Thermal stressed human immunodeficiency virus type 1 nucleocapsid protein NCp7 maintains nucleic acid-binding activity.

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is a small, highly basic nucleic acid (NA)-binding protein with two CCHC zinc-finger motifs. In this study, we report for the first time, to our knowledge, that thermal stressed HIV-1 NCp7 maintained NA-binding activity. About 41.3% of NCp7 remained soluble after incubated at 100 °C for 60 min, and heat-treated NCp7 maintained its abilities to bind to HIV-1 packaging signal (Psi) and the stem-loop 3 of the Psi. At high or very high degrees of sequence occupancy, NCp7 inhibited first-strand cDNA synthesis catalyzed by purified HIV-1 reverse transcriptase, and heat-treated NCp7 maintained the inhibition. Moreover, both EDTA-treated and H23K + H44K double mutant of NCp7 inhibited first-strand cDNA synthesis, demonstrating that the NA-binding activity of NCp7 at high NC:NA ratios is independent on its zinc-fingers. These results may benefit further investigations of the structural stability and function of NCp7 in viral replication.

Specific inter-domain interactions stabilize a compact HIV-1 Gag conformation.

HIV-1 Gag is a large multidomain poly-protein with flexible unstructured linkers connecting its globular subdomains. It is compact when in solution but assumes an extended conformation when assembled within the immature HIV-1 virion. Here, we use molecular dynamics (MD) simulations to quantitatively characterize the intra-domain interactions of HIV-1 Gag. We find that the matrix (MA) domain and the C-terminal subdomain CActd of the CA capsid domain can form a bound state. The bound state, which is held together primarily by interactions between complementary charged and polar residues, stabilizes the compact state of HIV-1 Gag. We calculate the depth of the attractive free energy potential between the MA/ CActd sites and find it to be about three times larger than the dimerization interaction between the CActd domains. Sequence analysis shows high conservation within the newly-found intra-Gag MA/CActd binding site, as well as its spatial proximity to other well known elements of Gag -such as CActd's SP1 helix region, its inositol hexaphosphate (IP6) binding site and major homology region (MHR), as well as the MA trimerization site. Our results point to a high, but yet undetermined, functional significance of the intra-Gag binding site. Recent biophysical experiments that address the binding specificity of Gag are interpreted in the context of the MA/CActd bound state, suggesting an important role in selective packaging of genomic RNA by Gag.

Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein.

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations. In this work, in order to obtain more potent NCp7 competitors, we designed a library of longer peptides (10-17 amino acids) whose sequences include most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities. Using an in vitro assay, the most active peptide (pE) was found to inhibit the NCp7 destabilizing activity, with a 50% inhibitory concentration in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide (CPP), pE was found to accumulate into HeLa cells, with low cytotoxicity. However, either formulated with a CPP or overexpressed in cells, pE did not show any antiviral activity. In vitro competition experiments revealed that its poor antiviral activity may be partly due to its sequestration by cellular RNAs. The selected peptide pE therefore appears to be a useful tool for investigating NCp7 properties and functions in vitro, but further work will be needed to design pE-derived peptides with antiviral activity.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).

Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates.

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.

Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer.

Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (-) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(-)PBS annealing mechanism by activating a loop-loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (-)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(-)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can 'freeze' PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC.

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5' thymine interacts with residues of the N-terminal zinc knuckle and the 3' guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA-protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids.

Predicting the binding mode of known NCp7 inhibitors to facilitate the design of novel modulators.

The HIV-1 nucleocapsid protein (NCp7) is an emerging target for antiretroviral therapy. Five hits have been reported to inhibit the NCp7-viral nucleic acids interaction at micromolar concentrations. We used two computationally refined structures of NCp7 as receptors to propose a reliable binding pose for these compounds, by means of computational methods. Theoretical binding modes are in agreement with available experimental data. Results lay the foundations for a rationale development of more effective NCp7 inhibitors.

Inhibition of HIV-1 replication by a bis-thiadiazolbenzene-1,2-diamine that chelates zinc ions from retroviral nucleocapsid zinc fingers.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid p7 (NCp7) protein holds two highly conserved "CCHC" zinc finger domains that are required for several phases of viral replication. Basic residues flank the zinc fingers, and both determinants are required for high-affinity binding to RNA. Several compounds were previously found to target NCp7 by reacting with the sulfhydryl group of cysteine residues from the zinc fingers. Here, we have identified an N,N'-bis(1,2,3-thiadiazol-5-yl)benzene-1,2-diamine (NV038) that efficiently blocks the replication of a wide spectrum of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) strains. Time-of-addition experiments indicate that NV038 interferes with a step of the viral replication cycle following the viral entry but preceding or coinciding with the early reverse transcription reaction, pointing toward an interaction with the nucleocapsid protein p7. In fact, in vitro, NV038 efficiently depletes zinc from NCp7, which is paralleled by the inhibition of the NCp7-induced destabilization of cTAR (complementary DNA sequence of TAR). A chemical model suggests that the two carbonyl oxygens of the esters in this compound are involved in the chelation of the Zn(2+) ion. This compound thus acts via a different mechanism than the previously reported zinc ejectors, as its structural features do not allow an acyl transfer to Cys or a thiol-disulfide interchange. This new lead and the mechanistic study presented provide insight into the design of a future generation of anti-NCp7 compounds.

How the HIV-1 nucleocapsid protein binds and destabilises the (-)primer binding site during reverse transcription.

The human immunodeficiency virus type 1 nucleocapsid protein (NCp7) plays an important role in the second strand transfer during reverse transcription. It promotes annealing of the 18-nucleotide complementary DNA primer-binding site (PBS) sequences at the 3' ends of (-)DNA and (+)DNA. NMR studies show that NCp7(12-55) and NCp7(1-55) interact at the 5' end of the loop of DeltaP(-)PBS, a (-)PBS derivative without the 3' protruding sequence, in a slow-exchange equilibrium. This interaction is mediated through the binding of the hydrophobic plateau (Val13, Phe16, Thr24, Ala25, Trp37, and Met46) on the zinc finger domain of both peptides to the 5-CTG-7 sequence of DeltaP(-)PBS. The stacking of the Trp37 aromatic ring with the G7 residue likely constitutes the determinant factor of the interaction. Although NCp7(12-55) does not melt the DeltaP(-)PBS stem-loop structure, it opens the loop and weakens the C5.G11 base pair next to the loop. Moreover, NCp7(12-55) was also found to bind but with lower affinity to the 10-CGG-12 sequence in an intermediate-exchange equilibrium on the NMR time scale. The loop modifications may favour a kissing interaction with the complementary (+)PBS loop. Moreover, the weakening of the upper base pair of the stem likely promotes the melting of the stem that is required to convert the kissing complex into the final (+/-)PBS extended duplex.

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation.

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs.