Stabilization of human immunodeficiency virus type 1 reverse transcriptase by site-directed mutagenesis.
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer containing 66 kDa p66 and 51 kDa p51 subunits. We previously showed that HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT have higher affinities for dTTP and template-primer (T/P) than Moloney murine leukemia virus RT, which is currently used for cDNA synthesis, suggesting that they might also be useful for cDNA synthesis (Konishi et al. Appl Biochem Biotechnol 2013, 169:77-87). Here, we have increased the thermostability of both HIV-1 M RT and HIV-1 O RT by site-directed mutagenesis. The Asp443 → Ala mutation, which abolishes RNase H activity, was introduced into the p66 subunits of HIV-1 M RT and HIV-1 O RT. The temperatures that reduced the initial activity by 50 % of the resulting mutants, HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51, were 44 and 52 °C, respectively, which were higher than those of wild-type HIV-1 M p66/p51 (42 °C) and HIV-1 O p66/p51 (48 °C). The highest temperature at which both HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51 exhibited cDNA synthesis activity was 68 °C, which was higher than for the wild-type enzymes (62 and 66 °C, respectively).
Kosaku
K
Nishimura
Mayu
M
Shinomura
Atsushi
A
Konishi
Kiyoshi
K
Yasukawa
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D017422:Sequence Analysis, DNA
D012367:RNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase