Abstract
Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products. To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared. Sequence preference was evident in positions P4 through P3' when compared to flanking sequences. Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond. The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes. Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag. In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon. The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site. Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.
Highlights
Analysis of Retroviral Protease CleavageSites Reveals Two Types of Cleavage Sites and the Structural Requirements of the P1 Amino Acid*
The larger products were present with all amino acid substitutions tested except Leu but were absent when the pol gene contained a mutation (D25I) that eliminates protease activity, suggesting they result from protease activity
Mature reverse transcriptase p51 and p64 were present with all mutations tested, many extra bands were evident. These bandswere present when the pol gene contained a mutation (D25I) that eliminated protease activity (PR-), suggesting they result from E . coli proteinase activity on the Pol precursor. These results show that substitutions that blocked processing at the mutated site reduced but did not eliminate protease activity
Summary
Selected discriminately by choosingonly sites proven by Searches of Retrouiral Protein Sequences-The sequence analysis amino-terminal sequencing of the protein products. In phosphate-buffered saline pH 7.2 (PBS) twice for 5 min, blocked The patterns of differences and similarities in the positions for 1 h in 3% gelatin inPBS,and exposed to 10 pl of anti-RT flanking the P1’ amino acid in the two types of PR cleavage monoclonal (Du Pont) or anti-PR polyclonal antibody in 10 ml of. Blots sites can be summarized as follows: P4, small amino acids in were washed three times for 5 min in PBS-Tw and reacted with a both types of sites (Pro, Ser, Thr, Gly, Ala); P3, generally. Sequence-To identify amino acid sequence determinants in amino acid, somePR cleavage site sequences contained amino the substrate that define PR specificity, adatabase containing acids other than Pro, Leu, Val, or Ala at the P1’ position. The cleavage sites were shared the general features of type 2 sites, polar
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