Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity

Abstract

The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant MasonPfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn, NC-polynucleotide complexes can be formed under conditions of low [NaCI] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 "C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 "C, 1 M NaCI, solution conditions considered t o be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nuc1eotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.

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

“…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. 96 For instance, the NCp7-assisted rate of a 149 bp DNA fragment was 100-200-fold faster than that obtained in conditions (68°C, 1 M NaCl) optimal for strand renaturation, suggesting that NCp7 strongly facilitates the nucleation of the duplex.…”

Retroviral Nucleocapsid Proteins Display Nonequivalent Levels of Nucleic Acid Chaperone Activity

“…Stronger binding to single-stranded versus double-stranded nucleic acids is characteristic of proteins that induce duplex destabilization (75). Assuming a similar site size of 6 nt (or 3 bp) (25,32,40,41,61,62,78,100,101,105,111) for binding to singlestranded and double-stranded sequences, the maximum duplex destabilization free energy by NC protein, ␦G, per base pair can be approximated according to equation 8 (75):…”

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.…”