Human Immunodeficiency Virus Type 1 NC Research Articles

RNA Structural Requirements for Nucleocapsid Protein-Mediated Extended Dimer Formation.

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC.

Identification and characterization of a new type of inhibitor against the human immunodeficiency virus type-1 nucleocapsid protein.

BackgroundThe human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) is an essential and multifunctional protein involved in multiple stages of the viral life cycle such as reverse transcription, integration of proviral DNA, and especially genome RNA packaging. For this reason, it has been considered as an attractive target for the development of new anti-HIV drugs. Although a number of inhibitors of NC have been reported thus far, the search for NC-specific and functional inhibitor(s) with a good antiviral activity continues.ResultsIn this study, we report the identification of A1752, a small molecule with inhibitory action against HIV-1 NC, which shows a strong antiviral efficacy and an IC50 around 1 μM. A1752 binds directly to HIV-1 NC, thereby inhibiting specific chaperone functions of NC including Psi RNA dimerization and complementary trans-activation response element (cTAR) DNA destabilization, and it also disrupts the proper Gag processing. Further analysis of the mechanisms of action of A1752 also showed that it generates noninfectious viral particles with defects in uncoating and reverse transcription in the infected cells.ConclusionsThese results demonstrate that A1752 is a specific and functional inhibitor of NC with a novel mode of action and good antiviral efficacy. Thus, this agent provides a new type of anti-HIV NC inhibitor candidate for further drug development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0218-9) contains supplementary material, which is available to authorized users.

NC-mediated nucleolar localization of retroviral gag proteins

The assembly and release of retrovirus particles from the cell membrane is directed by the Gag polyprotein. The Gag protein of Rous sarcoma virus (RSV) traffics through the nucleus prior to plasma membrane localization. We previously reported that nuclear localization of RSV Gag is linked to efficient packaging of viral genomic RNA, however the intranuclear activities of RSV Gag are not well understood. To gain insight into the properties of the RSV Gag protein within the nucleus, we examined the subnuclear localization and dynamic trafficking of RSV Gag. Restriction of RSV Gag to the nucleus by mutating its nuclear export signal (NES) in the p10 domain or interfering with CRM1-mediated nuclear export of Gag by leptomycin B (LMB) treatment led to the accumulation of Gag in nucleoli and discrete nucleoplasmic foci. Retention of RSV Gag in nucleoli was reduced with cis-expression of the 5′ untranslated RU5 region of the viral RNA genome, suggesting the psi (Ψ) packaging signal may alter the subnuclear localization of Gag. Fluorescence recovery after photobleaching (FRAP) demonstrated that the nucleolar fraction of Gag was highly mobile, indicating that there was rapid exchange with Gag proteins in the nucleoplasm. RSV Gag is targeted to nucleoli by a nucleolar localization signal (NoLS) in the NC domain, and similarly, the human immunodeficiency virus type 1 (HIV-1) NC protein also contains an NoLS consisting of basic residues. Interestingly, co-expression of HIV-1 NC or Rev with HIV-1 Gag resulted in accumulation of Gag in nucleoli. Moreover, a subpopulation of HIV-1 Gag was detected in the nucleoli of HeLa cells stably expressing the entire HIV-1 genome in a Rev-dependent fashion. These findings suggest that the RSV and HIV-1 Gag proteins undergo nucleolar trafficking in the setting of viral infection.

Nucleic Acid Chaperone Activity of the Yeast Ty3 Retrotransposon Nucleocapsid Protein

Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which facilitate the rearrangement of nucleic acid secondary structure. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) have been extensively studied, and nucleic acid aggregation, duplex destabilization, and rapid protein binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. We used single molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. Wild type Ty3 NC strongly aggregates both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), and melted DNA exhibits rapid reannealing in its presence. We also studied several Ty3 NC mutants to identify the roles of functional regions of the protein. We found that the N-terminal basic residues contribute to duplex stabilization, while the zinc finger at the C-terminus counteracts this effect. The mutants examined lack the rapid kinetics of wild type Ty3 NC, indicating that both the basic residues and the zinc finger are required for optimum chaperone activity, which is consistent with previous biochemical experiments. Ty3 NC therefore has a chaperone mechanism similar to that of HIV-1 NC. Although Ty3 NC does not exhibit the strong duplex destabilization of HIV-1 NC, this is consistent with the weaker secondary structure of the Ty3 long-terminal repeat region, which suggests that strong duplex destabilization is not needed for NC to facilitate minus-strand transfer during reverse transcription. This research was supported in part by funding from INSERM and ANRS (France).

