiated leucine or isoleucine were immunoprecipitated with the monoclonal antibody. The immunoprecipitates were fractionated by polyacrylamide gel electrophoresis and the p41 was eluted from the gel bands and subjected to amino-terminal radiolabeled amino acid sequencing by the semiautomated Edman degradation. Leucine residues occurred in cycles 7, 9, 12, 26, 33, and 34 among 40 cycles and isoleucine occurred in cycle 4 among 24 cycles analyzed. Comparison of the data with the deduced amino acid sequence of the env gene product of HTLV-III precisely placed gp41 in the COOH-terminal region of the env gene product. Gp160 is thus the primary env gene product and it is processed into gp120 and gp41.

The primary structure of the NH2-terminal region of the gag gene encoded internal membrane-associated protein p15 has been determined for both Rauscher and Moloney murine leukemia viruses. Peptides generated by endopeptidases and purified by HPLC were subjected to semi-automated Edman degradation. Dipeptides obtained with dipeptidyl carboxypeptidase were identified by gas chromatography-mass spectrometry. The amino acid sequence of the first 16-residue segment of Rauscher p15 is identical to the sequence of Moloney p15 except for a single amino acid substitution (Gly-* Asp) at position 13. Both proteins were found to have an acylated NH2 terminus. By mass spectroscopy, myristic acid [CH3(CH2)12COOH] was found to be bound through an amide linkage to the NH2-terminal glycyl residue in both pl5s. The results ofliquid chromatography show that the NH2-terminal myristyl group greatly contributes to the strong binding of these modified proteins and peptides to hydrophobic surfaces. Because p15 is known to be derived from the NH2-terminal region of a precursor polyprotein Pr659'9 by proteolytic cleavage in the assembled virus, it is suggested that myristylation in vivo takes place during the biosynthesis ofPr659'9. Preliminary data indicate that such modification ofgag precursor polyproteins may be common to mammalian retroviruses. The role of NH2-terminal myristyl acylation of Pr65"ag in virus assembly and the possibility ofsimilar NH2-terminal modifications ofgag-related fusion proteins of transforming viruses are discussed.Type C murine leukemia viruses (MuLV) are representatives of a morphologically and biochemically defined group of RNA viruses (family Retroviridae) which replicate via a DNA intermediate synthesized by the virus-encoded enzyme reverse transcriptase (for review see ref. 1). Viruses of the Retroviridae family have been associated with animal and human leukemia and other types of cancer (for review see refs. 2 and 3).The type C MuLVs are composed of a RNA and a protein inner core structure encapsulated by a lipoprotein envelope. The inner core structural proteins of type C MuLVs are synthesized as a precursor polyprotein designated Pr65'ag. In the process of virus assembly, Pr659ag is associated with the inner plasma membrane where it also forms a specific complex with viral RNA. During subsequent viral budding and maturation,

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