Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein.
Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These "selectively deleted" heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Delta13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Delta13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.
Cameron
C E
CE
Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Ghosh
M
M
Le Grice
S F
SF
Benkovic
S J
SJ
D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D017353:Gene Deletion; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016914:Ribonuclease H / Q000378:metabolism*
D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H