Molecular characterization of proteolytic processing of the Pol proteins of human foamy virus reveals novel features of the viral protease.
Spumaviruses, or foamy viruses, express a pol-specific transcript that codes for a Pol polyprotein that consists of the protease, reverse transcriptase, ribonuclease H, and the integrase domains. To delineate the proteolytic cleavage sites between the Pol subdomains, recombinant human foamy virus (HFV) Pol proteins were expressed, purified by affinity chromatography, and subjected to either HFV protease assays or autocatalytic processing. In control experiments, HFV protease-deficient mutant proteins in which the active site Asp was replaced by an Ala residue were used to rule out unspecific processing by nonviral proteases. Specific proteolytic cleavage products were isolated, and the cleavage sites were analyzed by amino acid sequencing. Peptides spanning the resulting cleavage sites were chemically synthesized and assayed with HFV protease, and the cleaved peptides were subjected to mass spectrometry. The cleavage site sequences obtained were in complete agreement with the amino-terminal sequences from amino acid sequencing of authentic cleavage products of the HFV Pol proteins. Analysis by fast-protein liquid chromatography of a short version of the active HFV protease revealed that the enzyme predominantly formed dimeric molecules.
Abteilungen Retroviral Gene Expression, Research Program Applied Tumorvirology, German Cancer Research Center, 69009 Heidelberg, Federal Republic of Germany.
K I
KI
Pfrepper
H R
HR
Rackwitz
M
M
Schnölzer
H
H
Heid
M
M
Löchelt
R M
RM
Flügel
D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015684:Gene Products, pol / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D016092:Spumavirus / Q000201:enzymology* / Q000235:genetics
D015684:Gene Products, pol; D019426:Integrases; D012194:RNA-Directed DNA Polymerase; D016282:Aspartic Acid Endopeptidases; C096363:proteinase, foamy virus