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

Abstract

During human immunodeficiency virus type 1 minusstrand 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 deadend 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) 1 that lead to conversion of the single-stranded RNA genome into an integration-competent linear doublestranded DNA (1). This process is facilitated by host and viral accessory proteins, one of which is the viral nucleocapsid protein (NC). Human immunodeficiency virus type 1 (HIV-1) NC is a small, highly basic, nucleic acid-binding protein with two zinc fingers, each containing the invariant CCHC metal ion-binding motif (2-5). NC functions as a nucleic acid chaperone in an ATP-independent manner (6) and catalyzes nucleic acid conformational rearrangements that lead to the formation of the most thermodynamically stable structure ). This activity is required for efficient reverse transcription and allows NC to promote intermolecular annealing of nucleic acids with significant stretches of base complementarity (7, 12-26), destabilization of secondary structures in RNA and DNA templates that are responsible for RT pausing (27-30), unwinding of primer tRNA (19, 31-34), primer placement (12,15,19,32,33,(35)(36)(37)(38), and the initiation step (39 -41).NC nucleic acid chaperone activity is also critical for the minus-strand (reviewed in Refs. 8, 10, and 11) and plus-strand (18,20,22,42) transfer events that are required to complete elongation of minus-and plus-strand DNAs and to generate the long te...

Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer

“…Secondary structure predictions by mFold (48,49) of A97h(30A) and A97h(30R) indicated that they folded into different, lower stability structures than the wild-type A97h. It has been reported that presence of a secondary structure in acceptor RNA reduces transfer efficiency, and removal of structure promotes transfer (19,30). Therefore, it is likely that structural changes made to construct the bipartite acceptors made them more effective for transfer.…”

“…For the acceptor template, secondary structure predictions by mFold (48,49) indicated the R region of the acceptor could form altered structures as varied lengths of U3 were attached, leading to different transfer efficiencies. This result suggests the structure and stability of the R region of the acceptor could also be influenced by regions outside of those directly involved in the base pairing associated with the long-transfer (30). Reverse transcription in HIV-1 is initiated by a tRNA Lys-3 primer.…”

“…The complex effect of R region on transfer was revealed by additional studies in vitro. Levin and co-workers (30) reported that truncating the R region of the acceptor actually led to efficient transfer. The explanation was that removal of secondary structures facilitated transfer.…”

Mechanisms of DNA binding determined in optical tweezers experiments

“…[124][125][126][127][128][129][130][131][132][133][134][135][136] This chaperone activity, facilitating restructuring of the nucleic acid complex, has been attributed to both an aggregating ability of NC and a facility for duplex destabilization. 125,131,[137][138][139][140][141][142][143][144][145] This destabilization, however, is weak, 143,144,146,147 and will not completely melt DNA without a complementary strand. 6,138,148,149 Stretching and relaxation cycles can also reveal information on the kinetics of protein association and dissociation.…”

Single-molecule stretching studies of RNA chaperones

“…Studies have shown that all these three steps need the participation of NC proteins, especially the minus stand transfer step. 66,[78][79][80] The effect of NC's chaperone activity on these processes is detailed in the review by Levin and Musier-Forsyth in this special issue.…”