Effect of Mg 2+ and Na + on the Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein: Implications for Reverse Transcription

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is an essential protein for retroviral replication. Among its numerous functions, NC is a nucleic acid (NA) chaperone protein that catalyzes NA rearrangements leading to the formation of thermodynamically more stable conformations. In vitro, NC chaperone activity is typically assayed under conditions of low or no Mg 2+, even though reverse transcription requires the presence of divalent cations. Here, the chaperone activity of HIV-1 NC was studied as a function of varying Na + and Mg 2+ concentrations by investigating the annealing of complementary DNA and RNA hairpins derived from the trans-activation response domain of the HIV genome. This reaction mimics the annealing step of the minus-strand transfer process in reverse transcription. Gel-shift annealing and sedimentation assays were used to monitor the annealing kinetics and aggregation activity of NC, respectively. In the absence of protein, a limited ability of Na + and Mg 2+ cations to facilitate hairpin annealing was observed, whereas NC stimulated the annealing 10 3- to 10 5-fold. The major effect of either NC or the cations is on the rate of bimolecular association of the hairpins. This effect is especially strong under conditions wherein NC induces NA aggregation. Titration with NC and NC/Mg 2+ competition studies showed that the annealing kinetics depends only on the level of NA saturation with NC. NC competes with Mg 2+ or Na + for sequence-nonspecific NA binding similar to a simple trivalent cation. Upon saturation, NC induces attraction between NA molecules corresponding to ∼ 0.3 kcal/mol/nucleotide, in agreement with an electrostatic mechanism of NC-induced NA aggregation. These data provide insights into the variable effects of NC's chaperone activity observed during in vitro studies of divalent metal-dependent reverse transcription reactions and suggest the feasibility of NC-facilitated proviral DNA synthesis within the mature capsid core.

HIV-1 Nucleocapsid Protein Switches the Pathway of Transactivation Response Element RNA/DNA Annealing from Loop–Loop “Kissing” to “Zipper”

The chaperone activity of HIV-1 (human immunodeficiency virus type 1) nucleocapsid protein (NC) facilitates multiple nucleic acid rearrangements that are critical for reverse transcription of the single-stranded RNA genome into double-stranded DNA. Annealing of the transactivation response element (TAR) RNA hairpin to a complementary TAR DNA hairpin is an essential step in the minus-strand transfer step of reverse transcription. Previously, we used truncated 27-nt mini-TAR RNA and DNA constructs to investigate this annealing reaction pathway in the presence and in the absence of HIV-1 NC. In this work, full-length 59-nt TAR RNA and TAR DNA constructs were used to systematically study TAR hairpin annealing kinetics. In the absence of NC, full-length TAR hairpin annealing is ∼ 10-fold slower than mini-TAR annealing. Similar to mini-TAR annealing, the reaction pathway for TAR in the absence of NC involves the fast formation of an unstable “kissing” loop intermediate, followed by a slower conversion to an extended duplex. NC facilitates the annealing of TAR by ∼ 10 5-fold by stabilizing the bimolecular intermediate (∼ 10 4-fold) and promoting the subsequent exchange reaction (∼ 10-fold). In contrast to the mini-TAR annealing pathway, wherein NC-mediated annealing can initiate through both loop–loop kissing and a distinct “zipper” pathway involving nucleation at the 3′-/5′-terminal ends, full-length TAR hairpin annealing switches predominantly to the zipper pathway in the presence of saturated NC.

