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Sample records for hiv-1 encoded peptide

  1. Specific binding of a basic peptide from HIV-1 Rev.

    PubMed Central

    Kjems, J; Calnan, B J; Frankel, A D; Sharp, P A

    1992-01-01

    Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents. Images PMID:1547776

  2. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    DOE PAGES

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; ...

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

  3. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    SciTech Connect

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.

  4. Albumin-conjugated C34 Peptide HIV-1 Fusion Inhibitor

    PubMed Central

    Stoddart, Cheryl A.; Nault, Geneviève; Galkina, Sofiya A.; Thibaudeau, Karen; Bakis, Peter; Bousquet-Gagnon, Nathalie; Robitaille, Martin; Bellomo, Maryanne; Paradis, Véronique; Liscourt, Patricia; Lobach, Alexandra; Rivard, Marie-Ève; Ptak, Roger G.; Mankowski, Marie K.; Bridon, Dominique; Quraishi, Omar

    2008-01-01

    Entry inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been the focus of much recent research. C34, a potent fusion inhibitor derived from the HR2 region of gp41, was engineered into a 1:1 human serum albumin conjugate through stable covalent attachment of a maleimido-C34 analog onto cysteine 34 of albumin. This bioconjugate, PC-1505, was designed to require less frequent dosing and less peptide than T-20 and was assessed for its antifusogenic activity both in vitro and in vivo in the SCID-hu Thy/Liv mouse model. PC-1505 was essentially equipotent to the original C34 peptide and to T-20 in vitro. In HIV-1-infected SCID-hu Thy/Liv mice, T-20 lost activity with infrequent dosing, whereas the antiviral potency of PC-1505 was sustained, and PC-1505 was active against T-20-resistant (“DIV”) virus with a G36D substitution in gp41. The in vivo results are the direct result of a significantly improved pharmacokinetic profile for the C34 peptide following albumin conjugation. Contrary to previous reports that the gp41 NHR trimer is poorly accessible to C34 fused to protein cargoes of increasing size (Hamburger, A. E., Kim, S., Welch, B. D., and Kay, M. S. (2005) J. Biol. Chem. 280, 12567–12572), these results are the first demonstration of the capacity for a large, endogenous serum protein to gain unobstructed access to the transient gp41 intermediates that exist during the HIV fusion process, and it supports further development of albumin conjugation as a promising approach to inhibit HIV-1 entry. PMID:18809675

  5. Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

    2015-04-21

    The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  6. Design and characterization of a peptide mimotope of the HIV-1 gp120 bridging sheet.

    PubMed

    Schiavone, Marco; Fiume, Giuseppe; Caivano, Antonella; de Laurentiis, Annamaria; Falcone, Cristina; Masci, Francesca Fasanella; Iaccino, Enrico; Mimmi, Selena; Palmieri, Camillo; Pisano, Antonio; Pontoriero, Marilena; Rossi, Annalisa; Scialdone, Annarita; Vecchio, Eleonora; Andreozzi, Concetta; Trovato, Maria; Rafay, Jan; Ferko, Boris; Montefiori, David; Lombardi, Angela; Morsica, Giulia; Poli, Guido; Quinto, Ileana; Pavone, Vincenzo; de Berardinis, Piergiuseppe; Scala, Giuseppe

    2012-01-01

    The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV(+) broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.

  7. Design of cell-permeable stapled peptides as HIV-1 integrase inhibitors.

    PubMed

    Long, Ya-Qiu; Huang, Shao-Xu; Zawahir, Zahrah; Xu, Zhong-Liang; Li, Huiyuan; Sanchez, Tino W; Zhi, Ying; De Houwer, Stephanie; Christ, Frauke; Debyser, Zeger; Neamati, Nouri

    2013-07-11

    HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions with the viral and cellular proteins. We have previously identified peptide IN inhibitors derived from the α-helical regions along the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon stapling of these peptides to stabilize their helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture. Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and cell permeability, these stapled peptides can serve as not only lead IN inhibitors but also prototypical biochemical probes or "nanoneedles" for the elucidation of HIV-1 IN dimerization and host cofactor interactions within their native cellular environment.

  8. Cell-permeable stapled peptides based on HIV-1 integrase inhibitors derived from HIV-1 gene products.

    PubMed

    Nomura, Wataru; Aikawa, Haruo; Ohashi, Nami; Urano, Emiko; Métifiot, Mathieu; Fujino, Masayuki; Maddali, Kasthuraiah; Ozaki, Taro; Nozue, Ami; Narumi, Tetsuo; Hashimoto, Chie; Tanaka, Tomohiro; Pommier, Yves; Yamamoto, Naoki; Komano, Jun A; Murakami, Tsutomu; Tamamura, Hirokazu

    2013-10-18

    HIV-1 integrase (IN) is an enzyme which is indispensable for the stable infection of host cells because it catalyzes the insertion of viral DNA into the genome and thus is an attractive target for the development of anti-HIV agents. Earlier, we found Vpr-derived peptides with inhibitory activity against HIV-1 IN. These Vpr-derived peptides are originally located in an α-helical region of the parent Vpr protein. Addition of an octa-arginyl group to the inhibitory peptides caused significant inhibition against HIV replication associated with an increase in cell permeability but also relatively high cytotoxicity. In the current study, stapled peptides, a new class of stabilized α-helical peptidomimetics were adopted to enhance the cell permeability of the above lead peptides. A series of stapled peptides, which have a hydrocarbon link formed by a ruthenium-catalyzed ring-closing metathesis reaction between successive turns of α-helix, were designed, synthesized, and evaluated for biological activity. In cell-based assays some of the stapled peptides showed potent anti-HIV activity comparable with that of the original octa-arginine-containing peptide (2) but with lower cytotoxicity. Fluorescent imaging experiments revealed that these stapled peptides are significantly cell permeable, and CD analysis showed they form α-helical structures, whereas the unstapled congeners form β-sheet structures. The application of this stapling strategy to Vpr-derived IN inhibitory peptides led to a remarkable increase in their potency in cells and a significant reduction of their cytotoxicity.

  9. Are Viral-Encoded MicroRNAs Mediating Latent HIV-1 Infection?

    PubMed Central

    WEINBERG, MARC S.; MORRIS, KEVIN V.

    2010-01-01

    The Human Immunodeficiency Virus type 1 (HIV-1), a member of the lentivirus subfamily, infects both dividing and nondividing cells and, following reverse transcription of the viral RNA genome, integrates into the host chromatin where it enters into a latent state. Many of the factors governing viral latency remain unresolved and current antiviral treatment regimens are largely ineffective at eliminating cellular reservoirs of latent virus. The recent identification of microRNA (miRNA) encoding sequences embedded in the HIV-1 genome, and the discovery of functional virus-derived miRNAs, suggests a role for RNA Interference (RNAi) in the regulation of HIV-1 gene expression. Recently, the mammalian RNAi machinery was shown to regulate gene expression epigenetically by transcriptional modulation, providing a direct link between RNAi and a mechanism for inducing latency. Interestingly, both HIV-1 Tat, and the host TAR RNA-binding protein (TRBP), bind to the transactivating response (TAR) RNA of HIV-1 and affect the function of RNAi in human cells. Specifically, TRBP, a cofactor in Tat-TAR interactions, is a vital component of Dicer-mediated dsRNA processing. These novel observations support a central role for HIV-1 and associated host factors in regulating cellular RNAi and viral gene expression through RNA directed processes. Thus, HIV-1 may have evolved mechanisms to exploit the RNAi pathway at both the transcriptional and posttranscriptional level to affect and/or maintain a latent infection. PMID:16629595

  10. Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

    PubMed

    Haußner, Christina; Damm, Dominik; Nirschl, Sandra; Rohrhofer, Anette; Schmidt, Barbara; Eichler, Jutta

    2017-01-26

    The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

  11. Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7

    PubMed Central

    Chen, Yaoqing; Cao, Luyang; Zhong, Maohua; Zhang, Yan; Han, Chen; Li, Qiaoli; Yang, Jingyi; Zhou, Dihan; Shi, Wei; He, Benxia; Liu, Fang; Yu, Jie; Sun, Ying; Cao, Yuan; Li, Yaoming; Li, Wenxin; Guo, Deying; Cao, Zhijian; Yan, Huimin

    2012-01-01

    For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC50 value of 2.76 µg/ml (1.65 µM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1. PMID:22536342

  12. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    SciTech Connect

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika; E-mail: hiokada@med.nagoya-cu.ac.jp

    2007-02-23

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1{sub IIIB} infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.

  13. Stapled HIV-1 Peptides Recapitulate Antigenic Structures and Engage Broadly Neutralizing Antibodies

    PubMed Central

    Bird, Gregory H.; Irimia, Adriana; Ofek, Gilad; Kwong, Peter D.; Wilson, Ian A.; Walensky, Loren D.

    2014-01-01

    Hydrocarbon stapling can restore bioactive, α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here, we explore the capacity of peptide stapling to generate high fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease-resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically-stabilized antigens for vaccination. PMID:25420104

  14. Stapled HIV-1 peptides recapitulate antigenic structures and engage broadly neutralizing antibodies.

    PubMed

    Bird, Gregory H; Irimia, Adriana; Ofek, Gilad; Kwong, Peter D; Wilson, Ian A; Walensky, Loren D

    2014-12-01

    Hydrocarbon stapling can restore bioactive α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here we explore the capacity of peptide stapling to generate high-fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane-proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically stabilized antigens for vaccination.

  15. DNA and modified vaccinia virus Ankara vaccines encoding multiple cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) are safe but weakly immunogenic in HIV-1-uninfected, vaccinia virus-naive adults.

    PubMed

    Gorse, Geoffrey J; Newman, Mark J; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C; Noonan, Elizabeth; Livingston, Brian D; Fuchs, Jonathan D; Kalams, Spyros A; Cassis-Ghavami, Farah L

    2012-05-01

    We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins.

  16. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase

    PubMed Central

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C.; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M.

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1–50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  17. HIV-1 fusion is blocked through binding of GB Virus C E2-derived peptides to the HIV-1 gp41 disulfide loop [corrected].

    PubMed

    Eissmann, Kristin; Mueller, Sebastian; Sticht, Heinrich; Jung, Susan; Zou, Peng; Jiang, Shibo; Gross, Andrea; Eichler, Jutta; Fleckenstein, Bernhard; Reil, Heide

    2013-01-01

    A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.

  18. HIV-1 Fusion Is Blocked through Binding of GB Virus C E2D Peptides to the HIV-1 gp41 Disulfide Loop

    PubMed Central

    Eissmann, Kristin; Mueller, Sebastian; Sticht, Heinrich; Jung, Susan; Zou, Peng; Jiang, Shibo; Gross, Andrea; Eichler, Jutta; Fleckenstein, Bernhard; Reil, Heide

    2013-01-01

    A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors. PMID:23349893

  19. HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma.

    PubMed

    Martellini, Julie A; Cole, Amy L; Svoboda, Pavel; Stuchlik, Olga; Chen, Li-Mei; Chai, Karl X; Gangrade, Bhushan K; Sørensen, Ole E; Pohl, Jan; Cole, Alexander M

    2011-01-20

    We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

  20. Mechanism of multivalent nanoparticle encounter with HIV-1 for potency enhancement of peptide triazole virus inactivation.

    PubMed

    Rosemary Bastian, Arangassery; Nangarlia, Aakansha; Bailey, Lauren D; Holmes, Andrew; Kalyana Sundaram, R Venkat; Ang, Charles; Moreira, Diogo R M; Freedman, Kevin; Duffy, Caitlin; Contarino, Mark; Abrams, Cameron; Root, Michael; Chaiken, Irwin

    2015-01-02

    Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.

  1. Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

    PubMed Central

    Chikaev, Anton N.; Bakulina, Anastasiya Yu.; Burdick, Ryan C.; Karpenko, Larisa I.; Pathak, Vinay K.; Ilyichev, Alexander A.

    2015-01-01

    The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies. PMID:25785734

  2. HIV-1 Drug Discovery: Targeting Folded RNA Structures With Branched Peptides

    PubMed Central

    Wynn, Jessica E.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) is an RNA virus that is prone to high rates of mutation. While the disease is managed with current antiretroviral therapies, drugs with a new mode of action are needed. A strategy towards this goal is aimed at targeting the native three-dimensional fold of conserved RNA structures. This perspective highlights medium-sized peptides and peptidomimetics used to target two conserved RNA structures of HIV-1. In particular, branched peptides have the capacity to bind in a multivalent fashion, utilizing a large surface area to achieve the necessary affinity and selectivity toward the target RNA. PMID:25958855

  3. Design, synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41.

    PubMed

    Tan, Jianjun; Su, Min; Zeng, Yi; Wang, Cunxin

    2016-01-15

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC50=53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus-cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.

  4. Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope.

    PubMed

    Palacios-Rodríguez, Yadira; Gazarian, Tatiana; Rowley, Merrill; Majluf-Cruz, Abraham; Gazarian, Karlen

    2007-02-01

    Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.

  5. Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors.

    PubMed

    Qasmi, D; de Rosny, E; René, L; Badet, B; Vergely, I; Boggetto, N; Reboud-Ravaux, M

    1997-04-01

    A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease. The N-glyoxylyl peptide CHOCO-Pro-Ile-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling. Increasing the length of the peptide sequence led to compounds acting only as substrates.

  6. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

    SciTech Connect

    Kong, Rui; Xu, Kai; Zhou, Tongqing; Acharya, Priyamvada; Lemmin, Thomas; Liu, Kevin; Ozorowski, Gabriel; Taft, Justin D.; Bailer, Robert T.; Cale, Evan M.; Chen, Lei; Choi, Chang W.; Chuang, Gwo-Yu; Doria-Rose, Nicole A.; Druz, Aliaksandr; Georgiev, Ivelin S.; Gorman, Jason; Huang, Jinghe; Joyce, M. Gordon; Louder, Mark K.; Ma, Xiaochu; McKee, Krisha; O'Dell, Sijy; Pancera, Marie; Yang, Yongping; Blanchard, Scott C.; Mothes, Walther; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Ward, Andrew B.; Mascola, John R.

    2016-05-13

    The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.

  7. A Truncated Nef Peptide from SIVcpz Inhibits the Production of HIV-1 Infectious Progeny

    PubMed Central

    Sabino Cunha, Marcela; Lima Sampaio, Thatiane; Peterlin, B. Matija; Jesus da Costa, Luciana

    2016-01-01

    Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity. PMID:27399760

  8. Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide.

    PubMed

    Deb, Arpan; Johnson, William A; Kline, Alexander P; Scott, Boston J; Meador, Lydia R; Srinivas, Dustin; Martin-Garcia, Jose M; Dörner, Katerina; Borges, Chad R; Misra, Rajeev; Hogue, Brenda G; Fromme, Petra; Mor, Tsafrir S

    2017-01-01

    Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

  9. Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide

    PubMed Central

    Deb, Arpan; Johnson, William A.; Kline, Alexander P.; Scott, Boston J.; Meador, Lydia R.; Srinivas, Dustin; Martin-Garcia, Jose M.; Dörner, Katerina; Borges, Chad R.; Misra, Rajeev; Hogue, Brenda G.; Fromme, Petra

    2017-01-01

    Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models. PMID:28225803

  10. Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

    PubMed

    Lottersberger, J; Salvetti, J L; Beltramini, L M; Tonarelli, G

    2004-01-01

    The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

  11. Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

    PubMed Central

    2010-01-01

    Background The presence of the cellular Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) protein is essential for integration of the Human immunodeficiency virus type 1 (HIV-1) cDNA and for efficient virus production. In the absence of LEDGF/p75 very little integration and virus production can be detected, as was demonstrated using LEDGF/p75-knokdown cells. Results Here we show that the failure to infect LEDGF/p75-knockdown cells has another reason aside from the lack of LEDGF/p75. It is also due to inhibition of the viral integrase (IN) enzymatic activity by an early expressed viral Rev protein. The formation of an inhibitory Rev-IN complex in virus-infected cells can be disrupted by the addition of three IN-derived, cell-permeable peptides, designated INr (IN derived-Rev interacting peptides) and INS (IN derived-integrase stimulatory peptide). The results of the present work confirm previous results showing that HIV-1 fails to infect LEDGF/p75-knockdown cells. However, in the presence of INrs and INS peptides, relatively high levels of viral cDNA integration as well as productive virus infection were obtained following infection by a wild type (WT) HIV-1 of LEDGF/p75-knockdown cells. Conclusions It appears that the lack of integration observed in HIV-1 infected LEDGF/p75-knockdown cells is due mainly to the inhibitory effect of Rev following the formation of a Rev-IN complex. Disruption of this inhibitory complex leads to productive infection in those cells. PMID:20678206

  12. Database-Guided Discovery of Potent Peptides to Combat HIV-1 or Superbugs.

    PubMed

    Wang, Guangshun

    2013-05-27

    Antimicrobial peptides (AMPs), small host defense proteins, are indispensable for the protection of multicellular organisms such as plants and animals from infection. The number of AMPs discovered per year increased steadily since the 1980s. Over 2,000 natural AMPs from bacteria, protozoa, fungi, plants, and animals have been registered into the antimicrobial peptide database (APD). The majority of these AMPs (>86%) possess 11-50 amino acids with a net charge from 0 to +7 and hydrophobic percentages between 31-70%. This article summarizes peptide discovery on the basis of the APD. The major methods are the linguistic model, database screening, de novo design, and template-based design. Using these methods, we identified various potent peptides against human immunodeficiency virus type 1 (HIV-1) or methicillin-resistant Staphylococcus aureus (MRSA). While the stepwise designed anti-HIV peptide is disulfide-linked and rich in arginines, the ab initio designed anti-MRSA peptide is linear and rich in leucines. Thus, there are different requirements for antiviral and antibacterial peptides, which could kill pathogens via different molecular targets. The biased amino acid composition in the database-designed peptides, or natural peptides such as θ-defensins, requires the use of the improved two-dimensional NMR method for structural determination to avoid the publication of misleading structure and dynamics. In the case of human cathelicidin LL-37, structural determination requires 3D NMR techniques. The high-quality structure of LL-37 provides a solid basis for understanding its interactions with membranes of bacteria and other pathogens. In conclusion, the APD database is a comprehensive platform for storing, classifying, searching, predicting, and designing potent peptides against pathogenic bacteria, viruses, fungi, parasites, and cancer cells.

  13. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

    DOE PAGES

    Kong, Rui; Xu, Kai; Zhou, Tongqing; ...

    2016-05-13

    The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showedmore » that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.« less

  14. The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation

    PubMed Central

    Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J.; Telenti, Amalio; de Bakker, Paul I.W.; Walker, Bruce D.; Jia, Xiaoming; McLaren, Paul J.; Ripke, Stephan; Brumme, Chanson J.; Pulit, Sara L.; Telenti, Amalio; Carrington, Mary; Kadie, Carl M.; Carlson, Jonathan M.; Heckerman, David; de Bakker, Paul I.W.; Pereyra, Florencia; de Bakker, Paul I.W.; Graham, Robert R.; Plenge, Robert M.; Deeks, Steven G.; Walker, Bruce D.; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M.; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P.; Guiducci, Candace; Gupta, Namrata; Carrington, Mary; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Pereyra, Florencia; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L.; Lemay, Paul; O’Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L.; Vine, Seanna; Addo, Marylyn M.; Allen, Todd M.; Altfeld, Marcus; Henn, Matthew R.; Le Gall, Sylvie; Streeck, Hendrik; Walker, Bruce D.; Haas, David W.; Kuritzkes, Daniel R.; Robbins, Gregory K.; Shafer, Robert W.; Gulick, Roy M.; Shikuma, Cecilia M.; Haubrich, Richard; Riddler, Sharon; Sax, Paul E.; Daar, Eric S.; Ribaudo, Heather J.; Agan, Brian; Agarwal, Shanu; Ahern, Richard L.; Allen, Brady L.; Altidor, Sherly; Altschuler, Eric L.; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J.; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C.; Benson, Anne M.; Berger, Judith; Bernard, Nicole F.; Bernard, Annette M.; Birch, Christopher; Bodner, Stanley J.; Bolan, Robert K.; Boudreaux, Emilie T.; Bradley, Meg; Braun, James F.; Brndjar, Jon E.; Brown, Stephen J.; Brown, Katherine; Brown, Sheldon T.; Burack, Jedidiah; Bush, Larry M.; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H.; Carmichael, J. Kevin; Casey, Kathleen K.; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T.; Chez, Nancy; Chirch, Lisa M.; Cimoch, Paul J.; Cohen, Daniel; Cohn, Lillian E.; Conway, Brian; Cooper, David A.; Cornelson, Brian; Cox, David T.; Cristofano, Michael V.; Cuchural, George; Czartoski, Julie L.; Dahman, Joseph M.; Daly, Jennifer S.; Davis, Benjamin T.; Davis, Kristine; Davod, Sheila M.; Deeks, Steven G.; DeJesus, Edwin; Dietz, Craig A.; Dunham, Eleanor; Dunn, Michael E.; Ellerin, Todd B.; Eron, Joseph J.; Fangman, John J.W.; Farel, Claire E.; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A.; French, Neel K.; Fuchs, Jonathan D.; Fuller, Jon D.; Gaberman, Jonna; Gallant, Joel E.; Gandhi, Rajesh T.; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C.; Gaultier, Cyril R.; Gebre, Wondwoosen; Gilman, Frank D.; Gilson, Ian; Goepfert, Paul A.; Gottlieb, Michael S.; Goulston, Claudia; Groger, Richard K.; Gurley, T. Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W. David; Harrigan, P. Richard; Hawkins, Trevor N.; Heath, Sonya; Hecht, Frederick M.; Henry, W. Keith; Hladek, Melissa; Hoffman, Robert P.; Horton, James M.; Hsu, Ricky K.; Huhn, Gregory D.; Hunt, Peter; Hupert, Mark J.; Illeman, Mark L.; Jaeger, Hans; Jellinger, Robert M.; John, Mina; Johnson, Jennifer A.; Johnson, Kristin L.; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C.; Kauffman, Carol A.; Khanlou, Homayoon; Killian, Robert K.; Kim, Arthur Y.; Kim, David D.; Kinder, Clifford A.; Kirchner, Jeffrey T.; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P. Todd; Kurisu, Wayne; Kwon, Douglas S.; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M.; Lee, David M.; Lee, Jean M.L.; Lee, Marah J.; Lee, Edward T.Y.; Lemoine, Janice; Levy, Jay A.; Llibre, Josep M.; Liguori, Michael A.; Little, Susan J.; Liu, Anne Y.; Lopez, Alvaro J.; Loutfy, Mono R.; Loy, Dawn; Mohammed, Debbie Y.; Man, Alan; Mansour, Michael K.; Marconi, Vincent C.; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N.; Martin, Harold L.; Mayer, Kenneth Hugh; McElrath, M. Juliana; McGhee, Theresa A.; McGovern, Barbara H.; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X.; Menezes, Prema; Mesa, Greg; Metroka, Craig E.; Meyer-Olson, Dirk; Miller, Andy O.; Montgomery, Kate; Mounzer, Karam C.; Nagami, Ellen H.; Nagin, Iris; Nahass, Ronald G.; Nelson, Margret O.; Nielsen, Craig; Norene, David L.; O’Connor, David H.; Ojikutu, Bisola O.; Okulicz, Jason; Oladehin, Olakunle O.; Oldfield, Edward C.; Olender, Susan A.; Ostrowski, Mario; Owen, William F.; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M.; Perlmutter, Aaron M.; Pierce, Michael N.; Pincus, Jonathan M.; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C.; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J.; Rhame, Frank S.; Richards, Constance Shamuyarira; Richman, Douglas D.; Robbins, Gregory K.; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C.; Rosenberg, Eric S.; Rosenthal, Daniel; Ross, Polly E.; Rubin, David S.; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R.; Sanchez, William C.; Sanjana, Veeraf M.; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M.; Shalit, Peter; Shay, William; Shirvani, Vivian N.; Silebi, Vanessa I.; Sizemore, James M.; Skolnik, Paul R.; Sokol-Anderson, Marcia; Sosman, James M.; Stabile, Paul; Stapleton, Jack T.; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F. Lisa; Stone, Valerie E.; Stone, David R.; Tambussi, Giuseppe; Taplitz, Randy A.; Tedaldi, Ellen M.; Telenti, Amalio; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A.; Trinh, Phuong D.; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J.; Vecino, Isabel; Vega, Vilma M.; Veikley, Wenoah; Wade, Barbara H.; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J.; Warner, Daniel A.; Weber, Robert D.; Webster, Duncan; Weis, Steve; Wheeler, David A.; White, David J.; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G.; Wout, Angelique van’t; Wright, David P.; Yang, Otto O.; Yurdin, David L.; Zabukovic, Brandon W.; Zachary, Kimon C.; Zeeman, Beth; Zhao, Meng

    2011-01-01

    Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA–viral peptide interaction as the major factor modulating durable control of HIV infection. PMID:21051598

  15. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

    PubMed

    Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J; Telenti, Amalio; de Bakker, Paul I W; Walker, Bruce D; Ripke, Stephan; Brumme, Chanson J; Pulit, Sara L; Carrington, Mary; Kadie, Carl M; Carlson, Jonathan M; Heckerman, David; Graham, Robert R; Plenge, Robert M; Deeks, Steven G; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P; Guiducci, Candace; Gupta, Namrata; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L; Lemay, Paul; O'Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L; Vine, Seanna; Addo, Marylyn M; Allen, Todd M; Altfeld, Marcus; Henn, Matthew R; Le Gall, Sylvie; Streeck, Hendrik; Haas, David W; Kuritzkes, Daniel R; Robbins, Gregory K; Shafer, Robert W; Gulick, Roy M; Shikuma, Cecilia M; Haubrich, Richard; Riddler, Sharon; Sax, Paul E; Daar, Eric S; Ribaudo, Heather J; Agan, Brian; Agarwal, Shanu; Ahern, Richard L; Allen, Brady L; Altidor, Sherly; Altschuler, Eric L; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C; Benson, Anne M; Berger, Judith; Bernard, Nicole F; Bernard, Annette M; Birch, Christopher; Bodner, Stanley J; Bolan, Robert K; Boudreaux, Emilie T; Bradley, Meg; Braun, James F; Brndjar, Jon E; Brown, Stephen J; Brown, Katherine; Brown, Sheldon T; Burack, Jedidiah; Bush, Larry M; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H; Carmichael, J Kevin; Casey, Kathleen K; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T; Chez, Nancy; Chirch, Lisa M; Cimoch, Paul J; Cohen, Daniel; Cohn, Lillian E; Conway, Brian; Cooper, David A; Cornelson, Brian; Cox, David T; Cristofano, Michael V; Cuchural, George; Czartoski, Julie L; Dahman, Joseph M; Daly, Jennifer S; Davis, Benjamin T; Davis, Kristine; Davod, Sheila M; DeJesus, Edwin; Dietz, Craig A; Dunham, Eleanor; Dunn, Michael E; Ellerin, Todd B; Eron, Joseph J; Fangman, John J W; Farel, Claire E; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A; French, Neel K; Fuchs, Jonathan D; Fuller, Jon D; Gaberman, Jonna; Gallant, Joel E; Gandhi, Rajesh T; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C; Gaultier, Cyril R; Gebre, Wondwoosen; Gilman, Frank D; Gilson, Ian; Goepfert, Paul A; Gottlieb, Michael S; Goulston, Claudia; Groger, Richard K; Gurley, T Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W David; Harrigan, P Richard; Hawkins, Trevor N; Heath, Sonya; Hecht, Frederick M; Henry, W Keith; Hladek, Melissa; Hoffman, Robert P; Horton, James M; Hsu, Ricky K; Huhn, Gregory D; Hunt, Peter; Hupert, Mark J; Illeman, Mark L; Jaeger, Hans; Jellinger, Robert M; John, Mina; Johnson, Jennifer A; Johnson, Kristin L; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C; Kauffman, Carol A; Khanlou, Homayoon; Killian, Robert K; Kim, Arthur Y; Kim, David D; Kinder, Clifford A; Kirchner, Jeffrey T; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P Todd; Kurisu, Wayne; Kwon, Douglas S; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M; Lee, David M; Lee, Jean M L; Lee, Marah J; Lee, Edward T Y; Lemoine, Janice; Levy, Jay A; Llibre, Josep M; Liguori, Michael A; Little, Susan J; Liu, Anne Y; Lopez, Alvaro J; Loutfy, Mono R; Loy, Dawn; Mohammed, Debbie Y; Man, Alan; Mansour, Michael K; Marconi, Vincent C; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N; Martin, Harold L; Mayer, Kenneth Hugh; McElrath, M Juliana; McGhee, Theresa A; McGovern, Barbara H; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X; Menezes, Prema; Mesa, Greg; Metroka, Craig E; Meyer-Olson, Dirk; Miller, Andy O; Montgomery, Kate; Mounzer, Karam C; Nagami, Ellen H; Nagin, Iris; Nahass, Ronald G; Nelson, Margret O; Nielsen, Craig; Norene, David L; O'Connor, David H; Ojikutu, Bisola O; Okulicz, Jason; Oladehin, Olakunle O; Oldfield, Edward C; Olender, Susan A; Ostrowski, Mario; Owen, William F; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M; Perlmutter, Aaron M; Pierce, Michael N; Pincus, Jonathan M; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J; Rhame, Frank S; Richards, Constance Shamuyarira; Richman, Douglas D; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C; Rosenberg, Eric S; Rosenthal, Daniel; Ross, Polly E; Rubin, David S; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R; Sanchez, William C; Sanjana, Veeraf M; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M; Shalit, Peter; Shay, William; Shirvani, Vivian N; Silebi, Vanessa I; Sizemore, James M; Skolnik, Paul R; Sokol-Anderson, Marcia; Sosman, James M; Stabile, Paul; Stapleton, Jack T; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F Lisa; Stone, Valerie E; Stone, David R; Tambussi, Giuseppe; Taplitz, Randy A; Tedaldi, Ellen M; Telenti, Amalio; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A; Trinh, Phuong D; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J; Vecino, Isabel; Vega, Vilma M; Veikley, Wenoah; Wade, Barbara H; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J; Warner, Daniel A; Weber, Robert D; Webster, Duncan; Weis, Steve; Wheeler, David A; White, David J; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G; van't Wout, Angelique; Wright, David P; Yang, Otto O; Yurdin, David L; Zabukovic, Brandon W; Zachary, Kimon C; Zeeman, Beth; Zhao, Meng

    2010-12-10

    Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

  16. Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA

    PubMed Central

    Harwig, Alex; Jongejan, Aldo; van Kampen, Antoine H. C.; Berkhout, Ben; Das, Atze T.

    2016-01-01

    Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3′ side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome. PMID:26984525

  17. Analysis of a subclass-restricted HIV-1 gp41 epitope by omission peptides.

    PubMed Central

    Mathiesen, T; Chiodi, F; Broliden, P A; Albert, J; Houghten, R A; Utter, G; Wahren, B; Norrby, E

    1989-01-01

    To define the amino acids involved in IgG subclass reactivity to two overlapping HIV-1 gp41 (E34/32; amino acid positions 582-613) peptides, sera from 18 HIV-infected individuals were studied. Peptides mimicking E34 but with single amino acid deletions or glycine substitutions were used to define the amino acid residues necessary for antibody binding. Two dominating immunogenic epitopes, containing highly hydrophilic amino acids, were found on the original peptide. Further analysis was undertaken with two corresponding omission sets of dodecapeptides representing halves of the complete E34 plus a terminal cystein peptide. The subclass reactivities usually differed between the patients with regard to the epitopes with which the different IgG subclasses reacted and also to the importance of different amino acids in antibody binding. The 600 glycine and the 601 lysine were involved in the binding of all IgG1, 2 and 4 and most IgG3. The development of E34/32-reactive IgM and IgG subclasses showed different patterns in four patients with primary HIV infections, contradicting the existence of a general pattern for the development of IgG subclasses to this peptide. The findings suggest that different progenitor clones are selected for synthesis of the different subclasses. PMID:2472353

  18. A survey of synthetic HIV-1 peptides with natural and chimeric sequences for differential reactivity with Zimbabwean, Tanzanian and Swedish HIV-1-positive sera.

    PubMed

    Blomberg, J; Lawoko, A; Pipkorn, R; Moyo, S; Malmvall, B E; Shao, J; Dash, R; Tswana, S

    1993-06-01

    The objective of this study was to determine whether the known sequence differences between African and non-African HIV-1 strains are reflected in the serological response. The authors investigated the antibody reactivity of 34 Swedish, 30 Tanzanian, and 42 Zimbabwean HIV-1 positive sera to 67 synthetic peptides with sequences from North American and African HIV-1 isolates, mostly derived from regions of gag and env known to be antigenic. Not all sera were tested against all peptides. The authors noted several results. Differences in frequency of reactivity were noted with peptides covering the entire third variable domain (V3), which is a primary neutralization determinant, and the carboxyl terminus of gp120, in 2 regions of gp41, and the carboxyl terminus of p24. In env, Tanzanian sera reacted preferentially with a V3 peptide from the strain JY1 (Zaire). Gradual substitutions in the central motif in V3 of ELI from GLGQ to GPGR, typical of many non-African strains, led to a gradual increase in reactivity of many Swedish sera, but did not affect Tanzanian and Zimbabwean sera, suggesting that the major epitopes recognized by these African sera are outside GPGR. V3 peptides from the MN and Z3 strains reacted with most sera, but missed 30% of those of Tanzanian origin. In the carboxyl terminus of gp120, both sets of African sera reacted preferentially with peptides from strains JY1 and MAL. Swedish sera reacted strongest with analogues from strains Z321 and HXB2. In gp41, Swedish sera showed a weak preference for reactivity with HXB2-derived peptides in the immunodominant region (amino acids 620-665). The differences in serological reactivity were as great between Zimbabwe and Tanzania as between the 2 African sets and the Swedish. The geographical differences in the pattern of reactivity with HIV peptides probably depend on both host and viral variation and may be developed into a seroepidemiological tool, useful for optimization of future HIV vaccines.

  19. The HIV-1 gp41 ectodomain is cleaved by matriptase to produce a chemotactic peptide that acts through FPR2.

    PubMed

    Wood, Matthew P; Cole, Amy L; Eade, Colleen R; Chen, Li-Mei; Chai, Karl X; Cole, Alexander M

    2014-07-01

    Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. Whereas this chemotactic activity has been reported, it is not known how these peptides could be produced under biological conditions. The heptad repeat 1 (HR1) region of gp41 is exposed to the extracellular environment and could therefore be susceptible to proteolytic processing into smaller peptides. Matriptase is a serine protease expressed at the surface of most epithelia, including the prostate and mucosal surfaces. Here, we present evidence that matriptase efficiently cleaves the HR1 portion of gp41 into a 22-residue chemotactic peptide MAT-1, the sequence of which is highly conserved across HIV-1 clades. We found that MAT-1 induced migration of primary neutrophils and monocytes, the latter of which act as a cellular reservoir of HIV during early stage infection. We then used formyl peptide receptor 1 (FPR1) and FPR2 inhibitors, along with HEK 293 cells, to demonstrate that MAT-1 can induce chemotaxis specifically using FPR2, a receptor found on the surface of monocytes, macrophages and neutrophils. These findings are the first to identify a proteolytic cleavage product of gp41 with chemotactic activity and highlight a potential role for matriptase in HIV-1 transmission and infection at epithelial surfaces and within tissue reservoirs of HIV-1.

  20. Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions

    PubMed Central

    2013-01-01

    Background We examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity. Results KR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions. Conclusions The antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion. PMID:24330857

  1. Identification of peptides from human pathogens able to cross-activate an HIV-1-gag-specific CD4+ T cell clone.

    PubMed

    Venturini, Sara; Allicotti, Gina; Zhao, Yindong; Simon, Richard; Burton, Dennis R; Pinilla, Clemencia; Poignard, Pascal

    2006-01-01

    Antigen recognition by T cells is degenerate both at the MHC and the TCR level. In this study, we analyzed the cross-reactivity of a human HIV-1 gag p24-specific CD4(+) T cell clone obtained from an HIV-1-seronegative donor using a positional scanning synthetic combinatorial peptide library (PS-SCL)-based biometrical analysis. A number of decapeptides able to activate the HIV-1 gag-specific clone were identified and shown to correspond to sequences found in other human pathogens. Two of these peptides activated the T cell clone with the same stimulatory potency as the original HIV-1 gag p24 peptide. These findings show that an HIV-1-specific human T helper clone can react efficiently with peptides from other pathogens and suggest that cellular immune responses identified as being specific for one human pathogen (HIV-1) could arise from exposure to other pathogens.

  2. A novel bispecific peptide HIV-1 fusion inhibitor targeting the N-terminal heptad repeat and fusion peptide domains in gp41.

    PubMed

    Jiang, Xifeng; Jia, Qiyan; Lu, Lu; Yu, Fei; Zheng, Jishen; Shi, Weiguo; Cai, Lifeng; Jiang, Shibo; Liu, Keliang

    2016-12-01

    HIV-1 fusion with the target cell is initiated by the insertion of the gp41 fusion peptide (FP) into the target cell membrane and the interaction between the gp41 N- and C-terminal heptad repeats (NHR and CHR), followed by the formation of the six-helix bundle (6-HB) fusion core. Therefore, both FP and NHR are important targets for HIV-1 fusion inhibitors. Here, we designed and synthesized a dual-target peptidic HIV-1 fusion inhibitor, 4HR-LBD-VIRIP, in which 4HR-LBD is able to bind to the gp41 NHR domain, while VIRIP is able to interact with gp41 FP. We found that 4HR-LBD-VIRIP is about tenfold more potent than 4HR-LBD and VIRIP in inhibiting HIV-1IIIB infection and HIV-1 envelope glycoprotein (Env)-mediated cell-cell fusion, suggesting that this dual-target HIV-1 fusion inhibitor possesses a strong synergistic antiviral effect. A biophysical analysis indicates that 4HR-LBD-VIRIP can interact with N70 peptide that contains the gp41 NHR and FP domains and binds with lipid membrane. This study provides a new approach for designing novel viral fusion inhibitors against HIV and other enveloped viruses with class I membrane fusion proteins.

  3. Ribosomally encoded cyclic peptide toxins from mushrooms.

    PubMed

    Walton, Jonathan D; Luo, Hong; Hallen-Adams, Heather

    2012-01-01

    The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis.

  4. The gp41659 - 671 HIV-1 antibody epitope: a structurally challenging small peptide

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Sagui, Celeste

    2014-03-01

    We present the results of extensive Molecular Dynamics (MD) simulations of the tridecapeptide corresponding to residues 659-671 of the envelope glycoprotein gp41 of HIV-1, which spans the 2F5 monoclonal antibody epitope ELDKWA. The most recent AMBER force fields ff99SB and ff12SB in both implicit and explicit solvents have been used for a cumulative time longer than 7.2 μs . We have analyzed the conformational ensembles of the peptide both with and without applied tensile restraints, and found that: (1) The amount of helical populations is important in aqueous solution, but this structure forms part of a flexible conformational ensemble with a rugged free energy landscape with shallow minima, which agrees well with the bulk of the experimental observations; (2) our results are more consistent with the experimental results than those from previous simulations; (3) under uniaxial tension, the disordered peptide first becomes fully helical before melting into turns, loops and 310-helices.

  5. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

    PubMed Central

    Pardi, Norbert; Secreto, Anthony J.; Shan, Xiaochuan; Debonera, Fotini; Glover, Joshua; Yi, Yanjie; Muramatsu, Hiromi; Ni, Houping; Mui, Barbara L.; Tam, Ying K.; Shaheen, Farida; Collman, Ronald G.; Karikó, Katalin; Danet-Desnoyers, Gwenn A.; Madden, Thomas D.; Hope, Michael J.; Weissman, Drew

    2017-01-01

    Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. PMID:28251988

  6. Encoded libraries of chemically modified peptides.

    PubMed

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries.

  7. Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition.

    PubMed

    Galatola, R; Cruz, A; Gómara, M J; Prat, J; Alsina, M A; Haro, I; Pujol, M

    2015-02-01

    The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30 mN m(-1)) and the HIV-1 FP (35 mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (GE). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive GE values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP-lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.

  8. Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera.

    PubMed

    Lawoko, A L; Johansson, B; Hjalmarsson, S; Christensson, B; Ljungberg, B; Al-Khalili, L; Sjölund, M; Pipkorn, R; Fenyö, E M; Blomberg, J

    1999-10-01

    IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.

  9. Combination of the CCL5-Derived Peptide R4.0 with Different HIV-1 Blockers Reveals Wide Target Compatibility and Synergic Cobinding to CCR5

    PubMed Central

    Secchi, Massimiliano; Vassena, Lia; Morin, Sébastien; Schols, Dominique

    2014-01-01

    R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission. PMID:25114130

  10. Combination of the CCL5-derived peptide R4.0 with different HIV-1 blockers reveals wide target compatibility and synergic cobinding to CCR5.

    PubMed

    Secchi, Massimiliano; Vassena, Lia; Morin, Sébastien; Schols, Dominique; Vangelista, Luca

    2014-10-01

    R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission.

  11. Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure.

    PubMed

    Gorny, Miroslaw K; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng

    2011-01-01

    Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

  12. Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.

    PubMed

    Dervillez, Xavier; Klaukien, Volker; Dürr, Ralf; Koch, Joachim; Kreutz, Alexandra; Haarmann, Thomas; Stoll, Michaela; Lee, Donghan; Carlomagno, Teresa; Schnierle, Barbara; Möbius, Kalle; Königs, Christoph; Griesinger, Christian; Dietrich, Ursula

    2010-10-01

    During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.

  13. Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA.

    PubMed

    Takahashi, T; Hamasaki, K; Ueno, A; Mihara, H

    2001-04-01

    In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.

  14. Reorientation of the helix of the tryptophan-rich gp41W peptide from HIV-1 at interfaces

    NASA Astrophysics Data System (ADS)

    Matar, Gladys; Benichou, Emmanuel; Nasir, Mehmet Nail; Harfouch, Yara El; Brevet, Pierre-François; Besson, Françoise

    2013-12-01

    The glycoprotein gp41 from the Human Immunodeficiency Virus type 1 (HIV-1) has an amino acid sequence enriched in tryptophan residues, the so-called gp41W peptide (i.e., KWASLWNWFNITNWLWYIK) and plays a crucial role in HIV-1 host cell infection. Using the coupling of Second Harmonic Generation targeting the tryptophan residues with lateral surface tension measurements, we investigate the interaction of gp41W with a neat air/water and a lipid/water interfaces. At the air/water interface, gp41W presents a well-defined orientation and this orientation is strongly modified at the lipid/water interface, depending on the surface pressure. These results show that this strategy is well suited to monitor tryptophan containing α-helices orientation at lipid/water interfaces.

  15. Nucleic acids encoding modified human immunodeficiency virus type 1 (HIV-1) group M consensus envelope glycoproteins

    DOEpatents

    Haynes, Barton F [Durham, NC; Gao, Feng [Durham, NC; Korber, Bette T [Los Alamos, NM; Hahn, Beatrice H [Birmingham, AL; Shaw, George M [Birmingham, AL; Kothe, Denise [Birmingham, AL; Li, Ying Ying [Hoover, AL; Decker, Julie [Alabaster, AL; Liao, Hua-Xin [Chapel Hill, NC

    2011-12-06

    The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralizes a wide spectrum of HIV primary isolates and/or to an immunogen that induces a T cell immune response. The invention also relates to a method of inducing anti-HIV antibodies, and/or to a method of inducing a T cell immune response, using such an immunogen. The invention further relates to nucleic acid sequences encoding the present immunogens.

  16. Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid.

    PubMed

    Bhattacharya, Shibani; Zhang, Hongtao; Debnath, Asim K; Cowburn, David

    2008-06-13

    The human immunodeficiency virus type 1 (HIV-1) capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study, we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1, for the capsid protein (K(d) approximately 1 mum) and demonstrated its ability to penetrate the cell membrane (Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., Heck, S., Goger, M., Cowburn, D., Freed, E. O., and Debnath, A. K. (2008) J. Mol. Biol. 378, 565-580). In cell-based assays, NYAD-1 colocalized with the Gag polyprotein during traffic to the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent-exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly.

  17. Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus

    PubMed Central

    Haggarty, Beth S.; Duong, Jennifer; Jordon, Andrea P. O.; Romano, Josephine; DeClercq, Joshua J.; Gregory, Philip D.; Riley, James L.; Holmes, Michael C.

    2016-01-01

    HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans. PMID:27855210

  18. Inhibition of HIV-1 enhancer-controlled transcription by artificial enhancer-binding peptides derived from bacteriophage 434 repressor.

    PubMed

    Caderas, G; Klauser, S; Liu, N; Bienz, A; Gutte, B

    1999-12-01

    An artificial HIV-1 enhancer-binding 42-residue peptide (R42) that had been derived from bacteriophage 434 repressor inhibited the cell-free in vitro transcription of HIV-1 enhancer-containing plasmids [Hehlgans, T., Stolz, M., Klauser, S., Cui, T., Salgam, P., Brenz Verca, S., Widmann, M., Leiser, A., Städler, K. & Gutte, B. (1993) FEBS Lett. 315, 51-55; Caderas, G. (1997) PhD Thesis, University of Zürich]. Here we show that, after N-terminal extension of R42 with a viral nuclear localization signal, the resulting nucR42 peptide was active in intact cells. NucR42 could be detected immunologically in nuclear extracts and produced a 60-70% reduction of the rate of transcription of an HIV-1 enhancer-carrying plasmid in COS-1 cells that had been cotransfected with the HIV enhancer plasmid, an expression plasmid for nucR42, and a control. NucR42 was also synthesized chemically and the synthetic product characterized by HPLC, mass spectrometry, and quantitative amino acid analysis. Band shift, footprint, and in vitro transcription assays in the presence of exogenous NF-kappaBp50 indicated that the binding sites of nucR42 and NF-kappaB on the HIV enhancers overlapped and that a relatively small excess of nucR42 sufficed to displace NF-kappaBp50. Band shift and in vitro transcription experiments showed also that exchange of the 434 repressor-derived nine-residue recognition helix of nucR42 for four glycines abolished the HIV enhancer binding specificity whereas leucine zipper- or retro-leucine zipper-mediated dimerization of R42 analogues increased it suggesting the potential application of such dimeric HIV enhancer-binding peptides as intracellular inhibitors of HIV replication.

  19. Site-Specific Polymer Attachment to HR2 Peptide Fusion Inhibitors against HIV-1 Decreases Binding Association Rates and Dissociation Rates Rather Than Binding Affinity.

    PubMed

    Danial, Maarten; Stauffer, Angela N; Wurm, Frederik R; Root, Michael J; Klok, Harm-Anton

    2017-03-15

    A popular strategy for overcoming the limited plasma half-life of peptide heptad repeat 2 (HR2) fusion inhibitors against HIV-1 is conjugation with biocompatible polymers such as poly(ethylene glycol) (PEG). However, despite improved resistance to proteolysis and reduced renal elimination, covalent attachment of polymers often causes a loss in therapeutic potency. In this study, we investigated the molecular origins of the loss in potency upon conjugation of linear, midfunctional, and hyperbranched PEG-like polymers to peptides that inhibit HIV-1-host cell membrane fusion. Fluorescence binding assays revealed that polymer conjugation imparted mass transport limitations that manifested as coexistent slower association and dissociation rates from the gp41 target on HIV-1. Furthermore, reduced association kinetics rather than affinity disruption was responsible for the loss in antiviral potency. Finally, the binding assays indicated that the unmodified HR2-derived peptide demonstrated diffusion-limited binding. The observed high potency of the unmodified peptide in HIV-1 inhibition assays was therefore attributed to rapid peptide conformational changes upon binding to the gp41 prehairpin structure. This study emphasizes that the view in which polymer ligation to therapeutic peptides inadvertently leads to loss in potency due to a loss in binding affinity requires scientific verification on a case-by-case basis and that high peptide potency may be due to rapid target-binding events.

