The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is modulated by residue 294 of the small subunit.
Reverse transcriptases (RTs) exhibit DNA polymerase and ribonuclease H (RNase H) activities. The RTs of human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2) are composed of two subunits, both sharing the same N-terminus (which encompasses the DNA polymerase domain). The smaller subunit lacks the C-terminal segment of the larger one, which contains the RNase H domain. The DNA polymerase domain of RTs resembles a right hand linked to the RNase H domain by a connection subdomain. Despite the high homology between HIV-1 and HIV-2 RTs, the RNase H activity of the latter is substantially lower than that of HIV-1 RT. The thumb subdomain of the small subunit controls the level of RNase H activity. We show here that Gln294, located in this thumb, is responsible for this difference in activity. A HIV-2 RT mutant, where Gln294 in the small subunit was replaced by a proline (present in HIV-1 RT), has an activity almost 10-fold higher than that of the wild-type RT. A comparative in vitro study of the kinetic parameters of the RNase H activity suggests that residue 294 affects the K(m) rather than the kcat value, influencing the affinity for the RNA.DNA substrate.
Sevilya
Z
Z
Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Loya
S
S
Adir
N
N
Hizi
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005973:Glutamine / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology; D015498:HIV-2 / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D005973:Glutamine; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H