The HIV-1 matrix protein p17 can be efficiently delivered by intranasal route in mice using the TLR 2/6 agonist MALP-2 as mucosal adjuvant.

The HIV-1 matrix protein p17 is a structural protein essential in the life cycle of HIV, by acting as a virokine/immunomodulator that supports viral replication and spreading. The presence of p17-specific antibodies and CTL responses correlates with slower progression to AIDS. Intranasal vaccination with p17 and the TLR2/6 agonist MALP-2 stimulates strong humoral and cellular immune responses at systemic and mucosal levels. The antibodies blocked p17 binding to its receptor, which is a critical step for the exertion of its virokine activity. Our results suggest that p17 and MALP-2 are attractive candidates for incorporation in mucosal vaccines against HIV/AIDS.

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: structural and mechanistic implications of p17 myristoylation.

The HIV-1 matrix protein p17, excised proteolytically from the N terminus of the Gag polyprotein, forms a protective shell attached to the inner surface of the plasma membrane of the virus. During the late stages of the HIV-1 replication cycle, the N-terminally myristoylated p17 domain targets the Gag polyprotein to the host-cell membrane for particle assembly. In the early stages of HIV-1 replication, however, some p17 molecules dissociate from the viral membrane to direct the preintegration complex to the host-cell nucleus. These two opposing targeting functions of p17 require that the protein be capable of reversible membrane interaction. It is postulated that a significant structural change in p17 triggered by proteolytic cleavage of the Gag polyprotein sequesters the N-terminal myristoyl group, resulting in a weaker membrane binding by the matrix protein than the Gag precursor. To test this "myristoyl switch" hypothesis, we obtained highly purified synthetic HIV-1 p17 of 131 amino acid residues and its N-myristoylated form in large quantity. Both forms of p17 were characterized by circular dichroism spectroscopy, protein chemical denaturation, and analytical centrifugal sedimentation. Our results indicate that although N-myristoylation causes no spectroscopically discernible conformational change in p17, it stabilizes the protein by 1 kcal/mol and promotes protein trimerization in solution. These findings support the premise that the myristoyl switch in p17 is triggered not by a structural change associated with proteolysis, but rather by the destabilization of oligomeric structures of membrane-bound p17 in the absence of downstream Gag subdomains.

Concerted action of multiple cis-acting sequences is required for Rev dependence of late human immunodeficiency virus type 1 gene expression.

Based on the human immunodeficiency virus type 1 (HIV-1) gag gene, subgenomic reporter constructs have been established allowing the contributions of different cis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimized gag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.

Generation of monoclonal antibodies specifically directed against the proximal zinc finger of HIV type 1 NCp7.

The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.

Analysis of the anti-HIV-1 activity of an anti-p17-derivative peptide (P30-52) monoclonal antibody.

The object of this study was to examine the possibility of immunotherapy using anti-human immunodeficiency virus type 1 (HIV-1) p17-derivative peptide monoclonal antibody (MAb), namely MAb 8H10. Previously we established MAb 8H10, and further characterization revealed that it inhibited the viral multiplication of the HIV-1-infected MT-4 cells, and that the inhibitory mechanism related to the decrease of p17 DNA of the infected cells. In the present study, based on the assumption that Hybridoma 8H10 (Hyb 8H10) is a source of this MAb, we examined how Hyb 8H10 influences the infected cells when the two are co-cultured using the transwell or by mixed culture. Hyb 8H10 did not influence the cell growth or viability of MT-4 cells, and MAb 8H10 was transferred to the cluster dish containing the infected cells. Furthermore, Hyb 8H10 could produce MAb 8H10 even when co-cultured with the infected MT-4 cells. However, the infectivity of the supernatant of the infected cells was temporarily enhanced when Hyb 8H10 was co-cultured without MAb 8H10, though it gradually reduced according to the increase in MAb 8H10 produced by Hyb 8H10. Though there were some problems, MAb 8H10 proved to be a strong candidate for immunotherapy against HIV.

Gag protein epitopes recognized by CD4(+) T-helper lymphocytes from equine infectious anemia virus-infected carrier horses.

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.

A binding shift assay for the zinc-bound and zinc-free HIV-1 nucleocapsid protein by capillary electrophoresis.

Affinity capillary electrophoresis was used to detect a shift in mobility when a zinc ion binds to the highly basic nucleocapsid protein (NCp7) of HIV-1. NCp7 contains two Cys-X2- Cys-X4-His-X4-Cys zinc fingers. With constant concentrations of NCp7 as a receptor and various concentrations of zinc as a ligand in the sample buffer and the electrophoresis buffer, we observed changes in electrophoretic mobilities of NCp7 protein when complexes were formed with zinc. Scatchard analysis of the mobility indicates the presence of at least two types of binding sites for zinc. At pH 6.0, one site is shown to bind zinc strongly with a binding constant Kb = 3.25 x 10(5) M-1 and the second site has a Kb = 1.8 x 10(5) M-1. The binding of zinc to the first zinc finger decreased the affinity of zinc for the second zinc finger approximately twofold. The Hill coefficient for this negative cooperativity is 0.9. A series of NCp7 mutants were also examined in the assay to determine their ability to bind zinc. This assay affords a quick method to observe a zinc ion binding to NCp7 and to calculate binding constants.

Synthesis and biological activities of fluorescent acridine-containing HIV-1 nucleocapsid proteins for investigation of nucleic acid-NCp7 interactions.

Specific interactions between the 72-amino acid nucleocapsid protein NCp7 of the human immunodeficiency virus, type 1 and the genomic RNA are essential for virus replication. Studies on the mechanism of action of NCp7 require a direct visualization of its complexes with nucleic acids and the determination of binding affinities. To facilitate these investigations, fluorescent NCp7 derivatives were developed by introduction in the NCp7 sequence of a non-natural amino acid, (S)-beta-(9-acridinyl)alanine (Aca) obtained by a chiral synthetic method. Three fluorescent NCp7 derivatives were obtained by introducing this amino acid at different positions. As shown by NMR, the three-dimensional structure of NCp7 is not altered by introduction of Aca. The fluorescent peptides were found to be as potent as their precursors in interacting with nucleic acids and in promoting HIV-1 genomic RNA dimerization. Moreover, because of their fluorescent properties, these NCp7s can be used at submicromolar concentrations to directly visualize and quantify protein-nucleic acid interactions in solution or after gel electrophoresis. This could facilitate the development of new antiviral agents aimed at inhibiting the functions of NCp7 and studies on the intracellular traffic of NCp7 within the preintegration complex.

Identification of HTLV-1-specific CTL directed against synthetic and naturally processed peptides in HLA-B*3501 transgenic mice.

Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-l ysyl- lysyl-lysine, which is a biocompatible, Th-epitopeindependent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after three in vitro stimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gagp19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.

Chemically synthesised human immunodeficiency virus P7 nucleocapsid protein can self-assemble into particles and binds to a specific site on the tRNA(Lys,3) primer.

The zinc-bound form of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, p7, aggregates into particles visible by electron microscopy. The HIV primer tRNA(Lys,3) forms similar high molecular weight complexes with p7 that are also detected by gel mobility shift assays. RNA oligonucleotides of the three stem-loop structures in tRNA(Lys,3) were assayed for the competitive inhibition of p7-tRNA(Lys,3) binding by the intensities of free tRNA(Lys,3) bands on native gels. This reveals that the p7 binds specifically to the central domain of tRNA(Lys,3) where the D and T psi C loops come together, but not the anticodon stem-loop.