  20. HIV-1 replication.

    PubMed

    Freed, E O

    2001-11-01

    In general terms, the replication cycle of lentiviruses, including HIV-1, closely resembles that of other retroviruses. There are, however, a number of unique aspects of HIV replication; for example, the HIVs and SIVs target receptors and coreceptors distinct from those used by other retroviruses. Lentiviruses encode a number of regulatory and accessory proteins not encoded by the genomes of the prototypical "simple" retroviruses. Of particular interest from the gene therapy perspective, lentiviruses possess the ability to productively infect some types of non-dividing cells. This chapter, while reiterating certain points discussed in Chapter 1, will attempt to focus on issues unique to HIV-1 replication. The HIV-1 genome encodes the major structural and non-structural proteins common to all replication-competent retroviruses (Fig. 1, and Chapter 1). From the 5'- to 3'-ends of the genome are found the gag (for group-specific antigen), pol (for polymerase), and env (for envelope glycoprotein) genes. The gag gene encodes a polyprotein precursor whose name, Pr55Gag, is based on its molecular weight. Pr55Gag is cleaved by the viral protease (PR) to the mature Gag proteins matrix (also known as MA or p17), capsid (CA or p24), nucleocapsid (NC or p7), and p6. Two spacer peptides, p2 and p1, are also generated upon Pr55Gag processing. The pol-encoded enzymes are initially synthesized as part of a large polyprotein precursor, Pr160GagPol, whose synthesis results from a rare frameshifting event during Pr55Gag translation. The individual pol-encoded enzymes, PR, reverse transcriptase (RT), and integrase (IN), are cleaved from Pr160GagPol by the viral PR. The envelope (Env) glycoproteins are also synthesized as a polyprotein precursor (Fig. 1). Unlike the Gag and Pol precursors, which are cleaved by the viral PR, the Env precursor, known as gp160, is processed by a cellular protease during Env trafficking to the cell surface, gp160 processing results in the generation of the

  1. Secretion modification region-derived peptide disrupts HIV-1 Nef's interaction with mortalin and blocks virus and Nef exosome release.

    PubMed

    Shelton, Martin N; Huang, Ming-Bo; Ali, Syed A; Powell, Michael D; Bond, Vincent C

    2012-01-01

    Nef is secreted from infected cells in exosomes and is found in abundance in the sera of HIV-infected individuals. Secreted exosomal Nef (exNef) induces apoptosis in uninfected CD4⁺ T cells and may be a key component of HIV pathogenesis. The exosomal pathway has been implicated in HIV-1 virus release, suggesting a possible link between these two viral processes. However, the underlying mechanisms and cellular components of exNef secretion have not been elucidated. We have previously described a Nef motif, the secretion modification region (SMR; amino acids 66 to 70), that is required for exNef secretion. In silico modeling data suggest that this motif can form a putative binding pocket. We hypothesized that the Nef SMR binds a cellular protein involved in protein trafficking and that inhibition of this interaction would abrogate exNef secretion. By using tandem mass spectrometry and coimmunoprecipitation with a novel SMR-based peptide (SMRwt) that blocks exNef secretion and HIV-1 virus release, we identified mortalin as an SMR-specific cellular protein. A second set of coimmunoprecipitation experiments with full-length Nef confirmed that mortalin interacts with Nef via Nef's SMR motif and that this interaction is disrupted by the SMRwt peptide. Overexpression and microRNA knockdown of mortalin revealed a positive correlation between exNef secretion levels and mortalin protein expression. Using antibody inhibition we demonstrated that the Nef/mortalin interaction is necessary for exNef secretion. Taken together, this work constitutes a significant step in understanding the underlying mechanism of exNef secretion, identifies a novel host-pathogen interaction, and introduces an HIV-derived peptide with antiviral properties.

  2. An extended CCR5-ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions

    PubMed Central

    Kessler, Naama; Arshava, Boris; Naider, Fred; Scherf, Tali; Anglister, Jacob

    2015-01-01

    The chemokine receptor CCR5 serves as a co-receptor for the Human Immunodefficiency Virus type-1, HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing the ECL1 and ECL3 were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study we determined that in aqueous buffer the segment Q188-Q194 in an elongated ECL2 peptide (R168 to K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two dimensional Saturation Transfer Difference NMR spectroscopy and dynamic filtering studies revealed the involvement of Y187, F189, W190 and F193 of the helical segment, in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule and therefore, could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. PMID:25703038

  3. Sequence conservation, HLA-E-Restricted peptide, and best-defined CTL/CD8+ epitopes in gag P24 (capsid) of HIV-1 subtype B

    NASA Astrophysics Data System (ADS)

    Prasetyo, Afiono Agung; Dharmawan, Ruben; Sari, Yulia; Sariyatun, Ratna

    2017-02-01

    Human immunodeficiency virus type 1 (HIV-1) remains a cause of global health problem. Continuous studies of HIV-1 genetic and immunological profiles are important to find strategies against the virus. This study aimed to conduct analysis of sequence conservation, HLA-E-restricted peptide, and best-defined CTL/CD8+ epitopes in p24 (capsid) of HIV-1 subtype B worldwide. The p24-coding sequences from 3,557 HIV subtype B isolates were aligned using MUSCLE and analysed. Some highly conserved regions (sequence conservation ≥95%) were observed. Two considerably long series of sequences with conservation of 100% was observed at base 349-356 and 550-557 of p24 (HXB2 numbering). The consensus from all aligned isolates was precisely the same as consensus B in the Los Alamos HIV Database. The HLA-E-restricted peptide in amino acid (aa) 14-22 of HIV-1 p24 (AISPRTLNA) was found in 55.9% (1,987/3,557) of HIV-1 subtype B worldwide. Forty-four best-defined CTL/CD8+ epitopes were observed, in which VKNWMTETL epitope (aa 181-189 of p24) restricted by B*4801 was the most frequent, as found in 94.9% of isolates. The results of this study would contribute information about HIV-1 subtype B and benefits for further works willing to develop diagnostic and therapeutic strategies against the virus.

  4. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    SciTech Connect

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-07-05

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  5. An HIV-1 antibody from an elite neutralizer implicates the fusion peptide as a site of vulnerability.

    PubMed

    van Gils, Marit J; van den Kerkhof, Tom L G M; Ozorowski, Gabriel; Cottrell, Christopher A; Sok, Devin; Pauthner, Matthias; Pallesen, Jesper; de Val, Natalia; Yasmeen, Anila; de Taeye, Steven W; Schorcht, Anna; Gumbs, Stephanie; Johanna, Inez; Saye-Francisco, Karen; Liang, Chi-Hui; Landais, Elise; Nie, Xiaoyan; Pritchard, Laura K; Crispin, Max; Kelsoe, Garnett; Wilson, Ian A; Schuitemaker, Hanneke; Klasse, Per Johan; Moore, John P; Burton, Dennis R; Ward, Andrew B; Sanders, Rogier W

    2016-11-14

    The induction by vaccination of broadly neutralizing antibodies (bNAbs) capable of neutralizing various HIV-1 viral strains is challenging, but understanding how a subset of HIV-infected individuals develops bNAbs may guide immunization strategies. Here, we describe the isolation and characterization of the bNAb ACS202 from an elite neutralizer that recognizes a new, trimer-specific and cleavage-dependent epitope at the gp120-gp41 interface of the envelope glycoprotein (Env), involving the glycan N88 and the gp41 fusion peptide. In addition, an Env trimer, AMC011 SOSIP.v4.2, based on early virus isolates from the same elite neutralizer, was constructed, and its structure by cryo-electron microscopy at 6.2 Å resolution reveals a closed, pre-fusion conformation similar to that of the BG505 SOSIP.664 trimer. The availability of a native-like Env trimer and a bNAb from the same elite neutralizer provides the opportunity to design vaccination strategies aimed at generating similar bNAbs against a key functional site on HIV-1.

  6. Durable cytotoxic immune responses against gp120 elicited by recombinant SV40 vectors encoding HIV-1 gp120 +/- IL-15.

    PubMed

    McKee, Hayley J; T'sao, Patricia Y; Vera, Maria; Fortes, Puri; Strayer, David S

    2004-08-23

    BACKGROUND: A vaccine that elicits durable, powerful anti-HIV immunity remains an elusive goal. In these studies we tested whether multiple treatments with viral vector-delivered HIV envelope antigen (gp120), with and without IL-15, could help to approach that goal. For this purpose, we used recombinant Tag-deleted SV40-derived vectors (rSV40s), since they do not elicit neutralizing antibody responses, and so can be given multiply without loss of transduction efficiency. METHODS: SV(gp120) carried the coding sequences for HIV-1NL4-3 Env, and SV(mIL-15) carried the cDNA for mouse IL-15. Singly, and in combination, these two vectors were given monthly to BALB/cJ mice. Cytotoxic immunity and cytotoxic memory were tested in direct cytotoxicity assays using unselected effector cells. Antibody vs. gp120 was measured in a binding assay. In both cases, targets were P815 cells that were stably transfected with gp120. RESULTS: Multiple injections of SV(gp120) elicited powerful anti-gp120 cytolytic activity (>70% specific lysis) by unselected spleen cells. Cells from multiply-immunized mice that were rested 1 year after their last injections still showed >60% gp120-specific lysis. Anti-gp120 antibody was first detected after 2 monthly injections of SV(gp120) and remained elevated thereafter. Adding SV(mIL-15) to the immunization regimen dramatically accelerated the development of memory cytolytic responses, with >/= 50% specific lysis seen 1 month after two treatments. IL-15 did not alter the development of antibody responses. CONCLUSIONS: Thus, rSV40s encoding antigens and immunostimulatory cytokines may be useful tools for priming and/or boosting immune responses against HIV.

  7. Structure and Immunogenicity of a Peptide Vaccine, Including the Complete HIV-1 gp41 2F5 Epitope

    PubMed Central

    Serrano, Soraya; Araujo, Aitziber; Apellániz, Beatriz; Bryson, Steve; Carravilla, Pablo; de la Arada, Igor; Huarte, Nerea; Rujas, Edurne; Pai, Emil F.; Arrondo, José L. R.; Domene, Carmen; Jiménez, María Angeles; Nieva, José L.

    2014-01-01

    The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development. PMID:24429284

  8. Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein

    SciTech Connect

    Davidson, Amy; Leeper, Thomas C.; Athanassiou, Zafiria; Patora-Komisarska, Krystyna; Karn, Jonathan; Robinson, John A.; Varani, Gabriele

    2009-07-21

    The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide–RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.

  9. Gene therapy with plasmids encoding IFN-β or IFN-α14 confers long-term resistance to HIV-1 in humanized mice

    PubMed Central

    Abraham, Sojan; Choi, Jang-Gi; Ortega, Nora M.; Zhang, Junli; Shankar, Premlata; Swamy, N. Manjunath

    2016-01-01

    Because endogenous interferon type I (IFN-I) produced by HIV-1 infection might complicate the analysis of therapeutically administered IFN-I, we tested different humanized mouse models for induction of IFN-I during HIV-1 infection. While HIV-1 induced high levels of IFN-α in BLT mice, IFN-I was undetectable following infection in the Hu-PBL mouse model, in which only T cells expand. We therefore tested the effect of treatment with Pegylated IFN-2 (pegasys), in Hu-PBL mice. Pegasys prevented CD4 T cell depletion and reduced the viral load for 10 days, but the effect waned thereafter. We next expressed IFN-I subsets (IFN-α2, −α6, −α8, −α14, and −β) in Hu-PBL mice by hydrodynamic injection of plasmids encoding them and 2 days later infected the mice with HIV-1. CD4 T cell depletion was prevented in all subtypes of IFN-I-expressing mice by day 10. However, at day 40 post-infection, protection was seen in IFN-β- and IFN-α14-expressing mice, but not the others. The viral load followed an inverse pattern and was highest in control mice and lowest in IFN-β- and IFN-α14-expressing mice until day 40 after infection. These results show that gene therapy with plasmids encoding IFN-β and −α14, but not the commonly used −α2, confers long-term suppression of HIV-1 replication. PMID:27729616

  10. Immunogenicity and virulence of attenuated vaccinia virus Tian Tan encoding HIV-1 muti-epitope genes, p24 and cholera toxin B subunit in mice.

    PubMed

    Du, Shouwen; Wang, Yuhang; Liu, Cunxia; Wang, Maopeng; Zhu, Yilong; Tan, Peng; Ren, Dayong; Li, Xiao; Tian, Mingyao; Yin, Ronglan; Li, Chang; Jin, Ningyi

    2015-07-01

    No effective prophylactic or therapeutic vaccine against HIV-1 in humans is currently available. This study analyzes the immunogenicity and safety of a recombinant attenuated vaccinia virus. A chimeric gene of HIV-1 multi-epitope genes containing CpG ODN and cholera toxin B subunit (CTB) was inserted into Chinese vaccinia virus Tian Tan strain (VTT) mutant strain. The recombinant virus rddVTT(-CCMp24) was assessed for immunogenicity and safety in mice. Results showed that the protein CCMp24 was expressed stably in BHK-21 infected with rddVTT(-CCMp24). And the recombinant virus induced the production of HIV-1 p24 specific immunoglobulin G (IgG), IL-2 and IL-4. The recombinant vaccine induced γ-interferon secretion against HIV peptides, and elicited a certain levels of immunological memory. Results indicated that the recombinant virus had certain immunogenicity to HIV-1. Additionally, the virulence of the recombinant virus was been attenuated in vivo of mice compared with wild type VTT (wtVTT), and the introduction of CTB and HIV Mp24 did not alter the infectivity and virulence of defective vaccinia virus.

  11. A peptide nucleic acid-aminosugar conjugate targeting transactivation response element of HIV-1 RNA genome shows a high bioavailability in human cells and strongly inhibits tat-mediated transactivation of HIV-1 transcription.

    PubMed

    Das, Indrajit; Désiré, Jérôme; Manvar, Dinesh; Baussanne, Isabelle; Pandey, Virendra N; Décout, Jean-Luc

    2012-07-12

    The 6-aminoglucosamine ring of the aminoglycoside antibiotic neomycin B (ring II) was conjugated to a 16-mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. For this purpose, we prepared the aminoglucosamine monomer 15 and attached it to the protected PNA prior to its cleavage from the solid support. We found that the resulting PNA-aminoglucosamine conjugate is stable under acidic conditions, efficiently taken up by the human cells and fairly distributed in both cytosol and nucleus without endosomal entrapment because cotreatment with endosome-disrupting agent had no effect on its cellular distribution. The conjugate displayed very high target specificity in vitro and strongly inhibited Tat mediated transactivation of HIV-1 LTR transcription in a cell culture system. The unique properties of this new class of PNA conjugate suggest it to be a potential candidate for therapeutic application.

  12. Neutron encoded labeling for peptide identification.

    PubMed

    Rose, Christopher M; Merrill, Anna E; Bailey, Derek J; Hebert, Alexander S; Westphall, Michael S; Coon, Joshua J

    2013-05-21

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, "Amino Acid Counter", which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis.

  13. Neutron Encoded Labeling for Peptide Identification

    PubMed Central

    Rose, Christopher M.; Merrill, Anna E.; Bailey, Derek J.; Hebert, Alexander S.; Westphall, Michael S.; Coon, Joshua J.

    2013-01-01

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, “Amino Acid Counter”, which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis. PMID:23638792

  14. Peptide-Derivatized SB105-A10 Dendrimer Inhibits the Infectivity of R5 and X4 HIV-1 Strains in Primary PBMCs and Cervicovaginal Histocultures

    PubMed Central

    Bon, Isabella; Lembo, David; Rusnati, Marco; Clò, Alberto; Morini, Silvia; Miserocchi, Anna; Bugatti, Antonella; Grigolon, Sonia; Musumeci, Giuseppina; Landolfo, Santo; Re, Maria Carla; Gibellini, Davide

    2013-01-01

    Peptide dendrimers are a class of molecules that exhibit a large array of biological effects including antiviral activity. In this report, we analyzed the antiviral activity of the peptide-derivatized SB105-A10 dendrimer, which is a tetra-branched dendrimer synthetized on a lysine core, in activated peripheral blood mononuclear cells (PBMCs) that were challenged with reference and wild-type human immunodeficiency virus type 1 (HIV-1) strains. SB105-A10 inhibited infections by HIV-1 X4 and R5 strains, interfering with the early phases of the viral replication cycle. SB105-A10 targets heparan sulfate proteoglycans (HSPGs) and, importantly, the surface plasmon resonance (SPR) assay revealed that SB105-A10 strongly binds gp41 and gp120, most likely preventing HIV-1 attachment/entry through multiple mechanisms. Interestingly, the antiviral activity of SB105-A10 was also detectable in an organ-like structure of human cervicovaginal tissue, in which SB105-A10 inhibited the HIV-1ada R5 strain infection without altering the tissue viability. These results demonstrated the strong antiviral activity of SB105-A10 and suggest a potential microbicide use of this dendrimer to prevent the heterosexual transmission of HIV-1. PMID:24116111

  15. Robust antigen-specific humoral immune responses to sublingually delivered adenoviral vectors encoding HIV-1 Env: association with mucoadhesion and efficient penetration of the sublingual barrier.

    PubMed

    Domm, William; Brooks, Lauren; Chung, Hung Li; Feng, Changyong; Bowers, William J; Watson, Gene; McGrath, James L; Dewhurst, Stephen

    2011-09-16

    The efficient induction of virus-specific mucosal antibodies is an important unmet objective in Human Immunodeficiency Virus Type-1 (HIV-1) vaccine research. One promising approach is sublingual (SL) immunization. We examined the effectiveness of SL delivery of two different viral vectors: (i) a recombinant adenovirus (rAd5), and (ii) a Herpes Simplex Virus Type-1 amplicon vector (HSV-1). Initial in vitro videomicroscopy experiments showed that rAd5 particles were trapped in saliva (i.e., that Ad5 was mucoadhesive) - unlike HSV-1 virions, which migrated freely in both saliva and water. In vivo imaging studies in mice revealed that only the rAd5 vector efficiently transduced the SL epithelium. Consistent with this, SL delivery of an rAd5 encoding HIV-1 envelope glycoprotein (Env) resulted in robust antigen-specific antibody responses in plasma and in vaginal washes, whereas SL delivery of a HSV-1 amplicon vector encoding HIV-1 Env failed to elicit Env-specific antibodies. In contrast, both vectors elicited equivalent humoral responses following intramuscular (IM) delivery. Finally, SL delivery of the rAd5:Env vector resulted in elevated levels of Env-specific serum IgA, and vaginal IgA and IgG, when compared to IM delivery of the same vector. These results findings shed light on vector properties (mucoadhesion, penetration of the sublingual barrier) which may be important for the induction of potent humoral immune responses following sublingual vector administration. Our data also show that SL delivery of an Env-encoding rAd5 vector can elicit a potent antigen-specific mucosal antibody response in the absence of adjuvant. Overall, these findings support the further exploration of the SL delivery route for HIV-1 vaccine delivery.

  16. Robust Antigen-Specific Humoral Immune Responses to Sublingually Delivered Adenoviral Vectors Encoding HIV-1 Env: Association with Mucoadhesion and Efficient Penetration of the Sublingual Barrier

    PubMed Central

    Domm, William; Brooks, Lauren; Chung, Hung Li; Feng, Changyong; Bowers, William J.; Watson, Gene; McGrath, James L.; Dewhurst, Stephen

    2011-01-01

    The efficient induction of virus-specific mucosal antibodies is an important unmet objective in Human Immunodeficiency Virus Type-1 (HIV-1) vaccine research. One promising approach is sublingual (SL) immunization. We examined the effectiveness of SL delivery of two different viral vectors: (i) a recombinant adenovirus (rAd5), and (ii) a Herpes Simplex Virus Type-1 amplicon vector (HSV-1). Initial in vitro videomicroscopy experiments showed that rAd5 particles were trapped in saliva (i.e., that Ad5 was mucoadhesive) - unlike HSV-1 virions, which migrated freely in both saliva and water. In vivo imaging studies in mice revealed that only the rAd5 vector efficiently transduced the SL epithelium. Consistent with this, SL delivery of an rAd5 encoding HIV-1 envelope glycoprotein (Env) resulted in robust antigen-specific antibody responses in plasma and in vaginal washes, whereas SL delivery of a HSV-1 amplicon vector encoding HIV-1 Env failed to elicit Env-specific antibodies. In contrast, both vectors elicited equivalent humoral responses following intramuscular (IM) delivery. Finally, SL delivery of the rAd5:Env vector resulted in elevated levels of Env-specific serum IgA, and vaginal IgA and IgG, when compared to IM delivery of the same vector. These results findings shed light on vector properties (mucoadhesion, penetration of the sublingual barrier) which may be important for the induction of potent humoral immune responses following sublingual vector administration. Our data also show that SL delivery of an Env-encoding rAd5 vector can elicit a potent antigen-specific mucosal antibody response in the absence of adjuvant. Overall, these findings support the further exploration of the SL delivery route for HIV-1 vaccine delivery. PMID:21801777

  17. Development of CD8+ T cells expressing two distinct receptors specific for MTB and HIV-1 peptides

    PubMed Central

    Hao, Pei-Pei; Zhang, Xiao-Bing; Luo, Wei; Zhou, Chao-Ying; Wen, Qian; Yang, Zhi; Liu, Su-Dong; Jiang, Zhen-Min; Zhou, Ming-Qian; Jin, Qi; Ma, Li

    2013-01-01

    The immune response in individuals co-infected with Mycobacterium tuberculosis (MTB) and the human immunodeficiency virus (MTB/HIV) gradually deteriorates, particularly in the cellular compartment. Adoptive transfer of functional effector T cells can confer protective immunity to immunodeficient MTB/HIV co-infected recipients. However, few such effector T cells exist in vivo, and their isolation and amplification to sufficient numbers is difficult. Therefore, enhancing immune responses against both pathogens is critical for treating MTB/HIV co-infected patients. One approach is adoptive transfer of T cell receptor (TCR) gene-modified T cells for the treatment of MTB/HIV co-infections because lymphocyte numbers and their functional avidity is significantly increased by TCR gene transfer. To generate bispecific CD8+ T cells, MTB Ag85B199–207 peptide-specific TCRs (MTB/TCR) and HIV-1 Env120–128 peptide-specific TCRs (HIV/TCR) were isolated and introduced into CD8+ T cells simultaneously using a retroviral vector. To avoid mispairing among exogenous and endogenous TCRs, and to improve the function and stability of the introduced TCRs, several strategies were employed, including introducing mutations in the MTB/TCR constant (C) regions, substituting part of the HIV/TCR C regions with CD3ζ, and linking gene segments with three different 2A peptides. Results presented in this report suggest that the engineered T cells possessed peptide-specific specificity resulting in cytokine production and cytotoxic activity. This is the first report describing the generation of engineered T cells specific for two different pathogens and provides new insights into TCR gene therapy for the treatment of immunocompromised MTB/HIV co-infected patients.

  18. Genetic mapping in human and mouse of the locus encoding TRBP, a protein that binds the TAR region of the human immunodeficiency virus (HIV-1)

    SciTech Connect

    Kozak, C.A.; Gatignol, A.; Graham, K.

    1995-01-01

    Productive infection with HIV-1, the virus responsible for AIDS, requires the involvement of host cell factors for completion of the replicative cycle, but the identification of these factors and elucidation of their specific functions has been difficult. A human cDNA, TRBP, was recently cloned and characterized as a positive regulator of gene expression that binds to the TAR region of the HIV-1 genome. Here we demonstrate that this factor is encoded by a gene, TARBP2, that maps to human chromosome 12 and mouse chromosome 15, and we also identify and map one human pseudogene (TARBP2P) and two mouse TRBP-related sequences. The map location of the expressed gene identifies it as a candidate for the previously identified factor encoded on human chromosome 12 that has been shown to be important for expression of HIV-1 genes. Western blotting indicates that despite high sequence conservation in human and mouse, the TARBP2 protein differs in apparent size in primate and rodent cells. 41 refs., 5 figs., 1 tab.

  19. Peptide and protein-based inhibitors of HIV-1 co-receptors

    PubMed Central

    von Recum, Horst A; Pokorski, Jonathan K

    2014-01-01

    Human immunodeficiency virus (HIV) afflicts an estimated 30 million people globally, making it a continuing pandemic. Despite major research efforts, the rate of new infections has remained relatively static over time. This article reviews an emerging strategy for the treatment of HIV, the inhibition of the co-receptors necessary for HIV entry, CCR5 and CXCR4. The aim of this article is to highlight potential therapeutics derived from peptides and proteins that show particular promise in HIV treatment. Molecules that act on CCR5, CXCR4 or on both receptors will be discussed herein. PMID:23856897

  20. Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin.

    PubMed

    Kumar, Santosh; Bose, Debojit; Suryawanshi, Hemant; Sabharwal, Harshana; Mapa, Koyeli; Maiti, Souvik

    2011-01-01

    Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT(m) = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K(a) = 16.2±0.6×10(7) M(-1) where enthalpic change ΔH = -13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = -2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K(a) = 3.1±0.4×10(7) M(-1)) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.

  1. Characterization of Gp41 polymorphisms in the fusion peptide domain and T-20 (Enfuvirtide) resistance-associated regions in Korean HIV-1 isolates.

    PubMed

    Jang, Dai-Ho; Yoon, Cheol-Hee; Choi, Byeong-Sun; Chung, Yoon-Seok; Kim, Hye-Young; Chi, Sung-Gil; Kim, Sung Soon

    2014-03-01

    HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naïve Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.

  2. HIV-1 fusion peptide decreases bending energy and promotes curved fusion intermediates.

    PubMed

    Tristram-Nagle, Stephanie; Nagle, John F

    2007-09-15

    A crucial step in human immunodeficiency virus (HIV) infection is fusion between the viral envelope and the T-cell membrane, which must involve intermediate membrane states with high curvature. Our main result from diffuse x-ray scattering is that the bending modulus K(C) is greatly reduced upon addition of the HIV fusion peptide FP-23 to lipid bilayers. A smaller bending modulus reduces the free energy barriers required to achieve and pass through the highly curved intermediate states and thereby facilitates fusion and HIV infection. The reduction in K(C) is by a factor of 13 for the thicker, stiffer 1,2-sn-dierucoylphosphatidylcholine bilayers and by a factor of 3 for 1,2-sn-dioleoylphosphatidylcholine bilayers. The reduction in K(C) decays exponentially with concentration of FP-23, and the 1/e concentration is <1 mol % peptide/lipid, which is well within the physiological range for a fusion site. A secondary result is, when FP-23 is added to the samples which consist of stacks of membranes, that the distance between membranes increases and eventually becomes infinite at full hydration (unbinding); we attribute this both to electrostatic repulsion of the positively charged arginine in the FP-23 and to an increase in the repulsive fluctuation interaction brought about by the smaller K(C). Although this latter interaction works against membrane fusion, our results show that the energy that it requires of the fusion protein machinery to bring the HIV envelope membrane and the target T-cell membrane into close contact is negligible.

  3. Human Endogenous Retrovirus K(HML-2) Gag- and Env-Specific T-Cell Responses Are Infrequently Detected in HIV-1-Infected Subjects Using Standard Peptide Matrix-Based Screening

    PubMed Central

    John, Vivek M.; Hunter, Diana V.; Martin, Eric; Mujib, Shariq; Mihajlovic, Vesna; Burgers, Peter C.; Luider, Theo M.; Gyenes, Gabor; Sheppard, Neil C.; SenGupta, Devi; Tandon, Ravi; Yue, Feng-Yun; Benko, Erika; Kovacs, Colin; Nixon, Douglas F.; Ostrowski, Mario A.

    2012-01-01

    T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15mer peptide mapping. PMID:22205657

  4. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

  5. Studies on the intermolecular forces involved in the antibody-antigen interactions, using V3 synthetic peptides and sera from HIV1 seropositive patients.

    PubMed

    Măgureanu, C G; Diaconu, C; Alexandrescu, R; Tirdei, G; Cernescu, C

    1994-01-01

    The nature of physical forces responsible for the antibody-antigen (Ab-Ag) reaction was analyzed in an original system, represented by synthetic peptides derived from the V3 consensus sequences of some HIV1 subtypes gp 120 and HIV1 positive human serum. For locating antigenic determines, flexibility, hydrophilicity and hydrophobicity profiles of the V3 peptides were analysed. The hydrophilicity indicates that V3 apex borders are involved in the first stage of the reaction. The flexibility and hydrophobicity suggest that the apex of the V3 loop (GPGR/Q) is involved in the stabilization of the complex by hydrophobic interactions. Further, we followed up the influence of the dielectric constant and of the pH upon the forces established between Ab and Ag. Modifications in the dielectric constant and pH reveal a significant contribution of electrostatic and van der Waals forces in securing the intermolecular complementarity. D2O produces the highest augmentation of the antibody affinity for the most hydrophilic peptides, while a very slight one was recorded for the most hydrophobic sequence. A high affinity of antibodies for the peptides MN, R and Z was registered at an acid pH, when their His residue was protonated. On the contrary, no influence was recorded in the case of the peptide A, which does not contain any His residue.

  6. Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

    PubMed Central

    Kassler, Kristin; Meier, Julia; Eichler, Jutta; Sticht, Heinrich

    2011-01-01

    The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4). Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120. PMID:22312332

  7. Structural and Functional Properties of Peptides Based on the N-terminus of HIV-1 gp41 and the C-terminus of the Amyloid-Beta Protein

    PubMed Central

    Gordon, Larry M.; Nisthal, Alex; Lee, Andy B.; Eskandari, Sepehr; Ruchala, Piotr; Jung, Chun-Ling; Waring, Alan J.; Mobley, Patrick W.

    2008-01-01

    Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26–42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer’s. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26–42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26–42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26–42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’). PMID:18515070

  8. Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

    DOE PAGES

    Heller, William T.; Rai, Durgesh K.

    2017-01-16

    HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

  9. Peptide Triazole Inactivators of HIV-1 Utilize a Conserved Two-Cavity Binding Site at the Junction of the Inner and Outer Domains of Env gp120

    PubMed Central

    Aneja, Rachna; Rashad, Adel A.; Li, Huiyuan; Sundaram, Ramalingam Venkat Kalyana; Duffy, Caitlin; Bailey, Lauren D.; Chaiken, Irwin

    2015-01-01

    We used coordinated mutagenesis, synthetic design, and flexible docking to investigate the structural mechanism of Env gp120 encounter by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated that the PT class of inhibitors suppresses binding at both CD4 and coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent irreversible virus inactivation. Despite these enticing anti-HIV-1 phenotypes, structural understanding of the PT–gp120 binding mechanism has been incomplete. Here we found that PT engages two inhibitor ring moieties at the junction between the inner and outer domains of the gp120 protein. The results demonstrate how combined occupancy of two gp120 cavities can coordinately suppress both receptor and coreceptor binding and conformationally entrap the protein in a destabilized state. The two-cavity model has common features with small molecule gp120 inhibitor binding sites and provides a guide for further design of peptidomimetic HIV-1 inactivators based on the PT pharmacophore. PMID:25860784

  10. Structure of Antibody F425-B4e8 in Complex With a V3 Peptide Reveals a New Binding Mode for Hiv-1 Neutralization

    SciTech Connect

    Bell, C.H.; Pantophlet, R.; Schiefner, A.; Cavacini, L.A.; Stanfield, R.L.; Burton, D.R.; Wilson, I.A.

    2009-05-11

    F425-B4e8 (B4e8) is a monoclonal antibody isolated from a human immunodeficiency virus type 1 (HIV-1)-infected individual that recognizes the V3 variable loop on the gp120 subunit of the viral envelope spike. B4e8 neutralizes a subset of HIV-1 primary isolates from subtypes B, C and D, which places this antibody among the very few human anti-V3 antibodies with notable cross-neutralizing activity. Here, the crystal structure of the B4e8 Fab fragment in complex with a 24-mer V3 peptide (RP142) at 2.8 A resolution is described. The complex structure reveals that the antibody recognizes a novel V3 loop conformation, featuring a five-residue alpha-turn around the conserved GPGRA apex of the beta-hairpin loop. In agreement with previous mutagenesis analyses, the Fab interacts primarily with V3 through side-chain contacts with just two residues, Ile(P309) and Arg(P315), while the remaining contacts are to the main chain. The structure helps explain how B4e8 can tolerate a certain degree of sequence variation within V3 and, hence, is able to neutralize an appreciable number of different HIV-1 isolates.

  11. Induction of cross clade reactive specific antibodies in mice by conjugates of HGP-30 (peptide analog of HIV-1(SF2) p17) and peptide segments of human beta-2-microglobulin or MHC II beta chain.

    PubMed

    Zimmerman, D H; Lloyd, J P; Heisey, D; Winship, M D; Siwek, M; Talor, E; Sarin, P S

    2001-09-14

    HGP-30, a 30 amino acid synthetic peptide homologous to a conserved region of HIV-1(SF2) p17 (aa86-115), has previously been shown to elicit both cellular and humoral immune responses when conjugated to KLH and adsorbed to alum. However, the free HGP-30 peptide is not immunogenic in animals. In order to improve the immunogenicity of HGP-30, peptide conjugates consisting of a modified HGP-30 sequence (m-HGP-30/aa82-111) and a peptide segment, residues 38-50, of the MHC I accessory molecule, human beta-2-microglobulin (beta-2-M), referred to as Peptide J, or a peptide from the MHC II beta chain (peptide G) were evaluated in mice. The effects of carriers and adjuvants on serum antibody titers, specificities to various HIV-1 clade peptides similar to HGP-30 and isotype patterns were examined. Peptides J or especially G conjugated to modified-HGP-30 (LEAPS 102 and LEAPS 101, respectively) generated comparable or better immune responses to modified HGP-30 than KLH conjugates as judged by the induction of: (1) similar antibody titers; (2) broader HIV clade antigen binding; and (3) antibody isotype response patterns indicative of a TH1 pathway (i.e. increased amounts of IgG2a and IgG2b antibodies). The ISA 51 and MPL(R)-SE adjuvants induced higher antibody responses than alum, with the ISA 51 being more potent. Immune responses to LEAPS 102, as compared to LEAPS 101, were weaker and slower to develop as determined by antibody titers and cross clade reactivity of the antibodies induced. Compared to KLH conjugates which induced significant anti-KLH antibody titers, minimal antibody responses were observed to peptide G, the more immunogenic conjugate, and peptide J. These results suggest that modified HGP-30 L.E.A.P.S. constructs may be useful as HIV vaccine candidates for preferential induction of TH1 directed cell mediated immune responses.

  12. Design of a modular tetrameric scaffold for the synthesis of membrane-localized D-peptide inhibitors of HIV-1 entry

    PubMed Central

    Francis, J. Nicholas; Redman, Joseph S.; Eckert, Debra M.; Kay, Michael S.

    2012-01-01

    The highly conserved HIV-1 gp41 “pocket” region is a promising target for inhibiting viral entry. PIE12-trimer is a protease-resistant trimeric D-peptide inhibitor that binds to this pocket and potently blocks HIV entry. PIE12-trimer also possesses a reserve of binding energy that provides it with a strong genetic barrier to resistance (“resistance capacitor”). Here we report the design of a modular scaffold employing PEGs of discrete lengths for the efficient optimization and synthesis of PIE12-trimer. This scaffold also allows us to conjugate PIE12-trimer to several membrane-localizing cargoes, resulting in dramatically improved potency and retention of PIE12-trimer’s ability to absorb the impact of resistance mutations. This scaffold design strategy should be of broad utility for the rapid prototyping of multimeric peptide inhibitors attached to potency- or pharmacokinetic-enhancing groups. PMID:22545664

  13. Induction of rapid apoptosis for class I MHC molecule-restricted CD8(+) HIV-1 gp160-specific murine activated CTLs by free antigenic peptide in vivo.

    PubMed

    Nakagawa, Yohko; Shimizu, Masumi; Norose, Yoshihiko; Takahashi, Megumi; Takahashi, Hidemi

    2013-01-01

    We have previously reported that the cytotoxic activity of murine CD8(+) CTLs specific for HIV-1 gp160 envelope protein was markedly inhibited in vitro by brief exposure to a free epitope peptide P18-I10 (aa: RGPGRAFVTI) using the epitope-specific CTL line (LINE-IIIB) or a clone (RT-1). We have also shown that recently stimulated P18-I10-specific murine CTLs rapidly fell into apoptosis in vitro after brief exposure to the free epitope peptide. In the present study, we examined whether similar inactivation or apoptosis of recently stimulated CTLs occurred in vivo by exposure to the free epitope peptide using TCR transgenic (Tg-RT-1) mice expressing TCRαβ genes of CTL clone RT-1. When the Tg mice were inoculated with recombinant vaccinia virus expressing HIV-1-IIIB gp160 genes followed by injection of P18-I10 epitope peptide, apparent reduction in the number of CTLs determined by flow cytometry using H-2D(d)/P18-I10 pentamer was observed within a few hours after the injection. Most of the H-2D(d)/P18-I10 pentamer-stained cells were positive for Annexin V and apoptosis was confirmed by microscopic analyses. Moreover, when mice were pretreated with immunosuppressive agents, such as cyclosporin A and tacrolimus (FK506), induction of apoptosis by P18-I10 was significantly inhibited and CTL cytotoxicity was maintained. These results suggest that the rapid loss of virus-specific CD8(+) CTLs might occur in vivo through apoptosis in the early stages of viral infection when activated CTLs may encounter viral epitope(s) released from virus-infected cells attacked by CTLs and we can prevent the loss by pretreatment with immunosuppressive agents.

  14. Depicting Binding-Mediated Translocation of HIV-1 Tat Peptides in Living Cells with Nanoscale Pens of Tat-Conjugated Quantum Dots

    PubMed Central

    Lin, Chien Y.; Huang, Jung Y.; Lo, Leu-Wei

    2017-01-01

    Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, a time-lapse single-particle light-sheet microscopy technique was implemented to obtain a parallel visualization of the translocating process of individual human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) peptide conjugated quantum dots (TatP-QDs) in complex cellular terrains. Here, TatP-QDs served as nanoscale dynamic pens, which depict remarkable trajectory aggregates of TatP-QDs on the cell surface. Spectral-embedding analysis of the trajectory aggregates revealed a manifold formed by isotropic diffusion and a fraction of directed movement, possibly caused by interaction between the Tat peptides and heparan sulfate groups on the plasma membrane. Further analysis indicated that the membrane deformation induced by Tat-peptide attachment increased with the disruption of the actin framework in cytochalasin D (cyto D)-treated cells, yielding higher interactions on the TatP-QDs. In native cells, the Tat peptides can remodel the actin framework to reduce their interaction with the local membrane environment. Characteristic hot spots for interaction were detected on the membrane, suggesting that a funnel passage may have formed for the Tat-coated particles. This finding offers valuable insight into the cellular delivery of nanoscale cargo, suggesting an avenue for direct therapeutic delivery. PMID:28208588

  15. Role of lipid structure in the humoral immune response in mice to covalent lipid-peptides from the membrane proximal region of HIV-1 gp41

    PubMed Central

    Watson, Douglas S.; Szoka, Francis C.

    2009-01-01

    The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to the MPR and neutralize primary HIV isolates. The 2F5 and 4E10 antibodies cross-react with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomal bilayers. In a comparison of eight lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide IgG titers (6.4 × 104) in sera of BALB/C mice. Two lipid anchors, palmitic acid and phosphatidylcholine, failed to elicit a detectable serum anti-peptide IgG response. Association with the liposomal vehicle contributed to the ability of a lipopeptide to elicit anti-peptide antibodies, but no other single factor, such as position of the lipid anchor, peptide helical content, lipopeptide partition coefficient, or presence of phosphate on the anchor clearly determined lipopeptide potency. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic (5.6 × 102 IgG titer in serum). Finally, attachment of CHEMS to a peptide spanning both the 2F5 and 4E10 epitopes elicited serum IgG antibodies that bound to each of the individual epitopes as well as to recombinant gp140. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine. PMID:19520200

  16. Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges

    PubMed Central

    Lakhashe, Samir K.; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B.; DiPasquale, Janet M.; Hemashettar, Girish; Yoon, John K.; Rasmussen, Robert A.; Yang, Feng; Lee, Sandra J.; Montefiori, David C.; Novembre, Francis J.; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R.; Robert-Guroff, Marjorie; Johnson, Welkin E.; Lieberman, Judy; Ruprecht, Ruth M.

    2011-01-01

    We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus >90%; these RM also had strong SIV Gag-specific proliferation of CD8+ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4+ T cells; the latter have been implicated as preferential virus targets in-vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. PMID:21693155

  17. HIV-1 encoded virus protein U (Vpu) solution structure of the 41-62 hydrophilic region containing the phosphorylated sites Ser52 and Ser56.

    PubMed

    Coadou, Gaël; Evrard-Todeschi, Nathalie; Gharbi-Benarous, Josyane; Benarous, Richard; Girault, Jean Pierre

    2002-03-08

    Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.

  18. C-terminal HIV-1 transframe p6* tetra-peptide blocks enhanced Gag cleavage incurred by leucine zipper replacement of a deleted p6* domain.

    PubMed

    Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

    2017-03-01

    HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C-terminus). The data indicate that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetra-peptide plays a role in preventing premature PR maturation.IMPORTANCE Supporting evidence is lacking for the assumption that p6* retards PR maturation in the context of virus assembly. We found that replacing p6* with a leucine-zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (a) p6

  19. Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

    PubMed Central

    Penn-Nicholson, Adam; Han, Dong P.; Kim, Soon J.; Park, Hanna; Ansari, Rais; Montefiori, David C.; Cho, Michael W.

    2008-01-01

    Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts. PMID:18068750

  20. Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

    SciTech Connect

    Penn-Nicholson, Adam; Han, Dong P.; Kim, Soon J.; Park, Hanna; Ansari, Rais; Montefiori, David C.; Cho, Michael W.

    2008-03-15

    Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts.

  1. Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag

    PubMed Central

    Xu, Wanghui; Zhao, Qing; Xing, Lei; Lin, Zhanglin

    2016-01-01

    The increasing demand for antigenic peptides in the development of novel serologic diagnostics and epitope-based vaccines requires rapid and reliable peptide synthesis techniques. Here we investigated a method for efficient recombinant expression and purification of medium- to large-sized antigenic peptides in E. coli. Previously we devised a streamlined protein expression and purification scheme based on a cleavable self-aggregating tag (cSAT), which comprised an intein molecule and a self-aggregating peptide ELK16. In this scheme, the target proteins were fused in the C-termini with cSAT and expressed as insoluble aggregates. After intein self-cleavage, target proteins were released into the soluble fraction with high yield and reasonable purity. We demonstrated the applicability of this scheme by preparing seven model viral peptides, with lengths ranging from 32 aa to 72 aa. By adding an N-terminal thioredoxin tag, we enhanced the yield of target peptides released from the aggregates. The purified viral peptides demonstrated high antigenic activities in ELISA and were successfully applied to dissecting the antigenic regions of influenza hemagglutinin. The cSAT scheme described here allows for the rapid and low-cost preparation of multiple antigenic peptides for immunological screening of a broad range of viral antigens. PMID:27808126

  2. Magic Angle Spinning NMR Reveals Sequence-Dependent Structural Plasticity, Dynamics, and the Spacer Peptide 1 Conformation in HIV-1 Capsid Protein Assemblies

    SciTech Connect

    Han, Yun; Hou, Guangjin; Suiter, Christopher L.; Ahn, Jinwoo; Byeon, In-Ja L.; Lipton, Andrew S.; Burton, Sarah D.; Hung, Ivan; Gorkov, Peter L.; Gan, Zhehong; Brey, William W.; Rice, David M.; Gronenborn, Angela M.; Polenova, Tatyana E.

    2013-11-27

    Maturation of HIV-1 virus into an infectious virion requires cleavage of the Gag polyprotein into its constituent domains and formation of a conical capsid core that encloses viral RNA and a small complement of proteins for replication. The final step of this process is the cleavage of the SP1 peptide from the CA-SP1 maturation intermediate, which triggers the condensation of the CA protein into a conical capsid. The mechanism of this step, including the conformation of the SP1 peptide in CA-SP1, is under intense debate. In this report, we examine the tubular assemblies of CA and the CA-SP1 maturation intermediate using Magic Angle Spinning NMR spectroscopy. At the magnetic fields of 19.9 T and above, tubular CA and CA-SP1 assemblies yield outstanding-quality 2D and 3D MAS NMR spectra, which are amenable to resonance assignments and detailed structural characterization. Dipolar- and scalar-based correlation experiments unequivocally indicate that SP1 peptide is in a random coil conformation and mobile in the assembled CA-SP1. Analysis of two sequence variants reveals that remarkably, the conformation of SP1 tail, of the functionally important CypA loop, and of the loop preceding helix 8 are sequence dependent and modulated by the residue variations at distal sites. These findings challenge the role of SP1 as a conformational switch in the maturation process and establish sequence-dependent conformational plasticity in CA.

  3. Structure Determination of An Anti-HIV-1 Fab 447-52d-Peptide Complex From An Expitaxially Twinned Data Set

    SciTech Connect

    Dhillon, A.K.; Stanfield, R.L.; Gorny, M.K.; Williams, C.; Zolla-Pazner, S.; Wilson, I.A.

    2009-05-14

    Although antibodies against the third variable loop (V3) of the HIV-1 viral envelope glycoprotein are among the first neutralizing antibodies to be detected in infected individuals, they are normally restricted in their specificity. X-ray crystallographic studies of V3-specific antibodies have contributed to a more thorough understanding of recognition of this epitope and of conserved features in the V3 loop that could potentially aid in the design of a multi-component vaccine. The human antibody 447-52D exhibits relatively broad neutralization of primary viral isolates compared with other V3-loop antibodies. A crystal structure of Fab 447-52D in complex with a V3 peptide (UG1033) was determined at 2.1 {angstrom} resolution. The structure was determined using an epitaxially twinned data set and in-house programs to detect and remove overlapping reflections. Although the processed data have lower than desired completeness and slightly higher than normal R values for the resolution, good-quality electron-density maps were obtained that enabled structure determination. The structure revealed an extended CDR H3 loop that forms a {beta}-sheet with the peptide, with the predominant contacts being main-chain hydrogen bonds. The V3 peptide and Fab show high structural homology with the previously reported structures of other Fab 447-52D complexes, reinforcing the idea that the V3 loop may adopt a small set of conserved structures, particularly around the crown of the {beta}-hairpin.

  4. Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design.

    PubMed

    Serrano, Soraya; Araujo, Aitziber; Apellániz, Beatriz; Bryson, Steve; Carravilla, Pablo; de la Arada, Igor; Huarte, Nerea; Rujas, Edurne; Pai, Emil F; Arrondo, José L R; Domene, Carmen; Jiménez, María Angeles; Nieva, José L

    2014-03-07

    The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.

  5. Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

    SciTech Connect

    Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T.

    2016-01-15

    Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.

  6. The current status and challenges in the development of fusion inhibitors as therapeutics for HIV-1 infection.

    PubMed

    Tan, Jian Jun; Ma, Xue Ting; Liu, Chang; Zhang, Xiao Yi; Wang, Cun Xin

    2013-01-01

    HIV-1 membrane fusion as a part of the process of viral entry in the target cells is facilitated by gp41 and gp120, which are encoded by Env gene of HIV-1. Based on the structure and the mechanism researches, new treatment options targeting HIV-1 entry process have been proposed. Enfuvirtide, which mimics amino acid sequences of viral envelope glycoprotein gp41, is the first HIV-1 fusion inhibitor approved by FDA. Although it fulfills vital functions by binding to gp41 and abolishing the membrane fusion reaction when used in combination, it could induce drug resistant virus variants. Currently, a number of design and modification schemes have been presented, a large number of prospective fusion peptides have emerged. For these fusion inhibitors, multiple mutations in gp41 have been associated with the loss of susceptibility to agents. This review reported the current developments and innovative designs of HIV-1 membrane fusion inhibitors.

  7. Structure and function of a cyanophage-encoded peptide deformylase

    PubMed Central

    Frank, Jeremy A; Lorimer, Don; Youle, Merry; Witte, Pam; Craig, Tim; Abendroth, Jan; Rohwer, Forest; Edwards, Robert A; Segall, Anca M; Burgin, Alex B

    2013-01-01

    Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities. PMID:23407310

  8. Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers

    PubMed Central

    Shchelokovskyy, P; Tristram-Nagle, S; Dimova, R

    2013-01-01

    The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer. PMID:23505334

  9. Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers

    NASA Astrophysics Data System (ADS)

    Shchelokovskyy, P.; Tristram-Nagle, S.; Dimova, R.

    2011-02-01

    The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer.

