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Sample records for 4e10-resistant hiv-1 isolated

  1. 4E10-Resistant HIV-1 Isolated from Four Subjects with Rare Membrane-Proximal External Region Polymorphisms

    PubMed Central

    Nakamura, Kyle J.; Gach, Johannes S.; Jones, Laura; Semrau, Katherine; Walter, Jan; Bibollet-Ruche, Frederic; Decker, Julie M.; Heath, Laura; Decker, William D.; Sinkala, Moses; Kankasa, Chipepo; Thea, Donald; Mullins, James; Kuhn, Louise; Zwick, Michael B.; Aldrovandi, Grace M.

    2010-01-01

    Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope. PMID:20352106

  2. Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors

    PubMed Central

    Toyoda, Mako; Ogata, Yoko; Mahiti, Macdonald; Maeda, Yosuke; Kuang, Xiaomei T.; Miura, Toshiyuki; Jessen, Heiko; Walker, Bruce D.; Brockman, Mark A.; Brumme, Zabrina L.

    2015-01-01

    ABSTRACT HIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified via in vitro mutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here, nef clones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 downregulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 downregulation ability and modestly impaired CXCR4 downregulation activity compared to those from CP and AP. Nef's ability to downregulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linked in vivo. Moreover, impairments in Nef's receptor downregulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune escape-associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks. IMPORTANCE HIV-1 Nef, a key factor for viral pathogenesis, downregulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and coreceptors CCR5

  3. GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

    PubMed Central

    Mohr, Emma L.; Xiang, Jinhua; McLinden, James H.; Kaufman, Thomas M.; Chang, Qing; Montefiori, David C.; Klinzman, Donna; Stapleton, Jack T.

    2012-01-01

    Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. PMID:20826757

  4. HERV-K–specific T cells eliminate diverse HIV-1/2 and SIV primary isolates

    PubMed Central

    Jones, R. Brad; Garrison, Keith E.; Mujib, Shariq; Mihajlovic, Vesna; Aidarus, Nasra; Hunter, Diana V.; Martin, Eric; John, Vivek M.; Zhan, Wei; Faruk, Nabil F.; Gyenes, Gabor; Sheppard, Neil C.; Priumboom-Brees, Ingrid M.; Goodwin, David A.; Chen, Lianchun; Rieger, Melanie; Muscat-King, Sophie; Loudon, Peter T.; Stanley, Cole; Holditch, Sara J.; Wong, Jessica C.; Clayton, Kiera; Duan, Erick; Song, Haihan; Xu, Yang; SenGupta, Devi; Tandon, Ravi; Sacha, Jonah B.; Brockman, Mark A.; Benko, Erika; Kovacs, Colin; Nixon, Douglas F.; Ostrowski, Mario A.

    2012-01-01

    The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1–infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)–specific CD8+ T cells obtained from HIV-1–infected human subjects responded to HIV-1–infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)–specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)–specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)–targeted HIV-1 vaccines and immunotherapeutics. PMID:23143309

  5. Neutralization of multiple laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) by antisera raised against gp120 from the MN isolate of HIV-1.

    PubMed Central

    Berman, P W; Matthews, T J; Riddle, L; Champe, M; Hobbs, M R; Nakamura, G R; Mercer, J; Eastman, D J; Lucas, C; Langlois, A J

    1992-01-01

    Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine. PMID:1602554

  6. 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

  7. The gp120 Protein Is a Second Determinant of Decreased Neurovirulence of Indian HIV-1C Isolates Compared to Southern African HIV-1C Isolates

    PubMed Central

    Rao, Vasudev R.; Neogi, Ujjwal; Eugenin, Eliseo; Prasad, Vinayaka R.

