The single-finger nucleocapsid protein of moloney murine leukemia virus binds and destabilizes the TAR sequences of HIV-1 but does not promote efficiently their annealing.

The retroviral nucleocapsid proteins (NCs) are small proteins with either one or two conserved zinc fingers flanked by basic domains. NCs play key roles during reverse transcription by chaperoning the obligatory strand transfers. In HIV-1, the first DNA strand transfer relies on the NCp7-promoted destabilization and subsequent annealing of the transactivation response element, TAR with its complementary cTAR sequence. NCp7 chaperone activity relies mainly on its two folded fingers. Since NCs with a unique zinc finger are encoded by gammaretroviruses such as the canonical Moloney murine leukemia virus (MoMuLV), our objective was to characterize, by fluorescence techniques, the binding and chaperone activities of the NCp10 protein of MoMuLV to the TAR sequences of HIV-1. The unique finger and the flanking 12-25 and 40-48 domains of NCp10 were found to bind and destabilize cTAR stem-loop almost as efficiently as the homologous NCp7 protein. The flanking domains were essential for properly positioning the finger and, notably, the Trp35 residue onto cTAR. Thus, the binding and destabilization determinants scattered on the two NCp7 fingers are encoded by the unique finger of NCp10 and its flanking domains. NCp10 also activates the cTAR/TAR annealing reaction, but less efficiently than NCp7, suggesting that the two NCp7 fingers promote in concert the rate-limiting nucleation of the duplex. Due to its ability to mimic NCp7, the simple structure of NCp10 might be useful to design peptidomimetics aimed at inhibiting HIV replication.

Investigating the mechanism of the nucleocapsid protein chaperoning of the second strand transfer during HIV-1 DNA synthesis.

Conversion of the human immunodeficiency virus type 1 (HIV-1) genomic RNA into the proviral DNA by reverse transcriptase involves two obligatory strand transfers that are chaperoned by the nucleocapsid protein (NC). The second strand transfer relies on the annealing of the (-) and (+) copies of the primer binding site, (-)PBS and (+) PBS, which fold into complementary stem-loops (SLs) with terminal single-stranded overhangs. To understand how NC chaperones their hybridization, we investigated the annealing kinetics of fluorescently labelled (+)PBS with various (-)PBS derivatives. In the absence of NC, the (+)/(-)PBS annealing was governed by a second-order pathway nucleated mainly by the single-stranded overhangs of the two PBS SLs. The annealing reaction appeared to be rate-limited by the melting of the stable G.C-rich stem subsequent to the formation of the partially annealed intermediate. A second pathway nucleated through the loops could be detected, but was very minor. NC(11-55), which consists primarily of the zinc finger domain, increased the (-)/(+) PBS annealing kinetics by about sixfold, by strongly activating the interaction between the PBS loops. NC(11-55) also activated (-)/(+) PBS annealing through the single-strand overhangs, but by a factor of only 2. Full-length NC(1-55) further increased the (-)/(+)PBS annealing kinetics by tenfold. The NC-promoted (-)/(+)PBS mechanism proved to be similar with extended (-)DNA molecules, suggesting that it is relevant in the context of proviral DNA synthesis. These findings favour the notion that the ubiquitous role of NC in the viral life-cycle probably relies on the ability of NC to chaperone nucleic acid hybridization via different mechanisms.

Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence.

The nucleocapsid protein NCp7 of HIV-1 possesses a nucleic acid chaperone activity that is critical in minus and plus strand transfer during reverse transcription. The minus strand transfer notably relies on the ability of NCp7 to destabilize the stable stem with five contiguous, double-stranded segments of both the TAR sequence at the 3' end of the viral genome and the complementary sequence, cTAR, in minus strong-stop DNA. In order to examine the nature and the extent of NCp7 destabilizing activity, we investigated, by absorbance and fluorescence spectroscopy, the interaction of TAR and cTAR with a (12-55)NCp7 peptide containing the zinc-finger motifs but lacking the ability to aggregate the oligonucleotides. The absorbance changes in the UV band of cTAR show that seven to eight base-pairs, on average, are melted per oligonucleotide at a ratio of one peptide to 7.5 nucleotides. In contrast, the melting of TAR does not exceed an average of one base-pair per oligonucleotide. This may be linked to the greater stability of TAR, since a strong correlation between NCp7 destabilizing effect and oligonucleotide stability was observed. The effect of (12-55)NCp7 on the stem terminus was investigated by using a cTAR molecule doubly labeled at the 3' and 5' ends by a donor/acceptor couple. In the absence of the peptide, about 80 % of the oligonucleotides are in a dark non-fluorescent state, having a close proximity of the two dyes. The remaining 20 % are distributed between three fluorescent species, having either the terminal segment, the two terminal segments or all segments of the stem melted. This is in line with a fraying mechanism wherein the stem terminus fluctuates rapidly between open and closed states. Addition of (12-55)NCp7 shifts the equilibrium toward the open species, suggesting that NC enhances fraying of the stem terminus. Taken together, our data suggest that NCp7 activates the transient opening of base-pairs in the least stable parts of the stem. Also, this activity of NCp7 was found to be dependent on the zinc-finger motifs, since no melting was observed with a fingerless NCp7 peptide.