  10. Differential Control of BST2 Restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists Encoded by HIV-1 Group M and O Strains.

    PubMed

    Bego, Mariana G; Cong, Lijun; Mack, Katharina; Kirchhoff, Frank; Cohen, Éric A

    2016-11-15

    BST2/tetherin is a type I interferon (IFN-I)-stimulated host factor that restricts the release of HIV-1 by entrapping budding virions at the cell surface. This membrane-associated protein can also engage and activate the plasmacytoid dendritic cell (pDC)-specific immunoglobulin-like transcript 7 (ILT7) inhibitory receptor to downregulate the IFN-I response by pDCs. Pandemic HIV-1 group M uses Vpu (M-Vpu) to counteract the two BST2 isoforms (long and short) that are expressed in human cells. M-Vpu efficiently downregulates surface long BST2, while it displaces short BST2 molecules away from viral assembly sites. We recently found that this attribute is used by M-Vpu to activate the BST2/ILT7-dependent negative-feedback pathway and to suppress pDC IFN-I responses during sensing of infected cells. However, whether this property is conserved in endemic HIV-1 group O, which has evolved Nef (O-Nef) to counteract specifically the long BST2 isoform, remains unknown. In the present study, we validated that O-Nefs have the capacity to downregulate surface BST2 and enhance HIV-1 particle release although less efficiently than M-Vpu. In contrast to M-Vpu, O-Nef did not efficiently enhance viral spread in T cell culture or displace short BST2 from viral assembly sites to prevent its occlusion by tethered HIV-1 particles. Consequently, O-Nef impairs the ability of BST2 to activate negative ILT7 signaling to suppress the IFN-I response by pDC-containing peripheral blood mononuclear cells (PBMCs) during sensing of infected cells. These distinctive features of BST2 counteraction by O-Nefs may in part explain the limited spread of HIV-1 group O in the human population.

  11. Structure determination of an anti-HIV-1 Fab 447-52D–peptide complex from an epitaxially twinned data set

    SciTech Connect

    Dhillon, Amandeep K.; Stanfield, Robyn L.; Gorny, Miroslaw K.; Williams, Constance; Zolla-Pazner, Susan; Wilson, Ian A.

    2008-07-01

    Separation of two individual lattices within an epitaxially twinned data set allowed the crystal structure of the V3-specific neutralizing antibody 447-52D in complex with a V3 peptide (UG1033) to be determined. The structure confirms that the neutralization breadth of Fab 447-52D is likely to be attributable to the extensive focus on main-chain hydrogen-bond interactions with the peptide that permit the recognition of a range of V3 sequences. Although antibodies against the third variable loop (V3) of the HIV-1 viral envelope glycoprotein are among the first neutralizing antibodies to be detected in infected individuals, they are normally restricted in their specificity. X-ray crystallographic studies of V3-specific antibodies have contributed to a more thorough understanding of recognition of this epitope and of conserved features in the V3 loop that could potentially aid in the design of a multi-component vaccine. The human antibody 447-52D exhibits relatively broad neutralization of primary viral isolates compared with other V3-loop antibodies. A crystal structure of Fab 447-52D in complex with a V3 peptide (UG1033) was determined at 2.1 Å resolution. The structure was determined using an epitaxially twinned data set and in-house programs to detect and remove overlapping reflections. Although the processed data have lower than desired completeness and slightly higher than normal R values for the resolution, good-quality electron-density maps were obtained that enabled structure determination. The structure revealed an extended CDR H3 loop that forms a β-sheet with the peptide, with the predominant contacts being main-chain hydrogen bonds. The V3 peptide and Fab show high structural homology with the previously reported structures of other Fab 447-52D complexes, reinforcing the idea that the V3 loop may adopt a small set of conserved structures, particularly around the crown of the β-hairpin.

  12. The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: a biophysical characterization.

    PubMed

    Garzón, María T; Lidón-Moya, María C; Barrera, Francisco N; Prieto, Alicia; Gómez, Javier; Mateu, Mauricio G; Neira, José L

    2004-06-01

    The type 1 HIV presents a conical capsid formed by approximately 1500 units of the capsid protein, CA. Homodimerization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second alpha-helix. Here, we show that an N-terminal-acetylated and C-terminal-amidated peptide, CAC1, comprising the sequence of the CA-C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA-C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size-exclusion chromatography provided evidence of the interaction between CAC1 and CA-C. The apparent dissociation constant of the heterocomplex formed by CA-C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl-labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 microM. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA-C and the intact capsid protein homodimers (10 microM).

  13. The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: A biophysical characterization

    PubMed Central

    Garzón, María T.; Lidón-Moya, María C.; Barrera, Francisco N.; Prieto, Alicia; Gómez, Javier; Mateu, Mauricio G.; Neira, José L.

    2004-01-01

    The type 1 HIV presents a conical capsid formed by ~1500 units of the capsid protein, CA. Homodimer-ization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second α-helix. Here, we show that an N-terminal-acetylated and C-terminal–amidated peptide, CAC1, comprising the sequence of the CA-C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA-C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size-exclusion chromatography provided evidence of the interaction between CAC1 and CA-C. The apparent dissociation constant of the heterocomplex formed by CA-C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl-labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 μM. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA-C and the intact capsid protein homodimers (10 μM). PMID:15152086

  14. Characterization of the HIV-1 TAR RNA-Tat peptide and drug interactions by on-line acoustic wave sensor

    NASA Astrophysics Data System (ADS)

    Tassew, Nardos Gobena

    This thesis presents the application of the thickness shear-mode (TSM) acoustic wave sensor to the study of RNA-protein and RNA-drug interactions at the solid-liquid interface. The binding of the human immunodeficiency virus-type 1 Tat protein to the trans-activation responsive RNA element (TAR) has been studied using this sensor. Data from such measurements show that the sensor is able to discriminate between different Tat peptides derived from the parent protein based on size. The effects of mutations introduced at specific sites in the protein and RNA on the TAR-Tat binding have also been examined in detail. Reduced level of response in acoustic parameters due to mutations was observed indicating that the decrease in binding in response to site specific mutations can be acoustically detected. Data from acoustic wave sensor measurements indicate that the TAR-Tat binding is also affected by ionic strength. Both the frequency and motional resistance signals show periodic responses when varying concentrations of salt are introduced on a TAR-modified surface. The binding of the two molecules seems to be a function of the response of the nucleic acid to salt concentrations. The kinetics of binding of Tat peptides to TAR RNA and to a bulge mutant analogue (MTAR) is also examined from the rate of change of the series resonant frequency. Results from such analysis illustrate longer Tat peptides formed more stable complexes with TAR RNA and exhibited increased discrimination between mutant and wild type TAR. The binding of two aminoglycoside antibiotics, neomycin and streptomycin, to TAR RNA and their effectiveness in preventing TAR-Tat complex formation has been studied in detail. Binding affinity is directly correlated with the inhibitory potency of these molecules and the TSM sensor shows that neomycin exhibits at least a ten fold greater affinity to TAR and that it is also a more potent inhibitor than streptomycin. The results from this research involving TAR-Tat and

  15. Development of HIV-1 fusion inhibitors targeting gp41.

    PubMed

    Lu, K; Asyifah, M R; Shao, F; Zhang, D

    2014-06-01

    The HIV-1 envelope protein glycoprotein 41 (gp41) is crucial in the HIV-1 infection process, therefore gp41 has emerged as an attractive target for drug design against AIDS. During the past few decades, tremendous efforts have been made on developing inhibitors that can prevent the HIV-1 entry process via suppressing functional gp41. In this review, the development of HIV-1 fusion inhibitors targeting gp41 including peptide inhibitors, small molecule inhibitors, vaccines and neutralized antibodies will be discussed.

  16. Selective induction of cell-mediated immunity and protection of rhesus macaques from chronic SHIV{sub KU2} infection by prophylactic vaccination with a conserved HIV-1 envelope peptide-cocktail

    SciTech Connect

    Nehete, Pramod N.; Nehete, Bharti P.; Hill, Lori; Manuri, Pallavi R.; Baladandayuthapani, Veerabhadran; Feng Lei; Simmons, Johnny; Sastry, K. Jagannadha

    2008-01-05

    Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-{gamma}-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activity against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV{sub KU2}. Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-{gamma} production, higher levels of vaccine-specific IFN-{gamma} producing CD4{sup +} cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.

  17. Targeting Multidrug-resistant Staphylococci with an anti-rpoA Peptide Nucleic Acid Conjugated to the HIV-1 TAT Cell Penetrating Peptide

    PubMed Central

    Abushahba, Mostafa FN; Mohammad, Haroon; Seleem, Mohamed N

    2016-01-01

    Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase α subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (α-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus. PMID:27434684

  18. High yields and soluble expression of superoxide dismutases in Escherichia coli due to the HIV-1 Tat peptide via increases in mRNA transcription

    PubMed Central

    Sun, Yangdong; Ye, Qiao; Wu, Min; Wu, Yonghong; Zhang, Chenggang; Yan, Weiqun

    2016-01-01

    This study aimed to validate the high yield and soluble expression of proteins carrying the transactivator of transcription (Tat) peptide tag, and further explored the potential mechanism by which the Tat tag increases expression. Escherichia coli superoxide dismutase (SOD) proteins, including SodA, SodB and SodC, were selected for analysis. As expected, the yields and the solubility of Tat-tagged proteins were higher than those of Tat-free proteins, and similar results were observed for the total SOD enzyme activity. Bacterial cells that overexpressed Tat-tagged proteins exhibited increased anti-paraquat activity compared with those expressing Tat-free proteins that manifested as SodA>SodC>SodB. When compared with an MG1655 wild-type strain, the growth of a ΔSodA mutant strain was found to be inhibited after paraquat treatment; the growth of ΔSodB and ΔSodC mutant strains was also slightly inhibited. The mRNA transcript level of genes encoding Tat-tagged proteins was higher than that of genes encoding Tat-free proteins. Furthermore, the α-helix and turn of Tat-tagged proteins were higher than those of Tat-free proteins, but the β-sheet and random coil content was lower. These results indicated that the incorporation of the Tat core peptide as a significant basic membrane transduction peptide in fusion proteins could increase mRNA transcripts and promote the high yield and soluble expression of heterologous proteins in E. coli. PMID:27741225

  19. Psychoneuroimmunology and HIV-1.

    ERIC Educational Resources Information Center

    Antoni, Michael H.; And Others

    1990-01-01

    Presents evidence describing benefits of behavioral interventions such as aerobic exercise training on both psychological and immunological functioning among high risk human immunodeficiency virus-Type 1 (HIV-1) seronegative and very early stage seropositive homosexual men. HIV-1 infection is cast as chronic disease for which early…

  20. A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

    PubMed Central

    Davis, Zachary B.; Cogswell, Andrew; Scott, Hamish; Mertsching, Amanda; Boucau, Julie; Wambua, Daniel; Le Gall, Sylvie; Planelles, Vicente; Campbell, Kerry S.; Barker, Edward

    2016-01-01

    Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94+ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94+ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL+ CD56dim NK cells, in contrast to the efficient responses by CD56bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94+ KIR2DL- NK cells may be uniquely beneficial. PMID:26828202

  1. NK cytotoxicity against CD4+ T cells during HIV-1 infection: A gp41 peptide induces the expression of an NKp44 ligand

    PubMed Central

    Vieillard, Vincent; Strominger, Jack L.; Debré, Patrice

    2005-01-01

    HIV infection leads to a state of chronic immune activation and progressive deterioration in immune function, manifested most recognizably by the progressive depletion of CD4+ T cells. A substantial percentage of natural killer (NK) cells from patients with HIV infection are activated and express the natural cytotoxicity receptor (NCR) NKp44. Here we show that a cellular ligand for NKp44 (NKp44L) is expressed during HIV-1 infection and is correlated with both the progression of CD4+ T cell depletion and the increase of viral load. CD4+ T cells expressing this ligand are highly sensitive to the NK lysis activity mediated by NKp44+ NK cells. The expression of NKp44L is induced by the linear motif NH2-SWSNKS-COOH of the HIV-1 envelope gp41 protein. This highly conserved motif appears critical to the sharp increase in NK lysis of CD4+ T cells from HIV-infected patients. These studies strongly suggest that induction of NKp44L plays a key role in the lysis of CD4+ T cells by activated NK cells in HIV infection and consequently provide a framework for considering how HIV-1 may use NK cell immune surveillance to trigger CD4+ T cells. Understanding this mechanism may help to develop future therapeutic strategies and vaccines against HIV-1 infection. PMID:16046540

  2. Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides.

    PubMed

    Thynne, Elisha; Saur, Isabel M L; Simbaqueba, Jaime; Ogilvie, Huw A; Gonzalez-Cendales, Yvonne; Mead, Oliver; Taranto, Adam; Catanzariti, Ann-Maree; McDonald, Megan C; Schwessinger, Benjamin; Jones, David A; Rathjen, John P; Solomon, Peter S

    2016-06-13

    In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.

  3. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules

    PubMed Central

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S. Y.

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This “shock” approach is then followed by “kill” of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  4. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules.

    PubMed

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells.

  5. Productive replication and evolution of HIV-1 in ferret cells.

    PubMed

    Fadel, Hind J; Saenz, Dyana T; Guevara, Rebekah; von Messling, Veronika; Peretz, Mary; Poeschla, Eric M

    2012-02-01

    A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1

  6. The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP.

    PubMed

    Cabrero, Pablo; Radford, Jonathan C; Broderick, Kate E; Costes, Laurence; Veenstra, Jan A; Spana, Eric P; Davies, Shireen A; Dow, Julian A T

    2002-12-01

    Dh, the gene that encodes a CRF-like peptide in Drosophila melanogaster, is described. The product of this gene is a 44-amino-acid peptide (Drome-DH(44)) with a sequence almost identical to the Musca domestica and Stomoxys calcitrans diuretic hormones. There are no other similar peptides encoded within the known Drosophila genomic sequence. Functional studies showed that the deduced peptide stimulated fluid production, and that this effect was mediated by cyclic AMP in principal cells only: there was no effect on the levels of either cyclic GMP or intracellular calcium. Stimulation also elevated levels of cyclic AMP (but not cyclic GMP) phosphodiesterase, a new mode of action for this class of hormone. The transcript was localised by in situ hybridisation, and the peptide by immunocytochemistry, to two groups of three neurones in the pars intercerebralis within the brain. These cells also express receptors for leucokinin, another major diuretic peptide, implying that the cells may be important in homeostatic regulation.

  7. Vpx-containing Dendritic Cell Vaccine Vectors Induce CTLs and Reactivate Latent HIV-1 in vitro

    PubMed Central

    Norton, Thomas D.; Miller, Elizabeth A.; Bhardwaj, Nina; Landau, Nathaniel R.

    2015-01-01

    Eradication of HIV-1 from an infected individual requires a means of inducing production of virus from latently infected cells and stimulating an immune response against the infected cells. We report the development of lentiviral vectors that transduce dendritic cells (DCs) to both induce production of virus from latently infected cells and stimulate antigen-specific CTLs. The vectors package Vpx, a lentiviral accessory protein that counteracts the SAMHD1-mediated block to DC transduction, allowing for long-term expression of vector-encoded proteins. The vectors encode influenza or HIV-1-derived epitopes fused via a self-cleaving peptide to CD40L that releases the peptide into the endoplasmic reticulum for entry into the antigen presentation pathway. Expression of CD40L caused transduced DCs to mature and produce Th1-skewing cytokines. The DCs presented antigen to CD8 T cells, enhancing antigen-specific CTLs. Coculture of the transduced DCs with latently infected cells induced high level virus production, an effect that was mediated by TNF-α. The ability of a DC vaccine to reactivate latent HIV-1 and stimulate an adaptive immune response provides a means to reduce the size of the latent reservoir in patients. This strategy can also be applied to develop DC vaccines for other diseases. PMID:25567537

  8. Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus.

    PubMed

    Becerril, B; Corona, M; Coronas, F I; Zamudio, F; Calderon-Aranda, E S; Fletcher, P L; Martin, B M; Possani, L D

    1996-02-01

    Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed.

  9. Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus.

    PubMed Central

    Becerril, B; Corona, M; Coronas, F I; Zamudio, F; Calderon-Aranda, E S; Fletcher, P L; Martin, B M; Possani, L D

    1996-01-01

    Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed. PMID:8611151

  10. Rhizobial peptidase HrrP cleaves host-encoded signaling peptides and mediates symbiotic compatibility.

    PubMed

    Price, Paul A; Tanner, Houston R; Dillon, Brett A; Shabab, Mohammed; Walker, Graham C; Griffitts, Joel S

    2015-12-08

    Legume-rhizobium pairs are often observed that produce symbiotic root nodules but fail to fix nitrogen. Using the Sinorhizobium meliloti and Medicago truncatula symbiotic system, we previously described several naturally occurring accessory plasmids capable of disrupting the late stages of nodule development while enhancing bacterial proliferation within the nodule. We report here that host range restriction peptidase (hrrP), a gene found on one of these plasmids, is capable of conferring both these properties. hrrP encodes an M16A family metallopeptidase whose catalytic activity is required for these symbiotic effects. The ability of hrrP to suppress nitrogen fixation is conditioned upon the genotypes of both the host plant and the hrrP-expressing rhizobial strain, suggesting its involvement in symbiotic communication. Purified HrrP protein is capable of degrading a range of nodule-specific cysteine-rich (NCR) peptides encoded by M. truncatula. NCR peptides are crucial signals used by M. truncatula for inducing and maintaining rhizobial differentiation within nodules, as demonstrated in the accompanying article [Horváth B, et al. (2015) Proc Natl Acad Sci USA, 10.1073/pnas.1500777112]. The expression pattern of hrrP and its effects on rhizobial morphology are consistent with the NCR peptide cleavage model. This work points to a symbiotic dialogue involving a complex ensemble of host-derived signaling peptides and bacterial modifier enzymes capable of adjusting signal strength, sometimes with exploitative outcomes.

  11. Rhizobial peptidase HrrP cleaves host-encoded signaling peptides and mediates symbiotic compatibility

    PubMed Central

    Price, Paul A.; Tanner, Houston R.; Dillon, Brett A.; Shabab, Mohammed; Walker, Graham C.; Griffitts, Joel S.

    2015-01-01

    Legume–rhizobium pairs are often observed that produce symbiotic root nodules but fail to fix nitrogen. Using the Sinorhizobium meliloti and Medicago truncatula symbiotic system, we previously described several naturally occurring accessory plasmids capable of disrupting the late stages of nodule development while enhancing bacterial proliferation within the nodule. We report here that host range restriction peptidase (hrrP), a gene found on one of these plasmids, is capable of conferring both these properties. hrrP encodes an M16A family metallopeptidase whose catalytic activity is required for these symbiotic effects. The ability of hrrP to suppress nitrogen fixation is conditioned upon the genotypes of both the host plant and the hrrP-expressing rhizobial strain, suggesting its involvement in symbiotic communication. Purified HrrP protein is capable of degrading a range of nodule-specific cysteine-rich (NCR) peptides encoded by M. truncatula. NCR peptides are crucial signals used by M. truncatula for inducing and maintaining rhizobial differentiation within nodules, as demonstrated in the accompanying article [Horváth B, et al. (2015) Proc Natl Acad Sci USA, 10.1073/pnas.1500777112]. The expression pattern of hrrP and its effects on rhizobial morphology are consistent with the NCR peptide cleavage model. This work points to a symbiotic dialogue involving a complex ensemble of host-derived signaling peptides and bacterial modifier enzymes capable of adjusting signal strength, sometimes with exploitative outcomes. PMID:26401024

  12. Induction of CD8+ cytotoxic T lymphocytes by immunization with syngeneic irradiated HIV-1 envelope derived peptide-pulsed dendritic cells.

    PubMed

    Takahashi, H; Nakagawa, Y; Yokomuro, K; Berzofsky, J A

    1993-08-01

    Based on the evidence that CD8+ cytotoxic T cells (CTL) precursors do not appear to distinguish between virus-infected cells and viral peptide-pulsed syngeneic cells, we have developed methods for priming class I MHC molecule restricted CD8+ CTL with such peptides without using any adjuvant. We were able to prime in vivo such CTL immunity lasting at least 6 months with a single i.v. injection of syngeneic 2200-3300 rad irradiated peptide-pulsed spleen cells, and even more efficiently with a very small number of irradiated class II MHC molecule expressing splenic dendritic cells (DC). No foreign serum source was necessary during the pulsing. Interestingly, we could not generate significant CTL activity with unirradiated or low dose (< 1100 rad) irradiated spleen cells. Because even purified DC required irradiation for optimal activity, because unirradiated B cells did not significantly inhibit the immunization with DC, and because B cell depletion did not substitute for irradiation, we believe that the effect of irradiation is more to determine homing of the cells than to eliminate interference by B cells. Intravenous immunization was much more effective than s.c. or i.p. immunization. CTL generated by this method could kill both peptide-pulsed syngeneic targets and targets endogenously expressing the whole gp160 gene. Moreover, we found that we could prime CD8+ CTL with the minimal 10-residue core peptide (RGPGRAFVTI) for optimal presentation by class I MHC molecules as efficiently as the original p18. These results suggested that DC bearing antigenic peptide may prime antigen-specific CD8+ CTL in vivo. These results offer useful information for development of synthetic peptide vaccines and immunotherapy.

  13. BAGEL3: automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides

    PubMed Central

    van Heel, Auke J.; de Jong, Anne; Montalbán-López, Manuel; Kok, Jan; Kuipers, Oscar P.

    2013-01-01

    Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl. PMID:23677608

  14. BAGEL3: Automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides.

    PubMed

    van Heel, Auke J; de Jong, Anne; Montalbán-López, Manuel; Kok, Jan; Kuipers, Oscar P

    2013-07-01

    Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl.

  15. Delivery of DNA HIV-1 Vaccine to the Liver Induces High and Long-lasting Humoral Immune Responses

    PubMed Central

    Raska, Milan; Moldoveanu, Zina; Novak, Jan; Hel, Zdenek; Bozja, Jadranka; Compans, Richard W.; Yang, Chinglai; Mestecky, Jiri

    2008-01-01

    The quality of immune responses induced by DNA vaccination depends on the site of DNA administration, the expression, and the properties of the encoded antigen. In the present study we demonstrate that intravenous hydrodynamic HIV-1 envelope DNA injection resulted in high levels of expression of HIV-1 envelope antigen in the liver. When compared to the administration of DNA by i.n., i.d., i.m., and i.splenic routes, hydrodynamic vaccination induced, upon DNA boosting, 40 times increase of HIV-1 envelope-specific antibodies over the preimmune levels. Hydrodynamic vaccination with 1 μg DNA induced higher humoral responses than 100 μg DNA given intramuscularly in the prime – boost regimen. High levels of envelope-specific IgG and IgA antibodies were induced in genital tract secretions after two doses of DNA followed by intranasal boosting with recombinant HIV-1 gp120 protein. Furthermore, two doses of 100 μg DNA generated interferon-gamma production in ~ 4.3 ± 1.7 % of CD8+ splenocytes after in vitro stimulation with HIV-1 envelope peptides. These results demonstrate that DNA vaccines targeted to tissues with high proteosynthetic activity, such as the liver, results in enhanced immune responses. PMID:18304708

  16. Core-Gene-Encoded Peptide Regulating Virulence-Associated Traits in Streptococcus mutans

    PubMed Central

    Kim, Jeong Nam; Stanhope, Michael J.

    2013-01-01

    Recently, high-coverage genome sequence of 57 isolates of Streptococcus mutans, the primary etiological agent of human dental caries, was completed. The SMU.1147 gene, encoding a 61-amino-acid (61-aa) peptide, was present in all sequenced strains of S. mutans but absent in all bacteria in current databases. Reverse transcription-PCR revealed that SMU.1147 is cotranscribed with scnK and scnR, which encode the histidine kinase and response regulator, respectively, of a two-component system (TCS). The C terminus of the SMU.1147 gene product was tagged with a FLAG epitope and shown to be expressed in S. mutans by Western blotting with an anti-FLAG antibody. A nonpolar mutant of SMU.1147 formed less biofilm in glucose-containing medium and grew slower than did the wild-type strain under aerobic and anaerobic conditions, at low pH, or in the presence of H2O2. Mutation of SMU.1147 dramatically reduced genetic competence and expression of comX and comY, compared to S. mutans UA159. The competence defect of the SMU.1147 mutant could not be overcome by addition of sigX-inducing peptide (XIP) in defined medium or by competence-stimulating peptide (CSP) in complex medium. Complementation with SMU.1147 on a plasmid restored all phenotypes. Interestingly, mutants lacking either one of the TCS components and a mutant lacking all three genes behaved like the wild-type strain for all phenotypes mentioned above, but all mutant strains grew slower than UA159 in medium supplemented with 0.3 M NaCl. Thus, the SMU.1147-encoded peptide affects virulence-related traits and dominantly controls quorum-sensing pathways for development of genetic competence in S. mutans. PMID:23603743

  17. Adaptation of HIV-1 to its human host.

    PubMed

    Wain, Louise V; Bailes, Elizabeth; Bibollet-Ruche, Frederic; Decker, Julie M; Keele, Brandon F; Van Heuverswyn, Fran; Li, Yingying; Takehisa, Jun; Ngole, Eitel Mpoudi; Shaw, George M; Peeters, Martine; Hahn, Beatrice H; Sharp, Paul M

    2007-08-01

    Human immunodeficiency virus type 1 (HIV-1) originated from three independent cross-species transmissions of simian immunodeficiency virus (SIVcpzPtt) infecting chimpanzees (Pan troglodytes troglodytes) in west central Africa, giving rise to pandemic (group M) and non-pandemic (groups N and O) clades of HIV-1. To identify host-specific adaptations in HIV-1 we compared the inferred ancestral sequences of HIV-1 groups M, N and O to 12 full length genome sequences of SIVcpzPtt and four of the outlying but closely related SIVcpzPts (from P. t. schweinfurthii). This analysis revealed a single site that was completely conserved among SIVcpzPtt strains but different (due to the same change) in all three groups of HIV-1. This site, Gag-30, lies within p17, the gag-encoded matrix protein. It is Met in SIVcpzPtt, underwent a conservative replacement by Leu in one lineage of SIVcpzPts but changed radically to Arg on all three lineages leading to HIV-1. During subsequent diversification this site has been conserved as a basic residue (Arg or Lys) in most lineages of HIV-1. Retrospective analysis revealed that Gag-30 had reverted to Met in a previous experiment in which HIV-1 was passaged through chimpanzees. To examine whether this substitution conferred a species specific growth advantage, we used site-directed mutagenesis to generate variants of these chimpanzee-adapted HIV-1 strains with Lys at Gag-30, and tested their replication in both human and chimpanzee CD4+ T lymphocytes. Remarkably, viruses encoding Met replicated to higher titers than viruses encoding Lys in chimpanzee T cells, but the opposite was found in human T cells. Taken together, these observations provide compelling evidence for host-specific adaptation during the emergence of HIV-1 and identify the viral matrix protein as a modulator of viral fitness following transmission to the new human host.

  18. Unusual Fusion Proteins of HIV-1

    PubMed Central

    Langer, Simon; Sauter, Daniel

    2017-01-01

    Despite its small genome size, the Human Immunodeficiency Virus 1 (HIV-1) is one of the most successful pathogens and has infected more than 70 million people worldwide within the last decades. In total, HIV-1 expresses 16 canonical proteins from only nine genes within its 10 kb genome. Expression of the structural genes gag, pol, and env, the regulatory genes rev and tat and the accessory genes vpu, nef, vpr, and vif enables assembly of the viral particle, regulates viral gene transcription, and equips the virus to evade or counteract host immune responses. In addition to the canonically expressed proteins, a growing number of publications describe the existence of non-canonical fusion proteins in HIV-1 infected cells. Most of them are encoded by the tat-env-rev locus. While the majority of these fusion proteins (e.g., TNV/p28tev, p186Drev, Tat1-Rev2, Tat^8c, p17tev, or Ref) are the result of alternative splicing events, Tat-T/Vpt is produced upon programmed ribosomal frameshifting, and a Rev1-Vpu fusion protein is expressed due to a nucleotide polymorphism that is unique to certain HIV-1 clade A and C strains. A better understanding of the expression and activity of these non-canonical viral proteins will help to dissect their potential role in viral replication and reveal how HIV-1 optimized the coding potential of its genes. The goal of this review is to provide an overview of previously described HIV-1 fusion proteins and to summarize our current knowledge of their expression patterns and putative functions. PMID:28119676

  19. A fully genetically encoded protein architecture for optical control of peptide ligand concentration

    NASA Astrophysics Data System (ADS)

    Schmidt, Daniel; Tillberg, Paul W.; Chen, Fei; Boyden, Edward S.

    2014-01-01

    Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.

  20. The choreography of HIV-1 proteolytic processing and virion assembly.

    PubMed

    Lee, Sook-Kyung; Potempa, Marc; Swanstrom, Ronald

    2012-11-30

    HIV-1 has been the target of intensive research at the molecular and biochemical levels for >25 years. Collectively, this work has led to a detailed understanding of viral replication and the development of 24 approved drugs that have five different targets on various viral proteins and one cellular target (CCR5). Although most drugs target viral enzymatic activities, our detailed knowledge of so much of the viral life cycle is leading us into other types of inhibitors that can block or disrupt protein-protein interactions. Viruses have compact genomes and employ a strategy of using a small number of proteins that can form repeating structures to enclose space (i.e. condensing the viral genome inside of a protein shell), thus minimizing the need for a large protein coding capacity. This creates a relatively small number of critical protein-protein interactions that are essential for viral replication. For HIV-1, the Gag protein has the role of a polyprotein precursor that contains all of the structural proteins of the virion: matrix, capsid, spacer peptide 1, nucleocapsid, spacer peptide 2, and p6 (which contains protein-binding domains that interact with host proteins during budding). Similarly, the Gag-Pro-Pol precursor encodes most of the Gag protein but now includes the viral enzymes: protease, reverse transcriptase (with its associated RNase H activity), and integrase. Gag and Gag-Pro-Pol are the substrates of the viral protease, which is responsible for cleaving these precursors into their mature and fully active forms (see Fig. 1A).

  1. HTLV-1 Tax activates HIV-1 transcription in latency models.

    PubMed

    Geddes, Victor Emmanuel Viana; José, Diego Pandeló; Leal, Fabio E; Nixon, Douglas F; Tanuri, Amilcar; Aguiar, Renato Santana

    2017-04-01

    HIV-1 latency is a major obstacle to HIV-1 eradication. Coinfection with HTLV-1 has been associated with faster progression to AIDS. HTLV-1 encodes the transactivator Tax which can activate both HTLV-1 and HIV-1 transcription. Here, we demonstrate that Tax activates HIV transcription in latent CD4(+) T cells. Tax promotes the activation of P-TEFb, releasing CDK9 and Cyclin T1 from inactive forms, promoting transcription elongation and reactivation of latent HIV-1. Tax mutants lacking interaction with the HIV-1-LTR promoter were not able to activate P-TEFb, with no subsequent activation of latent HIV. In HIV-infected primary resting CD4(+) T cells, Tax-1 reactivated HIV-1 transcription up to five fold, confirming these findings in an ex vivo latency model. Finally, our results confirms that HTLV-1/Tax hijacks cellular partners, promoting HIV-1 transcription, and this interaction should be further investigated in HIV-1 latency studies in patients with HIV/HTLV-1 co-infection.

  2. A Gene Encoding Antigenic Peptides of Human Squamous Cell Carcinoma Recognized by Cytotoxic T Lymphocytes

    PubMed Central

    Shichijo, Shigeki; Nakao, Masanobu; Imai, Yasuhisa; Takasu, Hideo; Kawamoto, Mayumi; Niiya, Fumihiko; Yang, Damu; Toh, Yuji; Yamana, Hideaki; Itoh, Kyogo

    1998-01-01

    Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients. PMID:9449708

  3. Maternal plasma and breastmilk viral loads are associated with HIV-1-specific cellular immune responses among HIV-1-exposed, uninfected infants in Kenya

    PubMed Central

    Liu, A Y; Lohman-Payne, B; Chung, M H; Kiarie, J; Kinuthia, J; Slyker, J; Richardson, B; Lehman, D; Farquhar, C; John-Stewart, G

    2015-01-01

    Infants exposed to maternal HIV-1 provide an opportunity to assess correlates of HIV-1-specific interferon (IFN)-γ responses and may be informative in the development of HIV-1 vaccines. HIV-1-infected women with CD4 counts 200–500 cells/mm3 were randomized to short-course zidovudine/nevirapine (ZDV/NVP) or highly active anti-retroviral therapy (HAART) between 2003 and 2005. Maternal plasma and breastmilk HIV-1 RNA and DNA were quantified during the first 6–12 months postpartum. HIV-1 gag peptide-stimulated enzyme-linked immunospot (ELISPOT) assays were conducted in HIV-1-exposed, uninfected infants (EU), and correlates were determined using regression and generalized estimating equations. Among 47 EU infants, 21 (45%) had ≥1 positive ELISPOT result during follow-up. Infants had a median response magnitude of 177 HIV-1-specific spot-forming units (SFU)/106 peripheral blood mononuclear cells (PBMC) [interquartile range (IQR) = 117–287] directed against 2 (IQR = 1–3) gag peptide pools. The prevalence and magnitude of responses did not differ by maternal anti-retroviral (ARV) randomization arm. Maternal plasma HIV-1 RNA levels during pregnancy (P = 0·009) and breastmilk HIV-1 DNA levels at 1 month (P = 0·02) were associated with a higher magnitude of infant HIV-1-specific ELISPOT responses at 1 month postpartum. During follow-up, concurrent breastmilk HIV-1 RNA and DNA (cell-free virus and cell-associated virus, respectively) each were associated positively with magnitude of infant HIV-1-specific responses (P = 0·01). Our data demonstrate the importance of antigenic exposure on the induction of infant HIV-1-specific cellular immune responses in the absence of infection. PMID:25652232

  4. Sequences encoding identical peptides for the analysis and manipulation of coding DNA

    PubMed Central

    Sánchez, Joaquín

    2013-01-01

    The use of sequences encoding identical peptides (SEIP) for the in silico analysis of coding DNA from different species has not been reported; the study of such sequences could directly reveal properties of coding DNA that are independent of peptide sequences. For practical purposes SEIP might also be manipulated for e.g. heterologous protein expression. We extracted 1,551 SEIP from human and E. coli and 2,631 SEIP from human and D. melanogaster. We then analyzed codon usage and intercodon dinucleotide tendencies and found differences in both, with more conspicuous disparities between human and E. coli than between human and D. melanogaster. We also briefly manipulated SEIP to find out if they could be used to create new coding sequences. We hence attempted replacement of human by E. coli codons via dicodon exchange but found that full replacement was not possible, this indicated robust species-specific dicodon tendencies. To test another form of codon replacement we isolated SEIP from human and the jellyfish green fluorescent protein (GFP) and we then re-constructed the GFP coding DNA with human tetra-peptide-coding sequences. Results provide proof-of-principle that SEIP may be used to reveal differences in the properties of coding DNA and to reconstruct in pieces a protein coding DNA with sequences from a different organism, the latter might be exploited in heterologous protein expression. PMID:23861567

  5. Sequences encoding identical peptides for the analysis and manipulation of coding DNA.

    PubMed

    Sánchez, Joaquín

    2013-01-01

    The use of sequences encoding identical peptides (SEIP) for the in silico analysis of coding DNA from different species has not been reported; the study of such sequences could directly reveal properties of coding DNA that are independent of peptide sequences. For practical purposes SEIP might also be manipulated for e.g. heterologous protein expression. We extracted 1,551 SEIP from human and E. coli and 2,631 SEIP from human and D. melanogaster. We then analyzed codon usage and intercodon dinucleotide tendencies and found differences in both, with more conspicuous disparities between human and E. coli than between human and D. melanogaster. We also briefly manipulated SEIP to find out if they could be used to create new coding sequences. We hence attempted replacement of human by E. coli codons via dicodon exchange but found that full replacement was not possible, this indicated robust species-specific dicodon tendencies. To test another form of codon replacement we isolated SEIP from human and the jellyfish green fluorescent protein (GFP) and we then re-constructed the GFP coding DNA with human tetra-peptide-coding sequences. Results provide proof-of-principle that SEIP may be used to reveal differences in the properties of coding DNA and to reconstruct in pieces a protein coding DNA with sequences from a different organism, the latter might be exploited in heterologous protein expression.

  6. Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Amino-Thiol Unnatural Amino Acids

    PubMed Central

    Frost, John R.; Jacob, Nicholas T.; Papa, Louis J.; Owens, Andrew E.

    2015-01-01

    A versatile method for orchestrating the formation of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side-chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro, or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in E. coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles. PMID:25933125

  7. Nanochemistry-based immunotherapy for HIV-1.

    PubMed

    Lori, F; Calarota, S A; Lisziewicz, J

    2007-01-01

    Highly active antiretroviral treatment (HAART), i.e. the combination of three or more drugs against human immunodeficiency virus type 1 (HIV-1), has greatly improved the clinical outcome of HIV-1-infected individuals. However, HAART is unable to reconstitute HIV-specific immunity and eradicate the virus. Several observations in primate models and in humans support the notion that cell-mediated immunity can control viral replication and slow disease progression. Thus, besides drugs, an immunotherapy that induces long-lasting HIV-specific T-cell responses could play a role in the treatment of HIV/AIDS. To induce such immune responses, DermaVir Patch has been developed. DermaVir consists of an HIV-1 antigen-encoding plasmid DNA that is chemically formulated in a nanoparticle. DermaVir is administered under a patch after a skin preparation that supports the delivery of the nanoparticle to Langerhans cells (LC). Epidermal LC trap and transport the nanomedicine to draining lymph nodes. While in transit, LC mature into dendritic cells (DC), which can efficiently present the DNA-encoded antigens to naïve T-cells for the induction of cellular immunity. Pre-clinical studies and Phase I clinical testing of DermaVir in HIV-1-infected individuals have demonstrated the safety and tolerability of DermaVir Patch. To further modulate cellular immunity, molecular adjuvants might be added into the nanoparticle. DermaVir Patch represents a new nanomedicine platform for immunotherapy of HIV/AIDS. In this review, the antiviral activity of DermaVir-induced cellular immunity is discussed. Furthermore, the action of some cytokines currently being tested as adjuvants are highlighted and the adjuvant effect of cytokine plasmid DNA included in the DermaVir nanoparticle is reviewed.

  8. Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8 in vitro and in vivo.

    PubMed

    Yu, Yongjiao; Fu, Lu; Jiang, Xiaoyu; Guan, Shanshan; Kuai, Ziyu; Kong, Wei; Shi, Yuhua; Shan, Yaming

    2016-12-01

    Despite unremitting efforts since the discovery of human immunodeficiency virus type 1 (HIV-1), an effective vaccine has not been generated. Viral vector-mediated transfer for expression of HIV-1 broadly neutralizing antibodies (BnAbs) is an attractive strategy. In this study, a recombinant adeno-associated virus 8 (rAAV8) vector was used to encode full-length antibodies against HIV-1 in 293T cells and Balb/c mice after gene transfer. The 10E8 or NIH45-46 BnAb was expressed from a single open reading frame by linking the heavy and light chains with a furin cleavage and a 2A self-processing peptide (F2A). The results showed that the BnAbs could be expressed in the 293T cell culture medium. A single intramuscular injection of rAAV8 led to long-term expression of BnAbs in Balb/c mice. The expressed antibodies in the supernatant of 293T cells and in Balb/c mice showed neutralization effects against HIV-1 pseudoviruses. Combined immunization of rAAV8 expressing 10E8 and rAAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization effects on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of rAAV8 expressing either antibody alone. Therefore, the combined immunization may be a potential vaccine approach against HIV-1.

  9. Gbeta subunit interacts with a peptide encoding region 956-982 of adenylyl cyclase 2. Cross-linking of the peptide to free Gbetagamma but not the heterotrimer.

    PubMed

    Weng, G; Li, J; Dingus, J; Hildebrandt, J D; Weinstein, H; Iyengar, R

    1996-10-25

    The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.

  10. Immunogenicity of a recombinant measles-HIV-1 clade B candidate vaccine.

    PubMed

    Stebbings, Richard; Février, Michèle; Li, Bo; Lorin, Clarisse; Koutsoukos, Marguerite; Mee, Edward; Rose, Nicola; Hall, Joanna; Page, Mark; Almond, Neil; Voss, Gerald; Tangy, Frédéric

    2012-01-01

    Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 10(5) TCID(50) of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.

  11. Design of dimerization inhibitors of HIV-1 aspartic proteinase: A computer-based combinatorial approach

    NASA Astrophysics Data System (ADS)

    Caflisch, Amedeo; Schramm, Hans J.; Karplus, Martin

    2000-02-01

    Inhibition of dimerization to the active form of the HIV-1 aspartic proteinase (HIV-1 PR) may be a way to decrease the probability of escape mutations for this viral protein. The Multiple Copy Simultaneous Search (MCSS) methodology was used to generate functionality maps for the dimerization interface of HIV-1 PR. The positions of the MCSS minima of 19 organic fragments, once postprocessed to take into account solvation effects, are in good agreement with experimental data on peptides that bind to the interface. The MCSS minima combined with an approach for computational combinatorial ligand design yielded a set of modified HIV-1 PR C-terminal peptides that are similar to known nanomolar inhibitors of HIV-1 PR dimerization. A number of N-substituted 2,5-diketopiperazines are predicted to be potential dimerization inhibitors of HIV-1 PR.

  12. Hyperthermia Stimulates HIV-1 Replication

    PubMed Central

    Roesch, Ferdinand; Meziane, Oussama; Kula, Anna; Nisole, Sébastien; Porrot, Françoise; Anderson, Ian; Mammano, Fabrizio; Fassati, Ariberto; Marcello, Alessandro; Benkirane, Monsef; Schwartz, Olivier

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity. PMID:22807676

  13. Glycans Flanking the Hypervariable Connecting Peptide between the A and B Strands of the V1/V2 Domain of HIV-1 gp120 Confer Resistance to Antibodies That Neutralize CRF01_AE Viruses

    PubMed Central

    O’Rourke, Sara M.; Sutthent, Ruengpung; Phung, Pham; Mesa, Kathryn A.; Frigon, Normand L.; To, Briana; Horthongkham, Navin; Limoli, Kay; Wrin, Terri; Berman, Phillip W.

    2015-01-01

    Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149. PMID:25793890

  14. Glycans flanking the hypervariable connecting peptide between the A and B strands of the V1/V2 domain of HIV-1 gp120 confer resistance to antibodies that neutralize CRF01_AE viruses.

    PubMed

    O'Rourke, Sara M; Sutthent, Ruengpung; Phung, Pham; Mesa, Kathryn A; Frigon, Normand L; To, Briana; Horthongkham, Navin; Limoli, Kay; Wrin, Terri; Berman, Phillip W

    2015-01-01

    Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.

  15. The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

    PubMed Central

    Szafron, Lukasz Michal; Balcerak, Anna; Grzybowska, Ewa Anna; Pienkowska-Grela, Barbara; Felisiak-Golabek, Anna; Podgorska, Agnieszka; Kulesza, Magdalena; Nowak, Natalia; Pomorski, Pawel; Wysocki, Juliusz; Rubel, Tymon; Dansonka-Mieszkowska, Agnieszka; Konopka, Bozena; Lukasik, Martyna; Kupryjanczyk, Jolanta

    2015-01-01

    CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene. PMID:25978564

  16. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

  17. HIV-1 subtypes in Yugoslavia.

    PubMed

    Stanojevic, Maja; Papa, Anna; Papadimitriou, Evagelia; Zerjav, Sonja; Jevtovic, Djordje; Salemovic, Dubravka; Jovanovic, Tanja; Antoniadis, Antonis

    2002-05-01

    To gain insight concerning the genetic diversity of HIV-1 viruses associated with the HIV-1 epidemic in Yugoslavia, 45 specimens from HIV-1-infected individuals were classified into subtypes by sequence-based phylogenetic analysis of the polymerase (pol) region of the viral genome. Forty-one of 45 specimens (91.2%) were identified as pol subtype B, 2 of 45 as subtype C (4.4%), 1 of 45 as CRF01_AE (2.2%), and 1 as CRF02_AG recombinant (2.2%). Nucleotide divergence among subtype B sequences was 4.8%. Results of this study show that among HIV-1-infected patients in Yugoslavia subtype B predominates (91.5%), whereas non-B subtypes are present at a low percentage, mostly related to travel abroad.

  18. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells.

    PubMed

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-11-01

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells.

  19. Encoding physico-chemical cues in synthetic hydrogels by triple helix assembly of collagen mimetic peptides

    NASA Astrophysics Data System (ADS)

    Stahl, Patrick

    The ECM is a complex natural system evolved to promote proliferation and differentiation of cells during tissue development. In order to create synthetic biomaterials for studying cell-scaffold interactions and ultimately for engineering tissues, scientists strive to recapitulate many characteristics of ECM by developing hydrogels that contain mechanical cues and biochemical signals such as adhesion moieties and cell growth factors. While synthetic hydrogels bypass limitations of naturally-derived materials (e.g. transfer of pathogens), nature provides inspiration to enhance the functionality of synthetic hydrogels through biomimetic approaches. The collagen triple helix is the basis for the supramolecular structure of collagen in the ECM, and its adaptation in collagen mimetic peptides (CMPs) has provided hybridization mechanisms that can be employed in the formation and functionalization of synthetic hydrogels. The aim of this dissertation is to develop novel poly(ethylene glycol) (PEG)-based hydrogels that employ CMP triple helix assembly as a non-covalent yet target-specific tool to encode physical and chemical cues into the hydrogel with spatial control. We demonstrate that multi-arm PEG functionalized with CMPs form hydrogels supported by physical crosslinks mediated by CMP triple helix. Particle tracking microrheology shows that these physical crosslinks are sensitive to temperature as well as addition of exogenous CMPs that can disrupt crosslinks by competing for triple helix formation. This physical crosslink disruption enables the modulation of bulk hydrogel elasticity and the introduction of local stiffness gradients in PEG-CMP hydrogels. We also present photopolymerized PEG diacrylate (PEGDA) hydrogels displaying CMPs that can be further conjugated to CMPs with bioactive moieties via triple helix hybridization. Encoding these hydrogels with cell-adhesive CMPs induces cell spreading and proliferation. We further demonstrate generation of gradients and

  20. HIV-1 Epidemiology, Genetic Diversity, and Primary Drug Resistance in the Tyumen Oblast, Russia

    PubMed Central

    Astakhova, Ekaterina M.; Gashnikova, Mariya P.; Bocharov, Evgeniy F.; Petrova, Svetlana V.; Pun'ko, Olga A.; Popkov, Alexander V.; Totmenin, Aleksey V.

    2016-01-01

    Introduction. Specific molecular epidemic features of HIV infection in Tyumen Oblast (TO), Russia, were studied. Methods. The genome sequences encoding HIV-1 protease-reverse transcriptase, integrase, and major envelope protein were examined for 72 HIV-1 specimens isolated from the TO resident infected in 2000–2015. Results. The recorded prevalence of HIV-1 subtype A (A1) is 93.1%; HIV-1 subtype B continues to circulate in MSM risk group (1.4%). Solitary instances of HIV-1 recombinant forms, CRF63_02A1 (1.4%) and CRF03_AB (1.4%), were detected as well as two cases of HIV-1 URF63_A1 (2.8%). Phylogenetic analysis showed no HIV-1 clustering according to the duration of infection and risk groups but revealed different epidemic networks confirming that HIV infection spread within local epidemic foci. A high incidence of CXCR4-tropic HIV-1 variants and a higher rate of secondary mutations influencing the virus fitness (K20R, L10V, and I) are observed among the virus specimens isolated from newly infected individuals. Conclusions. The current HIV-1 epidemic in TO develops within the local epidemic networks. Similar to the previous period, HIV-1 subtype A is predominant in TO with sporadic cases of importation of HIV-1 recombinant forms circulating in adjacent areas. PMID:27957489

  1. HIV-1 Epidemiology, Genetic Diversity, and Primary Drug Resistance in the Tyumen Oblast, Russia.

    PubMed

    Gashnikova, Natalya M; Astakhova, Ekaterina M; Gashnikova, Mariya P; Bocharov, Evgeniy F; Petrova, Svetlana V; Pun'ko, Olga A; Popkov, Alexander V; Totmenin, Aleksey V

    2016-01-01

    Introduction. Specific molecular epidemic features of HIV infection in Tyumen Oblast (TO), Russia, were studied. Methods. The genome sequences encoding HIV-1 protease-reverse transcriptase, integrase, and major envelope protein were examined for 72 HIV-1 specimens isolated from the TO resident infected in 2000-2015. Results. The recorded prevalence of HIV-1 subtype A (A1) is 93.1%; HIV-1 subtype B continues to circulate in MSM risk group (1.4%). Solitary instances of HIV-1 recombinant forms, CRF63_02A1 (1.4%) and CRF03_AB (1.4%), were detected as well as two cases of HIV-1 URF63_A1 (2.8%). Phylogenetic analysis showed no HIV-1 clustering according to the duration of infection and risk groups but revealed different epidemic networks confirming that HIV infection spread within local epidemic foci. A high incidence of CXCR4-tropic HIV-1 variants and a higher rate of secondary mutations influencing the virus fitness (K20R, L10V, and I) are observed among the virus specimens isolated from newly infected individuals. Conclusions. The current HIV-1 epidemic in TO develops within the local epidemic networks. Similar to the previous period, HIV-1 subtype A is predominant in TO with sporadic cases of importation of HIV-1 recombinant forms circulating in adjacent areas.

