Molecular biological characterization of the human foamy virus reverse transcriptase and ribonuclease H domains.
Foamy viruses form a separate group of retroviruses encoding a pol protein with at least four domains based on comparative sequence alignments. The polymerase and ribonuclease H domains of the human foamy virus (HFV) pol gene were expressed in Escherichia coli either individually or in combination. The histidine-tagged HFV fusion proteins were subsequently purified to near homogeneity by affinity Ni2+ chelate column chromatography. The polymerase and RNase H activities were characterized by performing conventional DNA polymerase and ribonuclease H assays and in situ gel assays. Six purified recombinant HFV proteins were enzymatically active either individually as DNA polymerase and ribonuclease H or as combined domains. The HFV enzymatic activities were characterized with respect to cation preferences and pH optima. Western blots with antibodies against the RNase H domain, in situ reverse transcriptase (RT), and RNase H gel assays showed that in HFV-infected cells pol proteins of 120 and 80 kDa were detectable. A novel activity band of 60 kDa was found in situ RT gel assays. Recombinant RNase H protein additionally purified by fast performance liquid chromatography was capable of removing the primer for minus-strand DNA synthesis when labeled tRNA(Lys1,2) model substrates were used. Specific cleavages occurred at the phosphodiester bonds one to three nucleotides 5' of the RNA-DNA junction. The results revealed biochemical properties of the HFV pol gene products that define functional domains of the HFV pol gene that are distinct but comparable to other retroviruses.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D015751:Genes, pol / Q000235:genetics; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D011817:Rabbits; D016914:Ribonuclease H / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D016092:Spumavirus / Q000201:enzymology*; D014764:Viral Proteins / Q000096:biosynthesis
D017931:DNA Primers; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H