Alteration of nucleic acid structure and stability modulates the efficiency of minus-strand transfer mediated by the HIV-1 nucleocapsid protein

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.     

Differing roles of the N- and C-terminal zinc fingers in human immunodeficiency virus nucleocapsid protein-enhanced nucleic acid annealing

The replication process of human immunodeficiency virus requires a number of nucleic acid annealing steps facilitated by the hybridization and helix-destabilizing activities of human immunodeficiency virus nucleocapsid (NC) protein. NC contains two CCHC zinc finger motifs numbered 1 and 2 from the N terminus. The amino acids surrounding the CCHC residues differ between the two zinc fingers. Assays were preformed to investigate the activities of the fingers by determining the effect of mutant and wild-type proteins on annealing of 42-nucleotide RNA and DNA complements. The mutants 1.1 NC and 2.2 NC had duplications of the N- and C-terminal zinc fingers in positions 1 and 2. The mutant 2.1 NC had the native zinc fingers with their positions switched. Annealing assays were completed with unstructured and highly structured oligonucleotide complements. 2.2 NC had a near wild-type level of annealing of unstructured nucleic acids, whereas it was completely unable to stimulate annealing of highly structured nucleic acids. In contrast, 1.1 NC was able to stimulate annealing of both unstructured and structured substrates, but to a lesser degree than the wild-type protein. Results suggest that finger 1 has a greater role in unfolding of strong secondary structures, whereas finger 2 serves an accessory role that leads to a further increase in the rate of annealing.     

Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1.

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.