  2. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

    PubMed Central

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  3. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

    PubMed

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-08-16

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

  4. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    SciTech Connect

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-11-15

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation

  5. Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA

    SciTech Connect

    Elton, T.S.; Dion, L.D.; Bost, K.L.; Oparil, S.; Blalock, J.E.

    1988-04-01

    The authors have generated a monospecific antibody to a synthetic peptide encoded by an RNA complementary to the mRNA for angiotensin II (AII) and determined whether this antibody recognizes the AII receptor. They demonstrate that the antibody competes specifically with /sup 125/I-labeled AII for the same binding site on rat adrenal membranes. Furthermore, they show this antibody inhibits the secretion of aldosterone from cultured rat adrenal cells, suggesting that the antibody recognizes the biologically relevant AII receptor. Finally, they demonstrate that antibody to the complementary peptide can be used to immunoaffinity-purify a protein of M/sub r/ 66,000 that specifically binds radiolabeled AII.

  6. Construction of a live-attenuated HIV-1 vaccine through genetic code expansion.

    PubMed

    Wang, Nanxi; Li, Yue; Niu, Wei; Sun, Ming; Cerny, Ronald; Li, Qingsheng; Guo, Jiantao

    2014-05-05

    A safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) is urgently needed to combat the worldwide AIDS pandemic, but still remains elusive. The fact that uncontrolled replication of an attenuated vaccine can lead to regaining of its virulence creates safety concerns precluding many vaccines from clinical application. We introduce a novel approach to control HIV-1 replication, which entails the manipulation of essential HIV-1 protein biosynthesis through unnatural amino acid (UAA*)-mediated suppression of genome-encoded blank codon. We successfully demonstrate that HIV-1 replication can be precisely turned on and off in vitro.

  7. Protein grafting of an HIV-1-inhibiting epitope

    NASA Astrophysics Data System (ADS)

    Sia, Samuel K.; Kim, Peter S.

    2003-08-01

    Protein grafting, the transfer of a binding epitope of one ligand onto the surface of another protein, is a potentially powerful technique for presenting peptides in preformed and active three-dimensional conformations. Its utility, however, has been limited by low biological activity of the designed ligands and low tolerance of the protein scaffolds to surface substitutions. Here, we graft the complete binding epitope (19 nonconsecutive amino acids with a solvent-accessible surface area of >2,000 Å2) of an HIV-1 C-peptide, which is derived from the C-terminal region of HIV-1 gp41 and potently inhibits HIV-1 entry into cells, onto the surface of a GCN4 leucine zipper. The designed peptide, named C34coil, displays a potent antiviral activity approaching that of the native ligand. Moreover, whereas the linear C-peptide is unstructured and sensitive to degradation by proteases, C34coil is well structured, conformationally stable, and exhibits increased resistance to proteolytic degradation compared with the linear peptide. In addition to being a structured antiviral inhibitor, C34coil may also serve as the basis for the development of an alternative class of immunogens. This study demonstrates that "one-shot" protein grafting, without subsequent rounds of optimization, can be used to create ligands with structural conformations and improved biomedical properties.

  8. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array

    NASA Astrophysics Data System (ADS)

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-beom; Choi, Heon-Jin

    2016-07-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes.

  9. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  10. Human cellular restriction factors that target HIV-1 replication

    PubMed Central

    Strebel, Klaus; Luban, Jeremy; Jeang, Kuan-Teh

    2009-01-01

    Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G), bone marrow stromal cell antigen 2 (BST-2), cyclophilin A, tripartite motif protein 5 alpha (Trim5α), and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions. PMID:19758442

  11. Diverse Ecological Strategies Are Encoded by Streptococcus pneumoniae Bacteriocin-Like Peptides

    PubMed Central

    Miller, Eric L.; Abrudan, Monica I.; Roberts, Ian S.; Rozen, Daniel E.

    2016-01-01

    The opportunistic pathogen Streptococcus pneumoniae is commonly carried asymptomatically in the human nasopharynx. Due to high rates of cocolonization with other pneumococcus strains, intraspecific competitive interactions partly determine the carriage duration of strains and thereby their potential to cause disease. These interactions may be mediated by bacteriocins, such as the type IIb bacteriocins encoded by the blp (bacteriocin-like peptide) locus. To understand blp diversity and evolution, we undertook a bioinformatic analysis of 4,418 pneumococcal genomes, including 168 newly sequenced genomes. We describe immense variation at all levels of genomic organization: Gene presence/absence, gene order, and allelic diversity. If we make the extreme and naive hypothesis that assumes all genes in this operon can assort randomly, this variation could lead to 1015 distinct bacteriocin-related phenotypes, each potentially representing a unique ecological strategy; however, we provide several explanations for why this extreme is not realized. Although rarefaction analysis indicates that the number of unique strategies is not saturated, even after sampling thousands of genomes, we show that the variation is neither unbounded nor random. We delimit three bacteriocin groups, which contain group-specific bacteriocins, immunity genes, and blp operon gene order, and argue that this organization places a constraint on realized ecological strategies. We additionally show that ecological strategy diversity is significantly constrained by pneumococcal phylogeny and clonal structure. By examining patterns of association between alleles within the blp operon, we show that bacteriocin genes, which were believed to function in pairs, can be found with a broad diversity of partner alleles and immunity genes; this overall lack of allelic fidelity likely contributes to the fluid structure of this operon. Our results clarify the diversity of antagonistic ecological strategies in the

  12. Novel twin streptolysin S-like peptides encoded in the sag operon homologue of beta-hemolytic Streptococcus anginosus.

    PubMed

    Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A; Nagamune, Hideaki

    2013-03-01

    Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.

  13. Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

    PubMed Central

    Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A.

    2013-01-01

    Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci. PMID:23292771

  14. Cloning and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic peptide domains.

    PubMed

    Lei, Weiwei; Zhang, Yong; Yu, Guoyu; Jiang, Ping; He, Yingying; Lee, Wenhui; Zhang, Yun

    2011-04-01

    The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs.

  15. Neutralizing antibodies decrease the envelope fluidity of HIV-1

    SciTech Connect

    Harada, Shinji Monde, Kazuaki; Tanaka, Yuetsu; Kimura, Tetsuya; Maeda, Yosuke; Yusa, Keisuke

    2008-01-05

    For successful penetration of HIV-1, the formation of a fusion pore may be required in order to accumulate critical numbers of fusion-activated gp41 with the help of fluidization of the plasma membrane and viral envelope. An increase in temperature to 40 {sup o}C after viral adsorption at 25 {sup o}C enhanced the infectivity by 1.4-fold. The enhanced infectivity was inhibited by an anti-CXCR4 peptide, T140, and anti-V3 monoclonal antibodies (0.5{beta} and 694/98-D) by post-attachment neutralization, but not by non-neutralizing antibodies (670-30D and 246-D) specific for the C5 of gp120 and cluster I of gp41, respectively. Anti-HLA-II and an anti-HTLV-I gp46 antibody, LAT27, neutralized the molecule-carrying HIV-1{sub C-2(MT-2)}. The anti-V3 antibodies suppressed the fluidity of the HIV-1{sub C-2} envelope, whereas the non-neutralizing antibodies did not. The anti-HLA-II antibody decreased the envelope fluidity of HIV-1{sub C-2(MT-2)}, but not that of HIV-1{sub C-2}. Therefore, fluidity suppression by these antibodies represents an important neutralization mechanism, in addition to inhibition of viral attachment.

  16. Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C

    PubMed Central

    Ghimire, Dibya; Timilsina, Uddhav; Srivastava, Tryambak Pratap; Gaur, Ritu

    2017-01-01

    HIV maturation inhibitors are an emerging class of anti-retroviral compounds that inhibit the viral protease-mediated cleavage of the Gag, CA-SP1 (capsid-spacer peptide 1) peptide to mature CA. The first-in-class maturation inhibitor bevirimat (BVM) displayed potent activity against HIV-1 clade B but was ineffective against other HIV-1 clades including clade C. Another pyridone-based maturation inhibitor, PF-46396 displayed potent activity against HIV-1 clade B. In this study, we aimed at determining the activity of PF-46396 against HIV-1 clade C. We employed various biochemical and virological assays to demonstrate that PF-46396 is effective against HIV-1 clade C. We observed a dose dependent accumulation of CA-SP1 intermediate in presence of the compound. We carried out mutagenesis in the CA- SP1 region of HIV-1 clade C Gag and observed that the mutations conferred resistance against the compound. Many mutations inhibited Gag processing thereby reducing virus release in the absence of the compound. However, presence of PF-46396 rescued these defects and enhanced virus release, replication capacity and infectivity of HIV-1 clade C. These results put together identify PF-46396 as a broadly active maturation inhibitor against HIV-1 clade B and C and help in rational designing of novel analogs with reduced toxicity and increased efficacy for its potential use in clinics. PMID:28252110

  17. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses.

    PubMed

    Smith, Heath A; Rekoske, Brian T; McNeel, Douglas G

    2014-03-26

    Plasmid DNA serves as a simple and easily modifiable form of antigen delivery for vaccines. The USDA approval of DNA vaccines for several non-human diseases underscores the potential of this type of antigen delivery method as a cost-effective approach for the treatment or prevention of human diseases, including cancer. However, while DNA vaccines have demonstrated safety and immunological effect in early phase clinical trials, they have not consistently elicited robust anti-tumor responses. Hence many recent efforts have sought to increase the immunological efficacy of DNA vaccines, and we have specifically evaluated several target antigens encoded by DNA vaccine as treatments for human prostate cancer. In particular, we have focused on SSX2 as one potential target antigen, given its frequent expression in metastatic prostate cancer. We have previously identified two peptides, p41-49 and p103-111, as HLA-A2-restricted SSX2-specific epitopes. In the present study we sought to determine whether the efficacy of a DNA vaccine could be enhanced by an altered peptide ligand (APL) strategy wherein modifications were made to anchor residues of these epitopes to enhance or ablate their binding to HLA-A2. A DNA vaccine encoding APL modified to increase epitope binding elicited robust peptide-specific CD8+ T cells producing Th1 cytokines specific for each epitope. Ablation of one epitope in a DNA vaccine did not enhance immune responses to the other epitope. These results demonstrate that APL encoded by a DNA vaccine can be used to elicit increased numbers of antigen-specific T cells specific for multiple epitopes simultaneously, and suggest this could be a general approach to improve the immunogenicity of DNA vaccines encoding tumor antigens.

  18. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    SciTech Connect

    Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

    2015-01-15

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.

  19. Low complexity regions (LCRs) contribute to the hypervariability of the HIV-1 gp120 protein.

    PubMed

    María Velasco, Ana; Becerra, Arturo; Hernández-Morales, Ricardo; Delaye, Luis; Jiménez-Corona, María Eugenia; Ponce-de-Leon, Samuel; Lazcano, Antonio

    2013-12-07

    Low complexity regions (LCRs) are sequences of nucleic acids or proteins defined by a compositional bias. Their occurrence has been confirmed in sequences of the three cellular lineages (Bacteria, Archaea and Eucarya), and has also been reported in viral genomes. We present here the results of a detailed computer analysis of the LCRs present in the HIV-1 glycoprotein 120 (gp120) encoded by the viral gene env. The analysis was performed using a sample of 3637 Env polyprotein sequences derived from 4117 completely sequenced and translated HIV-1 genomes available in public databases as of December 2012. We have identified 1229 LCRs located in four different regions of the gp120 protein that correspond to four of the five regions that have been identified as hypervariable (V1, V2, V4 and V5). The remaining 29 LCRs are found in the signal peptide and in the conserved regions C2, C3, C4 and C5. No LCR has been identified in the hypervariable region V3. The LCRs detected in the V1, V2, V4, and V5 hypervariable regions exhibit a high Asn content in their amino acid composition, which very likely correspond to glycosylation sites, which may contribute to the retroviral ability to avoid the immune system. In sharp contrast with what is observed in gp120 proteins lacking LCRs, the glycosylation sites present in LCRs tend to be clustered towards the center of the region forming well-defined islands. The results presented here suggest that LCRs represent a hitherto undescribed source of genomic variability in lentivirus, and that these repeats may represent an important source of antigenic variation in HIV-1 populations. The results reported here may exemplify the evolutionary processes that may have increased the size of primitive cellular RNA genomes and the role of LCRs as a source of raw material during the processes of evolutionary acquisition of new functions.

  20. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  1. HIV-1 production is specifically associated with human NMT1 long form in human NMT isozymes.

    PubMed

    Takamune, Nobutoki; Gota, Kayoko; Misumi, Shogo; Tanaka, Kenzo; Okinaka, Shigetaka; Shoji, Shozo

    2008-02-01

    The N-myristoylation of the N-terminal of human immunodeficiency virus type-1 (HIV-1) Pr55(gag) by human N-myristoyltransferase (hNMT) is a prerequisite modification for HIV-1 production. hNMT consists of multiple isozymes encoded by hNMT1 and hNMT2. The hNMT1 isozyme consists of long, medium, and short forms. Here, we investigated which isozyme is crucial for HIV-1 production. Human embryonic kidney (HEK) 293 cells transfected with infectious HIV-1 vectors were used as models of HIV-1-infected cells in this study. The significant reduction in HIV-1 production and the failure of the specific localization of Pr55(gag) in a detergent-resistant membrane fraction were dependent on the knockdown of the different forms of the hNMT1 isozyme but not of the hNMT2 isozyme. Additionally, the coexpression of an inactive mutant hNMT1 isozyme, namely the hNMT1 long form (hNMT1(L)), but not that of other hNMT mutants resulted in a significant reduction in HIV-1 production. These results strongly suggest that HIV-1 production is specifically associated with hNMT1, particularly hNMT1(L), but not with hNMT2 in vivo, contributing to the understanding of a step in HIV-1 replication.

  2. Two putative subunits of a peptide pump encoded in the human major histocompatibility complex class II region.

    PubMed Central

    Bahram, S; Arnold, D; Bresnahan, M; Strominger, J L; Spies, T

    1991-01-01

    The class II region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class I molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class I molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class II DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by gamma interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class I molecules, although a distinct function of PSF2 cannot be ruled out. Images PMID:1946428

  3. Polyepitope protein incorporated the HIV-1 mimotope recognized by monoclonal antibody 2G12.

    PubMed

    Karpenko, Larisa I; Scherbakova, Nadezhda S; Chikaev, Anton N; Tumanova, Olga Yu; Lebedev, Leonid R; Shalamova, Lyudmila A; Pyankova, Olga G; Ryzhikov, Alexander B; Ilyichev, Alexander A

    2012-04-01

    A major goal in HIV-1 vaccine research is to develop an immunogen that can elicit broadly neutralizing antibodies that efficiently neutralize a wide range of the HIV-1 subtypes. Using biopanning procedure we have selected linear peptide VGAFGSFYRLSVLQS mimicking the structure of discontinuous binding sites of broadly neutralizing antibodies 2G12 from phage peptide library. As a protein carrier, we used the earlier designed artificial polyepitope immunogen named TBI (T- and B-cell immunogen), which comprises B-cell and T-helper epitopes from the HIV-1 Env and Gag proteins. On the base of selected peptide mimotope VGAFGSFYRLSVLQS the artificial protein TBI-2g12 was constructed and its immunogenic properties was investigated. It was shown that the TBI-2g12 as well as the original TBI induces antibodies that recognize HIV-1 proteins and TBI protein using ELISA and immunoblotting. However only anti-TBI-2g12 serum recognized the synthetic peptide mimotope VGAFGSFYRLSVLQS, whereas the antibodies against original TBI don't recognize it. The neutralization assay demonstrated that serum antibodies of the mice immunized with TBI-2g12 possess virus neutralizing activity. The addition of selected peptide leads to inhibition neutralizing activity of anti- TBI-2g12 serum. We conclude from these results that immunogen TBI-2g12 containing the selected peptide VGAFGSFYRLSVLQS elicits HIV-1 neutralizing antibodies during immunization. Our data suggest that this immunogen may be useful in designing effective HIV-vaccine candidates.

  4. A mitogenic peptide amide encoded within the E peptide domain of the insulin-like growth factor IB prohormone.

    PubMed Central

    Siegfried, J M; Kasprzyk, P G; Treston, A M; Mulshine, J L; Quinn, K A; Cuttitta, F

    1992-01-01

    We have identified an amino acid sequence within the E peptide of the insulin-like growth factor IB (IGF-IB) precursor that is biologically active and designated this peptide insulin-like growth factor IB-(103-124) E1 amide (IBE1). Its existence was predicted by a flanking Gly-Lys-Lys-Lys, a signal sequence for sequential proteolytic cleavage and peptidyl C-terminal amidation. A synthetic analog of the predicted IBE1 peptide, designated Y-23-R-NH2, was generated with tyrosine added at position 0. This peptide at 2-20 nM had growth-promoting effects on both normal and malignant human bronchial epithelial cells. Y-23-R-NH2 bound to specific high-affinity receptors (Kd = 2.8 +/- 1.4 x 10(-11) M) present at 1-2 x 10(4) binding sites per cell. Ligand binding was not inhibited by recombinant insulin or recombinant IGF-I. Furthermore, a monoclonal antibody antagonist to the IGF-I receptor (alpha IR3) did not suppress the proliferative response induced by Y-23-R-NH2. In addition, C-terminal amidation was shown to be important in receptor recognition since the free-acid analog of IBE1 (Y-23-R-OH) did not effectively compete for binding and was not a potent agonist of proliferation. Immunoblot analysis of human lung tumor cell line extracts using an antibody raised against Y-23-R-NH2 detected a low molecular mass band of approximately 5 kDa, implying that a protein product is produced that has immunological similarity to IBE1. Extracts of human, mammalian, and avian livers analyzed on an immunoblot with the anti-Y-23-R-NH2 antibody contained proteins of approximately 21 kDa that were specifically recognized by the antiserum and presumably represent an IGF-I precursor molecule. This implies that in species where an IGF-I mRNA with homology to the human IGF-IB E domain has not yet been described, an alternate mRNA must be produced that contains a sequence similar to that of the midportion of the human IGF-IB E domain. Our findings demonstrate that IBE1 is a growth factor that

  5. Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide

    PubMed Central

    Hanson, Joshua M.; Gettel, Douglas L.; Tabaei, Seyed R.; Jackman, Joshua; Kim, Min Chul; Sasaki, Darryl Y.; Groves, Jay T.; Liedberg, Bo; Cho, Nam-Joon; Parikh, Atul N.

    2016-01-01

    The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity. PMID:26745420

  6. Inhibition of HIV-1 gp41 expression with hammerhead ribozymes.

    PubMed

    Fedoruk-Wyszomirska, Agnieszka; Szymański, Maciej; Głodowicz, Paweł; Gabryelska, Marta; Wyszko, Eliza; Estrin, William J; Barciszewski, Jan

    2015-10-01

    Despite great progress in the treatment of AIDS, HIV-1 remains one of the major concerns as a human pathogen. One of the therapeutic strategies against viral infections is the application of catalytic ribonucleic acids (ribozymes) that can significantly reduce expression of a target gene by site-specific hydrolysis of its mRNA. In the present paper, we report a study on the activity of several variants of hammerhead ribozymes targeting a conserved region within mRNA encoding HIV-1 envelope glycoprotein gp41. On the basis of the data from in vitro assays and gene silencing in the cultured cells, we propose a new hammerhead ribozyme targeting the gp41-encoding sequence that can be potentially used as a therapeutic agent in AIDS treatment. Moreover, we demonstrate that the hydrolytic activity of the ribozyme in the intracellular environment cannot be inferred solely from the results of in vitro experiments.

  7. Inhibition of HIV-1 by fusion inhibitors.

    PubMed

    Eggink, Dirk; Berkhout, Ben; Sanders, Rogier W

    2010-01-01

    The envelope glycoprotein complex (Env) is responsible for entry of the human immunodeficiency virus type 1 (HIV-1) into cells by mediating attachment to target cells and subsequent membrane fusion. Env consists of three gp120 subunits that mediate receptor and co-receptor attachment and three gp41 subunits responsible for membrane fusion. Several steps of the entry process can serve as drug targets. Receptor antagonists prevent attachment of gp120 to the receptor or co-receptor and conformational changes within gp41 required for membrane fusion can be inhibited by fusion inhibitors. Enfuvirtide (T20, Fuzeon) is a peptide based on the gp41 sequence and is the only approved fusion inhibitor. It prevents membrane fusion by competitively binding to gp41 and blocking the formation of the post-fusion structure. New generations of T20-like peptides have been developed with improved potency and stability. Besides T20 and derivatives, other fusion inhibitors have been developed that target different domains of gp41. Here we discuss the development of fusion inhibitors, their mode of action and their potential for incorporation in future drug regimens.

  8. HIV-1 Entry Inhibitors: Recent Development and Clinical Use

    PubMed Central

    Henrich, Timothy J.; Kuritzkes, Daniel R.

    2014-01-01

    Purpose of review This review provides an overview of HIV-1 entry inhibitors, with a focus on drugs in the later stages of clinical development. Recent findings Entry of HIV-1 into target cells involves viral attachment, co-receptor binding and fusion. Antiretroviral drugs that interact with each step in the entry process have been developed, but only two are currently approved for clinical use. The small molecule attachment inhibitor BMS-663068 has shown potent antiviral activity in early phase studies, and phase 2b trials are currently underway. The post-attachment inhibitor ibalizumab has shown antiviral activity in phase 1 and 2 trials; further studies, including subcutaneous delivery of drug to healthy individuals, are anticipated. The CCR5 antagonist maraviroc is approved for use in treatment-naïve and treatment-experienced patients. Cenicriviroc, a small-molecule CCR5 antagonist that also has activity as a CCR2 antagonist, has entered phase 2b studies. No CXCR4 antagonists are currently in clinical trials, but once daily, next-generation injectable peptide fusion inhibitors have entered human trials. Both maraviroc and ibalizumab are being studied for prevention of HIV-1 transmission and/or for use in nucleoside reverse transcriptase inhibitor-sparing antiretroviral regimens. Summary Inhibition of HIV-1 entry continues to be a promising target for antiretroviral drug development. PMID:23290628

  9. Dual-function sRNA encoded peptide SR1P modulates moonlighting activity of B. subtilis GapA

    PubMed Central

    Gimpel, Matthias; Brantl, Sabine

    2016-01-01

    ABSTRACT SR1 is a dual-function sRNA from B. subtilis that acts as a base-pairing regulatory RNA and as a peptide-encoding mRNA. Both functions of SR1 are highly conserved. Previously, we uncovered that the SR1 encoded peptide SR1P binds the glycolytic enzyme GapA resulting in stabilization of gapA mRNA. Here, we demonstrate that GapA interacts with RNases Y and J1, and this interaction was RNA-independent. About 1% of GapA molecules purified from B. subtilis carry RNase J1 and about 2% RNase Y. In contrast to the GapA/RNase Y interaction, the GapA/RNaseJ1 interaction was stronger in the presence of SR1P. GapA/SR1P-J1/Y displayed in vitro RNase activity on known RNase J1 substrates. Moreover, the RNase J1 substrate SR5 has altered half-lives in a ΔgapA strain and a Δsr1 strain, suggesting in vivo functions of the GapA/SR1P/J1 interaction. Our results demonstrate that the metabolic enzyme GapA moonlights in recruiting RNases while GapA bound SR1P promotes binding of RNase J1 and enhances its activity. PMID:27449348

  10. Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor

    SciTech Connect

    Dame, J.B.; Williams, J.L.; McCutchan, T.F.; Schneider, I.

    1987-11-17

    An antimalarial immunogenic stimulant is described comprising an immunogenic carrier and a peptide sequence of between 2 and 1000 consecutive repeats of a sequence Asn-X-Y-Pro, wherein X is Ala or Val and Y is Asn or Asp.

  11. GAD1 Encodes a Secreted Peptide That Regulates Grain Number, Grain Length, and Awn Development in Rice Domestication[OPEN

    PubMed Central

    Hua, Lei; Zhao, Xinhui; Zhang, Weifeng; Liu, Fengxia; Fu, Yongcai; Cai, Hongwei; Sun, Xianyou; Gu, Ping; Xie, Daoxin

    2016-01-01

    Cultivated rice (Oryza sativa) was domesticated from wild rice (Oryza rufipogon), which typically displays fewer grains per panicle and longer grains than cultivated rice. In addition, wild rice has long awns, whereas cultivated rice has short awns or lacks them altogether. These changes represent critical events in rice domestication. Here, we identified a major gene, GRAIN NUMBER, GRAIN LENGTH AND AWN DEVELOPMENT1 (GAD1), that regulates those critical changes during rice domestication. GAD1 is located on chromosome 8 and is predicted to encode a small secretary signal peptide belonging to the EPIDERMAL PATTERNING FACTOR-LIKE family. A frame-shift insertion in gad1 destroyed the conserved cysteine residues of the peptide, resulting in a loss of function, and causing the increased number of grains per panicle, shorter grains, and awnless phenotype characteristic of cultivated rice. Our findings provide a useful paradigm for revealing functions of peptide signal molecules in plant development and helps elucidate the molecular basis of rice domestication. PMID:27634315

  12. Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides

    SciTech Connect

    Robakis, N.K.; Ramakrishna, N.; Wolfe, G.; Wisniewski, H.M.

    1987-06-01

    Deposits of amyloid fibers are found in large numbers in the walls of blood vessels and in neuritic plaques in the brains of patients with Alzheimer disease and adults with Down syndrome. The authors used the amino acid sequence of the amyloid peptide to synthesize oligonucleotide probes specific for the gene encoding this peptide. When a human brain cDNA library was screened with this probe, a clone was found with a 1.7-kilobase insert that contains a long open reading frame coding for 412 amino acid residues including the 28 amino acids of the amyloid peptide. RNA gel blots revealed that a 3.3-kilobase mRNA species was present in the brains of individuals with Alzheimer disease, with Down syndrome, or with not apparent neurological disorders. Southern blots showed that homologous genes are present in the genomic DNA of humans, rabbits, sheep, hamsters, and mice, suggesting that this gene has been conserved through mammalian evolution. Localization of the corresponding genomic sequences on human chromosome 21 suggest a genetic relationship between Alzheimer disease and Down syndrome, and it may explain the early appearance of large numbers of neuritic plaques in adult Down syndrome patients.

  13. Cross-Reactive Potential of HIV-1 Subtype C-Infected Indian Individuals Against Multiple HIV-1 Potential T Cell Epitope Gag Variants.

    PubMed

    Negi, Neema; Vajpayee, Madhu; Singh, Ravinder; Sharma, Ashutosh; Murugavel, Kailapuri G; Ranga, Udaykumar; Thakar, Madhuri; Sreenivas, Vishnubhatla; Das, Bimal Kumar

    2016-12-01

    Vaccine immunogen with expanded T cell coverage for protection against HIV-1 diversity is the need of the hour. This study was undertaken to examine the ability of T cells to respond to a broad spectrum of potential T cell epitope (PTE) peptides containing variable as well as conserved sequences that would most accurately reflect immune responses to different circulating strains. Set of 320 PTE peptides were pooled in a matrix format that included 40 pools of 32 peptides per pool. These pools were used in interferon-γ enzyme-linked immunospot assay for screening and confirmation of HIV-1 PTE Gag-specific T cell immune responses in 34 HIV-1 seropositive Indian individuals. "Deconvolute This" software was used for result analysis. The dominant target in terms of magnitude and breadth of responses was observed to be the p24 subunit of Gag protein. Of the 34 study subjects, 26 (77%) showed a response to p24 PTE Gag peptides, 17 (50%) to p17, and 17 (50%) responded to p15 PTE peptides. The total breadth and magnitude of immune response ranged from 0.75 to 14.50 and 95.02 to 1,103 spot-forming cells/10(6) cells, respectively. Seventy-six peptides located in p24 Gag were targeted by 77% of the study subjects followed by 51 peptides in p17 Gag and 46 peptides in p15 Gag with multiple variants being recognized. Maximum study participants recognized PTE peptide sequence Gag271→285NKIVRMYSPVSILDI located in p24 Gag subunit. T cells from HIV-1-infected individuals can recognize multiple PTE peptide variants, although the magnitude of the responses can vary greatly across these variants.

  14. Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells

    PubMed Central

    2012-01-01

    Background The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs. Results Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication. Conclusions HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known. PMID:22458358

  15. Gp120 on HIV-1 Virions Lacks O-Linked Carbohydrate.

    PubMed

    Stansell, Elizabeth; Panico, Maria; Canis, Kevin; Pang, Poh-Choo; Bouché, Laura; Binet, Daniel; O'Connor, Michael-John; Chertova, Elena; Bess, Julian; Lifson, Jeffrey D; Haslam, Stuart M; Morris, Howard R; Desrosiers, Ronald C; Dell, Anne

    2015-01-01

    As HIV-1-encoded envelope protein traverses the secretory pathway, it may be modified with N- and O-linked carbohydrate. When the gp120s of HIV-1 NL4-3, HIV-1 YU2, HIV-1 Bal, HIV-1 JRFL, and HIV-1 JRCSF were expressed as secreted proteins, the threonine at consensus position 499 was found to be O-glycosylated. For SIVmac239, the corresponding threonine was also glycosylated when gp120 was recombinantly expressed. Similarly-positioned, highly-conserved threonines in the influenza A virus H1N1 HA1 and H5N1 HA1 envelope proteins were also found to carry O-glycans when expressed as secreted proteins. In all cases, the threonines were modified predominantly with disialylated core 1 glycans, together with related core 1 and core 2 structures. Secreted HIV-1 gp140 was modified to a lesser extent with mainly monosialylated core 1 O-glycans, suggesting that the ectodomain of the gp41 transmembrane component may limit the accessibility of Thr499 to glycosyltransferases. In striking contrast to these findings, gp120 on purified virions of HIV-1 Bal and SIV CP-MAC lacked any detectable O-glycosylation of the C-terminal threonine. Our results indicate the absence of O-linked carbohydrates on Thr499 as it exists on the surface of virions and suggest caution in the interpretation of analyses of post-translational modifications that utilize recombinant forms of envelope protein.

  16. Functional C‐TERMINALLY ENCODED PEPTIDE (CEP) plant hormone domains evolved de novo in the plant parasite Rotylenchulus reniformis

    PubMed Central

    Eves‐Van Den Akker, Sebastian; Lilley, Catherine J.; Yusup, Hazijah B.; Jones, John T.

    2016-01-01

    Summary Sedentary plant‐parasitic nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re‐differentiate into unique and metabolically active ‘feeding sites’. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C‐TERMINALLY ENCODED PEPTIDE (CEP) plant hormone mimics (RrCEPs), in the syncytia‐forming plant parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP‐encoding nematode genus identified to date (Meloidogyne), this suggests that CEPs probably evolved de novo in R. reniformis. We have characterized the first member of this large gene family (RrCEP1), demonstrating its significant up‐regulation during the plant–nematode interaction and expression in the effector‐producing pharyngeal gland cell. All internal CEP domains of multi‐domain RrCEPs are followed by di‐basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of up‐regulating plant nitrate transporter (AtNRT2.1) expression, whilst simultaneously reducing primary root elongation. When a non‐CEP‐containing, syncytia‐forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP‐rich environment, a smaller feeding site is produced. We hypothesize that CEPs of R. reniformis represent a two‐fold adaptation to sustained biotrophy in this species: (i) increasing host nitrate uptake, whilst (ii) limiting the size of the syncytial feeding site produced. PMID:26996971

  17. A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.

    PubMed Central

    Dropulić, B; Hĕrmánková, M; Pitha, P M

    1996-01-01

    Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8855316

  18. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    PubMed Central

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-01-01

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4+ T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4+ T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4+ T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. PMID:26184775

  19. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load.

    PubMed

    Addo, M M; Yu, X G; Rathod, A; Cohen, D; Eldridge, R L; Strick, D; Johnston, M N; Corcoran, C; Wurcel, A G; Fitzpatrick, C A; Feeney, M E; Rodriguez, W R; Basgoz, N; Draenert, R; Stone, David R; Brander, C; Goulder, P J R; Rosenberg, E S; Altfeld, M; Walker, B D

    2003-02-01

    Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

  20. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake.

    PubMed

    Zhang, Jian V; Ren, Pei-Gen; Avsian-Kretchmer, Orna; Luo, Ching-Wei; Rauch, Rami; Klein, Cynthia; Hsueh, Aaron J W

    2005-11-11

    Ghrelin, a circulating appetite-inducing hormone, is derived from a prohormone by posttranslational processing. On the basis of the bioinformatic prediction that another peptide also derived from proghrelin exists, we isolated a hormone from rat stomach and named it obestatin-a contraction of obese, from the Latin "obedere," meaning to devour, and "statin," denoting suppression. Contrary to the appetite-stimulating effects of ghrelin, treatment of rats with obestatin suppressed food intake, inhibited jejunal contraction, and decreased body-weight gain. Obestatin bound to the orphan G protein-coupled receptor GPR39. Thus, two peptide hormones with opposing action in weight regulation are derived from the same ghrelin gene. After differential modification, these hormones activate distinct receptors.

  1. Macrophages and HIV-1: An Unhealthy Constellation.

    PubMed

    Sattentau, Quentin J; Stevenson, Mario

    2016-03-09

    Lentiviruses have a long-documented association with macrophages. Abundant evidence exists for in vitro and, in a tissue-specific manner, in vivo infection of macrophages by the primate lentiviruses HIV-1 and SIV. However, macrophage contribution to aspects of HIV-1 and SIV pathogenesis, and their role in viral persistence in individuals on suppressive antiretroviral therapy, remains unclear. Here we discuss recent evidence implicating macrophages in HIV-1-mediated disease and highlight directions for further investigation.

  2. Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

    PubMed

    Chao, Lijun; Lu, Lu; Yang, Hengwen; Zhu, Yun; Li, Yuan; Wang, Qian; Yu, Xiaowen; Jiang, Shibo; Chen, Ying-Hua

    2013-01-01

    The HIV-1 envelope glycoprotein (Env) gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR) of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521) of the human POB1 (the partner of RalBP1), designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR) of gp41, and to the six-helix bundle (6-HB) formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

  3. HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA.

    PubMed

    Ren, Xiao-Xin; Wang, Hai-Bo; Li, Chuan; Jiang, Jin-Feng; Xiong, Si-Dong; Jin, Xia; Wu, Li; Wang, Jian-Hua

    2016-02-26

    HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.

  4. The HIV-1 epidemic in South Africa.

    PubMed

    Puren, A J

    2002-01-01

    The first reported cases of HIV-1 infection in South Africa occurred in 1982. Two distinct HIV-1 epidemic patterns were recognized. Initially the infection was prevalent in white males who had sex with males. The HIV-1 clade B was associated with this group. By 1989, the second epidemic was recognized primarily in the black population. Infections in this case were mainly heterosexual in origin. The HIV-1 clade involved was mainly C. The national HIV-1 sero-prevalence in antenatal attendees was less than 1% in 1990 and by 1994 this figure had risen to 7.5%. The most recent antenatal surveillance for HIV-1 sero-prevalence in 1999 revealed the following. The national prevalence rate for 1999 was 22.4% compared with the 1998 rate of 22.8%. The data highlighted the profound effect the epidemic had and will have on the disease burden in South Africa and by extension on the social and economic fronts. This view was emphasised by the impact HIV-1 infection had on tuberculosis. For example, sentinel surveys have attributed 44% of tuberculosis cases to HIV-1 infection. Moreover, the high prevalence of sexually transmitted infections will certainly exacerbate the HIV-1 epidemic.

  5. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    PubMed

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application.

  6. Genomic organization of the gene that encodes the precursor to EGF-related peptides, exogastrula-inducing peptides, of the sea urchin Anthocidaris crassispina.

    PubMed

    Haruguchi, Yoshiko; Horii, Keisuke; Suzuki, Gentaroh; Suyemitsu, Takashi; Ishihara, Katsutoshi; Yamasu, Kyo

    2002-04-12

    Exogastrula-inducing peptides (EGIPs) were identified in embryos of the sea urchin Anthocidaris crassispina as polypeptides with structural similarity to epidermal growth factor (EGF) that severely affect gastrulation of sea urchin embryos to induce exogastrulation. Here we have obtained genomic clones for the EGIP precursor gene (EGIP) and determined its genomic organization. The EGIP gene spans the length of 9 kb in the genome and is composed of seven exons and six introns. Each of the four EGF motifs in the precursor protein is encoded by a single exon, and all the exon boundaries are in phase 1, suggesting that EGIP have been generated during evolution by duplication of an exon encoding a single ancient EGIP sequence. The 5'-flanking sequence of EGIP from -4372 to +194 revealed the presence of multiple repeat sequences including direct and inverted repeats as well as two clusters of GGGG/CCCC elements. The function of the upstream flanking region of EGIP was examined by introducing the gene constructs into embryos in which different regions from the flanking DNA were placed upstream to the GFP reporter gene. Systematic deletion of the upstream DNA revealed the presence of potent enhancer activity between -372 and -210.

  7. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  8. Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede Scolopendra subspinipes mutilans.

    PubMed

    Liu, Wanhong; Luo, Feng; He, Jing; Cao, Zhijian; Miao, Lixia

    2012-01-01

    Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was successfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5' UTR and a 171 nt 3' UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

  9. Mass spectrometric identification, sequence evolution, and intraspecific variability of dimeric peptides encoded by cockroach akh genes.

    PubMed

    Sturm, Sebastian; Predel, Reinhard

    2015-02-01

    Neuropeptides are structurally the most diverse group of messenger molecules of the nervous system. Regarding neuropeptide identification, distribution, function, and evolution, insects are among the best studied invertebrates. Indeed, more than 100 neuropeptides are known from single species. Most of these peptides can easily be identified by direct tissue or cell profiling using MALDI-TOF MS. In these experiments, protein hormones with extensive post-translational modifications such as inter- and intramolecular disulfides are usually missed. It is evident that an exclusion of these bioactive molecules hinders the utilization of direct profiling methods in comprehensive peptidomic analyses. In the current study, we focus on the detection and structural elucidation of homo- and heterodimeric adipokinetic hormone precursor-related peptides (APRPs) of cockroaches. The physiological relevance of these molecules with highly conserved sequences in insects is still uncertain. Sequence similarities with vertebrate growth hormone-releasing factors have been reported, but remarkably, few data regarding APRP processing exist and these data are restricted to locusts. Here, we elucidated sequences of carbamidomethylated APRP monomers of different cockroaches by means of MALDI-TOF MS(2), and we were able to identify a surprisingly large number of APRP sequences, resulting either from intraspecific amino acid substitutions within the APRP sequences or C-terminal truncated APRPs.

  10. Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation.

    PubMed

    Kamata, Masakazu; Kim, Patrick Y; Ng, Hwee L; Ringpis, Gene-Errol E; Kranz, Emiko; Chan, Joshua; O'Connor, Sean; Yang, Otto O; Chen, Irvin S Y

    2015-07-31

    Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance.

  11. A comparison of the Genie and western blot assays in confirmatory testing for HIV-1 antibody.

    PubMed

    Chan, E L; Sidaway, F; Horsman, G B

    1996-03-01

    The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.

  12. Skin Electroporation of a Plasmid Encoding hCAP-18/LL-37 Host Defense Peptide Promotes Wound Healing

    PubMed Central

    Steinstraesser, Lars; Lam, Martin C; Jacobsen, Frank; Porporato, Paolo E; Chereddy, Kiran Kumar; Becerikli, Mustafa; Stricker, Ingo; Hancock, Robert EW; Lehnhardt, Marcus; Sonveaux, Pierre; Préat, Véronique; Vandermeulen, Gaëlle

    2014-01-01

    Host defense peptides, in particular LL-37, are emerging as potential therapeutics for promoting wound healing and inhibiting bacterial growth. However, effective delivery of the LL-37 peptide remains limiting. We hypothesized that skin-targeted electroporation of a plasmid encoding hCAP-18/LL-37 would promote the healing of wounds. The plasmid was efficiently delivered to full-thickness skin wounds by electroporation and it induced expression of LL-37 in the epithelium. It significantly accelerated reepithelialization of nondiabetic and diabetic wounds and caused a significant VEGFa and interleukin (IL)-6 induction. IL-6 was involved in LL-37–mediated keratinocyte migration in vitro and IL-6 neutralizing antibodies delivered to mice were able to suppress the wound healing activity of the hCAP-18/LL-37 plasmid. In a hindlimb ischemia model, electroporation of the hCAP-18/LL-37 plasmid increased blood perfusion, reduced muscular atrophy, and upregulated the angiogenic chemokines VEGFa and SDF-1a, and their receptors VEGF-R and CXCR-4. These findings demonstrate that a localized gene therapy with LL-37 is a promising approach for the treatment of wounds. PMID:24394186

  13. Lyme Disease-Causing Borrelia Species Encode Multiple Lipoproteins Homologous to Peptide-Binding Proteins of ABC-Type Transporters

    PubMed Central

    Kornacki, Jon A.; Oliver, Donald B.

    1998-01-01

    To identify cell envelope proteins of Borrelia burgdorferi, the causative agent of Lyme disease, we constructed a library of B. burgdorferi genes fused to the Escherichia coli phoA gene, which expresses enzymatically active alkaline phosphatase. One such gene, oppA-1, encodes a predicted polypeptide with significant similarities to various peptide-binding proteins of ABC-type transporters. Immediately downstream of oppA-1 are two genes, oppA-2 and oppA-3, whose predicted polypeptide products show strong similarities in their amino acid sequences to OppA-1, including a sequence that resembles the most highly conserved region in peptide-binding proteins. By labeling with [3H]palmitate, OppA-1, OppA-2, and OppA-3 were shown to be lipoproteins. DNA hybridization analysis showed that the oppA-1 oppA-2 oppA-3 region is located on the linear chromosome of B. burgdorferi, and the genes are conserved among different Borrelia species that cause Lyme disease (B. burgdorferi, B. garinii, and B. afzelli), suggesting that all three homologous genes are important to the maintenance of Lyme disease spirochetes in one or more of their hosts. PMID:9712756

  14. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes.

    PubMed

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S; Dewdney, Tamaria G; Reiter, Samuel J; Brunzelle, Joseph S; Kovari, Iulia A; Kovari, Ladislau C

    2013-01-18

    The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  15. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Dewdney, Tamaria G.; Reiter, Samuel J.; Brunzelle, Joseph S.; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  16. HIV-1-induced AIDS in monkeys.

    PubMed

    Hatziioannou, Theodora; Del Prete, Gregory Q; Keele, Brandon F; Estes, Jacob D; McNatt, Matthew W; Bitzegeio, Julia; Raymond, Alice; Rodriguez, Anthony; Schmidt, Fabian; Mac Trubey, C; Smedley, Jeremy; Piatak, Michael; KewalRamani, Vineet N; Lifson, Jeffrey D; Bieniasz, Paul D

    2014-06-20

    Primate lentiviruses exhibit narrow host tropism, reducing the occurrence of zoonoses but also impairing the development of optimal animal models of AIDS. To delineate the factors limiting cross-species HIV-1 transmission, we passaged a modified HIV-1 in pigtailed macaques that were transiently depleted of CD8(+) cells during acute infection. During adaptation over four passages in macaques, HIV-1 acquired the ability to antagonize the macaque restriction factor tetherin, replicated at progressively higher levels, and ultimately caused marked CD4(+) T cell depletion and AIDS-defining conditions. Transient treatment with an antibody to CD8 during acute HIV-1 infection caused rapid progression to AIDS, whereas untreated animals exhibited an elite controller phenotype. Thus, an adapted HIV-1 can cause AIDS in macaques, and stark differences in outcome can be determined by immunological perturbations during early infection.

  17. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.

    PubMed

    Yoder, Kristine E; Bundschuh, Ralf

    2016-07-12

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.

  18. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9

    PubMed Central

    Yoder, Kristine E.; Bundschuh, Ralf

    2016-01-01

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance. PMID:27404981

  19. Increased immunoglobulin G, but not M, binding to endogenous retroviral antigens in HIV-1 infected persons.

    PubMed

    Lawoko, A; Johansson, B; Rabinayaran, D; Pipkorn, R; Blomberg, J

    2000-12-01

    The modes of interaction between products of human endogenous retroviral (HERV) sequences and the immune system are largely unknown. In HIV infected persons, an exogenous retrovirus adds further complexity to the situation. Therefore, 14 synthetic peptides with sequences derived from conserved regions of various endogenous retroviruses (ERVs) and from related exogenous retroviruses were used to search for IgG and IgM antibodies that bind to such antigens in 15 HIV-1 seropositive and 17 seronegative immunosuppressed patients. IgG binding to three peptides, namely, the C-terminal half of murine leukemia virus (MLV) capsid protein, the conserved portion of HERV-H transmembrane protein, and the Pol region of human mouse mammary tumor virus (MMTV)-like (HML3) sequence, was observed in both groups. Binding was, however, more frequent and more firm in HIV-1 positive samples (P<0.0001, Wilcoxon rank sum test). IgM binding to the same peptides showed no significant differentiation between the two groups of patients. Binding to both immunoglobulin isotypes was sometimes variable over time in both groups. No correlation of either IgG or IgM peptide binding with progression to AIDS in HIV-1 infected individuals was observed. Inhibition studies using analogous endogenous and exogenous retroviral peptides, including HIV-1, demonstrated specificity of the IgG antibodies for a narrow range of MLV- and MMTV-like retroviral antigens, and excluded cross-reactivity of antibodies to HIV-1 as a cause of these observations. Thus, unlike IgG, IgM binding to retroviral antigens was ubiquitous. It is suggested that anti-HERV IgM belong to a class of natural antibodies and might serve as primers in the mediation of humoral immune responses to more or less related exogenous retroviruses. Increased IgG binding in HIV-1 infected individuals could result from such priming, or reflect higher HERV antigen expression.

  20. A turquoise mutant genetically separates expression of genes encoding phycoerythrin and its associated linker peptides.

    PubMed

    Seib, Laura Ort; Kehoe, David M

    2002-02-01

    During complementary chromatic adaptation (CCA), cyanobacterial light harvesting structures called phycobilisomes are restructured in response to ambient light quality shifts. Transcription of genes encoding components of the phycobilisome is differentially regulated during this process: red light activates cpcB2A2, whereas green light coordinately activates the cpeCDE and cpeBA operons. Three signal transduction components that regulate CCA have been isolated to date: a sensor-photoreceptor (RcaE) and two response regulators (RcaF and RcaC). Mutations in the genes encoding these components affect the accumulation of both cpcB2A2 and cpeBA gene products. We have isolated and characterized a new pigmentation mutant called Turquoise 1. We demonstrate that this mutant phenotype is due to a dramatic decrease in cpeBA transcript abundance and results from a lesion in the cpeR gene. However, in this mutant cpeCDE RNA levels remain near those found in wild-type cells. Our results show that the coordinate regulation of cpeBA and cpeCDE by green light can be uncoupled by the loss of CpeR, and we furnish the first genetic evidence that different regulatory mechanisms control these two operons. Sequence analysis of CpeR reveals that it shares limited sequence similarity to members of the PP2C class of protein serine/threonine phosphatases. We also demonstrate that cpeBA and cpeCDE retain light quality responsiveness in a mutant lacking the RcaE photoreceptor. This provides compelling evidence for the partial control of CCA through an as-yet-uncharacterized second light quality sensing system.