    2014-01-01

    Regional differences in neurovirulence have been documented among subtype/clade-C HIV-1 isolates in India and Southern Africa. We previously demonstrated that a C31S substitution in Clade-C Tat dicysteine motif reduces monocyte recruitment, cytokine induction and direct neurotoxicity. Therefore, this polymorphism is considered to be a causative factor for these differences in neurovirulence. We previously reported on the genotypic differences in Tat protein between clade-C and rest of the clades showing that approximately 90% of clade-C HIV-1 Tat sequences worldwide contained this C31S polymorphism, while 99% of non-clade C isolates lacked this Tat polymorphism at C31 residue (Ranga et al. (2004) J Virol 78∶2586–2590). Subsequently, we documented intra-clade-C differences in the frequency of Tat dicysteine variants between India and Southern Africa, as the basis for differential disease severity and showed the importance of the Tat dicysteine motif for neuropathogenesis using small animal models. We have now examined if determinants of neurovirulence besides Tat are different between the clade-C HIV-1 isolates from Southern Africa and India. Envelope glycoprotein gp120 is a well-documented contributor to neurotoxicity. We found that gp120 sequences of HIV-1 isolates from these two regions are genetically distinct. In order to delineate the contribution of gp120 to neurovirulence, we compared direct in vitro neurotoxicity of HIV-infected supernatants of a representative neurovirulent US clade-B isolate with two isolates each from Southern Africa and India using primary human neurons and SH-SY5Y neuroblastoma cells. Immunodepletion of gp120 of both US clade B and the Southern African clade C isolates revealed robust decreases in neurotoxicity, while that of the Indian isolates showed minimal effect on neurotoxicity. The gp120 as a cause of differential neurotoxicity was further confirmed using purified recombinant gp120 from HIV isolates from these regions. We

  8. Identification of Owl Monkey CD4 Receptors Broadly Compatible with Early-Stage HIV-1 Isolates.

    PubMed

    Meyerson, Nicholas R; Sharma, Amit; Wilkerson, Gregory K; Overbaugh, Julie; Sawyer, Sara L

    2015-08-01

    Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or they do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly, we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys (Aotus vociferans) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterized the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and found that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We found that the CD4 receptors encoded by two other species of owl monkeys (Aotus azarae and Aotus nancymaae) also serve as functional entry receptors for early-stage isolates of HIV-1. Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. In this study, we found that some individuals from captive owl

  9. Identification of Owl Monkey CD4 Receptors Broadly Compatible with Early-Stage HIV-1 Isolates

    PubMed Central

    Meyerson, Nicholas R.; Sharma, Amit; Wilkerson, Gregory K.

    2015-01-01

    ABSTRACT Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or they do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly, we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys (Aotus vociferans) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterized the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and found that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We found that the CD4 receptors encoded by two other species of owl monkeys (Aotus azarae and Aotus nancymaae) also serve as functional entry receptors for early-stage isolates of HIV-1. IMPORTANCE Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. In this study, we found that some individuals

  10. 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.

  11. Evolution of an attenuated HIV-1 isolate in an elite suppressor.

    PubMed

    Salgado, Maria; Gandhi, Shiv; Buckheit, Robert W; Berkenblit, Gail V; Blankson, Joel N

    2014-03-01

    Elite controllers or suppressors (ES) control viral replication without antiretroviral therapy. While many ES are infected with replication-competent virus, others have evidence of infection with attenuated isolates. Here we report a case of an ES infected with an HIV-1 isolate that contained a 38-base pair deletion in nef that led to a reading frame shift and a premature stop codon. Interestingly, clones amplified from plasma or cultured from CD4(+) T cells between 2006 and 2008 contained one of two separate compensatory deletions that restored the reading frame. A new insertion generated by duplication of adjacent sequences was found in isolates obtained in 2010 and this evolution was accompanied by the development of low level viremia. This article provides evidence of the evolution of an attenuated HIV-1 isolate toward greater virulence in an elite suppressor.

  12. Evolution of an Attenuated HIV-1 Isolate in an Elite Suppressor

    PubMed Central

    Salgado, Maria; Gandhi, Shiv; Buckheit, Robert W.; Berkenblit, Gail V.