Human T-Cell Lymphotropic Virus Type 1 Nucleocapsid Protein-Induced Structural Changes in Transactivation Response DNA Hairpin Measured by Single-Molecule Fluorescence Resonance Energy Transfer

Time-resolved single-molecule fluorescence spectroscopy was used to study the human T-cell lymphotropic virus type 1 (HTLV-1) nucleocapsid protein (NC) chaperone activity compared to that of the human immunodeficiency virus type 1 (HIV-1) NC protein. HTLV-1 NC contains two zinc fingers, each having a CCHC binding motif similar to HIV-1 NC. HIV-1 NC is required for recognition and packaging of the viral RNA and is also a nucleic acid chaperone protein that facilitates nucleic acid restructuring during reverse transcription. Because of similarities in structures between the two retroviruses, we have used single-molecule fluorescence energy transfer to investigate the chaperoning activity of the HTLV-1 NC protein. The results indicate that the HTLV-1 NC protein induces structural changes by opening the transactivation response (TAR) DNA hairpin to an even greater extent than HIV-1 NC. However, unlike HIV-1 NC, HTLV-1 NC does not chaperone the strand-transfer reaction involving TAR DNA. These results suggest that, despite its effective destabilization capability, HTLV-1 NC is not as effective at overall chaperone function as is its HIV-1 counterpart.

Rapid Kinetics of Protein–Nucleic Acid Interaction is a Major Component of HIV-1 Nucleocapsid Protein’s Nucleic Acid Chaperone Function

The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) plays an important role in the retroviral life cycle, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. Recent studies have identified duplex destabilization and nucleic acid aggregation as the two major components of NC's chaperone activity. In order to better understand the contribution of the functional domains of NC to these two activities, we used optical tweezers to stretch single lambda DNA molecules through the helix-coil transition in the presence of wild-type or mutant HIV-1 NC. Protein-induced duplex destabilization was measured directly as an average decrease of the force-induced melting free energy, while NC's ability to facilitate strand annealing was determined by the amount of hysteresis in the DNA stretch-relax cycle. By studying zinc-free variants of full-length and truncated NC, the relative contributions of NC's zinc fingers and N-terminal basic domain to the two major components of chaperone activity were elucidated. In addition, examination of NC variants containing mutations affecting one or both zinc finger motifs showed that effective strand annealing activity is correlated with NC's ability to rapidly bind and dissociate from nucleic acids. NC variants with slow on/off rates are inefficient in strand annealing, even though they may still be capable of high affinity nucleic acid binding, duplex destabilization, and/or nucleic acid aggregation. Taken together, these observations establish the rapid kinetics of protein–nucleic acid interaction as another major component of NC's chaperone function.

Specific Interactions Between HIV-1 Nucleocapsid Protein and the TAR Element

During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer.

Amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human APOBEC3G packaging.

APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.

Human immunodeficiency virus type 1 nucleocapsid protein nuclear localization mediates early viral mRNA expression.

An important aspect of the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection is the ability of the virus to replicate in the host vigorously without a latent phase and to kill cells with a dynamic turnover of 1.8 x 10(9) cells/day and 10.3 x 10(9) virions/24 h. The transcription of HIV-1 RNA in acute infection occurs at two stages; the transcription of viral spliced mRNA occurs early, and the transcription of viral genomic RNA occurs later. The HIV-1 Tat protein is translated from the early spliced mRNA and is critical for HIV-1 genomic RNA expression. The cellular transcription factors are important for HIV-1 early spliced mRNA expression. In this study we show that virion nucleocapsid protein (NC) has a role in expression of HIV-1 early spliced mRNA. The HIV-1 NC migrates from the cytoplasm to the nucleus and accumulates in the nucleus at 18 h postinfection. Mutations on HIV-1 NC zinc fingers change the pattern of early viral spliced mRNA expression and result in a delayed expression of early viral mRNA in HIV-infected cells. This delayed HIV-1 early spliced mRNA expression occurs after proviral DNA has been integrated into the cellular genome, as shown by a quantitative integration assay. These results show that virion NC plays an important role in inducing HIV-1 early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection.

Rapid CD4(+) T-cell loss induced by human immunodeficiency virus type 1(NC) in uninfected and previously infected chimpanzees.