  1. GADD45 proteins inhibit HIV-1 replication through specific suppression of HIV-1 transcription.

    PubMed

    Liang, Zhibin; Liu, Ruikang; Zhang, Hui; Zhang, Suzhen; Hu, Xiaomei; Tan, Juan; Liang, Chen; Qiao, Wentao

    2016-06-01

    GADD45 proteins are a group of stress-induced proteins and participate in various cellular pathways including cell cycle regulation, cell survival and death, DNA repair and demethylation. It was recently shown that HIV-1 infection induces the expression of GADD45 proteins. However, the effect of GADD45 on HIV-1 replication has not been studied. Here, we report that overexpression of GADD45 proteins reduces HIV-1 production through suppressing transcription from the HIV-1 LTR promoter. This inhibitory effect is specific to HIV-1, since GADD45 proteins neither inhibit the LTR promoters from other retroviruses nor reduce the production of these viruses. Knockdown of endogenous GADD45 modestly activates HIV-1 in the J-Lat A72 latency cell line, which suggests GADD45 proteins might play a role in maintaining HIV-1 latency.

  2. HIV-1 proteins in infected cells determine the presentation of viral peptides by HLA class I and class II molecules and the nature of the cellular and humoral antiviral immune responses--a review.

    PubMed

    Becker, Y

    1994-07-01

    The goals of molecular virology and immunology during the second half of the 20th century have been to provide the conceptual approaches and the tools for the development of safe and efficient virus vaccines for the human population. The success of the vaccination approach to prevent virus epidemics was attributed to the ability of inactivated and live virus vaccines to induce a humoral immune response and to produce antiviral neutralizing antibodies in the vaccinees. The successful development of antiviral vaccines and their application to most of the human population led to a marked decrease in virus epidemics around the globe. Despite this remarkable achievement, the developing epidemics of HIV-caused AIDS (accompanied by activation of latent herpesviruses in AIDS patients), epidemics of Dengue fever, and infections with respiratory syncytial virus may indicate that conventional approaches to the development of virus vaccines that induce antiviral humoral responses may not suffice. This may indicate that virus vaccines that induce a cellular immune response, leading to the destruction of virus-infected cells by CD8+ cytotoxic T cells (CTLs), may be needed. Antiviral CD8+ CTLs are induced by viral peptides presented within the peptide binding grooves of HLA class I molecules present on the surface of infected cells. Studies in the last decade provided an insight into the presentation of viral peptides by HLA class I molecules to CD8+ T cells. These studies are here reviewed, together with a review of the molecular events of virus replication, to obtain an overview of how viral peptides associate with the HLA class I molecules. A similar review is provided on the molecular pathway by which viral proteins, used as subunit vaccines or inactivated virus particles, are taken up by endosomes in the endosome pathway and are processed by proteolytic enzymes into peptides that interact with HLA class II molecules during their transport to the plasma membrane of antigen

  3. Ion Channel Activity of Vpu Proteins Is Conserved throughout Evolution of HIV-1 and SIV

    PubMed Central

    Greiner, Timo; Bolduan, Sebastian; Hertel, Brigitte; Groß, Christine; Hamacher, Kay; Schubert, Ulrich; Moroni, Anna; Thiel, Gerhard

    2016-01-01

    The human immunodeficiency virus type 1 (HIV-1) protein Vpu is encoded exclusively by HIV-1 and related simian immunodeficiency viruses (SIVs). The transmembrane domain of the protein has dual functions: it counteracts the human restriction factor tetherin and forms a cation channel. Since these two functions are causally unrelated it remains unclear whether the channel activity has any relevance for viral release and replication. Here we examine structure and function correlates of different Vpu homologs from HIV-1 and SIV to understand if ion channel activity is an evolutionary conserved property of Vpu proteins. An electrophysiological testing of Vpus from different HIV-1 groups (N and P) and SIVs from chimpanzees (SIVcpz), and greater spot-nosed monkeys (SIVgsn) showed that they all generate channel activity in HEK293T cells. This implies a robust and evolutionary conserved channel activity and suggests that cation conductance may also have a conserved functional significance. PMID:27916968

  4. Adenoviral gene delivery for HIV-1 vaccination.

    PubMed

    Vanniasinkam, T; Ertl, H C J

    2005-04-01

    The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.

  5. Ectopic expression of anti-HIV-1 shRNAs protects CD8{sup +} T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation

    SciTech Connect

    Kamata, Masakazu; Kim, Patrick Y.; Ng, Hwee L.; Ringpis, Gene-Errol E.; Kranz, Emiko; Chan, Joshua; O'Connor, Sean; Yang, Otto O.; Chen, Irvin S.Y.

    2015-07-31

    Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8{sup +} T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8{sup +} T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8{sup +} T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24{sup Gag} in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8{sup +} T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8{sup +} T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8{sup +} T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance. - Highlights: • Ectopic expression of CD4ζ CAR in CD8{sup +} T cells renders them susceptible to HIV-1 infection. • Co-expression of two anti-HIV-1 shRNAs protects CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection. • Protecting CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection suppresses its cytopathic effect.

  6. Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

    PubMed Central

    Ahmed, Tina; Borthwick, Nicola J.; Gilmour, Jill; Hayes, Peter; Dorrell, Lucy; Hanke, Tomáš

    2016-01-01

    Objective The specificity of CD8+ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8+ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4+ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. Design CD8+ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. Methods Frozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. Results We formally demonstrated that the vaccine-elicited inhibitory human CD8+ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. Conclusions These results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient

  7. Antiretroviral (HIV-1) activity of azulene derivatives.

    PubMed

    Peet, Julia; Selyutina, Anastasia; Bredihhin, Aleksei

    2016-04-15

    The antiretroviral activity of azulene derivatives was detected for the first time. A series of eighteen diversely substituted azulenes was synthesized and tested in vitro using HIV-1 based virus-like particles (VLPs) and infectious HIV-1 virus in U2OS and TZM-bl cell lines. Among the compounds tested, the 2-hydroxyazulenes demonstrated the most significant activity by inhibiting HIV-1 replication with IC50 of 2-10 and 8-20 μM for the VLPs and the infectious virus, respectively. These results indicate that azulene derivatives may be potentially useful candidates for the development of antiretroviral agents.

  8. Nucleic acids encoding mosaic HIV-1 gag proteins

    SciTech Connect

    Korber, Bette T.; Perkins, Simon; Bhattacharya, Tanmoy; Fischer, William M.; Theiler, James; Letvin, Norman; Haynes, Barton F.; Hahn, Beatrice H.; Yusim, Karina; Kuiken, Carla

    2016-11-15

    The disclosure generally relates to an immunogenic composition (e.g., a vaccine) and, in particular, to a polyvalent immunogenic composition, such as a polyvalent HIV vaccine, and to methods of using same.

  9. The APPEESFRS Peptide, Restricted by the HLA-B*35:01 Molecule, and the APPEESFRF Variant Derived from an Autologous HIV-1 Strain Induces Polyfunctional Responses in CD8+ T Cells

    PubMed Central

    Acevedo-Sáenz, Liliana; Carmona-Pérez, Liseth; Velilla-Hernández, Paula Andrea; Delgado, Julio C.; Rugeles L., María Teresa

    2015-01-01

    Abstract Numerous reports have focused on consensus peptides to determine CD8+ T-cell responses; however, few studies evaluated the functional profile using peptides derived from circulating strains of a specific region. We determined the effector profile and maturation phenotype of CD8+ T-cells targeting the consensus APPEESFRS (AS9) epitope and its variant APPEESFRF (AF9), previously identified. The free energy of binding, maturation phenotype, and polyfunctional profile of both peptides were similar. The magnitude of CD8+ T-cell responses to AF9 was greater than the one elicited by AS9, although the difference was not significant. The polyfunctional profile of AF9 was characterized by CD107a/interleukin-2 (IL-2)/macrophage inflammatory protein beta (MIP1β) and by interferon gamma (IFNγ)/MIP1β/tumor necrosis factor alpha (TNFα) in response to AS9. TNFα production was significantly higher in response to AF9 than to AS9, and there was a negative correlation between the absolute number of CD8+ T-cell-producing TNFα and the plasma human immunodeficiency virus (HIV) load, suggesting a role of this cytokine in the control of HIV replication. PMID:26309788

  10. Prevalence and persistence of antibody titers to recombinant HIV-1 core and matrix proteins in HIV-1 infection.

    PubMed

    Janvier, B; Mallet, F; Cheynet, V; Dalbon, P; Vernet, G; Besnier, J M; Choutet, P; Goudeau, A; Mandrand, B; Barin, F

    1993-08-01

    Numerous studies have established the correlation between antibodies to the core protein p24 of HIV-1 and the progression of the acquired immunodeficiency syndrome. In this study, we analyzed the immune response to two recombinant gag proteins, p24 and p17, in order to evaluate their diagnostic or prognostic significance. Immune response to the immunodominant domain of the transmembrane glycoprotein gp41 was used as a reference. Sera collected from individuals from France and Burundi (Central Africa) at various CDC stages of HIV-1 infection were tested using three sandwich enzyme-linked immunoassays developed with a synthetic peptide corresponding to the immunodominant domain of gp41, SP gp41, or recombinant p24 and p17 cloned and expressed in Escherichia coli. These assays allowed detection of titer antibodies to the three cited antigens. Antibodies to SP gp41 were detected in every HIV-1-positive patient from France and Burundi, generally at a high and stable level. Results obtained with p24 confirmed the value of antibodies to p24 as a prognostic marker only in European and North American populations, since the African population had very high levels of these antibodies even at an advanced stage of the disease. They also confirmed that initial antibody response to p24 is more predictive of outcome than antibody titer change over time. Although antibodies to p17 decline during progression to AIDS, they are frequently absent in French patients at early, asymptomatic stages and therefore could not be used as a prognostic marker. In contrast, antibodies to p17 are significantly less common in African patients with AIDS when compared with symptomless HIV-1-infected African individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Development of prophylactic vaccines against HIV-1.

    PubMed

    Schiffner, Torben; Sattentau, Quentin J; Dorrell, Lucy

    2013-07-17

    The focus of most current HIV-1 vaccine development is on antibody-based approaches. This is because certain antibody responses correlated with protection from HIV-1 acquisition in the RV144 phase III trial, and because a series of potent and broad spectrum neutralizing antibodies have been isolated from infected individuals. Taken together, these two findings suggest ways forward to develop a neutralizing antibody-based vaccine. However, understanding of the correlates of protection from disease in HIV-1 and other infections strongly suggests that we should not ignore CTL-based research. Here we review recent progress in the field and highlight the challenges implicit in HIV-1 vaccine design and some potential solutions.

  12. HIV-1 Eradication: Early Trials (and Tribulations).

    PubMed

    Spivak, Adam M; Planelles, Vicente

    2016-01-01

    Antiretroviral therapy (ART) has rendered HIV-1 infection a manageable illness for those with access to treatment. However, ART does not lead to viral eradication owing to the persistence of replication-competent, unexpressed proviruses in long-lived cellular reservoirs. The potential for long-term drug toxicities and the lack of access to ART for most people living with HIV-1 infection have fueled scientific interest in understanding the nature of this latent reservoir. Exploration of HIV-1 persistence at the cellular and molecular level in resting memory CD4(+) T cells, the predominant viral reservoir in patients on ART, has uncovered potential strategies to reverse latency. We review recent advances in pharmacologically based 'shock and kill' HIV-1 eradication strategies, including comparative analysis of early clinical trials.

  13. Immunogenicity and specificity of the candidate multi-epitope-vaccines against HIV-1.

    PubMed

    Lu, Y; Ding, J; Chen, Y H

    2001-11-01

    The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)2-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.

  14. Functional characteristics of the natural polymorphisms of HIV-1 gp41 in HIV-1 isolates from enfuvirtide-naïve Korean patients.

    PubMed

    Shin, YoungHyun; Yoon, Cheol-Hee; Yang, Hyo-Jin; Lim, Hoyong; Choi, Byeong-Sun; Kim, Sung Soon; Kang, Chun

    2016-06-01

    HIV-1 gp41 plays a key role in viral entry. The insertion of Thr at position 4 and Met/Val/Phe substitutions at position 7 are frequently observed in the fusion peptide (FP) motif of gp41 without major enfuvirtide resistance associated with mutation in heptad repeats 1/2 (HR1/2) of HIV-1 isolates from Korean patients. Here, the influence of these mutations on their biological function was evaluated by employing HIV-1 variants with mutant FPs as shown previously and with recombinant HIV-1 using the env genes of 20 HIV-1 isolates from Korean patients. In an infectivity assay, all FP mutants showed lower infectivity than the wild-type NL4-3. In particular, the substitutions at position 7 led to much greater reductions in infectivity than the insertions at position 4. Nevertheless, the replication kinetics of most mutants were similar to those of the wild type, except that the FP mutants with an Ile insertion at position 4 and a Phe substitution at position 7 showed reduced replication. Moreover, most point mutants showed lower IC50 values for enfuvirtide than the wild type, whereas the L7M substitution resulted in a slightly increased IC50 value. The infectivity using the HIV-1 env recombinant viruses decreased in 14 cases but increased slightly in six cases compared with the wild type. Most recombinants were more susceptible to enfuvirtide than the wild type, except for three recombinants that showed slight resistance. Our findings may help to explain the potential mechanisms corresponding to the natural polymorphism of gp41 and to predict the efficiency of enfuvirtide in treatment of HIV-1-infected patients in Korea.

  15. Substance abuse, HIV-1 and hepatitis.

    PubMed

    Parikh, Nirzari; Nonnemacher, Michael R; Pirrone, Vanessa; Block, Timothy; Mehta, Anand; Wigdahl, Brian

    2012-10-01

    During the course of human immunodeficiency virus type 1 (HIV-1) disease, the virus has been shown to effectively escape the immune response with the subsequent establishment of latent viral reservoirs in specific cell populations within the peripheral blood (PB) and associated lymphoid tissues, bone marrow (BM), brain, and potentially other end organs. HIV-1, along with hepatitis B and C viruses (HBV and HCV), are known to share similar routes of transmission, including intravenous drug use, blood transfusions, sexual intercourse, and perinatal exposure. Substance abuse, including the use of opioids and cocaine, is a significant risk factor for exposure to HIV-1 and the development of acquired immune deficiency syndrome, as well as HBV and HCV exposure, infection, and disease. Thus, coinfection with HIV-1 and HBV or HCV is common and may be impacted by chronic substance abuse during the course of disease. HIV- 1 impacts the natural course of HBV and HCV infection by accelerating the progression of HBV/HCV-associated liver disease toward end-stage cirrhosis and quantitative depletion of the CD4+ T-cell compartment. HBV or HCV coinfection with HIV-1 is also associated with increased mortality when compared to either infection alone. This review focuses on the impact of substance abuse and coinfection with HBV and HCV in the PB, BM, and brain on the HIV-1 pathogenic process as it relates to viral pathogenesis, disease progression, and the associated immune response during the course of this complex interplay. The impact of HIV-1 and substance abuse on hepatitis virus-induced disease is also a focal point.

  16. Cytolytic T lymphocytes (CTLs) from HIV-1 subtype C-infected Indian patients recognize CTL epitopes from a conserved immunodominant region of HIV-1 Gag and Nef.

    PubMed

    Thakar, Madhuri R; Bhonge, Leena S; Lakhashe, Samir K; Shankarkumar, U; Sane, Suvarna S; Kulkarni, Smita S; Mahajan, Bharati A; Paranjape, Ramesh S

    2005-09-01

    Analysis of the human immunodeficiency virus type 1 (HIV-1) cytolytic T lymphocyte (CTL) epitopes recognized by the targeted population is critical for HIV-1 vaccine design. Peripheral blood mononuclear cells from 47 Indian subjects at different stages of HIV-1 infection were tested for HIV-1 Gag-, Nef-, and Env-specific T cell responses by interferon (IFN)- gamma enzyme-linked immunospot (ELISPOT) assay, using pools of overlapping peptides. The Gag and Nef antigens were targeted by 83% and 36% of responders. Five immunodominant regions, 4 in Gag and 1 in Nef, were identified in the study; these regions are conserved across clades, including the African subtype C clade. Three antigenic regions were also found to be recognized by CTLs of the study participants. These regions were not identified as immunodominant regions in studies performed in Africa, which highlights the importance of differential clustering of responses within HIV-1 subtype C. Twenty-six putative epitopes--15 Gag (10 in p24 and 5 in p17), 10 Nef, and 1 Env (gp 41)--were predicted using a combination of peptide matrix ELISPOT assay and CTL epitope-prediction software. Ninety percent of the predicted epitopes were clustered in the conserved immunodominant regions of the Gag and Nef antigens. Of 26 predicted epitopes, 8 were promiscuous, 3 of which were highly conserved across clades. Three Gag and 4 Nef epitopes were novel. The identification of conserved epitopes will be important in the planning of an HIV-1 vaccine strategy for subtype C-affected regions.

  17. HIV-1 transmission linkage in an HIV-1 prevention clinical trial

    SciTech Connect

    Leitner, Thomas; Campbell, Mary S; Mullins, James I; Hughes, James P; Wong, Kim G; Raugi, Dana N; Scrensen, Stefanie

    2009-01-01

    HIV-1 sequencing has been used extensively in epidemiologic and forensic studies to investigate patterns of HIV-1 transmission. However, the criteria for establishing genetic linkage between HIV-1 strains in HIV-1 prevention trials have not been formalized. The Partners in Prevention HSV/HIV Transmission Study (ClinicaITrials.gov NCT00194519) enrolled 3408 HIV-1 serodiscordant heterosexual African couples to determine the efficacy of genital herpes suppression with acyclovir in reducing HIV-1 transmission. The trial analysis required laboratory confirmation of HIV-1 linkage between enrolled partners in couples in which seroconversion occurred. Here we describe the process and results from HIV-1 sequencing studies used to perform transmission linkage determination in this clinical trial. Consensus Sanger sequencing of env (C2-V3-C3) and gag (p17-p24) genes was performed on plasma HIV-1 RNA from both partners within 3 months of seroconversion; env single molecule or pyrosequencing was also performed in some cases. For linkage, we required monophyletic clustering between HIV-1 sequences in the transmitting and seroconverting partners, and developed a Bayesian algorithm using genetic distances to evaluate the posterior probability of linkage of participants sequences. Adjudicators classified transmissions as linked, unlinked, or indeterminate. Among 151 seroconversion events, we found 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) to have indeterminate transmissions. Nine (8.3%) were linked by consensus gag sequencing only and 8 (7.4%) required deep sequencing of env. In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage

  18. Exosomes: Implications in HIV-1 Pathogenesis.

    PubMed

    Madison, Marisa N; Okeoma, Chioma M

    2015-07-20

    Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. The role of exosomes in viral pathogenesis, especially human immunodeficiency virus type 1 [HIV-1] is beginning to unravel. Recent research reports suggest that exosomes from various sources play important but different roles in the pathogenesis of HIV-1. From these reports, it appears that the source of exosomes is the defining factor for the exosomal effect on HIV-1. In this review, we will describe how HIV-1 infection is modulated by exosomes and in turn how exosomes are targeted by HIV-1 factors. Finally, we will discuss potentially emerging therapeutic options based on exosomal cargos that may have promise in preventing HIV-1 transmission.

  19. Exosomes: Implications in HIV-1 Pathogenesis

    PubMed Central

    Madison, Marisa N.; Okeoma, Chioma M.

    2015-01-01

    Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. The role of exosomes in viral pathogenesis, especially human immunodeficiency virus type 1 [HIV-1] is beginning to unravel. Recent research reports suggest that exosomes from various sources play important but different roles in the pathogenesis of HIV-1. From these reports, it appears that the source of exosomes is the defining factor for the exosomal effect on HIV-1. In this review, we will describe how HIV-1 infection is modulated by exosomes and in turn how exosomes are targeted by HIV-1 factors. Finally, we will discuss potentially emerging therapeutic options based on exosomal cargos that may have promise in preventing HIV-1 transmission. PMID:26205405

  20. A structure-based approach to a synthetic vaccine for HIV-1.

    PubMed

    Cabezas, E; Wang, M; Parren, P W; Stanfield, R L; Satterthwait, A C

    2000-11-28

    The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH --> O=CRNH) with a hydrazone link (N-N=CH-CH(2)CH(2)) and scan constrained peptides for fit with neutralizing monoclonal antibodies (MAbs). To explore this approach, a V3 MAb 58.2 that potently neutralizes T-cell lab-adapted HIV-1(MN) was used to identify a cyclic peptide, [JHIGPGR(Aib)F(D-Ala)GZ]G-NH(2) (loop 5), that binds with >1000-fold higher affinity than the unconstrained peptide. NMR structural studies suggested that loop 5 stabilized beta-turns at GPGR and R(Aib)F(D-Ala) in aqueous solvent implying considerable conformational mimicry of a Fab 58.2 bound V3 peptide determined by X-ray crystallography [Stanfield, R. L. et al. (1999) Structure 142, 131-142]. Rabbit polyclonal antibodies (PAbs) generated to loop 5 but not to the corresponding uncyclized peptide bound the HIV-1(MN) envelope glycoprotein, gp120. When individual rabbit antisera were scanned with linear and cyclic peptides, further animal-to-animal differences in antibody populations were characterized. Loop 5 PAbs that most closely mimicked MAb 58.2 neutralized HIV-1(MN) with similar potency. These results demonstrate the remarkable effect that conformation can have on peptide affinity and immunogenicity and identify an approach that can be used to achieve these results. The implications for synthetic vaccine and HIV-1 vaccine research are discussed.

  1. A novel and rapid assay for HIV-1 protease detection using magnetic bead mediation.

    PubMed

    Esseghaier, Chiheb; Ng, Andy; Zourob, Mohammed

    2013-03-15

    A simple sensing assay was established for label-free detection of HIV-1 protease. HIV-1 protease peptide substrate conjugated to magnetic beads via its N-terminus is directly fixed onto the sensor gold surface through the sulphur atom of cysteine. Surface plasmon resonance (SPR) was used to study the peptide substrate cleavage efficiency of the protease with magnetic beads of different sizes (1 μm and 30 nm). Cyclic voltammetry and faradic impedance spectroscopy were employed in order to characterize the functionalized gold electrode. It was found that the nano-sized beads are a more efficient sensing probe for the protease. Electrochemical biosensing showed a gradual decrease in charge transfer resistance after injection of the HIV-1 protease. The experimental data established a detection limit of 10 pg/ml, as well as demonstrated a drug screening assay. This HIV-1 protease biosensor represents a new detection approach which will lead to low-cost point-of-care devices for sensitive HIV-1 diagnosis, as well as high-throughput drug screening platforms.

  2. Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1.

    PubMed

    Li, Hua; Liu, Zu-Qiang; Ding, Jian; Chen, Ying-Hua

    2002-11-01

    Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.

  3. Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection

    PubMed Central

    Lisanti, Antonella C.; Körner, Christian; Schiff, Abigail E.; Rosenberg, Eric S.; Allen, Todd M.; Altfeld, Marcus; Kwon, Douglas S.

    2017-01-01

    HIV-1 is able to evade innate antiviral responses during acute infection to establish a chronic systemic infection which, in the absence of antiretroviral therapy (ART), typically progresses to severe immunodeficiency. Understanding these early innate immune responses against HIV-1 and their mechanisms of failure is relevant to the development of interventions to better prevent HIV-1 transmission. Human beta defensins (HBDs) are antibacterial peptides but have recently also been associated with control of viral replication. HBD1 and 2 are expressed in PBMCs as well as intestinal tissue, but their expression in vivo during HIV-1 infection has not been characterized. We demonstrate that during acute HIV-1 infection, HBD1 but not HBD2 is highly upregulated in circulating monocytes but returns to baseline levels during chronic infection. HBD1 expression in monocytes can be induced by HIV-1 in vitro, although direct infection may not entirely account for the increase in HBD1 during acute infection. We provide evidence that HIV-1 triggers antiviral IFN-α responses, which act as a potent inducer of HBD1. Our results show the first characterization of induction of an HBD during acute and chronic viral infection in humans. HBD1 has been reported to have low activity against HIV-1 compared to other defensins, suggesting that in vivo induced defensins may not significantly contribute to the robust early antiviral response against HIV-1. These data provide important insight into the in vivo kinetics of HBD expression, the mechanism of HBD1 induction by HIV-1, and the role of HBDs in the early innate response to HIV-1 during acute infection. PMID:28253319

  4. Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate*

    PubMed Central

    Sneha Priya, Rangasamy; Veena, Menon; Kalisz, Irene; Whitney, Stephen; Priyanka, Dhopeshwarkar; LaBranche, Celia C.; Sri Teja, Mullapudi; Montefiori, David C.; Pal, Ranajit; Mahalingam, Sundarasamy; Kalyanaraman, Vaniambadi S.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines. PMID:25691567

  5. Artificial polyepitope HIV-1 immunogen containing mimotope of 2F5 epitope.

    PubMed

    Shcherbakova, Nadezhda S; Shcherbakov, Dmitry N; Bakulina, Anastasiya Yu; Karpenko, Larisa I; Ryzhikov, Alexander B; Ilyichev, Alexander A

    2016-01-01

    Constructing a vaccine against HIV-1, able to induce production of broadly neutralizing antibodies, is crucial. We report here the selection and characterization of RDWSFDRWSLSEFWL peptide mimotope that binds specifically to bNAbs 2F5. The peptide mimotope was selected from 15-mer phage-displayed peptide library by using Mab 2F5 as the selecting agent. The most abundant RDWSFDRWSLSEFWL peptide was inserted into a carrier, an artificial polyepitope immunogen - TBI (T- and B-cell immunogen). TBI-2F5 polyepitope immunogen that includes the mimotope of 2F5 epitope was constructed. It was shown that sera of mice immunized with TBI-2F5 protein recognized TBI protein as well as RDWSFDRWSLSEFWL peptide. The capacity of sera of immunized mice to neutralize HIV-1 was demonstrated using subtype B env-pseudoviruses of HIV-1 QH0692.42 and PVO.4. Based on these results, we conclude that peptide mimotope of 2F5 epitope RDWSFDRWSLSEFWL can be an essential component for a successful HIV-vaccine.

  6. Role of APOBEC3F Gene Variation in HIV-1 Disease Progression and Pneumocystis Pneumonia.

    PubMed

    An, Ping; Penugonda, Sudhir; Thorball, Christian W; Bartha, Istvan; Goedert, James J; Donfield, Sharyne; Buchbinder, Susan; Binns-Roemer, Elizabeth; Kirk, Gregory D; Zhang, Wenyan; Fellay, Jacques; Yu, Xiao-Fang; Winkler, Cheryl A

    2016-03-01

    Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant (p. I231V, with allele (V) frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) (RH = 0.53, 95% CI: 0.37-0.76). A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression.

  7. Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.

    PubMed

    Kepler, Thomas B; Liao, Hua-Xin; Alam, S Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Yandava, Chandri; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S Abdool; Cohen, Myron S; Walter, Emmanuel; Moody, M Anthony; Wu, Xueling; Altae-Tran, Han R; Georgiev, Ivelin S; Kwong, Peter D; Boyd, Scott D; Fire, Andrew Z; Mascola, John R; Haynes, Barton F

    2014-09-10

    Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.

  8. Role of APOBEC3F Gene Variation in HIV-1 Disease Progression and Pneumocystis Pneumonia

    PubMed Central

    An, Ping; Penugonda, Sudhir; Thorball, Christian W.; Bartha, Istvan; Goedert, James J.; Donfield, Sharyne; Buchbinder, Susan; Binns-Roemer, Elizabeth; Kirk, Gregory D.; Zhang, Wenyan; Fellay, Jacques; Yu, Xiao-Fang; Winkler, Cheryl A.

    2016-01-01

    Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant (p. I231V, with allele (V) frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) (RH = 0.53, 95% CI: 0.37–0.76). A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression. PMID:26942578

  9. Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene.

    PubMed Central

    Thiede, M A; Strewler, G J; Nissenson, R A; Rosenblatt, M; Rodan, G A

    1988-01-01

    A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene. Images PMID:3290897

  10. Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1–Seropositive Kenyan Men

    PubMed Central

    Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L.; Sanders, Eduard J.; Peshu, Norbert M.; Krieger, John N.; Muller, Charles H.; Coombs, Robert W.; Fredricks, David N.; Graham, Susan M.

    2017-01-01

    Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1–seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: −0.77, 95% CI: −1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1–infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission. PMID:27861240

  11. Virion-incorporated alpha-enolase suppresses the early stage of HIV-1 reverse transcription.

    PubMed

    Kishimoto, Naoki; Iga, Nozomi; Yamamoto, Kengo; Takamune, Nobutoki; Misumi, Shogo

    2017-03-04

    Human immunodeficiency virus type-1 (HIV-1) particles contain not only viral-encoded but also host-encoded proteins. Interestingly, several studies showed that host proteins play a critical role in viral infectivity, replication and/or immunoreactivity in the next target cells. Here, we show that alpha-enolase (ENO1) is incorporated into HIV-1 virions and the virion-incorporated ENO1 prevents the early stage of HIV-1 reverse transcription. We found that viral particles contain two isoforms of ENO1 with different isoelectric points by two-dimensional electrophoresis. Suppression of ENO1 expression by RNA interference in the HIV-1 producer cells decreased ENO1 incorporation into virions without altering the packaging of viral structural proteins and viral production but increased viral infectivity. Although the low-level-ENO1-packaging virus maintained comparable levels of reverse transcriptase activity, viral genomic RNA and tRNA(Lys3) packaging to the control virus, its levels of early cDNA products of reverse transcription were higher than those of the control virus. In contrast, the high-level-ENO1-packaging virus, which was produced from ENO1-overexpressing cells, showed decreased infectivity and the levels of early cDNA products. Taken together, these findings reveal a novel function of ENO1 as a negative regulation factor targeting HIV-1 reverse transcription.

  12. Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

    PubMed Central

    König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

    2008-01-01

    Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

  13. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1

    SciTech Connect

    Diaz-Griffero, Felipe; Vandegraaff, Nick; Li Yuan; McGee-Estrada, Kathleen; Stremlau, Matthew; Welikala, Sohanya; Si Zhihai; Engelman, Alan; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2006-08-01

    In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5{alpha} with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain.

  14. N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    PubMed Central

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

  15. Approaches for identification of HIV-1 entry inhibitors targeting gp41 pocket.

    PubMed

    Yu, Fei; Lu, Lu; Du, Lanying; Zhu, Xiaojie; Debnath, Asim K; Jiang, Shibo

    2013-01-11

    The hydrophobic pocket in the HIV-1 gp41 N-terminal heptad repeat (NHR) domain plays an important role in viral fusion and entry into the host cell, and serves as an attractive target for development of HIV-1 fusion/entry inhibitors. The peptide anti-HIV drug targeting gp41 NHR, T-20 (generic name: enfuvirtide; brand name: Fuzeon), was approved by the U.S. FDA in 2003 as the first HIV fusion/entry inhibitor for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, because T20 lacks the pocket-binding domain (PBD), it exhibits low anti-HIV-1 activity and short half-life. Therefore, several next-generation HIV fusion inhibitory peptides with PBD have been developed. They possess longer half-life and more potent antiviral activity against a broad spectrum of HIV-1 strains, including the T-20-resistant variants. Nonetheless, the clinical application of these peptides is still limited by the lack of oral availability and the high cost of production. Thus, development of small molecule compounds targeting the gp41 pocket with oral availability has been promoted. This review describes the main approaches for identification of HIV fusion/entry inhibitors targeting the gp41 pocket and summarizes the latest progress in developing these inhibitors as a new class of anti-HIV drugs.

  16. Macrophage polarization and HIV-1 infection.

    PubMed

    Cassol, Edana; Cassetta, Luca; Alfano, Massimo; Poli, Guido

    2010-04-01

    Polarization of MP into classically activated (M1) and alternatively activated (M2a, M2b, and M2c) macrophages is critical in mediating an effective immune response against invading pathogens. However, several pathogens use these activation pathways to facilitate dissemination and pathogenesis. Viruses generally induce an M1-like phenotype during the acute phase of infection. In addition to promoting the development of Th1 responses and IFN production, M1 macrophages often produce cytokines that drive viral replication and tissue damage. As shown for HIV-1, polarization can also alter macrophage susceptibility to infection. In vitro polarization into M1 cells prevents HIV-1 infection, and M2a polarization inhibits viral replication at a post-integration level. M2a cells also express high levels of C-type lectins that can facilitate macrophage-mediated transmission of HIV-1 to CD4(+) T cells. Macrophages are particularly abundant in mucosal membranes and unlike DCs, do not usually migrate to distal tissues. As a result, macrophages are likely to contribute to HIV-1 pathogenesis in mucosal rather than lymphatic tissues. In vivo polarization of MP is likely to span a spectrum of activation phenotypes that may change the permissivity to and alter the outcome of HIV-1 and other viral infections.

  17. HIV-1 Entry Inhbitors: An Overview

    PubMed Central

    Kuritzkes, Daniel R.

    2009-01-01

    Purpose of review This review provides an overview of HIV-1 entry inhibitors, with a focus on chemokine receptor antagonists. Recent findings Entry of HIV-1 into target cells is an ordered multi-step process involving attachment, co-receptor binding and fusion. Inhibitors of each step have been identified and shown to have antiviral activity in clinical trials. Phase 1-2 trials of monoclonal antibodies and small-molecule attachment inhibitors have demonstrated activity in HIV-1-infected subjects, but none has progressed to later phase clinical trials. The post-attachment inhibitor ibalizumab has shown activity in phase 1 and 2 trials; further studies are anticipated. The CCR5 antagonists maraviroc (now been approved for clinical use) and vicriviroc (in phase 3 trials) have shown significant benefit in controlled trials in treatment-experienced subjects; additional CCR5 antagonists are in various stages of clinical development. Targeting CXCR4 has proven to be more challenging. Although proof of concept has been demonstrated in phase 1-2 trials of two compounds, neither proved suitable for chronic administration. Little progress has been reported in developing longer acting or orally bioavailable fusion inhibitors. Summary ACCR5 antagonist and a fusion inhibitor are approved for use as HIV-1 entry inhibitors. Development of drugs targeting other steps in HIV-1 entry is ongoing. PMID:19339945

  18. Modulation of HIV-1 immunity by adjuvants

    PubMed Central

    Moody, M. Anthony

    2014-01-01

    Purpose of review To summarize the role of adjuvants in eliciting desirable antibody responses against HIV-1 with particular emphasis on both historical context and recent developments. Recent findings Increased understanding of the role of pattern recognition receptors such as Toll-like receptors in recruiting and directing the immune system has increased the variety of adjuvant formulations being tested in animal models and humans. Across all vaccine platforms, adjuvant formulations have been shown to enhance desirable immune responses such as higher antibody titers and increased functional activity. Although no vaccine formulation has yet succeeded in eliciting broad neutralizing antibodies against HIV-1, the ability of adjuvants to direct the immune response to immunogens suggests they will be critically important in any successful HIV-1 vaccine. Summary The parallel development of adjuvants along with better HIV-1 immunogens will be needed for a successful AIDS vaccine. Additional comparative testing will be required to determine the optimal adjuvant and immunogen regimen that can elicit antibody responses capable of blocking HIV-1 transmission. PMID:24670321

  19. HIV-1 associated dementia: symptoms and causes

    PubMed Central

    Ghafouri, Mohammad; Amini, Shohreh; Khalili, Kamel; Sawaya, Bassel E

    2006-01-01

    Despite the use of highly active antiretroviral therapy (HAART), neuronal cell death remains a problem that is frequently found in the brains of HIV-1-infected patients. HAART has successfully prevented many of the former end-stage complications of AIDS, however, with increased survival times, the prevalence of minor HIV-1 associated cognitive impairment appears to be rising among AIDS patients. Further, HIV-1 associated dementia (HAD) is still prevalent in treated patients as well as attenuated forms of HAD and CNS opportunistic disorders. HIV-associated cognitive impairment correlates with the increased presence in the CNS of activated, though not necessarily HIV-1-infected, microglia and CNS macrophages. This suggests that indirect mechanisms of neuronal injury and loss/death occur in HIV/AIDS as a basis for dementia since neurons are not themselves productively infected by HIV-1. In this review, we discussed the symptoms and causes leading to HAD. Outcome from this review will provide new information regarding mechanisms of neuronal loss in AIDS patients. PMID:16712719

  20. Technologies for the synthesis of mRNA-encoding libraries and discovery of bioactive natural product-inspired non-traditional macrocyclic peptides.

    PubMed

    Ito, Kenichiro; Passioura, Toby; Suga, Hiroaki

    2013-03-18

    In this review, we discuss emerging technologies for drug discovery, which yields novel molecular scaffolds based on natural product-inspired non-traditional peptides expressed using the translation machinery. Unlike natural products, these technologies allow for constructing mRNA-encoding libraries of macrocyclic peptides containing non-canonical sidechains and N-methyl-modified backbones. The complexity of sequence space in such libraries reaches as high as a trillion (>1012), affording initial hits of high affinity ligands against protein targets. Although this article comprehensively covers several related technologies, we discuss in greater detail the technical development and advantages of the Random non-standard Peptide Integration Discovery (RaPID) system, including the recent identification of inhibitors against various therapeutic targets.

  1. Cellular RNA helicases and HIV-1: insights from genome-wide, proteomic, and molecular studies.

    PubMed

    Chen, Chia-Yen; Liu, Xiang; Boris-Lawrie, Kathleen; Sharma, Amit; Jeang, Kuan-Teh

    2013-02-01

    RNA helicases are ubiquitous in plants and animals and function in many cellular processes. Retroviruses, such as human immunodeficiency virus (HIV-1), encode no RNA helicases in their genomes and utilize host cellular RNA helicases at various stages of their life cycle. Here, we briefly summarize the roles RNA helicases play in HIV-1 replication that have been identified recently, in part, through genome-wide screenings, proteomics, and molecular studies. Some of these helicases augment virus propagation while others apparently participate in antiviral defenses against viral replication.

  2. MAS NMR of HIV-1 protein assemblies

    NASA Astrophysics Data System (ADS)

    Suiter, Christopher L.; Quinn, Caitlin M.; Lu, Manman; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

    2015-04-01

    The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

  3. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation.

    PubMed

    Schur, Florian K M; Obr, Martin; Hagen, Wim J H; Wan, William; Jakobi, Arjen J; Kirkpatrick, Joanna M; Sachse, Carsten; Kräusslich, Hans-Georg; Briggs, John A G

    2016-07-29

    Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1.

  4. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.

    PubMed

    Martins, Laura J; Bonczkowski, Pawel; Spivak, Adam M; De Spiegelaere, Ward; Novis, Camille L; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Typsteen, Wim; Bosque, Alberto; Vanderkerckhove, Linos; Planelles, Vicente

    2016-02-01

    HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput.

  5. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus

    PubMed Central

    Martins, Laura J.; Bonczkowski, Pawel; Spivak, Adam M.; De Spiegelaere, Ward; Novis, Camille L.; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Typsteen, Wim; Vanderkerckhove, Linos

    2016-01-01

    Abstract HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4+ T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput. PMID:26171776

  6. Altered immunological reactivity in HIV-1-exposed uninfected neonates.

    PubMed

    Hygino, Joana; Lima, Patrícia G; Filho, Renato G S; Silva, Agostinho A L; Saramago, Carmen S M; Andrade, Regis M; Andrade, Daniel M; Andrade, Arnaldo F B; Brindeiro, Rodrigo; Tanuri, Amilcar; Bento, Cleonice A M

    2008-06-01

    This work aimed to evaluate immune events in HIV-1-exposed uninfected neonates born from mothers who control (G1) or not (G2) the plasma viral load, using unexposed neonates as controls. Cord blood from each neonate was collected, plasma and mononuclear cells were separated and the lymphoproliferation and cytokine pattern were evaluated. The results demonstrated that the in vitro lymphoproliferation induced by polyclonal activators was higher in the G2 neonates. Nevertheless, no cell culture responded to poll synthetic HIV-1 envelope peptides. The cytokine dosage in the plasma and supernatants of polyclonally-activated cultures demonstrated that, while IL-4 and IL-10 were the dominant cytokines produced in G1 and control groups, IFN-gamma and TNF-alpha were significantly higher in G2 neonates. Systemic levels of IL-10 observed among the G1 neonates were higher in those born from anti-retroviral treated mothers. In summary, our results indicate an altered immune responsiveness in neonates exposed in utero to HIV and support the role of maternal anti-retroviral treatment to attenuate it.

  7. HIV-1 matrix protein p17: a candidate antigen for therapeutic vaccines against AIDS.

    PubMed

    Fiorentini, Simona; Giagulli, Cinzia; Caccuri, Francesca; Magiera, Anna K; Caruso, Arnaldo

    2010-12-01

    The success in the development of anti-retroviral therapies (HAART) that contain human immunodeficiency virus type 1 (HIV-1) infection is challenged by the cost of this lifelong therapy and by its toxicity. Immune-based therapeutic strategies that boost the immune response against HIV-1 proteins or protein subunits have been recently proposed to control virus replication in order to provide protection from disease development, reduce virus transmission, and help limit the use of anti-retroviral treatments. HIV-1 matrix protein p17 is a structural protein that is critically involved in most stages of the life cycle of the retrovirus. Besides its well established role in the virus life cycle, increasing evidence suggests that p17 may also be active extracellularly in deregulating biological activities of many different immune cells that are directly or indirectly involved in AIDS pathogenesis. Thus, p17 might represent a promising target for developing a therapeutic vaccine as a contribution to combating AIDS. In this article we review the biological characteristics of HIV-1 matrix protein p17 and we describe why a synthetic peptide representative of the p17 functional epitope may work as a vaccine molecule capable of inducing anti-p17 neutralizing response against p17 derived from divergent HIV-1 strains.

  8. Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription

    PubMed Central

    Ammosova, Tatyana; Berro, Reem; Jerebtsova, Marina; Jackson, Angela; Charles, Sharroya; Klase, Zachary; Southerland, William; Gordeuk, Victor R; Kashanchi, Fatah; Nekhai, Sergei

    2006-01-01

    Background Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription. Results We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease. Conclusion Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription. PMID:17083724

  9. HIV-1 Capsid: The Multifaceted Key Player in HIV-1 infection

    PubMed Central

    Campbell, Edward M.; Hope, Thomas J.

    2016-01-01

    In a mature, infectious HIV-1 virion, the viral genome is housed within a conical capsid core comprised of the viral capsid (CA) protein. The CA protein, and the structure into which it assembles, facilitate virtually every step of infection through a series of interactions with multiple host cell factors. This review describes our understanding of the interactions between the viral capsid core and several cellular factors that enable efficient HIV-1 genome replication, timely core disassembly, nuclear import and the integration of the viral genome into the genome of the target cell. We then discuss how elucidating these interactions can reveal new targets for therapeutic interactions against HIV-1. PMID:26179359

  10. HIV-1 vaccines: challenges and new perspectives.

    PubMed

    Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

    2014-01-01

    The development of a safe and effective preventive HIV-1 vaccine remains a public health priority. Despite scientific difficulties and disappointing results, HIV-1 vaccine clinical development has, for the first time, established proof-of-concept efficacy against HIV-1 acquisition and identified vaccine-associated immune correlates of risk. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop correlated with decreased risk of HIV infection, while Env-specific IgA directly correlated with increased risk. The development of vaccine strategies such as improved envelope proteins formulated with potent adjuvants and DNA and vectors expressing mosaics, or conserved sequences, capable of eliciting greater breadth and depth of potentially relevant immune responses including neutralizing and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immunological memory, is now proceeding quickly. Additional human efficacy trials combined with other prevention modalities along with sustained funding and international collaboration remain key to bring an HIV-1 vaccine to licensure.

  11. HIV-1 transcription and latency: an update

    PubMed Central

    2013-01-01

    Combination antiretroviral therapy, despite being potent and life-prolonging, is not curative and does not eradicate HIV-1 infection since interruption of treatment inevitably results in a rapid rebound of viremia. Reactivation of latently infected cells harboring transcriptionally silent but replication-competent proviruses is a potential source of persistent residual viremia in cART-treated patients. Although multiple reservoirs may exist, the persistence of resting CD4+ T cells carrying a latent infection represents a major barrier to eradication. In this review, we will discuss the latest reports on the molecular mechanisms that may regulate HIV-1 latency at the transcriptional level, including transcriptional interference, the role of cellular factors, chromatin organization and epigenetic modifications, the viral Tat trans-activator and its cellular cofactors. Since latency mechanisms may also operate at the post-transcriptional level, we will consider inhibition of nuclear RNA export and inhibition of translation by microRNAs as potential barriers to HIV-1 gene expression. Finally, we will review the therapeutic approaches and clinical studies aimed at achieving either a sterilizing cure or a functional cure of HIV-1 infection, with a special emphasis on the most recent pharmacological strategies to reactivate the latent viruses and decrease the pool of viral reservoirs. PMID:23803414

  12. Multiple APOBEC3 Restriction Factors for HIV-1 and One Vif to Rule Them All

    PubMed Central

    Desimmie, Belete A.; Delviks-Frankenberry, Krista A.; Burdick, Ryan; Qi, Dongfei; Izumi, Taisuke; Pathak, Vinay K.

    2013-01-01

    Several members of the APOBEC3 family of cellular restriction factors provide intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection against HIV-1Δvif through hypermutation of the viral genome, inhibition of reverse transcription, and inhibition of viral DNA integration into the host genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif, which contains distinct domains that specifically interact with these APOBEC3 proteins to ensure their proteasomal degradation, allowing virus replication to proceed. Here, we review our current understanding of APOBEC3 structure, editing and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3 interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3 proteins to restriction and control of HIV-1 replication in infected patients. PMID:24189052

  13. Involvement of a small GTP binding protein in HIV-1 release

    PubMed Central

    Audoly, Gilles; Popoff, Michel R; Gluschankof, Pablo

    2005-01-01

    Background There is evidence suggesting that actin binding to HIV-1 encoded proteins, or even actin dynamics themselves, might play a key role in virus budding and/or release from the infected cell. A crucial step in the reorganisation of the actin cytoskeleton is the engagement of various different GTP binding proteins. We have thus studied the involvement of GTP-binding proteins in the final steps of the HIV-1 viral replication cycle. Results Our results demonstrate that virus production is abolished when cellular GTP binding proteins involved in actin polymerisation are inhibited with specific toxins. Conclusion We propose a new HIV budding working model whereby Gag interactions with pre-existing endosomal cellular tracks as well as with a yet non identified element of the actin polymerisation pathway are required in order to allow HIV-1 to be released from the infected cell. PMID:16080789

  14. Lysosomal and cytosolic sialic acid 9-O-acetylesterase activities can Be encoded by one gene via differential usage of a signal peptide-encoding exon at the N terminus.

    PubMed

    Takematsu, H; Diaz, S; Stoddart, A; Zhang, Y; Varki, A

    1999-09-03

    9-O-Acetylation is one of the most common modifications of sialic acids, and it can affect several sialic acid-mediated recognition phenomena. We previously reported a cDNA encoding a lysosomal sialic acid-specific 9-O-acetylesterase, which traverses the endoplasmic reticulum-Golgi pathway and localizes primarily to lysosomes and endosomes. In this study, we report a variant cDNA derived from the same gene that contains a different 5' region. This cDNA has a putative open reading frame lacking a signal peptide-encoding sequence and is thus a candidate for the previously described cytosolic sialic acid 9-O-acetylesterase activity. Epitope-tagged constructs confirm that the new sequence causes the protein product to be targeted to the cytosol and has esterase activity. Using reverse transcription-polymerase chain reaction to distinguish the two forms of message, we show that although the lysosomal sialic acid-specific 9-O-acetylesterase message has a widespread pattern of expression in adult mouse tissues, this cytosolic sialic acid 9-O-acetylesterase form has a rather restricted distribution, with the strongest expression in the liver, ovary, and brain. Using a polyclonal antibody directed against the 69-amino acid region common to both proteins, we confirmed that the expression of glycosylated and nonglycosylated polypeptides occurred in appropriate subcellular fractions of normal mouse tissues. Rodent liver polypeptides reacting to the antibody also co-purify with previously described lysosomal sialic acid esterase activity and at least a portion of the cytosolic activity. Thus, two sialic acid 9-O-acetylesterases found in very different subcellular compartments can be encoded by a single gene by differential usage of a signal peptide-encoding exon at the N terminus. The 5'-rapid amplification of cDNA ends results and the differences in tissue-specific expression suggest that expression of these two products may be differentially regulated by independent promoters.

