HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro.

Abstract

After a retrovirus particle is released from the cell, the dimeric genomic RNA undergoes a change in conformation. We have previously proposed that this change, termed maturation of the dimer, is due to the action of nucleocapsid (NC) protein on the RNA within the virus particle. We now report that treatment of a 345-base synthetic fragment of Harvey sarcoma virus RNA with recombinant or synthetic HIV-1 NC protein converts a less stable form of dimeric RNA to a more stable form. This phenomenon thus appears to reproduce the maturation of dimeric retroviral RNA in a completely defined system in vitro, To our knowledge, maturation of dimeric RNA within a retrovirus particle is the first example of action of an "RNA chaperone" protein in vivo. Studies with mutant NC proteins suggest that the activity depends upon basic amino acid residues flanking the Nterminal zinc finger and upon residues within the N-terminal finger, including an aromatic amino acid, but do not require the zinc finger structures themselves.After a retrovirus particle is released from the cell, the dimeric genomic RNA within the particle undergoes a change in conformation (1-3). This change, which renders the dimer more thermostable, has been referred to as "maturation" of the dimer; maturation requires the activity of the viral protease (PR) (2, 3).The requirement for PR activity suggested that RNA maturation results from interaction of one or more of the Gag proteins with the RNA after PR-mediated cleavage releases these proteins from the Gag polyprotein precursor. We have specifically suggested (2) that one of these cleavage products, the viral nucleocapsid (NC) protein, is responsible for maturation of the RNA, because (i) NC is known to be associated with the RNA in the nucleoprotein core of the mature particle (4-6), and (ii) NC is known to facilitate the formation of the optimal, most thermodynamically stable structures in nucleic acids (7,8).The nature of the difference between the immature and mature dimeric forms of viral RNA is not known; indeed, the structure of the dimeric RNA in retrovirus particles is very poorly understood. The two monomers are believed to be joined near their 5' ends (9). One important step in the analysis of these structures was the finding that relatively short RNA transcripts containing sequences from near the 5' end of retroviral genomes can dimerize spontaneously in vitro under conditions of high ionic strength (4, 5, 10).In the course of experiments designed to identify sequences from the Harvey sarcoma virus (HaSV) genome capable of dimerization in vitro, we noted (11) that a 345-base transcript could give rise to two distinct dimeric forms differing in thermostability: dimerization at 37°C produces a population of dimeric molecules of which some can be dissociated into monomers at temperatures near 45°C, whereas dimerization at 55°C produces dimers that are all stable to temperatures of 55°C or higher.We now report that incubation of the HaSV dimers formed at 37°C with recombinant or synthetic HIV...

Structure/Function Mapping of Amino Acids in the N-Terminal Zinc Finger of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein: Residues Responsible for Nucleic Acid Helix Destabilizing Activity

“…There are five amino acids which differ between the two finger sequences. These include (finger one to finger two): F to W, N to K, I to Q, A to M, and N to D at positions 16,17,24,25, and 27 of finger one, respectively. NC point mutants were constructed where the amino acid residues in finger one were incrementally replaced by those at the corresponding locations in finger two.…”

“…Of the 14 amino acids that compose each finger, five differ between the two zinc fingers (in the pNL4-3 clone of HIV-1): (finger one to finger two): phenylalanine to tryptophan (F to W), asparagine to lysine (N to K), isoleucine to glutamine (I to Q), alanine to methionine (A to M), and asparagine to aspartic acid (N to D) at positions 16,17,24,25, and 27 of finger one, respectively. Presumably these differences underlie the much stronger helix destabilizing activity of finger one.…”

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

“…NC has been shown to function in several steps of viral replication, including genomic RNA maturation, reverse transcription, and integration. NC displays nucleic acid chaperone activity, catalyzing the melting, reannealing, and folding of nucleic acid into their most stable conformations (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). All of these have important implications in strand transfer and reverse transcription.…”

Differing Roles of the N- and C-terminal Zinc Fingers in Human Immunodeficiency Virus Nucleocapsid Protein-enhanced Nucleic Acid Annealing

“…Recent studies have shown that NC may play a role in integration of the proviral DNA (18,(25)(26)(27). Also, NC sequences in the Gag precursor are a necessity for packaging of the RNA genome (28 -30) and the maturation of the genomic RNA dimer in the virion core (31,32). In vivo studies have specifically linked packaging activity to the zinc finger regions of the Gag (NC) precursor (33,34).…”