    2014-01-01

    Abstract Elite controllers or suppressors (ES) control viral replication without antiretroviral therapy. While many ES are infected with replication-competent virus, others have evidence of infection with attenuated isolates. Here we report a case of an ES infected with an HIV-1 isolate that contained a 38-base pair deletion in nef that led to a reading frame shift and a premature stop codon. Interestingly, clones amplified from plasma or cultured from CD4+ T cells between 2006 and 2008 contained one of two separate compensatory deletions that restored the reading frame. A new insertion generated by duplication of adjacent sequences was found in isolates obtained in 2010 and this evolution was accompanied by the development of low level viremia. This article provides evidence of the evolution of an attenuated HIV-1 isolate toward greater virulence in an elite suppressor. PMID:24117037

  13. Successful Isolation of Infectious and High Titer Human Monocyte-Derived HIV-1 from Two Subjects with Discontinued Therapy

    PubMed Central

    Zhu, Haiying; Andrus, Thomas; Ivanov, Sergei B.; Pan, Charlotte; Dolores, Jazel; Dann, Gregory C.; Zhou, Michael; Forte, Dominic; Yang, Zihuan; Holte, Sarah; Corey, Lawrence; Zhu, Tuofu

    2013-01-01

    Background HIV-1 DNA in blood monocytes is considered a viral source of various HIV-1 infected tissue macrophages, which is also known as “Trojan horse” hypothesis. However, whether these DNA can produce virions has been an open question for years, due to the inability of isolating high titer and infectious HIV-1 directly from monocytes. Results In this study, we demonstrated successful isolation of two strains of M-HIV-1 (1690 M and 1175 M) from two out of four study subjects, together with their in vivo controls, HIV-1 isolated from CD4+ T-cells (T-HIV-1), 1690 T and 1175 T. All M- and T- HIV-1 isolates were detected CCR5-tropic. Both M- HIV-1 exhibited higher levels of replication in monocyte-derived macrophages (MDM) than the two T- HIV-1. Consistent with our previous reports on the subject 1175 with late infection, compartmentalized env C2-V3-C3 sequences were identified between 1175 M and 1175 T. In contrast, 1690 M and 1690 T, which were isolated from subject 1690 with relatively earlier infection, showed homogenous env C2-V3-C3 sequences. However, multiple reverse transcriptase (RT) inhibitor resistance-associated variations were detected in the Gag-Pol region of 1690 M, but not of 1690 T. By further measuring HIV DNA intracellular copy numbers post-MDM infection, 1690 M was found to have significantly higher DNA synthesis efficiency than 1690 T in macrophages, indicating a higher RT activity, which was confirmed by AZT inhibitory assays. Conclusions These results suggested that the M- and T- HIV-1 are compartmentalized in the two study subjects, respectively. Therefore, we demonstrated that under in vitro conditions, HIV-1 infected human monocytes can productively release live viruses while differentiating into macrophages. PMID:23741458

  14. HIV-1 sequence variation between isolates from mother-infant transmission pairs

    SciTech Connect

    Wike, C.M.; Daniels, M.R.; Furtado, M.; Wolinsky, M.; Korber, B.; Hutto, C.; Munoz, J.; Parks, W.; Saah, A.

    1991-01-01

    To examine the sequence diversity of human immunodeficiency virus type 1 (HIV-1) between known transmission sets, sequences from the V3 and V4-V5 region of the env gene from 4 mother-infant pairs were analyzed. The mean interpatient sequence variation between isolates from linked mother-infant pairs was comparable to the sequence diversity found between isolates from other close contacts. The mean intrapatient variation was significantly less in the infants' isolates then the isolates from both their mothers and other characterized intrapatient sequence sets. In addition, a distinct and characteristic difference in the glycosylation pattern preceding the V3 loop was found between each linked transmission pair. These findings indicate that selection of specific genotypic variants, which may play a role in some direct transmission sets, and the duration of infection are important factors in the degree of diversity seen between the sequence sets.

