Replication of human immunodeficiency virus 1 (HIV-1) involves conversion of its single-stranded RNA genome to double-stranded DNA, which is integrated into the genome of the host. This conversion is catalyzed by reverse transcriptase (RT), which possesses DNA polymerase and RNase H domains. The available crystal structures suggest that at any given time the RNA/DNA substrate interacts with only one active site of the two domains of HIV-1 RT. Unknown is whether a simultaneous interaction of the substrate with polymerase and RNase H active sites is possible. Therefore, the mechanism of the coordination of the two activities is not fully understood. We performed molecular dynamics simulations to obtain a conformation of the complex in which the unwound RNA/DNA substrate simultaneously interacts with the polymerase and RNase H active sites. When the RNA/DNA hybrid was immobilized at the polymerase active site, RNase H cleavage occurred, experimentally verifying that the substrate can simultaneously interact with both active sites. These findings demonstrate the existence of a transient conformation of the HIV-1 RT substrate complex, which is important for modulating and coordinating the enzymatic activities of HIV-1 RT.

Institute of Biochemistry and Biophysics Polish Academy of Sciences 02 106 Warsaw Poland

Institute of Biophysics Academy of Sciences of the Czech Republic 612 65 Brno Czech Republic

Laboratory of Protein Structure International Institute of Molecular and Cell Biology 02 109 Warsaw Poland

Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacky University Olomouc 77146 Olomouc Czech Republic

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Abbink T.E., Berkhout B.. HIV-1 reverse transcription initiation: a potential target for novel antivirals. Virus Res. 2008; 134:4–18. PubMed

Basu V.P., Song M., Gao L., Rigby S.T., Hanson M.N., Bambara R.A.. Strand transfer events during HIV-1 reverse transcription. Virus Res. 2008; 134:19–38. PubMed

Cote M.L., Roth M.J.. Murine leukemia virus reverse transcriptase: structural comparison with HIV-1 reverse transcriptase. Virus Res. 2008; 134:186–202. PubMed PMC

Le Grice S.F.J., Nowotny M.. Nucleic Acid Polymerases. 2014; 30, NY: Springer-Verlag Berlin Heidelberg; 189–214.

Gopalakrishnan V., Peliska J.A., Benkovic S.J.. Human immunodeficiency virus type 1 reverse transcriptase: spatial and temporal relationship between the polymerase and RNase H activities. Proc. Natl. Acad. Sci. U.S.A. 1992; 89:10763–10767. PubMed PMC

Furfine E.S., Reardon J.E.. Reverse transcriptase. RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and RNA hydrolysis activities. J Biol. Chem. 1991; 266:406–412. PubMed

Gotte M., Maier G., Gross H.J., Heumann H.. Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals. J. Biol. Chem. 1998; 273:10139–10146. PubMed

Kati W.M., Johnson K.A., Jerva L.F., Anderson K.S.. Mechanism and fidelity of HIV reverse transcriptase. J. Biol. Chem. 1992; 267:25988–25997. PubMed

DeStefano J.J., Buiser R.G., Mallaber L.M., Myers T.W., Bambara R.A., Fay P.J.. Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. J. Biol. Chem. 1991; 266:7423–7431. PubMed

Driscoll M.D., Golinelli M.P., Hughes S.H.. In vitro analysis of human immunodeficiency virus type 1 minus-strand strong-stop DNA synthesis and genomic RNA processing. J. Virol. 2001; 75:672–686. PubMed PMC

Purohit V., Balakrishnan M., Kim B., Bambara R.A.. Evidence that HIV-1 reverse transcriptase employs the DNA 3΄ end-directed primary/secondary RNase H cleavage mechanism during synthesis and strand transfer. J. Biol. Chem. 2005; 280:40534–40543. PubMed

Purohit V., Roques B.P., Kim B., Bambara R.A.. Mechanisms that prevent template inactivation by HIV-1 reverse transcriptase RNase H cleavages. J. Biol. Chem. 2007; 282:12598–12609. PubMed

Schultz S.J., Zhang M.H., Champoux J.J.. Recognition of internal cleavage sites by retroviral RNases H. J. Mol. Biol. 2004; 344:635–652. PubMed

Wisniewski M., Balakrishnan M., Palaniappan C., Fay P.J., Bambara R.A.. Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H. Proc. Natl. Acad. Sci. U.S.A. 2000; 97:11978–11983. PubMed PMC

Wisniewski M., Balakrishnan M., Palaniappan C., Fay P.J., Bambara R.A.. The sequential mechanism of HIV reverse transcriptase RNase H. J. Biol. Chem. 2000; 275:37664–37671. PubMed

Hostomsky Z., Hostomska Z., Hudson G.O., Moomaw E.W., Nodes B.R.. Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. Proc. Natl. Acad. Sci. U.S.A. 1991; 88:1148–1152. PubMed PMC

Smith J.S., Gritsman K., Roth M.J.. Contributions of DNA polymerase subdomains to the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase. J. Virol. 1994; 68:5721–5729. PubMed PMC

Huang H.F., Chopra R., Verdine G.L., Harrison S.C.. Structure of a covalently trapped catalytic complex of HIV-I reverse transcriptase: implications for drug resistance. Science. 1998; 282:1669–1675. PubMed

Lapkouski M., Tian L., Miller J.T., Le Grice S.F.J., Yang W.. Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation. Nat. Struct. Mol. Biol. 2013; 20:230–236. PubMed PMC

Das K., Martinez S.E., Bandwar R.P., Arnold E.. Structures of HIV-1 RT-RNA/DNA ternary complexes with dATP and nevirapine reveal conformational flexibility of RNA/DNA: insights into requirements for RNase H cleavage. Nucleic Acids Res. 2014; 42:8125–8137. PubMed PMC

Cornell W.D., Cieplak P., Bayly C.I., Gould I.R., Merz K.M., Ferguson D.M., Spellmeyer D.C., Fox T., Caldwell J.W., Kollman P.A.. A 2nd generation force-field for the simulation of proteins, nucleic-acids, and organic-molecules. J. Am. Chem. Soc. 1995; 117:5179–5197.