To investigate the pathogenicity of a virus originating in a chimpanzee with AIDS (C499), two chimpanzees were inoculated with a plasma-derived isolate termed human immunodeficiency virus type 1(NC) (HIV-1(NC)). A previously uninfected chimpanzee, C534, experienced rapid peripheral CD4(+) T-cell loss to fewer than 26 cells/microl by 14 weeks after infection. CD4(+) T-cell depletion was associated with high plasma HIV-1 loads but a low virus burden in the peripheral lymph node. The second chimpanzee, C459, infected 13 years previously with HIV-1(LAV), experienced a more protracted course of peripheral CD4(+) T-cell loss after HIV-1(NC) inoculation, resulting in fewer than 200 cells/microl by 96 weeks postinoculation. The quantities of viral RNA in the plasma and peripheral lymph node from C459 were below the lower limits of detection prior to inoculation with HIV-1(NC) but were significantly and persistently increased after superinfection, with HIV-1(NC) representing the predominant viral genotype. These results show that viruses derived from C499 are more pathogenic for chimpanzees than any other HIV-1 isolates described to date.

Host cell-dependent replication of HIV-1 gag MA, CA, and NC mutants are independent of the functions of Vif and Vpu.

We have previously shown that some gag gene mutants of human immunodeficiency virus type 1 (HIV-1) display a replication-defect in a cell-dependent manner. We and others have also demonstrated that the requirement of vif and vpu genes for HIV-1 replication is cell-dependent. To determine whether the cell-dependent growth of the HIV-1 gag mutants is related to the functions of Vif and Vpu, double mutants of gag-vif and gag-vpu were constructed, and monitored for their replication in various cell lines. The results obtained showed that the mutations in gag do not affect the cell-dependent functions of Vif and Vpu.

DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.

Replacement of His 23 by Cys in a zinc finger of HIV-1 NC p7 led to a change in 1H NMR-derived 3D structure and to a loss of biological activity

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which is necessary for the formation of infectious virions, contains two zinc fingers of the Cys-X 2-Cys-X 4-His-X 4-Cys form. To elucidate the importance of this particular motif, well conserved in retroviruses and retroelements, we substituted the histidine residue by a cysteine in the first zinc binding domain 13VKCFNCGKEGHTARNCRA 30. The structures of the mutated and native zinc complexed peptides were studied by two-dimensional 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution. The nuclear Overhauser effects were used as constraints to determine the solution structures using DIANA software followed by AMBER energy refinement. The results show that native and mutant peptides fold into non-identical three-dimensional structures, probably accounting for the loss of retrovirus infectivity following the His-Cys point mutation.

Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA.

The nucleocapsid protein (NC) of all animal retroviruses, encoded by the gag gene, is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. NC is also thought to play one or more accessory roles in reverse transcription. Mature NC (p7NC) from human immunodeficiency virus type 1 (HIV-1) is a 71-amino acid, basic protein which contains two Cys3His Zn(II) retroviral-type zinc finger domains. Herein, we describe the subcloning and expression of HIV-1 NC, denoted NC71, from an inducible phage T7 RNA polymerase promoter in Escherichia coli. Purified NC71 can be reversibly reconstituted with 2 g.at Zn(II) determined by atomic absorption. Ultraviolet circulation dichroism spectroscopy has been used to characterize the complexes between highly purified NC71 and the RNA homopolynucleotide poly(A) and E. coli tRNA(mixed). On poly(A), Zn2 NC71 is characterized by an apparent site size n = 15 +/- 3 nucleotides and high affinity (Kapp = 3 x 10(7) M-1) and moderately cooperative (omega approximately 170 +/- 25) binding. A mixture of E. coli tRNA species (tRNA(mixed) was used to probe the conformational changes induced in tRNA upon binding of HIV-1 NC71. Two structural forms of tRNA(mixed), which differ in their degree of tertiary structure, were assayed for their susceptibility to denaturation by NC71. Five molar monomer equivalents of NC71 are required to denature the "inactive" tRNA in the absence of Mg2+. A Zn(II)-free, oxidized form of NC71 was also shown to unwind inactive tRNA with the same efficiency and stoichiometry. The detailed spectral changes which occur on NC-induced denaturation closely mimic temperature-induced denaturation of inactive tRNA(mixed). The prototype helix-destabilizing protein, T4 gene 32 protein, is unable to unwind this form of tRNA under the same conditions. The stoichiometry of unwinding of inactive tRNA by NC71 is consistent with the site size determined with poly(A). An "active" form of tRNA(mixed), prepared by thermal denaturation and refolding of the inactive form with Mg2+, proved less susceptible to both temperature and NC71-induced unwinding. The mechanistic implications of these findings on the reported biochemical activities of RNA:RNA annealing and replication primer tRNA positioning by NC are discussed.