Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein (2004)

During human immunodeficiency virus type 1 minus-strand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3′-ends of acceptor RNA and minus-strand strong-stop DNA ((-) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3′-end of (-) SSDNA and inhibits TAR-induced self-priming, a dead-end reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (-) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro. The results were correlated with extensive enzymatic probing and mFold analysis. As the length of (-) SSDNA was decreased, self-priming increased and was highest when the DNA contained little more than TAR DNA, even if NC and acceptor were both present; in contrast, truncations within TAR DNA led to a striking reduction or elimination of self-priming. However, destabilization of TAR DNA was not sufficient for successful strand transfer: the stability of acceptor RNA was also crucial, and little or no strand transfer occurred if the RNA was highly stable. Significantly, NC may not be required for in vitro strand transfer if (-) SSDNA and acceptor RNA are small, relatively unstructured molecules with low thermodynamic stabilities. Collectively, these findings demonstrate that for efficient NC-mediated minus-strand transfer, a delicate thermodynamic balance between the RNA and DNA reactants must be maintained. During human immunodeficiency virus type 1 minus-strand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3′-ends of acceptor RNA and minus-strand strong-stop DNA ((-) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3′-end of (-) SSDNA and inhibits TAR-induced self-priming, a dead-end reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (-) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro. The results were correlated with extensive enzymatic probing and mFold analysis. As the length of (-) SSDNA was decreased, self-priming increased and was highest when the DNA contained little more than TAR DNA, even if NC and acceptor were both present; in contrast, truncations within TAR DNA led to a striking reduction or elimination of self-priming. However, destabilization of TAR DNA was not sufficient for successful strand transfer: the stability of acceptor RNA was also crucial, and little or no strand transfer occurred if the RNA was highly stable. Significantly, NC may not be required for in vitro strand transfer if (-) SSDNA and acceptor RNA are small, relatively unstructured molecules with low thermodynamic stabilities. Collectively, these findings demonstrate that for efficient NC-mediated minus-strand transfer, a delicate thermodynamic balance between the RNA and DNA reactants must be maintained. Reverse transcription consists of a complex series of reactions catalyzed by the virion-associated enzyme reverse transcriptase (RT) 1The abbreviations used are: RT, reverse transcriptase; NC, nucleocapsid protein; HIV-1, human immunodeficiency virus type 1; (-) SSDNA, minus-strand strong-stop DNA; nt, nucleotide(s); R, repeat; SP, self-priming; MNase, micrococcal nuclease; TAR, trans-activation response element.1The abbreviations used are: RT, reverse transcriptase; NC, nucleocapsid protein; HIV-1, human immunodeficiency virus type 1; (-) SSDNA, minus-strand strong-stop DNA; nt, nucleotide(s); R, repeat; SP, self-priming; MNase, micrococcal nuclease; TAR, trans-activation response element. that lead to conversion of the single-stranded RNA genome into an integration-competent linear double-stranded DNA (1Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997Google Scholar). 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Biol. 2002; 318: 149-159Crossref PubMed Scopus (33) Google Scholar)) and in viral RNA (69Lee N. Gorelick R.J. Musier-Forsyth K. Nucleic Acids Res. 2003; 31: 4847-4855Crossref PubMed Scopus (27) Google Scholar). In this work, we have examined the effect of nucleic acid secondary structure upon the ability of NC to stimulate efficient minus-strand transfer and have focused on structural determinants in both (-) SSDNA and acceptor RNA. Our approach was to test a series of truncated (-) SSDNA and acceptor RNA constructs using an in vitro assay that mimics the biologically relevant events of minus-strand transfer. The results of these assays are interpreted in terms of nucleic acid stability as judged by enzymatic probing and mFold analysis of individual (-) SSDNA or acceptor RNA constructs. Surprisingly, we found that destabilization of (-) SSDNA alone and elimination of self-priming by disruption of the TAR DNA stem-loop did not necessarily result in an increase in strand transfer: destabilization of acceptor RNA structure was also required. Thus, NC mediates efficient strand transfer only if both (-) SSDNA and acceptor RNA are moderately structured. Collectively, these results demonstrate that a delicate balance must exist between the stabilities of intramolecular secondary structures in (-) SSDNA and acceptor RNA and the stability of the intermolecular strand transfer duplex. Proteins—HIV-1 RT was obtained from Worthington. Micrococcal nuclease (MNase) was purchased from U. S. Biochemical Corp. (Cleveland, OH). The following enzymes were obtained from Ambion Inc. (Austin, TX): DNase I, RNase T1, RNase A, RNase V1, S1 nuclease, and proteinase K. Taq DNA polymerase was obtained from Roche Applied Science. Wild-type HIV-1 NC was a generous gift from Dr. Robert Gorelick (SAIC Frederick, Inc., NCI-Frederick, Frederick, MD). The protein was prepared as described previously (28Wu W. Henderson L.E. Copeland T.D. Gorelick R.J. Bosche W.J. Rein A. Levin J.G. J. Virol. 1996; 70: 7132-7142Crossref PubMed Google Scholar). Plasmids and Synthetic (-) SSDNAs—Plasmid pJA has been described previously (51Guo J. Henderson L.E. Bess J. Kane B. Levin J.G. J. Virol. 1997; 71: 5178-5188Crossref PubMed Google Scholar); it contains the 3′-half of HIV-1 pNL4-3 DNA (70Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar), starting from the at and including of This was used for of the acceptor RNA by the DNA with Inc., DNA was by with with and in to a of 1 Synthetic (-) of were obtained from DNA The DNAs were by the by and in to of templates for transcription of acceptor RNA were prepared by of pJA (51Guo J. Henderson L.E. Bess J. Kane B. Levin J.G. J. Virol. 1997; 71: 5178-5188Crossref PubMed Google Scholar). In the primer a the primer the 3′-end of the RNA were obtained from or DNA The for the were as for and for and and for and The for the were as for and for and and for and and contained an at to transcription by RNA polymerase D.R. G.W. Nucleic Acids Res. PubMed Scopus Google Scholar). were with Taq DNA and the products were with or The DNA was in to a of RNA were in vitro using the following the by the the transcription products were with and were by a or by on a In in vitro strand transfer