The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is affected by the thumb subdomain of the small protein subunits.
Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227-427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT). We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244-322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.
Sevilya
Z
Z
Department of Cell Biology and Histology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel.
Loya
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Hughes
S H
SH
Hizi
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A
D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D015498:HIV-2 / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H