Author:

Keywords:

Anti-HIV Agents, DNA, Viral, Deoxyadenine Nucleotides, HIV Reverse Transcriptase, Models, Molecular, Nevirapine, Nucleic Acid Conformation, RNA, Viral, Reverse Transcriptase Inhibitors, Ribonuclease H, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, VIRUS REVERSE-TRANSCRIPTASE, HUMAN-IMMUNODEFICIENCY, CRYSTAL-STRUCTURE, RIBONUCLEASE-H, DNA-POLYMERASE, MOLECULAR-MECHANISMS, ANGSTROM RESOLUTION, ACTIVE-SITE, DOMAIN, SPECIFICITY, 05 Environmental Sciences, 06 Biological Sciences, 08 Information and Computing Sciences, Developmental Biology, 31 Biological sciences, 34 Chemical sciences, 41 Environmental sciences

Abstract:

In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2'-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal that conformational changes of an RNA/DNA and increased interactions with the RNase H domain, including the interaction of a 2'-OH with N474, help to position the RNA nearer to the active site. The structures and existing biochemical data suggest a nucleic acid conformation-induced mechanism for guiding cleavage of the RNA strand.