  15. Mutation of a Single Residue Renders Human Tetherin Resistant to HIV-1 Vpu-Mediated Depletion

    PubMed Central

    Schaller, Torsten; Verschoor, Ernst; Pillay, Deenan; Towers, Greg J.

    2009-01-01

    The recently identified restriction factor tetherin/BST-2/CD317 is an interferon-inducible trans-membrane protein that restricts HIV-1 particle release in the absence of the HIV-1 countermeasure viral protein U (Vpu). It is known that Tantalus monkey CV1 cells can be rendered non-permissive to HIV-1 release upon stimulation with type 1 interferon, despite the presence of Vpu, suggesting species-specific sensitivity of tetherin proteins to viral countermeasures such as Vpu. Here we demonstrate that Tantalus monkey tetherin restricts HIV-1 by nearly two orders of magnitude, but in contrast to human tetherin the Tantalus protein is insensitive to HIV-1 Vpu. We have investigated tetherin's sensitivity to Vpu using positive selection analyses, seeking evidence for evolutionary conflict between tetherin and viral countermeasures. We provide evidence that tetherin has undergone positive selection during primate evolution. Mutation of a single amino acid (showing evidence of positive selection) in the trans-membrane cap of human tetherin to that in Tantalus monkey (T45I) substantially impacts on sensitivity to HIV-1 Vpu, but not on antiviral activity. Finally, we provide evidence that cellular steady state levels of tetherin are substantially reduced by Vpu, and that the T45I mutation abrogates this effect. This study provides evidence that tetherin is important in protecting mammals against viral infection, and that the HIV-1 Vpu–mediated countermeasure is specifically adapted to act against human tetherin. It also emphasizes the power of selection analyses to illuminate the molecular details of host–virus interactions. This work suggests that tetherin binding agents might protect it from viral encoded countermeasures and thus make powerful antivirals. PMID:19461879

  16. Cyclophilin A is required for the replication of group M human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIV(CPZ)GAB but not group O HIV-1 or other primate immunodeficiency viruses.

    PubMed Central

    Braaten, D; Franke, E K; Luban, J

    1996-01-01

    The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds to cyclophilin A and incorporates this cellular peptidyl prolyl-isomerase into virions. Disruption of cyclophilin A incorporation, either by gag mutations or by cyclosporine A, inhibits virion infectivity, indicating that cyclophilin A plays an essential role in the HIV-1 life cycle. Using assays for packaging of cyclophilin A into virions and for viral replication sensitivity to cyclosporine A, as well as information gleaned from the alignment of Gag residues encoded by representative viral isolates, we demonstrate that of the five lineages of primate immunodeficiency viruses, only HIV-1 requires cyclophilin A for replication. Cloned viral isolates from clades A, B, and D of HIV-1 group M, as well as a phylogenetically related isolate from chimpanzee, all require cyclophilin A for replication. In contrast, the replication of two outlier (group O) HIV-1 isolates is unaffected by concentrations of cyclosporine A which disrupt cyclophilin A incorporation into virions, indicating that these viruses are capable of replicating independently of cyclophilin A. These studies identify the first phenotypic difference between HIV-1 group M and group O and are consistent with phylogenetic studies suggesting that the two HIV-1 groups were introduced into human populations via separate zoonotic transmission events. PMID:8676442

  17. HIV-1 target cells in the CNS.

    PubMed

    Joseph, Sarah B; Arrildt, Kathryn T; Sturdevant, Christa B; Swanstrom, Ronald

    2015-06-01

    HIV-1 replication in the central nervous system (CNS) is typically limited by the availability of target cells. HIV-1 variants that are transmitted and dominate the early stages of infection almost exclusively use the CCR5 coreceptor and are well adapted to entering, and thus infecting, cells expressing high CD4 densities similar to those found on CD4+ T cells. While the "immune privileged" CNS is largely devoid of CD4+ T cells, macrophage and microglia are abundant throughout the CNS. These cells likely express CD4 densities that are too low to facilitate efficient entry or allow sustained replication by most HIV-1 isolates. Examination of CNS viral populations reveals that late in disease the CNS of some individuals contains HIV-1 lineages that have evolved the ability to enter cells expressing low levels of CD4 and are well-adapted to entering macrophages. These macrophage-tropic (M-tropic) viruses are able to maintain sustained replication in the CNS for many generations, and their presence is associated with severe neurocognitive impairment. Whether conditions such as pleocytosis are necessary for macrophage-tropic viruses to emerge in the CNS is unknown, and extensive examinations of macrophage-tropic variants have not revealed a genetic signature of this phenotype. It is clear, however, that macrophage tropism is rare among HIV-1 isolates and is not transmitted, but is important due to its pathogenic effects on hosts. Prior to the evolution of macrophage-tropic variants, the viruses that are predominately infecting T cells (R5 T cell-tropic) may infect macrophages at a low level and inefficiently, but this could contribute to the reservoir.

  18. HIV-1 target cells in the CNS

    PubMed Central

    Joseph, Sarah B.; Arrildt, Kathryn T.; Sturdevant, Christa B.; Swanstrom, Ronald

    2014-01-01

    HIV-1 replication in the central nervous system (CNS) is typically limited by the availability of target cells. HIV-1 variants that are transmitted and dominate the early stages of infection almost exclusively use the CCR5 coreceptor and are well adapted to entering, and thus infecting, cells expressing high CD4 densities similar to those found on CD4+ T cells. While the “immune privileged” CNS is largely devoid of CD4+ T cells, macrophage and microglia are abundant throughout the CNS. These cells likely express CD4 densities that are too low to facilitate efficient entry or allow sustained replication by most HIV-1 isolates. Examination of CNS viral populations reveals that late in disease the CNS of some individuals contains HIV-1 lineages that have evolved the ability to enter cells expressing low levels of CD4 and are well-adapted to entering macrophages. These macrophage-tropic (M-tropic) viruses are able to maintain sustained replication in the CNS for many generations, and their presence is associated with severe neurocognitive impairment. Whether conditions such as pleocytosis are necessary for macrophage-tropic viruses to emerge in the CNS is unknown, and extensive examinations of macrophage-tropic variants have not revealed a genetic signature of this phenotype. It is clear, however, that macrophage tropism is rare among HIV-1 isolates and is not transmitted, but is important due to its pathogenic effects on hosts. Prior to the evolution of macrophage-tropic variants, the viruses that are predominately infecting T cells (R5 T cell-tropic) may infect macrophages at a low level and inefficiently, but this could contribute to the reservoir. PMID:25236812

  19. HIV-1 gp41 fusion intermediate: a target for HIV therapeutics.

    PubMed

    Pan, Chungen; Liu, Shuwen; Jiang, Shibo

    2010-02-01

    Human immunodeficiency virus (HIV)-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4) and a coreceptor (CXCR4 or CCR5), followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR) peptide with the N-heptad repeat (NHR) trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

  20. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic.

    PubMed

    Cassan, Elodie; Arigon-Chifolleau, Anne-Muriel; Mesnard, Jean-Michel; Gross, Antoine; Gascuel, Olivier

    2016-10-11

    Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame -2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of ∼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins-an expected outcome with overlaps in frame -2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic.

  1. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic

    PubMed Central

    Cassan, Elodie; Arigon-Chifolleau, Anne-Muriel; Mesnard, Jean-Michel; Gross, Antoine; Gascuel, Olivier

    2016-01-01

    Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame −2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of ∼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp. Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins—an expected outcome with overlaps in frame −2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic. PMID:27681623

  2. A Minor Subset of Super Elongation Complexes Plays a Predominant Role in Reversing HIV-1 Latency.

    PubMed

    Li, Zichong; Lu, Huasong; Zhou, Qiang

    2016-02-01

    Promoter-proximal pausing by RNA polymerase II (Pol II) is a key rate-limiting step in HIV-1 transcription and latency reversal. The viral Tat protein recruits human super elongation complexes (SECs) to paused Pol II to overcome this restriction. Despite the recent progress in understanding the functions of different subsets of SECs in controlling cellular and Tat-activated HIV transcription, little is known about the SEC subtypes that help reverse viral latency in CD4(+) T cells. Here, we used the CRISPR-Cas9 genome-editing tool to knock out the gene encoding the SEC subunit ELL2, AFF1, or AFF4 in Jurkat/2D10 cells, a well-characterized HIV-1 latency model. Depletion of these proteins drastically reduced spontaneous and drug-induced latency reversal by suppressing HIV-1 transcriptional elongation. Surprisingly, a low-abundance subset of SECs containing ELL2 and AFF1 was found to play a predominant role in cooperating with Tat to reverse latency. By increasing the cellular level/activity of these Tat-friendly SECs, we could potently activate latent HIV-1 without using any drugs. These results implicate the ELL2/AFF1-SECs as an important target in the future design of a combinatorial therapeutic approach to purge latent HIV-1.

  3. Galectin-1 promotes HIV-1 infectivity in macrophages through stabilization of viral adsorption

    SciTech Connect

    Mercier, Simon; St-Pierre, Christian; Pelletier, Isabelle; Ouellet, Michel; Tremblay, Michel J. Sato, Sachiko

    2008-02-05

    Following primary infection with human immunodeficiency virus type-1 (HIV-1), macrophages are thought to play an important role, as they are one of the first target cells the virus encounters and can also sustain a significant production of viruses over extended periods of time. While the interaction between the primary cellular receptor CD4 and the virus-encoded external envelope glycoprotein gp120 initiates the infection process, it has been suggested that various host factors are exploited by HIV-1 to facilitate adsorption onto the cell surface. Macrophages and other cells found at the infection site can secrete a soluble mammalian lectin, galectin-1, which binds to {beta}-galactoside residues through its carbohydrate recognition domain. Being a dimer, galectin-1 can cross-link ligands expressed on different constituents to mediate adhesion between cells or between cells and pathogens. We report here that galectin-1, but not galectin-3, increased HIV-1 infectivity in monocyte-derived macrophages (MDMs). This phenomenon was likely due to an enhancement of virus adsorption kinetics, which facilitates HIV-1 entry. The fusion inhibitors T-20 and TAK779 remained effective at reducing infection even in the presence of galectin-1, indicating that the galectin-1-mediated effect is occurring at a step prior to fusion. Together, our data suggest that galectin-1 can facilitate HIV-1 infection in MDMs by promoting early events of the virus replicative cycle (i.e. adsorption)

  4. A Minor Subset of Super Elongation Complexes Plays a Predominant Role in Reversing HIV-1 Latency

    PubMed Central

    Li, Zichong; Lu, Huasong

    2016-01-01

    Promoter-proximal pausing by RNA polymerase II (Pol II) is a key rate-limiting step in HIV-1 transcription and latency reversal. The viral Tat protein recruits human super elongation complexes (SECs) to paused Pol II to overcome this restriction. Despite the recent progress in understanding the functions of different subsets of SECs in controlling cellular and Tat-activated HIV transcription, little is known about the SEC subtypes that help reverse viral latency in CD4+ T cells. Here, we used the CRISPR-Cas9 genome-editing tool to knock out the gene encoding the SEC subunit ELL2, AFF1, or AFF4 in Jurkat/2D10 cells, a well-characterized HIV-1 latency model. Depletion of these proteins drastically reduced spontaneous and drug-induced latency reversal by suppressing HIV-1 transcriptional elongation. Surprisingly, a low-abundance subset of SECs containing ELL2 and AFF1 was found to play a predominant role in cooperating with Tat to reverse latency. By increasing the cellular level/activity of these Tat-friendly SECs, we could potently activate latent HIV-1 without using any drugs. These results implicate the ELL2/AFF1-SECs as an important target in the future design of a combinatorial therapeutic approach to purge latent HIV-1. PMID:26830226

  5. Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo.

    PubMed

    Lu, Ching-Lan; Murakowski, Dariusz K; Bournazos, Stylianos; Schoofs, Till; Sarkar, Debolina; Halper-Stromberg, Ariel; Horwitz, Joshua A; Nogueira, Lilian; Golijanin, Jovana; Gazumyan, Anna; Ravetch, Jeffrey V; Caskey, Marina; Chakraborty, Arup K; Nussenzweig, Michel C

    2016-05-20

    Antiretroviral drugs and antibodies limit HIV-1 infection by interfering with the viral life cycle. In addition, antibodies also have the potential to guide host immune effector cells to kill HIV-1-infected cells. Examination of the kinetics of HIV-1 suppression in infected individuals by passively administered 3BNC117, a broadly neutralizing antibody, suggested that the effects of the antibody are not limited to free viral clearance and blocking new infection but also include acceleration of infected cell clearance. Consistent with these observations, we find that broadly neutralizing antibodies can target CD4(+) T cells infected with patient viruses and can decrease their in vivo half-lives by a mechanism that requires Fcγ receptor engagement in a humanized mouse model. The results indicate that passive immunotherapy can accelerate elimination of HIV-1-infected cells.

  6. Picomolar dichotomous activity of gnidimacrin against HIV-1.

    PubMed

    Huang, Li; Ho, Phong; Yu, Jie; Zhu, Lei; Lee, Kuo-Hsiung; Chen, Chin-Ho

    2011-01-01

    Highly active antiretroviral therapy (HAART) has offered a promising approach for controlling HIV-1 replication in infected individuals. However, with HARRT, HIV-1 is suppressed rather than eradicated due to persistence of HIV-1 in latent viral reservoirs. Thus, purging the virus from latent reservoirs is an important strategy toward eradicating HIV-1 infection. In this study, we discovered that the daphnane diterpene gnidimacrin, which was previously reported to have potent anti-cancer cell activity, activated HIV-1 replication and killed persistently-infected cells at picomolar concentrations. In addition to its potential to purge HIV-1 from latently infected cells, gnidimacrin potently inhibited a panel of HIV-1 R5 virus infection of peripheral blood mononuclear cells (PBMCs) at an average concentration lower than 10 pM. In contrast, gnidimacrin only partially inhibited HIV-1 ×4 virus infection of PBMCs. The strong anti-HIV-1 R5 virus activity of gnidimacrin was correlated with its effect on down-regulation of the HIV-1 coreceptor CCR5. The anti-R5 virus activity of gnidimacrin was completely abrogated by a selective protein kinase C beta inhibitor enzastaurin, which suggests that protein kinase C beta plays a key role in the potent anti-HIV-1 activity of gnidimacrin in PBMCs. In summary, these results suggest that gnidimacrin could activate latent HIV-1, specifically kill HIV-1 persistently infected cells, and inhibit R5 viruses at picomolar concentrations.

  7. Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy.

    PubMed

    Imamichi, Hiromi; Dewar, Robin L; Adelsberger, Joseph W; Rehm, Catherine A; O'Doherty, Una; Paxinos, Ellen E; Fauci, Anthony S; Lane, H Clifford

    2016-08-02

    Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5'LTR-to-3'LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of "defective" proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not "silent," but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.

  8. Effective Cytotoxic T Lymphocyte Targeting of Persistent HIV-1 during Antiretroviral Therapy Requires Priming of Naive CD8+ T Cells

    PubMed Central

    Smith, Kellie N.; Mailliard, Robbie B.; Piazza, Paolo A.; Fischer, Will; Korber, Bette T.; Fecek, Ronald J.; Ratner, Deena; Gupta, Phalguni; Mullins, James I.

    2016-01-01

    ABSTRACT Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8+ T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8+ T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4+ T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8+ T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection. PMID:27247230

  9. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

    PubMed Central

    Kula, Anna; Marcello, Alessandro

    2012-01-01

    Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function. PMID:24832221

  10. Overexpression of Peptide-Encoding OsCEP6.1 Results in Pleiotropic Effects on Growth in Rice (O. sativa)

    PubMed Central

    Sui, Zhipeng; Wang, Tianya; Li, Hongjian; Zhang, Ming; Li, Yangyang; Xu, Ruibin; Xing, Guofang; Ni, Zhongfu; Xin, Mingming

    2016-01-01

    Plant peptide hormones play an important role in regulating plant developmental programs via cell-to-cell communication in a non-cell autonomous manner. To characterize the biological relevance of C-TERMINALLY ENCODED PEPTIDE (CEP) genes in rice, we performed a genome-wide search against public databases using a bioinformatics approach and identified six additional CEP members. Expression analysis revealed a spatial-temporal pattern of OsCEP6.1 gene in different tissues and at different developmental stages of panicle. Interestingly, the expression level of the OsCEP6.1 was also significantly up-regulated by exogenous cytokinin. Application of a chemically synthesized 15-amino acid OsCEP6.1 peptide showed that OsCEP6.1 had a negative role in regulating root and seedling growth, which was further confirmed by transgenic lines. Furthermore, the constitutive expression of OsCEP6.1 was sufficient to lead to panicle architecture and grain size variations. Scanning electron microscopy analysis revealed that the phenotypic variation of OsCEP6.1 overexpression lines resulted from decreased cell size but not reduced cell number. Moreover, starch accumulation was not significantly affected. Taken together, these data suggest that the OsCEP6.1 peptide might be involved in regulating the development of panicles and grains in rice. PMID:26973672

  11. Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy

    SciTech Connect

    Mangin, M.; Webb, A.C.; Dreyer, B.E.; Posillico, J.T.; Ikeda, K.; Weir, E.C.; Stewart, A.F.; Bander, N.H.; Milstone, L.; Barton, D.E.

    1988-01-01

    Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome that appears to be mediated in many instances by a parathyroid hormone-like peptide. Poly(A)/sup +/ RNA from a human renal carcinoma associated with this syndrome was enriched by preparative electrophoresis and used to construct an enriched cDNA library in phage lambdagt10. The library was screened with a codon-preference oligonucleotide synthesized on the basis of a partial N-terminal amino acid sequence from a human tumor-derived peptide, and a 2.0 kilo-base cDNA was identified. The cDNA encodes a 177 amino acid protein consisting of a 36 amino acid leader sequence and a 141 amino acid mature peptide. The first 13 amino acids of the deduced sequence of the mature peptide display strong homology to human PTH, with complete divergence thereafter. RNA blot-hybridization analysis revealed multiple transcripts in mRNA from tumors associated with the humor syndrome and also in mRNA from normal human keratinocytes. Southern blot analysis of genomic DNA from humans and rodents revealed a simple pattern compatible with a single-copy gene. The gene has been mapped to chromosome 12.

  12. The HIV-1 Entry Process: A Stoichiometric View.

    PubMed

    Brandenberg, Oliver F; Magnus, Carsten; Regoes, Roland R; Trkola, Alexandra

    2015-12-01

    HIV-1 infection starts with fusion of the viral and the host cell membranes, a process mediated by the HIV-1 envelope glycoprotein trimer. The number of trimers required to complete membrane fusion, referred to as HIV-1 entry stoichiometry, remains under debate. A precise definition of HIV-1 entry stoichiometry is important as it reflects the efficacy of the viral entry process and steers the infectivity of HIV-1 virion populations. Initial estimates suggested a unanimous entry stoichiometry across HIV-1 strains while recent findings showed that HIV-1 strains can differ in entry stoichiometry. Here, we review current analyses of HIV-1 entry stoichiometry and point out future research directions to further define the interplay between entry stoichiometry, virus entry fitness, transmission, and susceptibility to antibody neutralization.

  13. Transplanting Supersites of HIV-1 Vulnerability

    PubMed Central

    Yang, Yongping; Gorman, Jason; Ofek, Gilad; Srivatsan, Sanjay; Druz, Aliaksandr; Lees, Christopher R.; Lu, Gabriel; Soto, Cinque; Stuckey, Jonathan; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Kwon, Peter D.

    2014-01-01

    One strategy for isolating or eliciting antibodies against a specific target region on the envelope glycoprotein trimer (Env) of the human immunodeficiency virus type 1 (HIV-1) involves the creation of site transplants, which present the target region on a heterologous protein scaffold with preserved antibody-binding properties. If the target region is a supersite of HIV-1 vulnerability, recognized by a collection of broadly neutralizing antibodies, this strategy affords the creation of “supersite transplants”, capable of binding (and potentially eliciting) antibodies similar to the template collection of effective antibodies. Here we transplant three supersites of HIV-1 vulnerability, each targeted by effective neutralizing antibodies from multiple donors. To implement our strategy, we chose a single representative antibody against each of the target supersites: antibody 10E8, which recognizes the membrane-proximal external region (MPER) on the HIV-1 gp41 glycoprotein; antibody PG9, which recognizes variable regions one and two (V1V2) on the HIV-1 gp120 glycoprotein; and antibody PGT128 which recognizes a glycopeptide supersite in variable region 3 (glycan V3) on gp120. We used a structural alignment algorithm to identify suitable acceptor proteins, and then designed, expressed, and tested antigenically over 100-supersite transplants in a 96-well microtiter-plate format. The majority of the supersite transplants failed to maintain the antigenic properties of their respective template supersite. However, seven of the glycan V3-supersite transplants exhibited nanomolar affinity to effective neutralizing antibodies from at least three donors and recapitulated the mannose9-N-linked glycan requirement of the template supersite. The binding of these transplants could be further enhanced by placement into self-assembling nanoparticles. Essential elements of the glycan V3 supersite, embodied by as few as 3 N-linked glycans and ∼25 Env residues, can be segregated

  14. A gene encoding a peptide with similarity to the plant IDA signaling peptide (AtIDA) is expressed most abundantly in the root-knot nematode (Meloidogyne incognita) soon after root infection.

    PubMed

    Tucker, Mark L; Yang, Ronghui

    2013-06-01

    Small peptides play important roles in intercellular signaling. Inflorescence deficient in abscission (ida) is an Arabidopsis mutant that does not abscise (shed) its flower petals. The IDA gene encodes a small, secreted peptide that putatively binds to two redundant receptor-like kinases (HAESA and HAESA-like2) that initiate a signal transduction pathway. We identified IDA-like (IDL) genes in the genomic sequence for Meloidogyne incognita and Meloidogyne hapla. No orthologous sequences were found in any other genus of nematodes. Transcript for both M. incognita and M. hapla IDLs were found in total RNA isolated from infected root systems of tomato, Solanum lycopersicum. Five and three prime RACE of RNA from M. incognita infected tomato roots revealed a sequence of 392 nt that includes a poly (A) tail of 39 nt. The open reading frame encodes a 47 aa protein with a putative 25 aa N-terminal signal peptide. Expression of MiIDL1 is very low in eggs and pre-parasitic J2 and rapidly increases in the first four days post inoculation (dpi) and then declines at approximately 14 dpi. A proposed role for the root-knot nematode IDL is discussed.

  15. Conserved Structural Elements in the V3 Crown of HIV-1 gp120

    SciTech Connect

    Jiang, X.; Burke, V; Totrov, M; Williams, C; Cardozo, T; Gorny, M; Zolla-Pazner, S; Kong, X

    2010-01-01

    Binding of the third variable region (V3) of the HIV-1 envelope glycoprotein gp120 to the cell-surface coreceptors CCR5 or CXCR4 during viral entry suggests that there are conserved structural elements in this sequence-variable region. These conserved elements could serve as epitopes to be targeted by a vaccine against HIV-1. Here we perform a systematic structural analysis of representative human anti-V3 monoclonal antibodies in complex with V3 peptides, revealing that the crown of V3 has four conserved structural elements: an arch, a band, a hydrophobic core and the peptide backbone. These are either unaffected by or are subject to minimal sequence variation. As these regions are targeted by cross-clade neutralizing human antibodies, they provide a blueprint for the design of vaccine immunogens that could elicit broadly cross-reactive protective antibodies.

  16. Therapeutics for HIV-1 reactivation from latency.

    PubMed

    Sgarbanti, Marco; Battistini, Angela

    2013-08-01

    Intensive combined antiretroviral therapy successfully suppresses HIV-1 replication and AIDS disease progression making infection manageable, but it is unable to eradicate the virus that persists in long-lived, drug-insensitive and immune system-insensitive reservoirs thus asking for life-long treatments with problems of compliance, resistance, toxicity and cost. These limitations and recent insights into latency mechanisms have fueled a renewed effort in finding a cure for HIV-1 infection. Proposed eradication strategies involve reactivation of the latent reservoir upon induction of viral transcription followed by the elimination of reactivated virus-producing cells by viral cytopathic effect or host immune response. Several molecules identified by mechanism-directed approaches or in large-scale screenings have been proposed as latency reversing agents. Some of them have already entered clinical testing in humans but with mixed or unsatisfactory results.

  17. Initial HIV-1 antigen-specific CD8+ T cells in acute HIV-1 infection inhibit transmitted/founder virus replication.

    PubMed

    Freel, Stephanie A; Picking, Ralph A; Ferrari, Guido; Ding, Haitao; Ochsenbauer, Christina; Kappes, John C; Kirchherr, Jennifer L; Soderberg, Kelly A; Weinhold, Kent J; Cunningham, Coleen K; Denny, Thomas N; Crump, John A; Cohen, Myron S; McMichael, Andrew J; Haynes, Barton F; Tomaras, Georgia D

    2012-06-01

    CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8(+) T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8(+) responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8(+) T cells during AHI. Autologous and heterologous CD8(+) T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8(+) T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8(+) antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8(+)-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8(+) T cell-mediated inhibition of virus replication. CD8(+) T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies.

  18. In vivo SELEX of single-stranded domains in the HIV-1 leader RNA.

    PubMed

    van Bel, Nikki; Das, Atze T; Berkhout, Ben

    2014-02-01

    The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two

  19. Population genomics of intrapatient HIV-1 evolution

    PubMed Central

    Zanini, Fabio; Brodin, Johanna; Thebo, Lina; Lanz, Christa; Bratt, Göran; Albert, Jan; Neher, Richard A

    2015-01-01

    Many microbial populations rapidly adapt to changing environments with multiple variants competing for survival. To quantify such complex evolutionary dynamics in vivo, time resolved and genome wide data including rare variants are essential. We performed whole-genome deep sequencing of HIV-1 populations in 9 untreated patients, with 6-12 longitudinal samples per patient spanning 5-8 years of infection. The data can be accessed and explored via an interactive web application. We show that patterns of minor diversity are reproducible between patients and mirror global HIV-1 diversity, suggesting a universal landscape of fitness costs that control diversity. Reversions towards the ancestral HIV-1 sequence are observed throughout infection and account for almost one third of all sequence changes. Reversion rates depend strongly on conservation. Frequent recombination limits linkage disequilibrium to about 100bp in most of the genome, but strong hitch-hiking due to short range linkage limits diversity. DOI: http://dx.doi.org/10.7554/eLife.11282.001 PMID:26652000

  20. HIV-1 Transmission Networks Across South Korea.

    PubMed

    Ahn, Mi Young; Wertheim, Joel O; Kim, Woo Joo; Kim, Shin-Woo; Lee, Jin Soo; Ann, Hea Won; Jeon, Yongduk; Ahn, Jin Young; Song, Je Eun; Oh, Dong Hyun; Kim, Yong Chan; Kim, Eun Jin; Jung, In Young; Kim, Moo Hyun; Jeong, Wooyoung; Jeong, Su Jin; Ku, Nam Su; Kim, June Myung; Smith, Davey M; Choi, Jun Yong

    2017-03-27

    Molecular epidemiology can help clarify the properties and dynamics of HIV-1 transmission networks in both global and regional scales. We studied 143 HIV-1-infected individuals recruited from four medical centers of three cities in South Korea between April 2013 and May 2014. HIV-1 env V3 sequence data were generated (337-793 bp) and analyzed using a pairwise distance-based clustering approach to infer putative transmission networks. Participants whose viruses were ≤2.0% divergent according to Tamura-Nei 93 genetic distance were defined as clustering. We collected demographic, risk, and clinical data and analyzed these data in relation to clustering. Among 143 participants, we identified nine putative transmission clusters of different sizes (range 2-4 participants). The reported risk factor of participants were concordant in only one network involving two participants, that is, both individuals reported homosexual sex as their risk factor. The participants in the other eight networks did not report concordant risk factors, although they were phylogenetically linked. About half of the participants refused to report their risk factor. Overall, molecular epidemiology provides more information to understand local transmission networks and the risks associated with these networks.

  1. Alkyl Amine Bevirimat Derivatives Are Potent and Broadly Active HIV-1 Maturation Inhibitors

    PubMed Central

    Urano, Emiko; Ablan, Sherimay D.; Mandt, Rebecca; Pauly, Gary T.; Sigano, Dina M.; Schneider, Joel P.; Martin, David E.; Nitz, Theodore J.; Wild, Carl T.

    2015-01-01

    Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1. PMID:26482309

  2. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

    SciTech Connect

    Di Nunzio, Francesca; Fricke, Thomas; Miccio, Annarita; Valle-Casuso, Jose Carlos; Perez, Patricio; Souque, Philippe; Rizzi, Ermanno; Severgnini, Marco; Mavilio, Fulvio; Charneau, Pierre; Diaz-Griffero, Felipe

    2013-05-25

    The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration. - Highlights: ► We studied the role of Nup98 and Nup153 in HIV-1 infection. ► Nup98 binds the HIV-1 core and is involved in HIV-1 integration. ► Nup153 binds the HIV-1 core and is involved in HIV-1 nuclear import. ► Depletion of Nup153 decreased the integration of HIV-1 in transcriptionally active sites.

  3. Enzymatic Triggered Release of an HIV-1 Entry Inhibitor from Prostate Specific Antigen Degradable Microparticles

    PubMed Central

    Clark, Meredith R.; Aliyar, Hyder A.; Lee, Chang-won; Jay, Julie I.; Gupta, Kavita M.; Watson, Karen M.; Stewart, Russell J.; Buckheit, Robert W.; Kiser, Patrick F.

    2011-01-01

    This paper describes the design, construction and characterization of the first anti-HIV drug delivery system that is triggered to release its contents in the presence of human semen. Microgel particles were synthesized with a crosslinker containing a peptide substrate for the seminal serine protease prostate specific antigen (PSA) and were loaded with the HIV-1 entry inhibitor sodium poly(styrene-4-sulfonate) (pSS). The particles were composed of N-2-hydroxyproplymethacrylamide and bis-methacrylamide functionalized peptides based on the PSA substrates GISSFYSSK and GISSQYSSK. Exposure to human seminal plasma (HSP) degraded the microgel network and triggered the release of the entrapped antiviral polymer. Particles with the crosslinker composed of the substrate GISSFYSSK showed 17 times faster degradation in seminal plasma than that of the crosslinker composed of GISSQYSSK. The microgel particles containing 1 mol% GISSFYSSK peptide crosslinker showed complete degradation in 30 hours in the presence of HSP at 37 °C and pSS released from the microgels within 30 minutes reached a concentration of 10 µg/mL, equivalent to the published IC90 for pSS. The released pSS inactivated HIV-1 in the presence of HSP. The solid phase synthesis of the crosslinkers, preparation of the particles by inverse microemulsion polymerization, HSP-triggered release of pSS and inactivation of HIV-1 studies are described. PMID:21511017

  4. Novel recombinant engineered gp41 N-terminal heptad repeat trimers and their potential as anti-HIV-1 therapeutics or microbicides.

    PubMed

    Chen, Xi; Lu, Lu; Qi, Zhi; Lu, Hong; Wang, Ji; Yu, Xiaoxia; Chen, Yinghua; Jiang, Shibo

    2010-08-13

    Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

  5. Suppression of HIV-1 Infectivity by Human Glioma Cells.

    PubMed

    Hoque, Sheikh Ariful; Tanaka, Atsushi; Islam, Salequl; Ahsan, Gias Uddin; Jinno-Oue, Atsushi; Hoshino, Hiroo

    2016-05-01

    HIV-1 infection to the central nervous system (CNS) is very common in AIDS patients. The predominant cell types infected in the brain are monocytes and macrophages, which are surrounded by several HIV-1-resistant cell types, such as astrocytes, oligodendrocytes, neurons, and microvascular cells. The effect of these HIV-1-resistant cells on HIV-1 infection is largely unknown. In this study, we examined the stability of HIV-1 cultured with several human glioblastoma cell lines, for example, NP-2, U87MG, T98G, and A172, to determine whether these HIV-1-resistant brain cells could enhance or suppress HIV-1 infection and thus modulate HIV-1 infection in the CNS. The HIV-1 titer was determined using the MAGIC-5A indicator cell line as well as naturally occurring CD4(+) T cells. We found that the stability of HIV-1 incubated with NP-2 or U87MG cells at 37°C was significantly shorter (half-life, 2.5-4 h) compared to that of HIV-1 incubated with T98G or A172 cells or in culture medium without cells (half-life, 8-18 h). The spent culture media (SCM) of NP-2 and U87MG cells had the ability to suppress both R5- and X4-HIV-1 infection by inhibiting HIV-1 attachment to target cells. This inhibitory effect was eliminated by the treatment of the SCM with chondroitinase ABC but not heparinase, suggesting that the inhibitory factor(s) secreted by NP-2 and U87MG cells was chiefly mediated by chondroitin sulfate (CS) or CS-like moiety. Thus, this study reveals that some but not all glioma cells secrete inhibitory molecules to HIV-1 infection that may contribute in lowering HIV-1 infection in the CNS in vivo.

  6. Identifying the Important HIV-1 Recombination Breakpoints

    PubMed Central

    Fan, Jun; Simon-Loriere, Etienne; Arts, Eric J.; Negroni, Matteo; Robertson, David L.

    2008-01-01

    Recombinant HIV-1 genomes contribute significantly to the diversity of variants within the HIV/AIDS pandemic. It is assumed that some of these mosaic genomes may have novel properties that have led to their prevalence, particularly in the case of the circulating recombinant forms (CRFs). In regions of the HIV-1 genome where recombination has a tendency to convey a selective advantage to the virus, we predict that the distribution of breakpoints—the identifiable boundaries that delimit the mosaic structure—will deviate from the underlying null distribution. To test this hypothesis, we generate a probabilistic model of HIV-1 copy-choice recombination and compare the predicted breakpoint distribution to the distribution from the HIV/AIDS pandemic. Across much of the HIV-1 genome, we find that the observed frequencies of inter-subtype recombination are predicted accurately by our model. This observation strongly indicates that in these regions a probabilistic model, dependent on local sequence identity, is sufficient to explain breakpoint locations. In regions where there is a significant over- (either side of the env gene) or under- (short regions within gag, pol, and most of env) representation of breakpoints, we infer natural selection to be influencing the recombination pattern. The paucity of recombination breakpoints within most of the envelope gene indicates that recombinants generated in this region are less likely to be successful. The breakpoints at a higher frequency than predicted by our model are approximately at either side of env, indicating increased selection for these recombinants as a consequence of this region, or at least part of it, having a tendency to be recombined as an entire unit. Our findings thus provide the first clear indication of the existence of a specific portion of the genome that deviates from a probabilistic null model for recombination. This suggests that, despite the wide diversity of recombinant forms seen in the viral

  7. Fluorescence-based characterization of genetically encoded peptides that fold in live cells: progress toward a generic hairpin scaffold

    NASA Astrophysics Data System (ADS)

    Cheng, Zihao; Campbell, Robert E.

    2007-02-01

    Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold based on a single β-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant β-hairpin peptides to be rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a 16mer tryptophan zipper β-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer β-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the 20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we experimentally demonstrate that the in vitro determined β-hairpin stabilities are well correlated with in vivo β-hairpin stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer β-hairpin peptides through the use of fluorescence-based library screening.

  8. HIV-1 Phylogenetic analysis shows HIV-1 transits through the meninges to brain and peripheral tissues

    PubMed Central

    Lamers, Susanna L.; Gray, Rebecca R.; Salemi, Marco; Huysentruyt, Leanne C.; McGrath, Michael

    2010-01-01

    Brain infection by the human immunodeficiency virus type 1 (HIV-1) has been investigated in many reports with a variety of conclusions concerning the time of entry and degree of viral compartmentalization. To address these diverse findings, we sequenced HIV-1 gp120 clones from a wide range of brain, peripheral and meningeal tissues from five patients who died from several HIV-1 associated disease pathologies. High-resolution phylogenetic analysis confirmed previous studies that showed a significant degree of compartmentalization in brain and peripheral tissue subpopulations. Some intermixing between the HIV-1 subpopulations was evident, especially in patients that died from pathologies other than HIV-associated dementia. Interestingly, the major tissue harboring virus from both the brain and peripheral tissues was the meninges. These results show that 1) HIV-1 is clearly capable of migrating out of the brain, 2) the meninges are the most likely primary transport tissues, and 3) infected brain macrophages comprise an important HIV reservoir during highly active antiretroviral therapy. PMID:21055482

  9. Phenotypic Correlates of HIV-1 Macrophage Tropism

    PubMed Central

    Arrildt, Kathryn T.; LaBranche, Celia C.; Joseph, Sarah B.; Dukhovlinova, Elena N.; Graham, William D.; Ping, Li-Hua; Schnell, Gretja; Sturdevant, Christa B.; Kincer, Laura P.; Mallewa, Macpherson; Heyderman, Robert S.; Van Rie, Annelies; Cohen, Myron S.; Spudich, Serena; Price, Richard W.; Montefiori, David C.

    2015-01-01

    ABSTRACT HIV-1 is typically CCR5 using (R5) and T cell tropic (T-tropic), targeting memory CD4+ T cells throughout acute and chronic infections. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize the phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation. IMPORTANCE HIV-1 typically replicates in CD4+ T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses

  10. HIV-1 Integrates Widely throughout the Genome of the Human Blood Fluke Schistosoma mansoni

    PubMed Central

    Mann, Victoria H.; Dubrovsky, Larisa; Yan, Hong-bin; Huckvale, Thomas; Protasio, Anna V.; Pushkarsky, Tatiana; Iordanskiy, Sergey; Bukrinsky, Michael I.

    2016-01-01

    Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate. PMID:27764257

  11. HIV-1 Integrates Widely throughout the Genome of the Human Blood Fluke Schistosoma mansoni.

    PubMed

    Suttiprapa, Sutas; Rinaldi, Gabriel; Tsai, Isheng J; Mann, Victoria H; Dubrovsky, Larisa; Yan, Hong-Bin; Holroyd, Nancy; Huckvale, Thomas; Durrant, Caroline; Protasio, Anna V; Pushkarsky, Tatiana; Iordanskiy, Sergey; Berriman, Matthew; Bukrinsky, Michael I; Brindley, Paul J

    2016-10-01

    Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.

  12. Calmodulin disrupts the structure of the HIV-1 MA protein†

    PubMed Central

    Chow, John Y. H.; Jeffries, Cy M.; Kwan, Ann H.; Guss, J. Mitchell; Trewhella, Jill

    2010-01-01

    The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium -sensing calmodulin (CaM) which is up-regulated upon HIV-1 infection. The nature of the CaM-MA interaction has been the subject of structural studies using peptides based on the MA sequence that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca2+-dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two tryptophans at the N-terminus of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, while chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the target-protein binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N-and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA looses ~20% of its α-helical content upon CaM binding. Thus CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions. PMID:20488189

  13. HIV-1 Vpr accelerates viral replication during acute infection by exploitation of proliferating CD4+ T cells in vivo.

    PubMed

    Sato, Kei; Misawa, Naoko; Iwami, Shingo; Satou, Yorifumi; Matsuoka, Masao; Ishizaka, Yukihito; Ito, Mamoru; Aihara, Kazuyuki; An, Dong Sung; Koyanagi, Yoshio

    2013-01-01

    The precise role of viral protein R (Vpr), an HIV-1-encoded protein, during HIV-1 infection and its contribution to the development of AIDS remain unclear. Previous reports have shown that Vpr has the ability to cause G2 cell cycle arrest and apoptosis in HIV-1-infected cells in vitro. In addition, vpr is highly conserved in transmitted/founder HIV-1s and in all primate lentiviruses, which are evolutionarily related to HIV-1. Although these findings suggest an important role of Vpr in HIV-1 pathogenesis, its direct evidence in vivo has not been shown. Here, by using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrated that Vpr causes G2 cell cycle arrest and apoptosis predominantly in proliferating CCR5(+) CD4(+) T cells, which mainly consist of regulatory CD4(+) T cells (Tregs), resulting in Treg depletion and enhanced virus production during acute infection. The Vpr-dependent enhancement of virus replication and Treg depletion is observed in CCR5-tropic but not CXCR4-tropic HIV-1-infected mice, suggesting that these effects are dependent on the coreceptor usage by HIV-1. Immune activation was observed in CCR5-tropic wild-type but not in vpr-deficient HIV-1-infected humanized mice. When humanized mice were treated with denileukin diftitox (DD), to deplete Tregs, DD-treated humanized mice showed massive activation/proliferation of memory T cells compared to the untreated group. This activation/proliferation enhanced CCR5 expression in memory CD4(+) T cells and rendered them more susceptible to CCR5-tropic wild-type HIV-1 infection than to vpr-deficient virus. Taken together, these results suggest that Vpr takes advantage of proliferating CCR5(+) CD4(+) T cells for enhancing viremia of CCR5-tropic HIV-1. Because Tregs exist in a higher cycling state than other T cell subsets, Tregs appear to be more vulnerable to exploitation by Vpr during acute HIV-1 infection.

  14. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

    PubMed

    Sanders, Rogier W; van Gils, Marit J; Derking, Ronald; Sok, Devin; Ketas, Thomas J; Burger, Judith A; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C; Julien, Jean-Philippe; Rakasz, Eva G; Seaman, Michael S; Guttman, Miklos; Lee, Kelly K; Klasse, Per Johan; LaBranche, Celia; Schief, William R; Wilson, Ian A; Overbaugh, Julie; Burton, Dennis R; Ward, Andrew B; Montefiori, David C; Dean, Hansi; Moore, John P

    2015-07-10

    A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.

  15. HIV-1-Specific CD8 T Cells Exhibit Limited Cross-Reactivity during Acute Infection.

    PubMed

    Du, Victor Y; Bansal, Anju; Carlson, Jonathan; Salazar-Gonzalez, Jesus F; Salazar, Maria G; Ladell, Kristin; Gras, Stephanie; Josephs, Tracy M; Heath, Sonya L; Price, David A; Rossjohn, Jamie; Hunter, Eric; Goepfert, Paul A

    2016-04-15

    Prior work has demonstrated that HIV-1-specific CD8 T cells can cross-recognize variant epitopes. However, most of these studies were performed in the context of chronic infection, where the presence of viral quasispecies makes it difficult to ascertain the true nature of the original antigenic stimulus. To overcome this limitation, we evaluated the extent of CD8 T cell cross-reactivity in patients with acute HIV-1 clade B infection. In each case, we determined the transmitted founder virus sequence to identify the autologous epitopes restricted by individual HLA class I molecules. Our data show that cross-reactive CD8 T cells are infrequent during the acute phase of HIV-1 infection. Moreover, in the uncommon instances where cross-reactive responses were detected, the variant epitopes were poorly recognized in cytotoxicity assays. Molecular analysis revealed that similar antigenic structures could be cross-recognized by identical CD8 T cell clonotypes mobilized in vivo, yet even subtle differences in a single TCR-accessible peptide residue were sufficient to disrupt variant-specific reactivity. These findings demonstrate that CD8 T cells are highly specific for autologous epitopes during acute HIV-1 infection. Polyvalent vaccines may therefore be required to provide optimal immune cover against this genetically labile pathogen.

  16. A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription.

    PubMed Central

    Bohjanen, P R; Colvin, R A; Puttaraju, M; Been, M D; Garcia-Blanco, M A

    1996-01-01

    Linear TAR RNA has previously been used as a decoy to inhibit HIV-1 transcription in vitro and HIV-1 replication in vivo. A 48 nucleotide circular RNA containing the stem, bulge and loop of the HIV-1 TAR element was synthesized using the self-splicing activity of a group I permuted intron-exon and was tested for its ability to function as a TAR decoy in vitro. This small circular TAR molecule was exceptionally stable in HeLa nuclear extracts, whereas a similar linear TAR molecule was rapidly degraded. The TAR circle bound specifically to Tfr38, a peptide containing the TAR-binding region of Tat. The ability of Tat to trans-activate transcription from the HIV-1 promoter in vitro was efficiently inhibited by circular TAR RNA but not by TAR circles that contained either bulge or loop mutations. TAR circles did not inhibit transactivation exclusively by binding to Tat since this inhibition was not reversed by adding excess Tat to the transcription reaction. Together, these data suggest that TAR circles act as decoys that inhibit transactivation by binding to Tat and at least one cellular factor. These data also demonstrate the utility of small circular RNA molecules as tools for biochemical studies. PMID:8871552

  17. The HIV-1 capsid protein as a drug target: recent advances and future prospects.

    PubMed

    Domenech, Rosa; Neira, José L

    2013-12-01

    HIV-1, the agent responsible for AIDS, belongs to the retrovirus family. Assembly of the immature HIV-1 capsid occurs through the controlled polymerization of the Gag polyprotein, which is transported to the plasma membrane of infected cells, where morphogenesis of the immature, non-infectious virion occurs. Moreover, the mature capsid of HIV-1 is formed by the assembly of copies of the capsid protein (CA), which results, among other proteins, from cleavage of Gag. The C-terminal domain of CA (CTD) can homodimerize, and most of the dimerization interface is formed by a single α-helix from each monomer. Assembly of the HIV-1 capsid critically depends on CA-CA interactions, including CTD interaction with itself and with the N-terminal domain of CA (NTD). This review will report on recent advances for the search of small organic compounds and peptides that have been designed in the last four years to hamper CA assembly. Most of the molecules have been proved to interact with CA; such molecules aim to disrupt and/or alter the oligomerization capability of CTD and/or NTD.

  18. Antibodies elicited by yeast glycoproteins recognize HIV-1 virions and potently neutralize virions with high mannose N-glycans

    PubMed Central

    Zhang, Hong; Fu, Hu; Luallen, Robert J.; Liu, Bingfen; Lee, Fang-Hua; Doms, Robert W.; Geng, Yu

    2015-01-01

    The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125–130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine. PMID:26277072

  19. Nesfatin-1-Like Peptide Encoded in Nucleobindin-1 in Goldfish is a Novel Anorexigen Modulated by Sex Steroids, Macronutrients and Daily Rhythm

    PubMed Central

    Sundarrajan, Lakshminarasimhan; Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Ramesh, Naresh; Canosa, Luis Fabián; Unniappan, Suraj

    2016-01-01

    Nesfatin-1 is an 82 amino acid anorexigen encoded in a secreted precursor nucleobindin-2 (NUCB2). NUCB2 was named so due to its high sequence similarity with nucleobindin-1 (NUCB1). It was recently reported that NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP) in mice. Here, we aimed to characterize NLP in fish. RT- qPCR showed NUCB1 expression in both central and peripheral tissues. Western blot analysis and/or fluorescence immunohistochemistry determined NUCB1/NLP in the brain, pituitary, testis, ovary and gut of goldfish. NUCB1 mRNA expression in goldfish pituitary and gut displayed a daily rhythmic pattern of expression. Pituitary NUCB1 mRNA expression was downregulated by estradiol, while testosterone upregulated its expression in female goldfish brain. High carbohydrate and fat suppressed NUCB1 mRNA expression in the brain and gut. Intraperitoneal injection of synthetic rat NLP and goldfish NLP at 10 and 100 ng/g body weight doses caused potent inhibition of food intake in goldfish. NLP injection also downregulated the expression of mRNAs encoding orexigens, preproghrelin and orexin-A, and upregulated anorexigen cocaine and amphetamine regulated transcript mRNA in goldfish brain. Collectively, these results provide the first set of results supporting the anorectic action of NLP, and the regulation of tissue specific expression of goldfish NUCB1. PMID:27329836

  20. ADAR1 and PACT contribute to efficient translation of transcripts containing HIV-1 trans-activating response (TAR) element

    PubMed Central

    Chukwurah, Evelyn; Handy, Indhira

    2017-01-01

    Human immunodeficiency virus type 1 (HIV-1) has evolved various measures to counter the host cell's innate antiviral response during the course of infection. Interferon (IFN)-stimulated gene products are produced following HIV-1 infection to limit viral replication, but viral proteins and RNAs counteract their effect. One such mechanism is specifically directed against the IFN-induced Protein Kinase PKR, which is centrally important to the cellular antiviral response. In the presence of viral RNAs, PKR is activated and phosphorylates the translation initiation factor eIF2α. This shuts down the synthesis of both host and viral proteins, allowing the cell to mount an effective antiviral response. PACT (protein activator of PKR) is a cellular protein activator of PKR, primarily functioning to activate PKR in response to cellular stress. Recent studies have indicated that during HIV-1 infection, PACT's normal cellular function is compromised and that PACT is unable to activate PKR. Using various reporter systems and in vitro kinase assays, we establish in this report that interactions between PACT, ADAR1 and HIV-1-encoded Tat protein diminish the activation of PKR in response to HIV-1 infection. Our results highlight an important pathway by which HIV-1 transcripts subvert the host cell's antiviral activities to enhance their translation. PMID:28167698

  1. Isolation, cloning, and expression mapping of a gene encoding an antidiuretic hormone and other CAPA-related peptides in the disease vector, Rhodnius prolixus.

    PubMed

    Paluzzi, Jean-Paul; Russell, William K; Nachman, Ronald J; Orchard, Ian

    2008-09-01

    After a blood meal, Rhodnius prolixus undergoes a rapid diuresis to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas' disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresis in R. prolixus is under the neurohormonal control of serotonin and peptidergic diuretic hormones, and thus, diuretic hormones play an important role in the transmission of Chagas' disease. Although diuretic hormones may be degraded or excreted, resulting in the termination of diuresis, it would also seem appropriate, given the high rates of secretion, that a potent antidiuretic factor could be present and act to prevent excessive loss of water and salts after the postgorging diuresis. Despite the medical importance of R. prolixus, no genes for any neuropeptides have been cloned, including obviously, those that control diuresis. Here, using molecular biology in combination with matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry, we determined the sequence of the CAPA gene and CAPA-related peptides in R. prolixus, which includes a peptide with anti-diuretic activity. We have characterized the expression of mRNA encoding these peptides in various developmental stage and also examined the tissue-specific distribution in fifth-instars. The expression is localized to numerous bilaterally paired cell bodies within the central nervous system. In addition, our results show that RhoprCAPA gene expression is also associated with the testes, suggesting a novel role for this family of peptides in reproduction.

  2. HIV-1 Accessory Proteins: Vpu and Vif

    PubMed Central

    Andrew, Amy; Strebel, Klaus

    2014-01-01

    HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by functionally inactivating the host factor BST-2. This chapter described techniques used for the characterization of Vif and Vpu and their functional interaction with host factors. Many of the techniques are, however, applicable to the functional analysis of other viral proteins. PMID:24158820

  3. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    SciTech Connect

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang; Bencze, Krisztina Z.; Koupparis, Kyriacos; O’Connor, Carrie E.; Kovari, Iulia A.; Spaller, Mark R.; Kovari, Ladislau C.

    2013-09-06

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  4. Methamphetamine inhibits HIV-1 replication in CD4+ T cells by modulating anti-HIV-1 miRNA expression.

    PubMed

    Mantri, Chinmay K; Mantri, Jyoti V; Pandhare, Jui; Dash, Chandravanu

    2014-01-01

    Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis.

  5. Methamphetamine Inhibits HIV-1 Replication in CD4+ T Cells by Modulating Anti–HIV-1 miRNA Expression

    PubMed Central

    Mantri, Chinmay K.; Mantri, Jyoti V.; Pandhare, Jui; Dash, Chandravanu

    2015-01-01

    Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4+ T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4+ T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti–HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4+ T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis. PMID:24434277

  6. Zinc coupling potentiates anti-HIV-1 activity of baicalin.

    PubMed

    Wang, Qian; Wang, Yu-Tian; Pu, Shao-Ping; Zheng, Yong-Tang

    2004-11-12

    Baicalin (BA) has been shown with anti-HIV-1 activity. Zinc is a nutrient element. The anti-HIV-1 activity of zinc complex of baicalin (BA-Zn) in vitro was studied and compared with the anti-HIV-1 activities between BA and BA-Zn in the present study. Our results suggested that BA-Zn has lower cytotoxicity and higher anti-HIV-1 activity compared with those of BA in vitro. The CC50s of BA-Zn and BA were 221.52 and 101.73 microM, respectively. The cytotoxicity of BA-Zn was about 1.2-fold lower than that of BA. The BA and BA-Zn inhibited HIV-1 induced syncytium formation, HIV-1 p24 antigen and HIV-1 RT production. The EC50s of BA-Zn on inhibiting HIV-1 induced syncytium formation (29.08 microM) and RT production (31.17 microM) were lower than those of BA (43.27 and 47.34 microM, respectively). BA-Zn was more effective than BA in inhibiting the activities of recombinant RT and HIV-1 entry into host cells. Zinc coupling enhanced the anti-HIV-1 activity of baicalin.

  7. Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen.

    PubMed

    Prado, Silvia; Beltrán, Manuela; Coiras, Mayte; Bedoya, Luis M; Alcamí, José; Gallego, José

    2016-05-01

    New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE-Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs.

  8. Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat.

    PubMed

    Nukuzuma, Souichi; Kameoka, Masanori; Sugiura, Shigeki; Nakamichi, Kazuo; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu

    2009-11-01

    Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter. In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.

  9. High Degree of HIV-1 Group M (HIV-1M) Genetic Diversity within Circulating Recombinant Forms: Insight into the Early Events of HIV-1M Evolution.

    PubMed

    Tongo, Marcel; Dorfman, Jeffrey R; Martin, Darren P

    2015-12-09

    The existence of various highly divergent HIV-1 lineages and of recombination-derived sequence tracts of indeterminate origin within established circulating recombinant forms (CRFs) strongly suggests that HIV-1 group M (HIV-1M) diversity is not fully represented under the current classification system. Here we used a fully exploratory screen for recombination on a set of 480 near-full-length genomes representing the full known diversity of HIV-1M. We decomposed recombinant sequences into their constituent parts and then used maximum-likelihood phylogenetic analyses of this mostly recombination-free data set to identify rare divergent sequence lineages that fall outside the major named HIV-1M taxonomic groupings. We found that many of the sequence fragments occurring within CRFs (including CRF04_cpx, CRF06_cpx, CRF11_cpx, CRF18_cpx, CRF25_cpx, CRF27_cpx, and CRF49_cpx) are in fact likely derived from divergent unclassified parental lineages that may predate the current subtypes, even though they are presently identified as derived from currently defined HIV-1M subtypes. Our evidence suggests that some of these CRFs are descended predominantly from what were or are major previously unidentified HIV-1M lineages that were likely epidemiologically relevant during the early stages of the HIV-1M epidemic. The restriction of these divergent lineages to the Congo basin suggests that they were less infectious and/or simply not present at the time and place of the initial migratory wave that triggered the global epidemic.IMPORTANCE HIV-1 group M (HIV-1M) likely spread to the rest of the world from the Congo basin in the mid-1900s (N. R. Faria et al., Science 346:56-61, 2014, http://dx.doi.org/10.1126/science.1256739) and is today the principal cause of the AIDS pandemic. Here, we show that large sequence fragments from several HIV-1M circulating recombinant forms (CRFs) are derived from divergent parental lineages that cannot reasonably be classified within the nine

  10. High Degree of HIV-1 Group M (HIV-1M) Genetic Diversity within Circulating Recombinant Forms: Insight into the Early Events of HIV-1M Evolution

    PubMed Central

    2015-01-01

    ABSTRACT The existence of various highly divergent HIV-1 lineages and of recombination-derived sequence tracts of indeterminate origin within established circulating recombinant forms (CRFs) strongly suggests that HIV-1 group M (HIV-1M) diversity is not fully represented under the current classification system. Here we used a fully exploratory screen for recombination on a set of 480 near-full-length genomes representing the full known diversity of HIV-1M. We decomposed recombinant sequences into their constituent parts and then used maximum-likelihood phylogenetic analyses of this mostly recombination-free data set to identify rare divergent sequence lineages that fall outside the major named HIV-1M taxonomic groupings. We found that many of the sequence fragments occurring within CRFs (including CRF04_cpx, CRF06_cpx, CRF11_cpx, CRF18_cpx, CRF25_cpx, CRF27_cpx, and CRF49_cpx) are in fact likely derived from divergent unclassified parental lineages that may predate the current subtypes, even though they are presently identified as derived from currently defined HIV-1M subtypes. Our evidence suggests that some of these CRFs are descended predominantly from what were or are major previously unidentified HIV-1M lineages that were likely epidemiologically relevant during the early stages of the HIV-1M epidemic. The restriction of these divergent lineages to the Congo basin suggests that they were less infectious and/or simply not present at the time and place of the initial migratory wave that triggered the global epidemic. IMPORTANCE HIV-1 group M (HIV-1M) likely spread to the rest of the world from the Congo basin in the mid-1900s (N. R. Faria et al., Science 346:56–61, 2014, http://dx.doi.org/10.1126/science.1256739) and is today the principal cause of the AIDS pandemic. Here, we show that large sequence fragments from several HIV-1M circulating recombinant forms (CRFs) are derived from divergent parental lineages that cannot reasonably be classified within the

  11. Nucleoprotein complex intermediates in HIV-1 integration

    PubMed Central

    Li, Min; Craigie, Robert

    2012-01-01

    Integration of retroviral DNA into the host genome is an essential step in the viral replication cycle. The viral DNA, made by reverse transcription in the cytoplasm, forms part of a large nucleoprotein complex called the preintegration complex (PIC). The viral integrase protein is the enzyme within the PIC that is responsible for integrating the viral DNA into the host genome. Integrase is tightly associated with the viral DNA within the PIC as demonstrated by functional assays. Integrase protein catalyzes the key DNA cutting and joining steps of integration in vitro with DNA substrates that mimic the ends of the viral DNA. Under most in vitro assay conditions the stringency of the reaction is relaxed; most products result from “half-site” integration in which only one viral DNA end is integrated into one strand of target DNA rather than concerted integration of pairs of DNA as occurs with PICs and in vivo. Under these relaxed conditions catalysis appears to occur without formation of the highly stable nucleoprotein complexes that is characteristic of the association of integrase with viral DNA in the PIC. Here we describe methods for the assembly of nucleoprotein complex intermediates in HIV-1 DNA integration from purified HIV-1 integrase and substrates that mimic the viral DNA ends. PMID:19232539

  12. Fucoidans as potential inhibitors of HIV-1.

    PubMed

    Prokofjeva, Maria M; Imbs, Tatyana I; Shevchenko, Natalya M; Spirin, Pavel V; Horn, Stefan; Fehse, Boris; Zvyagintseva, Tatyana N; Prassolov, Vladimir S

    2013-08-19

    The antiviral activity of different structure fucoidans (α-l-fucans and galactofucans) was studied using two model viral systems based on a lentiviral vectors and a replication competent Moloney murine leukemia virus (Mo-MuLV). It was found that investigated fucoidans have no cytotoxic effects on Jurkat and SC-1cell at the concentration range of 0.001-100 µg/mL. Fucoidans with different efficiency suppressed transduction of Jurkat cell line by pseudo-HIV-1 particles carrying the envelope protein of HIV-1 and infection of SC-1 cells by Mo-MuLV. According to our data, all natural fucoidans can be considered as potential anti-HIV agents regardless of their carbohydrate backbone and degree of sulfating, since their activity is shown at low concentrations (0.001-0.05 µg/mL). High molecular weight fucoidans isolated from Saccharina cichorioides (1.3-α-l-fucan), and S. japonica (galactofucan) were the most effective inhibitors.

  13. Cyclophilin B enhances HIV-1 infection

    SciTech Connect

    DeBoer, Jason; Madson, Christian J.; Belshan, Michael

    2016-02-15

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

  14. Structural Basis of Potent and Broad HIV-1 Fusion Inhibitor CP32M*

    PubMed Central

    Yao, Xue; Chong, Huihui; Zhang, Chao; Qiu, Zonglin; Qin, Bo; Han, Ruiyun; Waltersperger, Sandro; Wang, Meitian; He, Yuxian; Cui, Sheng

    2012-01-01

    CP32M is a newly designed peptide fusion inhibitor possessing potent anti-HIV activity, especially against T20-resistant HIV-1 strains. In this study, we show that CP32M can efficiently inhibit a large panel of diverse HIV-1 variants, including subtype B′, CRF07_BC, and CRF01_AE recombinants and naturally occurring or induced T20-resistant viruses. To elucidate its mechanism of action, we determined the crystal structure of CP32M complexed with its target sequence. Differing from its parental peptide, CP621-652, the 621VEWNEMT627 motif of CP32M folds into two α-helix turns at the N terminus of the pocket-binding domain, forming a novel layer in the six-helix bundle structure. Prominently, the residue Asn-624 of the 621VEWNEMT627 motif is engaged in the polar interaction with a hydrophilic ridge that borders the hydrophobic pocket on the N-terminal coiled coil. The original inhibitor design of CP32M provides several intra- and salt bridge/hydrogen bond interactions favoring the stability of the helical conformation of CP32M and its interactions with N-terminal heptad repeat (NHR) targets. We identified a novel salt bridge between Arg-557 on the NHR and Glu-648 of CP32M that is critical for the binding of CP32M and resistance against the inhibitor. Therefore, our data present important information for developing novel HIV-1 fusion inhibitors for clinical use. PMID:22679024

  15. Decline in CTL and antibody responses to HIV-1 p17 and p24 antigens in HIV-1-infected hemophiliacs irrespective of disease progression. A 5-year follow-up study.

    PubMed

    O'Toole, C M; Lowdell, M W; Chargelegue, D; Colvin, B T

    1992-08-01

    CTL and antibody responses to HIV-1 p17 and p24 antigens were monitored from 1986-1991, in 4 hemophiliacs. The patients had been infected with HIV-1 between 1980 and 1984. Two patients have remained asymptomatic while two progressed to AIDS in 1990. CTL were boosted by culturing with peptides from p17 aa 86-115, or p24 aa 265-279; and aa 270-373 or PHA. Lysis was measured on autologous or allogeneic targets pulsed with peptides or infected with recombinant vaccinia virus carrying HIV-1 gag or influenza A matrix genes. Antibodies to p17 and p24 were tested by ELISA using peptides and by Western blotting. High levels of CTL activity to p17 and p24 antigens could be generated only with lymphocytes from the two asymptomatic patients between 1986 and 1989, but these responses were absent in 1990 and 1991. Antibodies to p17 peptides disappeared in parallel with CTL activity. Antibodies to some p24 peptides also declined but most patients retained activity to others. In all patients a > or = 3-fold increase in CD8+ cell numbers occurred over time and accompanied the decline of CTL and antibody responses. The loss of CTL and p17 antibodies occurred irrespective of whether patients remained asymptomatic or progressed to AIDS in the intervening two years.

  16. Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: trans-splicing may play an important role in the diversification of scorpion venom peptides.

    PubMed

    Zeng, Xian-Chun; Luo, Feng; Li, Wen-Xin

    2006-04-01

    A novel cDNA clone (named BmKT-u) which is a hybrid molecule of the 5'-terminal region of BmKT' cDNA and the 3'-terminal region of an undocumented cDNA (named BmKu), was isolated from a cDNA library made from the venom gland of scorpion Buthus martensii Karsch. BmKT-u codes for a 30 amino acid residue precursor peptide composed of a 20-residue signal sequence, and a putative 10-residue novel mature peptide. Northern blot hybridization showed BmKT-u cDNA is generated from a transcript. RT-PCR experiments excluded the possibility that BmKT-u cDNA is an artifact generated during reverse transcription. Genomic amplifications performed with three pairs of BmKT-u gene-specific primers showed the BmKT-u gene does not exist in the genome of the scorpion as a single transcriptional unit. Genomic cloning for BmKT' showed that the BmKT' gene contains an intron of 509 bp inserted into the region encoding the C-terminal region of the signal peptide. A sequence alignment comparison of the cDNA of BmKT-u with genomic BmKT' revealed that the junction site of the hybrid molecule is located at the 5'-splicing site of the intron. The data suggest that the BmKT-u transcript is a naturally occurring mature mRNA that is generated by trans-splicing. Trans-splicing may contribute to the diversity of venom peptides from venomous animals.

  17. Broadly neutralizing antibodies: An approach to control HIV-1 infection.

    PubMed

    Yaseen, Mahmoud Mohammad; Yaseen, Mohammad Mahmoud; Alqudah, Mohammad Ali

    2017-01-02

    Although available antiretroviral therapy (ART) has changed human immunodeficiency virus (HIV)-1 infection to a non-fatal chronic disease, the economic burden of lifelong therapy, severe adverse ART effects, daily ART adherence, and emergence of ART-resistant HIV-1 mutants require prospecting for alternative therapeutic modalities. Indeed, a growing body of evidence suggests that broadly neutralizing anti-HIV-1 antibodies (BNAbs) may offer one such feasible alternative. To evaluate their therapeutic potential in established HIV-1 infection, we sought to address recent advances in pre-clinical and clinical investigations in this area of HIV-1 research. In addition, we addressed the obstacles that may impede the success of such immunotherapeutic approach, suggested strategic solutions, and briefly compared this approach with the currently used ART to open new insights for potential future passive immunotherapy for HIV-1 infection.

  18. miRNA-encoded peptides (miPEPs): A new tool to analyze the roles of miRNAs in plant biology

    PubMed Central

    Couzigou, Jean-Malo; Lauressergues, Dominique; Bécard, Guillaume; Combier, Jean-Philippe

    2015-01-01

    MicroRNAs (miRNAs) are short RNA molecules negatively regulating the expression of many important genes in plants and animals. We have recently shown that plant primary transcripts of miRNAs encode peptides (miPEPs) able to increase specifically the transcription of their associated miRNA.1 We discuss here the possibility of using miPEPs as a new tool for functional analysis of single members of miRNA families in plants, including in non-model plants, that could avoid transgenic transformation and minimize artifactual interpretation. We also raise several fundamental and crucial questions that need to be address for a deeper understanding of the cellular and molecular mechanisms underlining the regulatory activity of miPEPs. PMID:26400469

  19. miRNA-encoded peptides (miPEPs): A new tool to analyze the roles of miRNAs in plant biology.

    PubMed

    Couzigou, Jean-Malo; Lauressergues, Dominique; Bécard, Guillaume; Combier, Jean-Philippe

    2015-01-01

    MicroRNAs (miRNAs) are short RNA molecules negatively regulating the expression of many important genes in plants and animals. We have recently shown that plant primary transcripts of miRNAs encode peptides (miPEPs) able to increase specifically the transcription of their associated miRNA.(1) We discuss here the possibility of using miPEPs as a new tool for functional analysis of single members of miRNA families in plants, including in non-model plants, that could avoid transgenic transformation and minimize artifactual interpretation. We also raise several fundamental and crucial questions that need to be address for a deeper understanding of the cellular and molecular mechanisms underlining the regulatory activity of miPEPs.

  20. Immunological Characterization of Plant-Based HIV-1 Gag/Dgp41 Virus-Like Particles

    PubMed Central

    Kessans, Sarah A.; Linhart, Mark D.; Meador, Lydia R.; Kilbourne, Jacquelyn; Hogue, Brenda G.; Fromme, Petra; Matoba, Nobuyuki; Mor, Tsafrir S.

    2016-01-01

    It is widely anticipated that a prophylactic vaccine may be needed to control the HIV/AIDS epidemic worldwide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although a recent clinical trial has shown promising results. Recent studies have focused on highly conserved domains within HIV-1 such as the membrane proximal external region (MPER) of the envelope glycoprotein, gp41. MPER has been shown to play critical roles in mucosal transmission of HIV-1, though this peptide is poorly immunogenic on its own. Here we provide evidence that plant-produced HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (Dgp41) provides an effective platform to display MPER for use as an HIV vaccine candidate. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR—a fusion of MPER and the B-subunit of cholera toxin) were investigated in BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens were elicited when systemically primed with VLPs. These responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a boosting response against Gag and gp41 when boosted with either candidate. Importantly, the VLPs also induced Gag-specific CD4 and CD8 T-cell responses. This report on the immunogenicity of plant-based Gag/Dgp41 VLPs may represent an important milestone on the road towards a broadly efficacious and inexpensive subunit vaccine against HIV-1. PMID:26986483

  1. HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles

    PubMed Central

    LeGoff, Jérôme; Bouhlal, Hicham; Lecerf, Maxime; Klein, Christophe; Hocini, Hakim; Si-Mohamed, Ali; Muggeridge, Martin; Bélec, Laurent

    2007-01-01

    Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1. Methods We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD. Results We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells. Conclusion Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2. PMID:17207276

  2. Membrane structure correlates to function of LLP2 on the cytoplasmic tail of HIV-1 gp41 protein.

    PubMed

    Boscia, Alexander L; Akabori, Kiyotaka; Benamram, Zachary; Michel, Jonathan A; Jablin, Michael S; Steckbeck, Jonathan D; Montelaro, Ronald C; Nagle, John F; Tristram-Nagle, Stephanie

    2013-08-06

    Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (-1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.

  3. Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

    PubMed Central

    Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

    2016-01-01

    The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains. PMID:27349805

  4. Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

    NASA Astrophysics Data System (ADS)

    Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

    2016-06-01

    The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.

  5. The Structural Interface between HIV-1 Vif and Human APOBEC3H.

    PubMed

    Ooms, Marcel; Letko, Michael; Simon, Viviana

    2017-03-01

    Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif β-sheet (β2-β5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif β-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV

  6. HIV-1 variants in South and South-East Asia.

    PubMed

    Tsuchie, H; Saraswathy, T S; Sinniah, M; Vijayamalar, B; Maniar, J K; Monzon, O T; Santana, R T; Paladin, F J; Wasi, C; Thongcharoen, P

    1995-01-01

    HIV spread in South and South-East Asia is most alarming, and genetic variability of HIV-1 is an important consideration in vaccine development. In this study, we examined the third variable (V3) region of env gene of HIV-1 variants prevalent in Thailand, Malaysia, India, and the Philippines. By phylogenetic tree analyses, an HIV-1 variant from an injecting drug user (IDU) in Thailand belonged to subtype B, and HIV-1 variants from 2 IDUs in Malaysia were classified into 2 subtypes, B and E. One HIV-1 variant from a male homosexual in the Philippines belonged to subtype B. Out of 8 HIV-1 variants from sexually transmitted disease patients in India, 7 belonged to subtype C, and one to subtype A. Although the total number of individuals examined in this study was limited, 4 HIV-1 subtypes were found in South and South-East Asia and large international movements of HIV-1-infected individuals in this region could induce global dissemination of these HIV-1 variants.

  7. Broad activation of latent HIV-1 in vivo

    PubMed Central

    Barton, Kirston; Hiener, Bonnie; Winckelmann, Anni; Rasmussen, Thomas Aagaard; Shao, Wei; Byth, Karen; Lanfear, Robert; Solomon, Ajantha; McMahon, James; Harrington, Sean; Buzon, Maria; Lichterfeld, Mathias; Denton, Paul W.; Olesen, Rikke; Østergaard, Lars; Tolstrup, Martin; Lewin, Sharon R.; Søgaard, Ole Schmeltz; Palmer, Sarah

    2016-01-01

    The ‘shock and kill' approach to cure human immunodeficiency virus (HIV) includes transcriptional induction of latent HIV-1 proviruses using latency-reversing agents (LRAs) with targeted immunotherapy to purge infected cells. The administration of LRAs (panobinostat or vorinostat) to HIV-1-infected individuals on antiretroviral therapy induces a significant increase in cell-associated unspliced (CA-US) HIV-1 RNA from CD4+ T cells. However, it is important to discern whether the increases in CA-US HIV-1 RNA are due to limited or broad activation of HIV-1 proviruses. Here we use single-genome sequencing to find that the RNA transcripts observed following LRA administration are genetically diverse, indicating activation of transcription from an extensive range of proviruses. Defective sequences are more frequently found in CA HIV-1 RNA than in HIV-1 DNA, which has implications for developing an accurate measure of HIV-1 reservoir size. Our findings provide insights into the effects of panobinostat and vorinostat as LRAs for latent HIV-1. PMID:27605062

  8. Genome editing strategies: potential tools for eradicating HIV-1/AIDS

    PubMed Central

    Khalili, Kamel; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

    2015-01-01

    Current therapy for controlling HIV-1 infection and preventing AIDS progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or “sterile” cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including ZFNs, TALENs, and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS. PMID:25716921

  9. Antiretroviral Therapy Fails to Restore Levels of HIV-1 Restriction miRNAs in PBMCs of HIV-1-infected MSM

    PubMed Central

    Liu, Man-Qing; Zhao, Min; Kong, Wen-Hua; Peng, Jin-Song; Wang, Fang; Qiu, Hong-Yan; Zhu, Ze-Rong; Tang, Li; Sang, Ming; Wu, Jian-Guo; Ho, Wen-Zhe; Zhou, Wang

    2015-01-01

    Abstract A number of cellular microRNAs (miRNAs) have been identified to have the ability to inhibit HIV-1 replication. In this study, we examined the impact of combination antiretroviral therapy (cART) on the expression of HIV-1 restriction miRNAs in peripheral blood mononuclear cells of HIV-1–infected men who have sex with men (MSM). Compared with male healthy donors, HIV-infected MSM had significantly lower levels of 9 HIV-1 restriction miRNAs. The treatment of HIV-1–infected MSM with cART, however, failed to restore the levels of these miRNAs in peripheral blood mononuclear cells. These observations suggest that the suppression of the cellular restriction miRNAs by HIV-1 may attribute to the virus latency during cART. PMID:26579828

  10. Antiretroviral Therapy Fails to Restore Levels of HIV-1 Restriction miRNAs in PBMCs of HIV-1-infected MSM.

    PubMed

    Liu, Man-Qing; Zhao, Min; Kong, Wen-Hua; Peng, Jin-Song; Wang, Fang; Qiu, Hong-Yan; Zhu, Ze-Rong; Tang, Li; Sang, Ming; Wu, Jian-Guo; Ho, Wen-Zhe; Zhou, Wang

    2015-11-01

    A number of cellular microRNAs (miRNAs) have been identified to have the ability to inhibit HIV-1 replication. In this study, we examined the impact of combination antiretroviral therapy (cART) on the expression of HIV-1 restriction miRNAs in peripheral blood mononuclear cells of HIV-1-infected men who have sex with men (MSM). Compared with male healthy donors, HIV-infected MSM had significantly lower levels of 9 HIV-1 restriction miRNAs. The treatment of HIV-1-infected MSM with cART, however, failed to restore the levels of these miRNAs in peripheral blood mononuclear cells. These observations suggest that the suppression of the cellular restriction miRNAs by HIV-1 may attribute to the virus latency during cART.

  11. Transcriptional Bursting from the HIV-1 Promoter is a Significant Source of Stochastic Noise in HIV-1 Gene Expression

    SciTech Connect

    Singh, A; Razooky, B; Cox, Chris D.; Simpson, Michael L; Weinberger, Leor S.

    2010-01-01

    Analysis of noise in gene expression has proven a powerful approach for analyzing gene regulatory architecture. To probe the regulatory mechanisms controlling expression of HIV-1, we analyze noise in gene-expression from HIV-1 s long terminal repeat (LTR) promoter at different HIV-1 integration sites across the human genome. Flow cytometry analysis of GFP expression from the HIV-1 LTR shows high variability (noise) at each integration site. Notably, the measured noise levels are inconsistent with constitutive gene expression models. Instead, quantification of expression noise indicates that HIV-1 gene expression occurs through randomly timed bursts of activity from the LTR and that each burst generates an average of 2 10 mRNA transcripts before the promoter returns to an inactive state. These data indicate that transcriptional bursting can generate high variability in HIV-1 early gene products, which may critically influence the viral fate-decision between active replication and proviral latency.

  12. [Sensitivity of the COBAS AmpliScreen™ HIV-1 test v1.5 for HIV-1 detection].

    PubMed

    Gomez, Lucía P; Balangero, Marcos C; Castro, Gonzalo; Kademian, Silvia; Mangeaud, Arnaldo; Barbas, María G; Cudolá, Analía; de León, Juan F; Carrizo, Horacio; Gallego, Sandra V

    2014-01-01

    The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants.

  13. HIV-1 p6-Another viral interaction partner to the host cellular protein cyclophilin A.

    PubMed

    Solbak, Sara M Ø; Reksten, Tove R; Röder, Rene; Wray, Victor; Horvli, Ole; Raae, Arnt J; Henklein, Petra; Henklein, Peter; Fossen, Torgils

    2012-04-01

    The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D (1)H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl-prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme-substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.

  14. Designing a soluble near full-length HIV-1 gp41 trimer.

    PubMed

    Gao, Guofen; Wieczorek, Lindsay; Peachman, Kristina K; Polonis, Victoria R; Alving, Carl R; Rao, Mangala; Rao, Venigalla B

    2013-01-04

    The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.

  15. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    PubMed Central

    2011-01-01

    Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP. PMID:21929758

  16. Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA*

    PubMed Central

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-01-01

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

  17. FCGR2C polymorphisms associate with HIV-1 vaccine protection in RV144 trial.

    PubMed

    Li, Shuying S; Gilbert, Peter B; Tomaras, Georgia D; Kijak, Gustavo; Ferrari, Guido; Thomas, Rasmi; Pyo, Chul-Woo; Zolla-Pazner, Susan; Montefiori, David; Liao, Hua-Xin; Nabel, Gary; Pinter, Abraham; Evans, David T; Gottardo, Raphael; Dai, James Y; Janes, Holly; Morris, Daryl; Fong, Youyi; Edlefsen, Paul T; Li, Fusheng; Frahm, Nicole; Alpert, Michael D; Prentice, Heather; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Robb, Merlin L; O'Connell, Robert J; Haynes, Barton F; Michael, Nelson L; Kim, Jerome H; McElrath, M Juliana; Geraghty, Daniel E

    2014-09-01

    The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1-specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor-mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT-carrying vaccine recipients only. These data suggest a potent role of Fc-γ receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.

  18. Overlapping Regions in HIV-1 Genome Act as Potential Sites for Host–Virus Interaction

    PubMed Central

    Saha, Deeya; Podder, Soumita; Ghosh, Tapash C.

    2016-01-01

    More than a decade, overlapping genes in RNA viruses became a subject of research which has explored various effect of gene overlapping on the evolution and function of viral genomes like genome size compaction. Additionally, overlapping regions (OVRs) are also reported to encode elevated degree of protein intrinsic disorder (PID) in unspliced RNA viruses. With the aim to explore the roles of OVRs in HIV-1 pathogenesis, we have carried out an in-depth analysis on the association of gene overlapping with PID in 35 HIV1- M subtypes. Our study reveals an over representation of PID in OVR of HIV-1 genomes. These disordered residues endure several vital, structural features like short linear motifs (SLiMs) and protein phosphorylation (PP) sites which are previously shown to be involved in massive host–virus interaction. Moreover, SLiMs in OVRs are noticed to be more functionally potential as compared to that of non-overlapping region. Although, density of experimentally verified SLiMs, resided in 9 HIV-1 genes, involved in host–virus interaction do not show any bias toward clustering into OVR, tat and rev two important proteins mediates host–pathogen interaction by their experimentally verified SLiMs, which are mostly localized in OVR. Finally, our analysis suggests that the acquisition of SLiMs in OVR is mutually exclusive of the occurrence of disordered residues, while the enrichment of PPs in OVR is solely dependent on PID and not on overlapping coding frames. Thus, OVRs of HIV-1 genomes could be demarcated as potential molecular recognition sites during host–virus interaction. PMID:27867372

  19. Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

    PubMed

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-07-05

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

  20. Novel pseudosymmetric inhibitors of HIV-1 protease

    SciTech Connect

    Faessler, A.; Roesel, J.; Gruetter, M.; Tintelnot-Blomley, M.; Alteri, E.; Bold, G.; Lang, M.

    1993-12-31

    Taking into account the unique C-2 symmetric nature of the HIV-1 protease homodimer, the authors have designed and synthesized novel inhibitors featuring an almost symmetric structure. Compounds containing the easily accessible Phe[CH(OH)CH{sub 2}N(NH)]Cha dipeptide isostere as a nonhydrolyzable replacement of the scissile amide bond of the natural substrate are potent inhibitors in vitro with IC{sub 50} values of 9 to 50 nM. The antiviral activity depends mainly on the nature of the anylated valine residues linked to the dipeptide mimic. In this series, CGP 53820 combines both high potency and excellent specificity. Its predicted symmetric binding pattern is illustrated by the X-ray structure analysis performed with the corresponding enzyme-inhibitor complex.

  1. Allosteric inhibition of HIV-1 integrase activity

    PubMed Central

    Engelman, Alan; Kessl, Jacques J.; Kvaratskhelia, Mamuka

    2013-01-01

    HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development. PMID:23647983

  2. Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

    PubMed Central

    BRADRICK, THOMAS D.; MARINO, JOHN P.

    2004-01-01

    Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (Δ TAR-ap23 and Δ TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP ΔTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD ~ 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD ~ 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the ΔTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD ~ 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD ~ 4 μM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions. PMID:15273324

  3. Chimpanzee GB virus C and GB virus A E2 envelope glycoproteins contain a peptide motif that inhibits human immunodeficiency virus type 1 replication in human CD4⁺ T-cells.

    PubMed

    McLinden, James H; Stapleton, Jack T; Klinzman, Donna; Murthy, Krishna K; Chang, Qing; Kaufman, Thomas M; Bhattarai, Nirjal; Xiang, Jinhua

    2013-04-01

    GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31 % at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4(+) Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus-HIV interactions.

  4. Fine-mapping classical HLA variation associated with durable host control of HIV-1 infection in African Americans.

    PubMed

    McLaren, Paul J; Ripke, Stephan; Pelak, Kimberly; Weintrob, Amy C; Patsopoulos, Nikolaos A; Jia, Xiaoming; Erlich, Rachel L; Lennon, Niall J; Kadie, Carl M; Heckerman, David; Gupta, Namrata; Haas, David W; Deeks, Steven G; Pereyra, Florencia; Walker, Bruce D; de Bakker, Paul I W

    2012-10-01

    A small proportion of human immunodeficiency virus-1 (HIV-1) infected individuals, termed HIV-1 controllers, suppress viral replication to very low levels in the absence of therapy. Genetic investigations of this phenotype have strongly implicated variation in the class I major histocompatibility complex (MHC) region as key to HIV-1 control. We collected sequence-based classical class I HLA genotypes at 4-digit resolution in HIV-1-infected African American controllers and progressors (n = 1107), and tested them for association with host control using genome-wide single nucleotide polymorphism data to account for population structure. Several classical alleles at HLA-B were associated with host control, including B*57:03 [odds ratio (OR) = 5.1; P= 3.4 × 10(-18)] and B*81:01 (OR = 4.8; P= 1.3 × 10(-9)). Analysis of variable amino acid positions demonstrates that HLA-B position 97 is the most significant association with host control in African Americans (omnibus P = 1.2 × 10(-21)) and explains the signal of several HLA-B alleles, including B*57:03. Within HLA-B, we also identified independent effects at position 116 (omnibus P= 2.8 × 10(-15)) in the canonical F pocket, position 63 in the B pocket (P= 1.5 × 10(-3)) and the non-pocket position 245 (P= 8.8 × 10(-10)), which is thought to influence CD8-binding kinetics. Adjusting for these HLA-B effects, there is evidence for residual association in the MHC region. These results underscore the key role of HLA-B in affecting HIV-1 replication, likely through the molecular interaction between HLA-B and viral peptides presented by infected cells, and suggest that sites outside the peptide-binding pocket also influence HIV-1 control.

  5. Fine-mapping classical HLA variation associated with durable host control of HIV-1 infection in African Americans

    PubMed Central

    McLaren, Paul J.; Ripke, Stephan; Pelak, Kimberly; Weintrob, Amy C.; Patsopoulos, Nikolaos A.; Jia, Xiaoming; Erlich, Rachel L.; Lennon, Niall J.; Kadie, Carl M.; Heckerman, David; Gupta, Namrata; Haas, David W.; Deeks, Steven G.; Pereyra, Florencia; Walker, Bruce D.; de Bakker, Paul I. W.

    2012-01-01

    A small proportion of human immunodeficiency virus-1 (HIV-1) infected individuals, termed HIV-1 controllers, suppress viral replication to very low levels in the absence of therapy. Genetic investigations of this phenotype have strongly implicated variation in the class I major histocompatibility complex (MHC) region as key to HIV-1 control. We collected sequence-based classical class I HLA genotypes at 4-digit resolution in HIV-1-infected African American controllers and progressors (n = 1107), and tested them for association with host control using genome-wide single nucleotide polymorphism data to account for population structure. Several classical alleles at HLA-B were associated with host control, including B*57:03 [odds ratio (OR) = 5.1; P= 3.4 × 10–18] and B*81:01 (OR = 4.8; P= 1.3 × 10−9). Analysis of variable amino acid positions demonstrates that HLA-B position 97 is the most significant association with host control in African Americans (omnibus P = 1.2 × 10−21) and explains the signal of several HLA-B alleles, including B*57:03. Within HLA-B, we also identified independent effects at position 116 (omnibus P= 2.8 × 10−15) in the canonical F pocket, position 63 in the B pocket (P= 1.5 × 10−3) and the non-pocket position 245 (P= 8.8 × 10−10), which is thought to influence CD8-binding kinetics. Adjusting for these HLA-B effects, there is evidence for residual association in the MHC region. These results underscore the key role of HLA-B in affecting HIV-1 replication, likely through the molecular interaction between HLA-B and viral peptides presented by infected cells, and suggest that sites outside the peptide-binding pocket also influence HIV-1 control. PMID:22718199

  6. Rapid and simple screening and supplemental testing for HIV-1 and HIV-2 infections in west Africa.

    PubMed

    Brattegaard, K; Kouadio, J; Adom, M L; Doorly, R; George, J R; De Cock, K M

    1993-06-01

    Researchers from an AIDS research project took blood samples from 1000 consecutive women during childbirth at a maternal and child health center in Abidjan, Cote d'Ivoire, and from 185 hospitalized patients to compare the results of a combination of synthetic peptide-based rapid tests (product names, Testpack and Genie), which check for HIV-1 and HIV-2 antibodies, with those of the Western Blot-based test. They also wanted to see whether the rapid test-based strategy could replace the Western Blot-based test as a supplemental test. The Western Blot indicated the HIV-1 and/or HIV-2 prevalence to be 13% among the new mothers and 78% among the hospitalized patients for an overall prevalence of 23%. 3.3% of all people were positive for both HIV-1 and HIV-2. 17.4% tested positive for just HIV-1. 2.1% were positive for HIV-2. The rapid tests had a sensitivity of 99.6% and a specificity of 99.9%. The positive predictive value was 99.6% and the negative predictive value was 99.9%. The rapid tests identified 4% of the HIV-2 positive samples and 1% of the HIV-1 samples to be dually reactive. These findings demonstrated that rapid synthetic peptide-based assays reliably detect HIV-1 and HIV-2 antibodies and can be supplemental tests. High quality HIV serology can be performed in a setting without running water and electricity which was the case in this study. A further advantage of this strategy is that each test takes only 10 minutes. These tests would have significant effects on HIV testing and counseling, diagnosis, and screening of blood for transfusion in rural areas of developing countries.

  7. Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.

    PubMed Central

    Strisovsky, K.; Tessmer, U.; Langner, J.; Konvalinka, J.; Kräusslich, H. G.

    2000-01-01

    Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme. PMID:11045610

  8. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

    PubMed

    Teixeira, Cátia; Barbault, Florent; Couesnon, Thierry; Gomes, José R B; Gomes, Paula; Maurel, François

    2016-01-01

    HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules.

  9. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

    PubMed Central

    Teixeira, Cátia; Barbault, Florent; Couesnon, Thierry; Gomes, José R. B.; Gomes, Paula; Maurel, François

    2016-01-01

    HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules. PMID:26785380

  10. Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides.

    PubMed Central

    Palker, T J; Clark, M E; Langlois, A J; Matthews, T J; Weinhold, K J; Randall, R R; Bolognesi, D P; Haynes, B F

    1988-01-01

    A synthetic peptide (SP-10-IIIB) with an amino acid sequence [Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro -Pro-Gly-(Tyr); amino acids 303-321] from the human immunodeficiency virus (HIV) isolate human T-cell lymphotropic virus type III (HTLV-III) HTLV-IIIB envelope glycoprotein gp120 was coupled to tetanus toxoid and used to raise goat antibodies to HIV gp120. Goat anti-SP-10-IIIB serum bound to the surface of HTLV-IIIB-infected CEM T cells but not to the surface of HTLV-IIIRF-infected or uninfected CEM T cells. Anti-SP-10-IIIB antibodies also selectively bound to gp120 from lysates of HTLV-IIIB cells in immunoblot assays. Twenty-one percent of sera (28 of 175) from patients seropositive for HIV contained antibodies that reacted with SP-10-IIIB in RIA. Human anti-SP-10-IIIB antibodies affinity purified from acquired immunodeficiency syndrome (AIDS) patient serum bound to HTLV-IIIB-infected cells and immunoprecipitated gp120. Goat antibodies to SP-10-IIIB neutralized HTLV-IIIB (80% neutralization titer of 1/600), inhibited HTLV-IIIB-induced syncytium formation, but did not neutralize HIV isolates HTLV-IIIRF or HTLV-IIIMN or inhibit syncytium formation with these isolates. Also, goat antiserum to an homologous synthetic peptide [SP-10-IIIRF(A), (Cys)-Arg-Lys-Ser-Ile-Thr-Lys-Gly-Pro-Gly-Arg-Val-Ile-Tyr] from gp120 of HIV isolate HTLV-IIIRF inhibited syncytium formation by HTLV-IIIRF, but did not inhibit syncytium formation by HTLV-IIIB or by HTLV-IIIMN. Thus, the amino acid sequences of SP-10-IIIB and SP-10-IIIRF(A) define homologous regions of gp120 that are important in type-specific virus neutralization. The identification of these type-specific neutralizing epitopes should facilitate the design of a polyvalent, synthetic vaccine for AIDS. Images PMID:2450351

  11. Consistent inhibition of HIV-1 replication in CD4+ T cells by acyclovir without detection of human herpesviruses.

    PubMed

    McMahon, Moira A; Parsons, Teresa L; Shen, Lin; Siliciano, Janet D; Siliciano, Robert F

    2011-05-01

    Acyclovir, a nucleoside analog, is thought to be specific for the human herpesviruses because it requires a virally encoded enzyme to phosphorylate it to acyclovir monophosphate. Recently, acyclovir triphosphate was shown to be a direct inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Here, we showed that acyclovir is an inhibitor of HIV-1 replication in CD4(+) T cells from cord blood that have undetectable levels of the eight human herpesviruses. Additionally, acyclovir phosphates were detected by reverse-phase-high performance liquid chromatography (RP-HPLC) and quantified in a primer extension assay from cord blood. The data support acyclovir as an inhibitor of HIV-1 replication in herpesvirus-negative cells.

  12. Host-Specific Adaptation of HIV-1 Subtype B in the Japanese Population

    PubMed Central

    Chikata, Takayuki; Carlson, Jonathan M.; Tamura, Yoshiko; Borghan, Mohamed Ali; Naruto, Takuya; Hashimoto, Masao; Murakoshi, Hayato; Le, Anh Q.; Mallal, Simon; John, Mina; Gatanaga, Hiroyuki; Oka, Shinichi; Brumme, Zabrina L.

    2014-01-01

    ABSTRACT The extent to which HIV-1 clade B strains exhibit population-specific adaptations to host HLA alleles remains incompletely known, in part due to incomplete characterization of HLA-associated HIV-1 polymorphisms (HLA-APs) in different global populations. Moreover, it remains unknown to what extent the same HLA alleles may drive significantly different escape pathways across populations. As the Japanese population exhibits distinctive HLA class I allele distributions, comparative analysis of HLA-APs between HIV-1 clade B-infected Japanese and non-Asian cohorts could shed light on these questions. However, HLA-APs remain incompletely mapped in Japan. In a cohort of 430 treatment-naive Japanese with chronic HIV-1 clade B infection, we identified 284 HLA-APs in Gag, Pol, and Nef using phylogenetically corrected methods. The number of HLA-associated substitutions in Pol, notably those restricted by HLA-B*52:01, was weakly inversely correlated with the plasma viral load (pVL), suggesting that the transmission and persistence of B*52:01-driven Pol mutations could modulate the pVL. Differential selection of HLA-APs between HLA subtype members, including those differing only with respect to substitutions outside the peptide-binding groove, was observed, meriting further investigation as to their mechanisms of selection. Notably, two-thirds of HLA-APs identified in Japan had not been reported in previous studies of predominantly Caucasian cohorts and were attributable to HLA alleles unique to, or enriched in, Japan. We also identified 71 cases where the same HLA allele drove significantly different escape pathways in Japan versus predominantly Caucasian cohorts. Our results underscore the distinct global evolution of HIV-1 clade B as a result of host population-specific cellular immune pressures. IMPORTANCE Cytotoxic T lymphocyte (CTL) escape mutations in HIV-1 are broadly predictable based on the HLA class I alleles expressed by the host. Because HLA allele

  13. Nonhuman TRIM5 Variants Enhance Recognition of HIV-1-Infected Cells by CD8+ T Cells

    PubMed Central

    Jimenez-Moyano, Esther; Ruiz, Alba; Kløverpris, Henrik N.; Rodriguez-Plata, Maria T.; Peña, Ruth; Blondeau, Caroline; Selwood, David L.; Izquierdo-Useros, Nuria; Moris, Arnaud; Clotet, Bonaventura; Goulder, Philip; Towers, Greg J.

    2016-01-01

    ABSTRACT Tripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type 1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two nonhuman TRIM5 orthologs, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8+ T cells. We illustrate how TRIM5 restriction improves CD8+ T-cell-mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the nonimmunosuppressive analog of cyclosporine (CsA), sarcosine-3(4-methylbenzoate)–CsA (SmBz-CsA), we found a significant reduction in CD107a/MIP-1β expression in HIV-1-specific CD8+ T cells. This finding underscores the direct link between TRIM5 restriction and activation of CD8+ T-cell responses. Interestingly, cells expressing RhT5 induced stronger CD8+ T-cell responses through the specific recognition of the HIV-1 capsid by the immune system. The underlying mechanism of this process may involve TRIM5-specific capsid recruitment to cellular proteasomes and increase peptide availability for loading and presentation of HLA class I antigens. In summary, we identified a novel function for nonhuman TRIM5 variants in cellular immunity. We hypothesize that TRIM5 can couple innate viral sensing and CD8+ T-cell activation to increase species barriers against retrovirus infection. IMPORTANCE New therapeutics to tackle HIV-1 infection should aim to combine rapid innate viral sensing and cellular immune recognition. Such strategies could prevent seeding of the viral reservoir and the immune damage that occurs during acute infection. The nonhuman TRIM5 variants, rhesus TRIM5α (RhT5) and TRIM

  14. Sparse Representation for Prediction of HIV-1 Protease Drug Resistance.

    PubMed

    Yu, Xiaxia; Weber, Irene T; Harrison, Robert W

    2013-01-01

    HIV rapidly evolves drug resistance in response to antiviral drugs used in AIDS therapy. Estimating the specific resistance of a given strain of HIV to individual drugs from sequence data has important benefits for both the therapy of individual patients and the development of novel drugs. We have developed an accurate classification method based on the sparse representation theory, and demonstrate that this method is highly effective with HIV-1 protease. The protease structure is represented using our newly proposed encoding method based on Delaunay triangulation, and combined with the mutated amino acid sequences of known drug-resistant strains to train a machine-learning algorithm both for classification and regression of drug-resistant mutations. An overall cross-validated classification accuracy of 97% is obtained when trained on a publically available data base of approximately 1.5×10(4) known sequences (Stanford HIV database http://hivdb.stanford.edu/cgi-bin/GenoPhenoDS.cgi). Resistance to four FDA approved drugs is computed and comparisons with other algorithms demonstrate that our method shows significant improvements in classification accuracy.

  15. The hemK gene in Escherichia coli encodes the N5-glutamine methyltransferase that modifies peptide release factors

    PubMed Central

    Heurgué-Hamard, Valérie; Champ, Stéphanie; Engström, Åke; Ehrenberg, Måns; Buckingham, Richard H.