Perez A., Marchan I., Svozil D., Sponer J., Cheatham T.E., Laughton C.A., Orozco M.. Refinenement of the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers. Biophys. J. 2007; 92:3817–3829. PubMed PMC

Zgarbova M., Otyepka M., Sponer J., Mladek A., Banas P., Cheatham T.E., Jurecka P.. Refinement of the Cornell et al. nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles. J. Chem. Theory Comput. 2011; 7:2886–2902. PubMed PMC

Zgarbova M., Luque F.J., Sponer J., Cheatham T.E., Otyepka M., Jurecka P.. Toward improved description of DNA backbone: revisiting epsilon and zeta torsion force field parameters. J. Chem. Theory Comput. 2013; 9:2339–2354. PubMed PMC

Hornak V., Abel R., Okur A., Strockbine B., Roitberg A., Simmerling C.. Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins. 2006; 65:712–725. PubMed PMC

Maier J.A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K.E., Simmerling C.. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015; 11:3696–3713. PubMed PMC

Himmel D.M., Myshakina N.S., Ilina T., Van Ry A., Ho W.C., Parniak M.A., Arnold E.. Structure of a dihydroxycoumarin active-site inhibitor in complex with the RNase H domain of HIV-1 reverse transcriptase and structure-activity analysis of inhibitor analogs. J. Mol. Biol. 426:2617–2631. PubMed PMC

Krepl M., Havrila M., Stadlbauer P., Banas P., Otyepka M., Pasulka J., Stefl R., Sponer J.. Can we execute stable microsecond-scale atomistic simulations of protein-RNA complexes. J. Chem. Theory Comput. 2015; 11:1220–1243. PubMed

Nowotny M., Gaidamakov S.A., Crouch R.J., Yang W.. Crystal structures of RNase H bound to an RNA/DNA hybrid: Substrate specificity and metal-dependent catalysis. Cell. 2005; 121:1005–1016. PubMed

Nowotny M., Gaidamakov S.A., Ghirlando R., Cerritelli S.M., Crouch R.J., Yang W.. Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV reverse transcription (vol 28, pg 264, 2007). Mol. Cell. 2007; 28:513–513. PubMed

Dash C., Yi-Brunozzi H.Y., Le Grice S.F.. Two modes of HIV-1 polypurine tract cleavage are affected by introducing locked nucleic acid analogs into the (-) DNA template. J. Biol. Chem. 2004; 279:37095–37102. PubMed

Huang H., Harrison S.C., Verdine G.L.. Trapping of a catalytic HIV reverse transcriptase*template:primer complex through a disulfide bond. Chem. Biol. 2000; 7:355–364. PubMed

Banerjee A., Santos W.L., Verdine G.L.. Structure of a DNA glycosylase searching for lesions. Science. 2006; 311:1153–1157. PubMed

Rittinger K., Divita G., Goody R.S.. Human-immunodeficiency-virus reverse-transcriptase substrate-induced conformational-changes and the mechanism of inhibition by non-nucleoside inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1995; 92:8046–8049. PubMed PMC

Beilhartz G.L., Gotte M.. HIV-1 ribonuclease H: structure, catalytic mechanism and inhibitors. Viruses. 2010; 2:900–926. PubMed PMC

Li A., Li J., Johnson K.A.. HIV-1 Reverse transcriptase polymerase and RNase H active sites work simultaneously and independently. J. Biol. Chem. 2016; 291:26566–26585. PubMed PMC

Li A., Gong S., Johnson K.A.. Rate-limiting pyrophosphate release by HIV reverse transcriptase improves fidelity. J. Biol. Chem. 2016; 291:26554–26565. PubMed PMC

Sevilya Z., Loya S., Adir N., Hizi A.. 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. Nucleic Acids Res. 2003; 31:1481–1487. PubMed PMC

Roda R.H., Balakrishnan M., Hanson M.N., Wohrl B.M., Le Grice S.F.J., Roques B.P., Gorelick R.J., Bambara R.A.. Role of the reverse transcriptase, nucleocapsid protein, and template structure in the two-step transfer mechanism in retroviral recombination. J. Biol. Chem. 2003; 278:31536–31546. PubMed

Nowak E., Potrzebowski W., Konarev P.V., Rausch J.W., Bona M.K., Svergun D.I., Bujnicki J.M., Le Grice S.F., Nowotny M.. Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res. 2013; 41:3874–3887. PubMed PMC

Luo G.X., Taylor J.. Template switching by reverse transcriptase during DNA synthesis. J. Virol. 1990; 64:4321–4328. PubMed PMC

Pullen K.A., Rattray A.J., Champoux J.J.. The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse transcriptase. J. Biol. Chem. 1993; 268:6221–6227. PubMed

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