Proteolytic Cleavages, Post-translational Modifications, and Microheterogeneity of HIV-1ₘₙ Gag Proteins

Abstract

The Gag proteins of Human Immunodeficiency Virus Type 1 (MN isolate) were purified by reverse phase high pressure liquid chromatography. The complete amino acid sequence of each Gag protein was determined. Each protein was examined for post-translational modifications. The results show that gag precursor polyproteins are efficiently cleaved by the viral protease into six products designated p17, p24, p2, p7, pl and p6 and their order of occurrence in the precursor is p17-p24-p2-p7-pl-p6. Two amino acid sequence variants of the p17 Gag protein were observed. The major p17 form contained an isoleucine residue at position 34 and a valine residue at position 45. The minor p17 form contained a valine residue at position 34 and an isoleucine residue at position 45. Two variants of the p24 Gag protein were also observed. The major p24 form contained an alanine residue at position 86, whereas, the minor p24 form contained a valine residue at position 86. These two variants forms of the p17 and p24 Gag protein indicated that the gag proteins are derived from two closely related gag precursors. Approximately 50 to 60% of all the gag proteins are obtained from a single precursor. Approximately 20 to 30% of the total are derived from a second gag precursor. Data related to other gag precursors were also noted. Other amino acid differences observed for the p17 and p24 Gag protein are indicated in the text. The p1 protein differed from the predicted sequence at position 13 where a proline residue was observed and at position 14 where a glycine residue was observed. The other Gag proteins (p2, p6, and p7) purified were found to be identical to the nucleotide deduced amino acid sequence. A comparison of the most abundant gag precursor with the amino acid sequence deduced from the gag open reading frame of the MN provirus showed differences at a total of twelve positions. Post-translational modifications were observed on the p17 and the p24 Gag proteins. Greater than 90% of the total p17 protein was myristylated at its N-terminal. The data was consistent with two sites of phosphorylation on the p24 protein.