    2002-01-01

    Class 1 peptide release factors (RFs) in Escherichia coli are N5-methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N5-methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N5-methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature. PMID:11847124

  16. Novel HIV-1 Therapeutics through Targeting Altered Host Cell Pathways

    PubMed Central

    Coley, William; Kehn-Hall, Kylene; Van Duyne, Rachel; Kashanchi, Fatah

    2009-01-01

    The emergence of drug-resistant human immunodeficiency virus type I (HIV-1) strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse-transcriptase, viral protease, or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we will discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host’s proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be performed to better design therapeutics against HIV-1. PMID:19732026

  17. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies.

    PubMed

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-11-18

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  18. Serum IgD behaviour in HIV-1 infected patients.

    PubMed

    Raiteri, R; Albonico, M; Deiana, R; Marietti, G; Sinicco, A

    1991-01-01

    From September 1987 to February 1990, repeated tests were performed in 325 HIV-1 infected subjects at different clinical stages using a radial immunodiffusion method to determine serum IgD behaviour in HIV-1 infection. Four patients had acute HIV-1 infection, 72 asymptomatic infection, 163 PGL, 49 ARC and 37 AIDS. During the study, 57 seropositive patients developed AIDS. The correlation between serum IgD and the clinical stage of HIV-1 infection, CD4+ and CD8+ lymphocyte levels, CD4+/CD8+ ratio, HIV-1 (p24) antigenemia and reactivity to core proteins, IgG, IgA, IgM isotypes and serum beta 2-microglobulin concentration. A significant correlation was noted between HIV-1 (p24) antigenemia, the disappearance of the antibodies reactivity to core proteins and IgD levels in ARC patients. A progressive increase of serum IgD before the occurrence of the symptomatic stage of HIV-1 infection was observed in HIV-1 infected patients who developed AIDS.

  19. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

    PubMed Central

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment. PMID:27869733

  20. Iron chelators ICL670 and 311 inhibit HIV-1 transcription

    SciTech Connect

    Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina; Kurantsin-Mills, Joseph; Niu, Xiaomei; Charles, Sharroya; Richardson, Des R.; Ray, Patricio E.; Gordeuk, Victor R.; Nekhai, Sergei

    2007-10-25

    HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics.

  1. Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

    PubMed Central

    Nomaguchi, Masako; Yokoyama, Masaru; Kono, Ken; Nakayama, Emi E.; Shioda, Tatsuo; Doi, Naoya; Fujiwara, Sachi; Saito, Akatsuki; Akari, Hirofumi; Miyakawa, Kei; Ryo, Akihide; Ode, Hirotaka; Iwatani, Yasumasa; Miura, Tomoyuki; Igarashi, Tatsuhiko

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells. PMID:23966385

  2. Modulation of cytokine release and gene expression by the immunosuppressive domain of gp41 of HIV-1.

    PubMed

    Denner, Joachim; Eschricht, Magdalena; Lauck, Michael; Semaan, Marwan; Schlaermann, Philipp; Ryu, Hyunmi; Akyüz, Levent

    2013-01-01

    The transmembrane envelope protein gp41 of the human immunodeficiency virus HIV-1 plays an important role during infection allowing fusion of the viral and cellular membrane. In addition, there is increasing evidence that gp41 may contribute to the immunodeficiency induced by HIV-1. Recombinant gp41 and a synthetic peptide corresponding to a highly conserved domain in gp41, the immunosuppressive (isu) domain, have been shown to inhibit mitogen-induced activation of human peripheral blood mononuclear cells (PBMCs) and to increase release of IL-6 and IL-10 from these cells. We recently reported that a single mutation in the isu domain of gp41 abrogated the immunosuppressive properties and that HIV-1 sequences containing such abrogating mutations had never been isolated from infected individuals. Here, we studied the influence of the isu peptide on the release of 66 cytokines and the expression of 27,000 genes in PBMCs. Incubation of PBMCs with isu peptide homopolymers increased the expression of 16 cytokines among them IL-6 and IL-10, and decreased that of IL-2 and CXCL9. Interestingly, the extend of cytokine modulation was donor-dependent. Among the genes up-regulated were IL-6, IL-8, IL-10 but also MMP-1, TREM-1 and IL-1beta. Most importantly, genes involved in innate immunity such as FCN1 and SEPP1 were found down-regulated. Many changes in cytokine expression demonstrated in our experiments were also found in HIV-1 infected individuals. These data indicate that the isu domain of gp41 has a broad impact on gene expression and cytokine release and therefore may be involved in HIV-1 induced immunopathogenesis.

  3. AIDS in rural Africa: a paradigm for HIV-1 prevention.

    PubMed

    Hudson, C P

    1996-07-01

    Networks of concurrent sexual partnerships may be the primary cause of epidemic spread of HIV-1 in parts of sub-Saharan Africa. This pattern of sexual behaviour increases the likelihood that individuals experiencing primary HIV-1 infection transmit the virus to other persons. Networks of concurrent partnerships are likely to be important in both the early ('epidemic') and late ('endemic') phases of HIV-1 transmission. Interventions should aim to break the sexual networks, whatever the stage of the epidemic. However, prevention of transmission in the endemic phase also requires a greater awareness of early clinical manifestations of HIV-1 infection in the general population. Such awareness, coupled with the availability of condoms and access to HIV-1 testing facilities, may reduce transmission in discordant couples.

  4. AIDS-protective HLA-B*27/B*57 and chimpanzee MHC class I molecules target analogous conserved areas of HIV-1/SIVcpz.

    PubMed

    de Groot, Natasja G; Heijmans, Corrine M C; Zoet, Yvonne M; de Ru, Arnoud H; Verreck, Frank A; van Veelen, Peter A; Drijfhout, Jan W; Doxiadis, Gaby G M; Remarque, Edmond J; Doxiadis, Ilias I N; van Rood, Jon J; Koning, Frits; Bontrop, Ronald E

    2010-08-24

    In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-B*27/B*57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-B*27/B*57, they target similar conserved areas of HIV-1/SIV(cpz). In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HIV-1/SIV(cpz) Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic.

  5. PQBP1 is a Proximal Sensor of the cGAS-dependent Innate Response to HIV-1

    PubMed Central

    Yoh, Sunnie M.; Schneider, Monika; Akleh, Rana E.; Olivieri, Kevin C.; De Jesus, Paul D.; Ruan, Chunhai; de Castro, Elisa; Ruiz, Pedro A.; Germanaud, David; des Portes, Vincent; García-Sastre, Adolfo; König, Renate; Chanda, Sumit K.

    2015-01-01

    Summary Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning Syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection. PMID:26046437

  6. Structure-based design of ligands for protein basic domains: application to the HIV-1 Tat protein.

    PubMed

    Filikov, A V; James, T L

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calormetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  7. Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

    NASA Astrophysics Data System (ADS)

    Filikov, Anton V.; James, Thomas L.

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  8. The Mediterranean scorpion Mesobuthus gibbosus (Scorpiones, Buthidae): transcriptome analysis and organization of the genome encoding chlorotoxin-like peptides

    PubMed Central

    2014-01-01

    Background Transcrof toxin genes of scorpion species have been published. Up to this moment, no information on the gene characterization of M. gibbosus is available. Results This study provides the first insight into gene expression in venom glands from M. gibbosus scorpion. A cDNA library was generated from the venom glands and subsequently analyzed (301 clones). Sequences from 177 high-quality ESTs were grouped as 48 Mgib sequences, of those 48 sequences, 40 (29 “singletons” and 11 “contigs”) correspond with one or more ESTs. We identified putative precursor sequences and were grouped them in different categories (39 unique transcripts, one with alternative reading frames), resulting in the identification of 12 new toxin-like and 5 antimicrobial precursors (transcripts). The analysis of the gene families revealed several new components categorized among various toxin families with effect on ion channels. Sequence analysis of a new KTx precursor provides evidence to validate a new KTx subfamily (α-KTx 27.x). A second part of this work involves the genomic organization of three Meg-chlorotoxin-like genes (ClTxs). Genomic DNA sequence reveals close similarities (presence of one same-phase intron) with the sole genomic organization of chlorotoxins ever reported (from M. martensii). Conclusions Transcriptome analysis is a powerful strategy that provides complete information of the gene expression and molecular diversity of the venom glands (telson). In this work, we generated the first catalogue of the gene expression and genomic organization of toxins from M. gibbosus. Our result represents a relevant contribution to the knowledge of toxin transcripts and complementary information related with other cell function proteins and venom peptide transcripts. The genomic organization of the chlorotoxin genes may help to understand the diversity of this gene family. PMID:24746279

  9. HIV-1 seroprevalence in an inner-city public hospital.

    PubMed Central

    Nagachinta, T.; Brown, C. P.; Cheng, F.; Temple, W.; Kerndt, P. R.; Janssen, R. S.

    1994-01-01

    In a hospital-based seroprevalence survey for human immunodeficiency virus type 1 (HIV-1) infection, a stratified sampling method based on age and gender was used to collect 5429 blood samples at an inner-city hospital. Sentinel Hospital Surveillance System (SHSS) criteria developed by the Centers for Disease Control and Prevention were used to classify patient diagnoses into two categories by the likelihood of being associated with HIV-1 infection. The two categories were those with high likelihood of association with HIV-1 (SHSS-ineligible) and those with low likelihood of association with HIV-1 infection (SHSS-eligible). Of the 5429 blood samples, 4262 were SHSS-eligible and 1167 were SHSS-ineligible. After personal identifies were removed, specimens were tested by ELISA and confirmed by Western blot analysis. The overall prevalence rate of HIV-1 infection was 0.98%. The seroprevalence rate was almost 2.6 times higher in high-association patients compared with low-association patients (1.89% versus 0.73%, P < .001). Results from this study indicate a high unsuspected HIV-1 seroprevalence rate in a subpopulation (SHSS-eligible) considered to have diagnoses with low likelihood of association with HIV-1 infection. These patients may better approximate HIV-1 seroprevalence in the general population of the area served by the hospital than would a sample of all patients. Monitoring HIV-1 seroprevalence in the SHSS-eligible group will be a useful measure for community serosurveillance for HIV-1 infection. PMID:8046762

  10. Sexually Transmitted Infections among HIV-1-Discordant Couples

    PubMed Central

    Guthrie, Brandon L.; Kiarie, James N.; Morrison, Susan; John-Stewart, Grace C.; Kinuthia, John; Whittington, William L. H.; Farquhar, Carey

    2009-01-01

    Introduction More new HIV-1 infections occur within stable HIV-1-discordant couples than in any other group in Africa, and sexually transmitted infections (STIs) may increase transmission risk among discordant couples, accounting for a large proportion of new HIV-1 infections. Understanding correlates of STIs among discordant couples will aid in optimizing interventions to prevent HIV-1 transmission in these couples. Methods HIV-1-discordant couples in which HIV-1-infected partners were HSV-2-seropositive were tested for syphilis, chlamydia, gonorrhea, and trichomoniasis, and HIV-1-uninfected partners were tested for HSV-2. We assessed sociodemographic, behavioral, and biological correlates of a current STI. Results Of 416 couples enrolled, 16% were affected by a treatable STI, and among these both partners were infected in 17% of couples. A treatable STI was found in 46 (11%) females and 30 (7%) males. The most prevalent infections were trichomoniasis (5.9%) and syphilis (2.6%). Participants were 5.9-fold more likely to have an STI if their partner had an STI (P<0.01), and STIs were more common among those reporting any unprotected sex (OR = 2.43; P<0.01) and those with low education (OR = 3.00; P<0.01). Among HIV-1-uninfected participants with an HSV-2-seropositive partner, females were significantly more likely to be HSV-2-seropositive than males (78% versus 50%, P<0.01). Conclusions Treatable STIs were common among HIV-1-discordant couples and the majority of couples affected by an STI were discordant for the STI, with relatively high HSV-2 discordance. Awareness of STI correlates and treatment of both partners may reduce HIV-1 transmission. Trial Registration ClinicalTrials.gov NCT00194519 PMID:20011596

  11. Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes.

    PubMed

    Gao, F; Bailes, E; Robertson, D L; Chen, Y; Rodenburg, C M; Michael, S F; Cummins, L B; Arthur, L O; Peeters, M; Shaw, G M; Sharp, P M; Hahn, B H

    1999-02-04

    The human AIDS viruses human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) represent cross-species (zoonotic) infections. Although the primate reservoir of HIV-2 has been clearly identified as the sooty mangabey (Cercocebus atys), the origin of HIV-1 remains uncertain. Viruses related to HIV-1 have been isolated from the common chimpanzee (Pan troglodytes), but only three such SIVcpz infections have been documented, one of which involved a virus so divergent that it might represent a different primate lentiviral lineage. In a search for the HIV-1 reservoir, we have now sequenced the genome of a new SIVcpzstrain (SIVcpzUS) and have determined, by mitochondrial DNA analysis, the subspecies identity of all known SIVcpz-infected chimpanzees. We find that two chimpanzee subspecies in Africa, the central P. t. troglodytes and the eastern P. t. schweinfurthii, harbour SIVcpz and that their respective viruses form two highly divergent (but subspecies-specific) phylogenetic lineages. All HIV-1 strains known to infect man, including HIV-1 groups M, N and O, are closely related to just one of these SIVcpz lineages, that found in P. t. troglodytes. Moreover, we find that HIV-1 group N is a mosaic of SIVcpzUS- and HIV-1-related sequences, indicating an ancestral recombination event in a chimpanzee host. These results, together with the observation that the natural range of P. t. troglodytes coincides uniquely with areas of HIV-1 group M, N and O endemicity, indicate that P. t. troglodytes is the primary reservoir for HIV-1 and has been the source of at least three independent introductions of SIVcpz into the human population.

  12. Tat is required for efficient HIV-1 reverse transcription.

    PubMed Central

    Harrich, D; Ulich, C; García-Martínez, L F; Gaynor, R B

    1997-01-01

    The ability of human immunodeficiency virus-1 (HIV-1) to undergo efficient reverse transcription is dependent on a number of parameters. These include the binding of the tRNA(3)(Lys) to the HIV-1 primer binding site and the subsequent interaction with the heterodimeric reverse transcriptase. Recently, we demonstrated that TAR RNA was also necessary for efficient HIV-1 reverse transcription. Given the fact that the Tat protein is involved in the activation of HIV-1 gene expression in conjunction with TAR, we wished to determine whether Tat might also be involved in the control of HIV-1 reverse transcription. HIV-1 virions deleted in the tat gene were unable to initiate reverse transcription efficiently upon infection of peripheral blood mononuclear cells (PBMCs). This defect was not due to decreased amounts of genomic RNA, reverse transcriptase or other HIV-1 proteins which were incorporated into the virion. Following transfection of wild-type but not mutant tat genes into cell lines producing HIV-1 lacking tat, the virions produced could be complemented for defects in reverse transcription upon subsequent infection of PBMCs. In contrast, the defect in reverse transcription seen with HIV-1 lacking the tat gene could not be complemented when the target cells rather than the producer cells contained tat. Viruses lacking tat were also defective in endogenous assays of reverse transcription, although these viruses contained similar levels of reverse transcriptase. These results indicate that the Tat protein, in addition to regulating the level of gene expression, is also important for efficient HIV-1 reverse transcription. PMID:9135139

  13. Evaluation in macaques of HIV-1 DNA vaccines containing primate CpG motifs and fowlpoxvirus vaccines co-expressing IFNgamma or IL-12.

    PubMed

    Dale, C Jane; De Rose, Robert; Wilson, Kim M; Croom, Hayley A; Thomson, Scott; Coupar, Barbara E H; Ramsay, Alistair; Purcell, Damian F J; Ffrench, Rosemary; Law, Matthew; Emery, Sean; Cooper, David A; Ramshaw, Ian A; Boyle, David B; Kent, Stephen J

    2004-11-25

    Induction of HIV-specific T-cell responses by vaccines may facilitate efficient control of HIV. Plasmid DNA vaccines and recombinant fowlpoxvirus (rFPV) vaccines are promising HIV-1 vaccine candidates, although either vaccine alone may be insufficient to protect against HIV-1. A consecutive immunisation strategy involving priming with DNA and boosting with rFPV vaccines encoding multiple common HIV-1 antigens was further evaluated in 30 macaques. The DNA vaccine vector included CpG immunostimulatory molecules, and rFPV vaccines were compared with rFPV vaccines co-expressing the pro-T cell cytokines IFNgamma or IL-12. Vaccines expressed multiple HIV-1 genes, mutated to remove active sites of the HIV proteins. The vaccines were well tolerated, and a significant enhancement of DNA-vaccine primed HIV-1 specific T lymphocyte responses was observed following rFPV boosting. Co-expression of IFNgamma or IL-12 by the rFPV vaccines did not further enhance immune responses. Non-sterilising protection from a non-pathogenic HIV-1 challenge was observed. This study provides evidence of a safe, optimised, strategy for the generation of T-cell mediated immunity to HIV-1.

  14. HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin

    PubMed Central

    Romani, Bizhan; Kamali Jamil, Razieh; Hamidi-Fard, Mojtaba; Rahimi, Pooneh; Momen, Seyed Bahman; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2016-01-01

    HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter. PMID:27550312

  15. Connectivity and HIV-1 infection: role of CD4(+) T-cell counts and HIV-1 RNA copy number.

    PubMed

    Padierna-Olivos, L; Moreno-Altamirano, M M; Sánchez-Colón, S; Massó-Rojas, F; Sánchez-García, F J

    2000-12-01

    Following primary infection with human immunodeficiency virus (HIV)-1, antibodies against specific HIV-1 epitopes are elicited. However, non-HIV-1 specific antibodies, including autoantibodies, also arise. In fact, it has been proposed that such autoantibodies have an important role in the pathogenesis of HIV-1 infection. Because an imbalance in connectivity has been associated with autoimmune processes, we investigated the connectivity status of HIV-1-infected individuals. Moreover, we tested the possible role of viral load and CD4(+) T-cell counts, in connectivity, because these parameters appear to be important in the prognosis of HIV-1 infection. Results show that indeed, there is an alteration in connectivity in these patients, both for immunoglobulin (Ig)G and IgM, which is an immune alteration not previously identified in HIV-1 infection. In addition, our results show that viral load and CD4(+) T-cell counts are both equally important in defining the characteristic pattern of connectivity in HIV-1-infected individuals, and that neither is independently responsible for alterations in patient connectivity status.

  16. Activation of latent HIV-1 expression by protein kinase C agonists. A novel therapeutic approach to eradicate HIV-1 reservoirs.

    PubMed

    Sánchez-Duffhues, Gonzalo; Vo, Minh Q; Pérez, Moisés; Calzado, Marco A; Moreno, Santiago; Appendino, Giovanni; Muñoz, Eduardo

    2011-03-01

    The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication in patients treated with highly active antiretroviral therapy. The molecular mechanisms by which integrated HIV-1 is repressed during latency have been partially identified in different models of HIV-1 latency, and the involvement of multiple processes has been demonstrated. Therefore, several molecular targets amenable to pharmacological manipulation have emerged to antagonize HIV-1 latency in the viral reservoirs. In this context, it has been suggested that successful depletion of such latent reservoirs will require a combination of therapeutic agents that can specifically and efficiently act on cells harbouring latent HIV-1 provirus. HIV-1 reactivation therapy is a potential therapeutic option to purge the viral reservoirs. The goal of this therapy is to enhance the transcriptional activity of the latent HIV-1 without inducing the polyclonal activation of non-infected cells. In this sense natural or semisynthetic protein kinase C agonists lacking tumour-promoter activities clearly fulfil this criterion, thereby opening new research avenues to purge HIV-1 reservoirs. In this review article, we have succinctly summarized the known effects of "natural products", focusing on phorboids like prostratin and ingenols, macrolides like bryostatin 1, and macrocyclic polyesters like ingols and jatrophanes. A comprehensive view on the molecular mechanisms underlying the principle of HIV-1 reactivation from latency is provided, discussing the combination of "natural products" with other experimental or conventional therapeutics.

  17. HIV-1–specific CD4+ T lymphocyte turnover and activation increase upon viral rebound

    PubMed Central

    Scriba, Thomas J.; Zhang, Hua-Tang; Brown, Helen L.; Oxenius, Annette; Tamm, Norbert; Fidler, Sarah; Fox, Julie; Weber, Jonathan N.; Klenerman, Paul; Day, Cheryl L.; Lucas, Michaela; Phillips, Rodney E.

    2005-01-01

    HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1–specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned. PMID:15668739

  18. Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody.

    PubMed Central

    Lescar, J.; Brynda, J.; Rezacova, P.; Stouracova, R.; Riottot, M. M.; Chitarra, V.; Fabry, M.; Horejsi, M.; Sedlacek, J.; Bentley, G. A.

    1999-01-01

    The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity. PMID:10631984

  19. Definition of the viral targets of protective HIV-1-specific T cell responses

    PubMed Central

    2011-01-01

    Background The efficacy of the CTL component of a future HIV-1 vaccine will depend on the induction of responses with the most potent antiviral activity and broad HLA class I restriction. However, current HIV vaccine designs are largely based on viral sequence alignments only, not incorporating experimental data on T cell function and specificity. Methods Here, 950 untreated HIV-1 clade B or -C infected individuals were tested for responses to sets of 410 overlapping peptides (OLP) spanning the entire HIV-1 proteome. For each OLP, a "protective ratio" (PR) was calculated as the ratio of median viral loads (VL) between OLP non-responders and responders. Results For both clades, there was a negative relationship between the PR and the entropy of the OLP sequence. There was also a significant additive effect of multiple responses to beneficial OLP. Responses to beneficial OLP were of significantly higher functional avidity than responses to non-beneficial OLP. They also had superior in-vitro antiviral activities and, importantly, were at least as predictive of individuals' viral loads than their HLA class I genotypes. Conclusions The data thus identify immunogen sequence candidates for HIV and provide an approach for T cell immunogen design applicable to other viral infections. PMID:22152067

  20. HLA class II genes modulate vaccine-induced antibody responses to affect HIV-1 acquisition.

    PubMed

    Prentice, Heather A; Tomaras, Georgia D; Geraghty, Daniel E; Apps, Richard; Fong, Youyi; Ehrenberg, Philip K; Rolland, Morgane; Kijak, Gustavo H; Krebs, Shelly J; Nelson, Wyatt; DeCamp, Allan; Shen, Xiaoying; Yates, Nicole L; Zolla-Pazner, Susan; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Ferrari, Guido; McElrath, M Juliana; Montefiori, David C; Bailer, Robert T; Koup, Richard A; O'Connell, Robert J; Robb, Merlin L; Michael, Nelson L; Gilbert, Peter B; Kim, Jerome H; Thomas, Rasmi

    2015-07-15

    In the RV144 vaccine trial, two antibody responses were found to correlate with HIV-1 acquisition. Because human leukocyte antigen (HLA) class II-restricted CD4(+) T cells are involved in antibody production, we tested whether HLA class II genotypes affected HIV-1-specific antibody levels and HIV-1 acquisition in 760 individuals. Indeed, antibody responses correlated with acquisition only in the presence of single host HLA alleles. Envelope (Env)-specific immunoglobulin A (IgA) antibodies were associated with increased risk of acquisition specifically in individuals with DQB1*06. IgG antibody responses to Env amino acid positions 120 to 204 were higher and were associated with decreased risk of acquisition and increased vaccine efficacy only in the presence of DPB1*13. Screening IgG responses to overlapping peptides spanning Env 120-204 and viral sequence analysis of infected individuals defined differences in vaccine response that were associated with the presence of DPB1*13 and could be responsible for the protection observed. Overall, the underlying genetic findings indicate that HLA class II modulated the quantity, quality, and efficacy of antibody responses in the RV144 trial.

  1. HLA class II genes modulate vaccine-induced antibody responses to affect HIV-1 acquisition

    PubMed Central

    Prentice, Heather A.; Tomaras, Georgia D.; Geraghty, Daniel E.; Apps, Richard; Fong, Youyi; Ehrenberg, Philip K.; Rolland, Morgane; Kijak, Gustavo H.; Krebs, Shelly J.; Nelson, Wyatt; DeCamp, Allan; Shen, Xiaoying; Yates, Nicole L.; Zolla-Pazner, Susan; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Ferrari, Guido; Juliana McElrath, M.; Montefiori, David C.; Bailer, Robert T.; Koup, Richard A.; O’Connell, Robert J.; Robb, Merlin L.; Michael, Nelson L.; Gilbert, Peter B.; Kim, Jerome H.; Thomas, Rasmi

    2016-01-01

    In the RV144 vaccine trial, two antibody responses were found to correlate with HIV-1 acquisition. Because human leukocyte antigen (HLA) class II–restricted CD4+ T cells are involved in antibody production, we tested whether HLA class II genotypes affected HIV-1–specific antibody levels and HIV-1 acquisition in 760 individuals. Indeed, antibody responses correlated with acquisition only in the presence of single host HLA alleles. Envelope (Env)–specific immunoglobulin A (IgA) antibodies were associated with increased risk of acquisition specifically in individuals with DQB1*06. IgG antibody responses to Env amino acid positions 120 to 204 were higher and were associated with decreased risk of acquisition and increased vaccine efficacy only in the presence of DPB1*13. Screening IgG responses to overlapping peptides spanning Env 120–204 and viral sequence analysis of infected individuals defined differences in vaccine response that were associated with the presence of DPB1*13 and could be responsible for the protection observed. Overall, the underlying genetic findings indicate that HLA class II modulated the quantity, quality, and efficacy of antibody responses in the RV144 trial. PMID:26180102

  2. Cell signaling pathways and HIV-1 therapeutics.

    PubMed

    He, Johnny J

    2011-06-01

    Host-virus interactions permeate every aspect of both virus life cycle and host response and involve host cell macromolecular machinery and viral elements. It is these intimate interactions that mandate the outcomes of the infection and pathogenesis. It is also these intimate interactions that lay the foundation for the development of pharmaceutical interventions. HIV-1 is no exception in these regards. In the first two decades, HIV/AIDS research has led to the successful development of a number of antiviral inhibitors and the landmark formulation of the suppressive therapy. It has become apparent that this therapy does not offer a complete solution to cure and eradicate the virus. Meanwhile, this therapy has changed the overall landscape of HIV-associated neurological disorders to a more common and prevalent form so-called minor cognitive motor disorder. Thus, there is an important and continued need for new anti-HIV therapeutics. We believe that this is an excellent opportunity to compile and present the latest works being done during the last few years in this exciting field of HIV-host interactions, particularly cell signaling pathways. We hope that this special issue composed of one brief report, eight thematic reviews, and two original articles will serve to foster the exchange of new scientific ideas on HIV-host interactions and anti-HIV therapy and eventually contribute to HIV/AIDS eradication.

  3. Possible applications for replicating HIV 1 vectors

    PubMed Central

    Das, Atze T; Jeeninga, Rienk E; Berkhout, Ben

    2010-01-01

    Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV 1 variant as a safe live attenuated virus vaccine, the construction of mini HIV variants as cancer selective viruses for virotherapy against leukemia, and the use of a conditionally live anti HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication competent HIV based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models. PMID:20582153

  4. Possible applications for replicating HIV 1 vectors.

    PubMed

    Das, Atze T; Jeeninga, Rienk E; Berkhout, Ben

    2010-05-01

    Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV 1 variant as a safe live attenuated virus vaccine, the construction of mini HIV variants as cancer selective viruses for virotherapy against leukemia, and the use of a conditionally live anti HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication competent HIV based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models.

  5. Quantitative Analysis of HIV-1 Preintegration Complexes

    PubMed Central

    Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

    2009-01-01

    Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

  6. Characterization of a Streptococcus mutans intergenic region containing a small toxic peptide and its cis-encoded antisense small RNA antitoxin.

    PubMed

    Koyanagi, Stephanie; Lévesque, Céline M

    2013-01-01

    Toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a protein toxin and an antitoxin, which may be in the form of either a labile protein or an antisense small RNA. Here we describe, to the best of our knowledge, the first functional chromosomal type I TA system in streptococci. Our model organism is the oral pathogen Streptococcus mutans. Our results showed that the genome of S. mutans UA159 reference strain harbors a previously unannotated Fst-like toxin (Fst-Sm) and its cis-encoded small RNA antitoxin (srSm) converging towards the end of the toxin gene in IGR176, a small intergenic region of 318 nt. Fst-Sm is a small hydrophobic peptide of 32 amino acid residues with homology to the Fst toxin family. Transcripts of ∼200 nt and ∼70 nt specific to fst-Sm mRNA and srSm RNA, respectively, were detected by Northern blot analysis throughout S. mutans growth. The toxin mRNA was considerably more stable than its cognate antitoxin. The half-life of srSm RNA was determined to be ∼30 min, while fst-Sm mRNA had a half-life of ∼90 min. Both fst-Sm and srSm RNAs were transcribed across direct tandem repeats providing a region of complementarity for inhibition of toxin translation. Overproduction of Fst-Sm had a toxic effect on E. coli and S. mutans cells which can be neutralized by coexpression of srSm RNA. Deletion of fst-Sm/srSm locus or overexpression of Fst-Sm/srSm had no effect on S. mutans cell growth in liquid medium and no differences in the total biofilm biomass were noted. In contrast, mild-overproduction of Fst-Sm/srSm type I TA system decreases the levels of persister cells tolerant to bacterial cell wall synthesis inhibitors.

  7. HIV-1 molecular epidemiology among newly diagnosed HIV-1 individuals in Hebei, a low HIV prevalence province in China

    PubMed Central

    Lu, Xinli; Kang, Xianjiang; Liu, Yongjian; Cui, Ze; Guo, Wei; Zhao, Cuiying; Li, Yan; Chen, Suliang; Li, Jingyun; Zhang, Yuqi; Zhao, Hongru

    2017-01-01

    New human immunodeficiency virus type 1 (HIV-1) diagnoses are increasing rapidly in Hebei. The aim of this study presents the most extensive HIV-1 molecular epidemiology investigation in Hebei province in China thus far. We have carried out the most extensive systematic cross-sectional study based on newly diagnosed HIV-1 positive individuals in 2013, and characterized the molecular epidemiology of HIV-1 based on full length gag-partial pol gene sequences in the whole of Hebei. Nine HIV-1 genotypes based on full length gag-partial pol gene sequence were identified among 610 newly diagnosed naïve individuals. The four main genotypes were circulating recombinant form (CRF)01_AE (53.4%), CRF07_BC (23.4%), subtype B (15.9%), and unique recombinant forms URFs (4.9%). Within 1 year, three new genotypes (subtype A1, CRF55_01B, CRF65_cpx), unknown before in Hebei, were first found among men who have sex with men (MSM). All nine genotypes were identified in the sexually contracted HIV-1 population. Among 30 URFs, six recombinant patterns were revealed, including CRF01_AE/BC (40.0%), CRF01_AE/B (23.3%), B/C (16.7%), CRF01_AE/C (13.3%), CRF01_AE/B/A2 (3.3%) and CRF01_AE/BC/A2 (3.3%), plus two potential CRFs. This study elucidated the complicated characteristics of HIV-1 molecular epidemiology in a low HIV-1 prevalence northern province of China and revealed the high level of HIV-1 genetic diversity. All nine HIV-1 genotypes circulating in Hebei have spread out of their initial risk groups into the general population through sexual contact, especially through MSM. This highlights the urgency of HIV prevention and control in China. PMID:28178737

  8. Anti-HIV-1 Activity of Flavonoid Myricetin on HIV-1 Infection in a Dual-Chamber In Vitro Model

    PubMed Central

    Pasetto, Silvana; Pardi, Vanessa; Murata, Ramiro Mendonça

    2014-01-01

    HIV infection by sexual transmission remains an enormous global health concern. More than 1 million new infections among women occur annually. Microbicides represent a promising prevention strategy that women can easily control. Among emerging therapies, natural small molecules such as flavonoids are an important source of new active substances. In this study we report the in vitro cytotoxicity and anti-HIV-1 and microbicide activity of the following flavonoids: Myricetin, Quercetin and Pinocembrin. Cytotoxicity tests were conducted on TZM-bl, HeLa, PBMC, and H9 cell cultures using 0.01–100 µM concentrations. Myricetin presented the lowest toxic effect, with Quercetin and Pinocembrin relatively more toxic. The anti-HIV-1 activity was tested with TZM-bl cell plus HIV-1 BaL (R5 tropic), H9 and PBMC cells plus HIV-1 MN (X4 tropic), and the dual tropic (X4R5) HIV-1 89.6. All flavonoids showed anti-HIV activity, although Myricetin was more effective than Quercetin or Pinocembrin. In TZM-bl cells, Myricetin inhibited ≥90% of HIV-1 BaL infection. The results were confirmed by quantification of HIV-1 p24 antigen in supernatant from H9 and PBMC cells following flavonoid treatment. In H9 and PBMC cells infected by HIV-1 MN and HIV-1 89.6, Myricetin showed more than 80% anti-HIV activity. Quercetin and Pinocembrin presented modest anti-HIV activity in all experiments. Myricetin activity was tested against HIV-RT and inhibited the enzyme by 49%. Microbicide activities were evaluated using a dual-chamber female genital tract model. In the in vitro microbicide activity model, Myricetin showed promising results against different strains of HIV-1 while also showing insignificant cytotoxic effects. Further studies of Myricetin should be performed to identify its molecular targets in order to provide a solid biological foundation for translational research. PMID:25546350

  9. Structures of HLA-A*1101 complexed with immunodominant nonamer and decamer HIV-1 epitopes clearly reveal the presence of a middle, secondary anchor residue.

    PubMed

    Li, Lenong; Bouvier, Marlene

    2004-05-15

    HLA-A*1101 is one of the most common human class I alleles worldwide. An increased frequency of HLA-A*1101 has been observed in cohorts of female sex workers from Northern Thailand who are highly exposed to HIV-1 and yet have remained persistently seronegative. In view of this apparent association of HLA-A*1101 with resistance to acquisition of HIV-1 infection, and given the importance of eliciting strong CTL responses to control and eliminate HIV-1, we have determined the crystal structure of HLA-A*1101 complexed with two immunodominant HIV-1 CTL epitopes: the nonamer reverse transcriptase(313-321) (AIFQSSMTK) and decamer Nef(73-82) (QVPLRPMTYK) peptides. The structures confirm the presence of primary anchor residues P2-Ile/-Val and P9-/P10-Lys, and also clearly reveal the presence of secondary anchor residues P6-Ser for reverse transcriptase and P7-Met for Nef. The overall backbone conformation of both peptides is defined as two bulges that are separated by a more buried middle residue. In this study, we discuss how this topology may offer functional advantages in the selection and presentation of HIV-1 CTL epitopes by HLA-A*1101. Overall, this structural analysis permits a more accurate definition of the peptide-binding motif of HLA-A*1101, the characterization of its antigenic surface, and the correlation of molecular determinants with resistance to HIV-1 infection. These studies are relevant for the rational design of HLA-A*1101-restricted CTL epitopes with improved binding and immunological properties for the development of HIV-1 vaccines.

  10. The Complex Interaction between Methamphetamine Abuse and HIV-1 pathogenesis

    PubMed Central

    Passaro, Ryan Colby; Pandhare, Jui; Qian, Han-Zhu; Dash, Chandravanu

    2016-01-01

    The global HIV/AIDS pandemic has claimed the lives of an estimated 35 million people. A significant barrier for combating this global pandemic is substance use since it is associated with HIV transmission, delayed diagnosis/initiation of therapy, and poor adherence to therapy. Clinical studies also suggest a link between substance use and HIV-disease progression/AIDS-associated mortality. Methamphetamine (METH) use is one of the fastest-growing substance use problems in the world. METH use enhances high-risk sexual behaviors, therefore increases the likelihood of HIV-1 acquisition. METH use is also associated with higher viral loads, immune dysfunction, and antiretroviral resistance. Moreover, METH use has also been correlated with rapid progression to AIDS. However, direct effects of METH on HIV-1 disease progression remains poorly understood because use of METH and other illicit drugs is often associated with reduced/non adherence to ART. Nevertheless, in vitro studies demonstrate that METH increases HIV-1 replication in cell cultures and animal models. Thus, it has been proposed that METH’s potentiating effects on HIV-1 replication may in part contribute to the worsening of HIV-1 pathogenesis. However, our recent data demonstrate that METH inhibits HIV-1 replication in CD4+ T cells and challenges this paradigm. Thus, the goal of this review is to systematically examine the published literature to better understand the complex interaction between METH abuse and HIV-1 disease progression. PMID:25850893

  11. Candidate antibody-based therapeutics against HIV-1.

    PubMed

    Gong, Rui; Chen, Weizao; Dimitrov, Dimiter S

    2012-06-01

    Antibody-based therapeutics have been successfully used for the treatment of various diseases and as research tools. Several well characterized, broadly neutralizing monoclonal antibodies (bnmAbs) targeting HIV-1 envelope glycoproteins or related host cell surface proteins show sterilizing protection of animals, but they are not effective when used for therapy of an established infection in humans. Recently, a number of novel bnmAbs, engineered antibody domains (eAds), and multifunctional fusion proteins have been reported which exhibit exceptionally potent and broad neutralizing activity against a wide range of HIV-1 isolates from diverse genetic subtypes. eAds could be more effective in vivo than conventional full-size antibodies generated by the human immune system. Because of their small size (12∼15 kD), they can better access sterically restricted epitopes and penetrate densely packed tissue where HIV-1 replicates than the larger full-size antibodies. HIV-1 possesses a number of mechanisms to escape neutralization by full-size antibodies but could be less likely to develop resistance to eAds. Here, we review the in vitro and in vivo antiviral efficacies of existing HIV-1 bnmAbs, summarize the development of eAds and multispecific fusion proteins as novel types of HIV-1 inhibitors, and discuss possible strategies to generate more potent antibody-based candidate therapeutics against HIV-1, including some that could be used to eradicate the virus.

  12. Defining the roles for Vpr in HIV-1-associated neuropathogenesis.

    PubMed

    James, Tony; Nonnemacher, Michael R; Wigdahl, Brian; Krebs, Fred C

    2016-08-01

    It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing that the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection.

  13. HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies.

    PubMed

    Sliepen, Kwinten; Sanders, Rogier W

    2016-01-01

    The long pursuit for a vaccine against human immunodeficiency virus 1 (HIV-1) has recently been boosted by a number of exciting developments. An HIV-1 subunit vaccine ideally should elicit potent broadly neutralizing antibodies (bNAbs), but raising bNAbs by vaccination has proved extremely difficult because of the characteristics of the HIV-1 envelope glycoprotein complex (Env). However, the isolation of bNAbs from HIV-1-infected patients demonstrates that the human humoral immune system is capable of making such antibodies. Therefore, a focus of HIV-1 vaccinology is the elicitation of bNAbs by engineered immunogens and by using vaccination strategies aimed at mimicking the bNAb maturation pathways in HIV-infected patients. Important clues can also be taken from the successful subunit vaccines against hepatitis B virus and human papillomavirus. Here, we review the different types of HIV-1 immunogens and vaccination strategies that are being explored in the search for an HIV-1 vaccine that induces bNAbs.

  14. Identification of Siglec-1 null individuals infected with HIV-1.

    PubMed

    Martinez-Picado, Javier; McLaren, Paul J; Erkizia, Itziar; Martin, Maureen P; Benet, Susana; Rotger, Margalida; Dalmau, Judith; Ouchi, Dan; Wolinsky, Steven M; Penugonda, Sudhir; Günthard, Huldrych F; Fellay, Jacques; Carrington, Mary; Izquierdo-Useros, Nuria; Telenti, Amalio

    2016-08-11

    Siglec-1/CD169 is a myeloid-cell surface receptor critical for HIV-1 capture and infection of bystander target cells. To dissect the role of SIGLEC1 in natura, we scan a large population genetic database and identify a loss-of-function variant (Glu88Ter) that is found in ∼1% of healthy people. Exome analysis and direct genotyping of 4,233 HIV-1-infected individuals reveals two Glu88Ter homozygous and 97 heterozygous subjects, allowing the analysis of ex vivo and in vivo consequences of SIGLEC1 loss-of-function. Cells from these individuals are functionally null or haploinsufficient for Siglec-1 activity in HIV-1 capture and trans-infection ex vivo. However, Siglec-1 protein truncation does not have a measurable impact on HIV-1 acquisition or AIDS outcomes in vivo. This result contrasts with the known in vitro functional role of Siglec-1 in HIV-1 trans-infection. Thus, it provides evidence that the classical HIV-1 infectious routes may compensate for the lack of Siglec-1 in fuelling HIV-1 dissemination within infected individuals.

  15. Identification of Siglec-1 null individuals infected with HIV-1

    PubMed Central

    Martinez-Picado, Javier; McLaren, Paul J.; Erkizia, Itziar; Martin, Maureen P.; Benet, Susana; Rotger, Margalida; Dalmau, Judith; Ouchi, Dan; Wolinsky, Steven M.; Penugonda, Sudhir; Günthard, Huldrych F.; Fellay, Jacques; Carrington, Mary; Izquierdo-Useros, Nuria; Telenti, Amalio

    2016-01-01

    Siglec-1/CD169 is a myeloid-cell surface receptor critical for HIV-1 capture and infection of bystander target cells. To dissect the role of SIGLEC1 in natura, we scan a large population genetic database and identify a loss-of-function variant (Glu88Ter) that is found in ∼1% of healthy people. Exome analysis and direct genotyping of 4,233 HIV-1-infected individuals reveals two Glu88Ter homozygous and 97 heterozygous subjects, allowing the analysis of ex vivo and in vivo consequences of SIGLEC1 loss-of-function. Cells from these individuals are functionally null or haploinsufficient for Siglec-1 activity in HIV-1 capture and trans-infection ex vivo. However, Siglec-1 protein truncation does not have a measurable impact on HIV-1 acquisition or AIDS outcomes in vivo. This result contrasts with the known in vitro functional role of Siglec-1 in HIV-1 trans-infection. Thus, it provides evidence that the classical HIV-1 infectious routes may compensate for the lack of Siglec-1 in fuelling HIV-1 dissemination within infected individuals. PMID:27510803

  16. Plausibility of HIV-1 Infection of Oral Mucosal Epithelial Cells

    PubMed Central

    Herzberg, M.C.; Vacharaksa, A.; Gebhard, K.H.; Giacaman, R.A.; Ross, K.F.

    2011-01-01

    The AIDS pandemic continues. Little is understood about how HIV gains access to permissive cells across mucosal surfaces, yet such knowledge is crucial to the development of successful topical anti-HIV-1 agents and mucosal vaccines. HIV-1 rapidly internalizes and integrates into the mucosal keratinocyte genome, and integrated copies of HIV-1 persist upon cell passage. The virus does not appear to replicate, and the infection may become latent. Interactions between HIV-1 and oral keratinocytes have been modeled in the context of key environmental factors, including putative copathogens and saliva. In keratinocytes, HIV-1 internalizes within minutes; in saliva, an infectious fraction escapes inactivation and is harbored and transferable to permissive target cells for up to 48 hours. When incubated with the common oral pathogen Porphyromonas gingivalis, CCR5− oral keratinocytes signal through protease-activated receptors and Toll-like receptors to induce expression of CCR5, which increases selective uptake of infectious R5-tropic HIV-1 into oral keratinocytes and transfer to permissive cells. Hence, oral keratinocytes—like squamous keratinocytes of other tissues—may be targets for low-level HIV-1 internalization and subsequent dissemination by transfer to permissive cells. PMID:21441479

  17. Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

    PubMed Central

    Zhang, Baoshan; McKee, Krisha; Longo, Nancy S.; Yang, Yongping; Huang, Jinghe; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Alam, S. Munir; Haynes, Barton F.; Mullikin, James C.; Connors, Mark; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.

    2016-01-01

    Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization. PMID:27299673

  18. Host SAMHD1 Protein Promotes HIV-1 Recombination in Macrophages*

    PubMed Central

    Nguyen, Laura A.; Kim, Dong-Hyun; Daly, Michele B.; Allan, Kevin C.; Kim, Baek

    2014-01-01

    Template switching can occur during the reverse transcription of HIV-1. Deoxynucleotide triphosphate (dNTP) concentrations have been biochemically shown to impact HIV-1 reverse transcriptase (RT)-mediated strand transfer. Lowering the dNTP concentrations promotes RT pausing and RNA template degradation by RNase H activity of the RT, subsequently leading to strand transfer. Terminally differentiated/nondividing macrophages, which serve as a key HIV-1 reservoir, contain extremely low dNTP concentrations (20–50 nm), which results from the cellular dNTP hydrolyzing sterile α motif and histidine aspartic domain containing protein 1 (SAMHD1) protein, when compared with activated CD4+ T cells (2–5 μm). In this study, we first observed that HIV-1 template switching efficiency was nearly doubled in human primary macrophages when compared with activated CD4+ T cells. Second, SAMHD1 degradation by viral protein X (Vpx), which elevates cellular dNTP concentrations, decreased HIV-1 template switching efficiency in macrophages to the levels comparable with CD4+ T cells. Third, differentiated SAMHD1 shRNA THP-1 cells have a 2-fold increase in HIV-1 template switching efficiency. Fourth, SAMHD1 degradation by Vpx did not alter HIV-1 template switching efficiency in activated CD4+ T cells. Finally, the HIV-1 V148I RT mutant that is defective in dNTP binding and has DNA synthesis delay promoted RT stand transfer when compared with wild type RT, particularly at low dNTP concentrations. Here, we report that SAMHD1 regulation of the dNTP concentrations influences HIV-1 template switching efficiency, particularly in macrophages. PMID:24352659

  19. Genome editing strategies: potential tools for eradicating HIV-1/AIDS.

    PubMed

    Khalili, Kamel; Kaminski, Rafal; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

    2015-06-01

    Current therapy for controlling human immunodeficiency virus (HIV-1) infection and preventing acquired immunodeficiency syndrome (AIDS) progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells, which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or "sterile" cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS.

  20. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  1. Hypocretin/Orexin Peptides Alter Spike Encoding by Serotonergic Dorsal Raphe Neurons through Two Distinct Mechanisms That Increase the Late Afterhyperpolarization

    PubMed Central

    Ishibashi, Masaru; Gumenchuk, Iryna; Miyazaki, Kenichi; Inoue, Takafumi; Ross, William N.

    2016-01-01

    Orexins (hypocretins) are neuropeptides that regulate multiple homeostatic processes, including reward and arousal, in part by exciting serotonergic dorsal raphe neurons, the major source of forebrain serotonin. Here, using mouse brain slices, we found that, instead of simply depolarizing these neurons, orexin-A altered the spike encoding process by increasing the postspike afterhyperpolarization (AHP) via two distinct mechanisms. This orexin-enhanced AHP (oeAHP) was mediated by both OX1 and OX2 receptors, required Ca2+ influx, reversed near EK, and decayed with two components, the faster of which resulted from enhanced SK channel activation, whereas the slower component decayed like a slow AHP (sAHP), but was not blocked by UCL2077, an antagonist of sAHPs in some neurons. Intracellular phospholipase C inhibition (U73122) blocked the entire oeAHP, but neither component was sensitive to PKC inhibition or altered PKA signaling, unlike classical sAHPs. The enhanced SK current did not depend on IP3-mediated Ca2+ release but resulted from A-current inhibition and the resultant spike broadening, which increased Ca2+ influx and Ca2+-induced-Ca2+ release, whereas the slower component was insensitive to these factors. Functionally, the oeAHP slowed and stabilized orexin-induced firing compared with firing produced by a virtual orexin conductance lacking the oeAHP. The oeAHP also reduced steady-state firing rate and firing fidelity in response to stimulation, without affecting the initial rate or fidelity. Collectively, these findings reveal a new orexin action in serotonergic raphe neurons and suggest that, when orexin is released during arousal and reward, it enhances the spike encoding of phasic over tonic inputs, such as those related to sensory, motor, and reward events. SIGNIFICANCE STATEMENT Orexin peptides are known to excite neurons via slow postsynaptic depolarizations. Here we elucidate a significant new orexin action that increases and prolongs the postspike

  2. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

    PubMed

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-04-25

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

  3. In Vivo SELEX of Single-Stranded Domains in the HIV-1 Leader RNA

    PubMed Central

    van Bel, Nikki; Das, Atze T.

    2014-01-01

    ABSTRACT The 5′ untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE in vivo PMID:24335293