Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

Abstract

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (−) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5′ end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

Biophysical studies of the nucleic acid chaperone properties of the HIV-1 nucleocapsid protein

“…41,48,95,[114][115][116] The efficiency of the minus strand transfer reaction was initially envisioned as resulting from the extent of homology between the two complementary sequences, [117][118][119][120][121][122] although the structure of the reactants was suspected to play a role, too. 117,118,[123][124][125][126][127] This assumption was confirmed with the relationship between the NA thermostability and the NC-mediated minus-strand transfer efficiency. 118 In fact, more than the overall stability of the NA sequence, the stability of local structures [127][128][129][130] appeared to be of particular importance to modulate the strand transfer, 118 suggesting that a fine protein.…”

“…117,118,[123][124][125][126][127] This assumption was confirmed with the relationship between the NA thermostability and the NC-mediated minus-strand transfer efficiency. 118 In fact, more than the overall stability of the NA sequence, the stability of local structures [127][128][129][130] appeared to be of particular importance to modulate the strand transfer, 118 suggesting that a fine protein. 29,32,48,49,[93][94][95] The largest annealing promotion by NCp7 is observed with folded RNAs.…”

Investigating the Mechanism of the Nucleocapsid Protein Chaperoning of the Second Strand Transfer during HIV-1 DNA Synthesis

“…46 Our conclusion is in agreement also with data for TAR RNA showing that the local structure at the nucleation site is critical for NC chaperone activity in minus-strand transfer. 61 The ability of NC to chaperone annealing reactions via several mechanisms appears to be of critical importance, because it should allow NC to direct the hybridization of a large set of complementary nucleic acid sequences. The annealing mechanism seems to depend on the oligonucleotide sequence.…”

“…The annealing mechanism seems to depend on the oligonucleotide sequence. In fact, while NC nucleates the cTAR/TAR duplex mainly via the lower part of TAR and cTAR stems, 47,58,59,61 it chaperones the annealing of mini-TAR duplexes 21,47 and DIS homodimers 55,56 through kissing intermediates. Since the DIS and the upper half of the cTAR stem (mini-TAR) are more stable than the lower half of the cTAR stem, 46,56 the NC-chaperoned mechanism depends on the stability of the nucleation site.…”

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

“…The explanation was that removal of secondary structures facilitated transfer. In some cases, the local secondary structure rather than the overall conformation of acceptor is more of a determinant of minus strand transfer efficiency (31). Considered together, the results suggest the length, structure, and sequence of the R region are all important factors in the transfer reaction.…”