  15. HIV-1 sequence variation between isolates from mother-infant transmission pairs

    SciTech Connect

    Wike, C.M.; Daniels, M.R.; Furtado, M.; Wolinsky, M.; Korber, B.; Hutto, C.; Munoz, J.; Parks, W.; Saah, A.

    1991-12-31

    To examine the sequence diversity of human immunodeficiency virus type 1 (HIV-1) between known transmission sets, sequences from the V3 and V4-V5 region of the env gene from 4 mother-infant pairs were analyzed. The mean interpatient sequence variation between isolates from linked mother-infant pairs was comparable to the sequence diversity found between isolates from other close contacts. The mean intrapatient variation was significantly less in the infants` isolates then the isolates from both their mothers and other characterized intrapatient sequence sets. In addition, a distinct and characteristic difference in the glycosylation pattern preceding the V3 loop was found between each linked transmission pair. These findings indicate that selection of specific genotypic variants, which may play a role in some direct transmission sets, and the duration of infection are important factors in the degree of diversity seen between the sequence sets.

  16. Virulence factors of Candida albicans isolates from the oral cavities of HIV-1-positive patients.

    PubMed

    Menezes, Tatiany O A; Gillet, Luciana C S; Menezes, Sílvio A F; Feitosa, Rosimar N M; Ishak, Marluísa O G; Ishak, Ricardo; Marques-da-Silva, Sílvia H; Vallinoto, Antonio C R

    2013-06-01

    The present study assessed the phenotypic aspects of oral-cavity Candida albicans isolates from 300 HIV-1- positive patients, relating the most commonly investigated virulence factors (enzyme typing and germ-tube formation) to the most common morphotypes. The samples were seeded into specific media for isolation and subsequent identification using the automated Vitek 2 system. The following assays were performed for phenotypic characterization: morphotyping, germ-tube formation and enzyme typing. Out of 300 collected samples, 144 tested positive for yeasts of the Candida genus, 98 (32.7 %) of which were identified as C. albicans. The latter samples were attributed to seven different morphotypes; the three most common morphotypes were 7208 (49 %), 7308 (14.3 %) and 3208 (13.3 %). All of the C. albicans isolate samples formed germ tubes and produced the enzymes proteinase and phospholipase, with an activity classified as intermediate to high. Due to the identification of virulence factors among the analyzed samples, monitoring of HIV-1-positive patients colonized by different morphotypes must be established because these morphotypes are extremely pathogenic and can trigger severe fungal infections.

  17. Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals.

    PubMed

    Konadu, Kateena Addae; Huang, Ming Bo; Roth, William; Armstrong, Wendy; Powell, Michael; Villinger, Francois; Bond, Vincent

    2016-01-05

    Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

  18. Epidemiology and genetic characterization of HIV-1 isolates in the general population of Djibouti (Horn of Africa).

    PubMed

    Maslin, Jérôme; Rogier, Christophe; Berger, Franck; Khamil, Mohamed Ali; Mattera, Didier; Grandadam, Marc; Caron, Mélanie; Nicand, Elizabeth

    2005-06-01

    During a national survey in 2002 in Djibouti, serum samples were collected using a valid sampling scheme from 2423 Djiboutians representing the general population of urban and rural districts. The HIV-1 seroprevalence was 2%. The HIV-1 polymerase gene from 53 untreated patients was amplified. Phylogenetic analysis of 34 isolates revealed a majority of subtype C (73%) as well as other subtypes, including CRF02_AG recombinants (18%), subtype D (6%), and subtype A (3%).

  19. Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

    PubMed Central

    Borges, Andrew Rosa; Wieczorek, Lindsay; Johnson, Benitra; Benesi, Alan J.; Brown, Bruce K.; Kensinger, Richard D.; Krebs, Fred C.; Wigdahl, Brian; Blumenthal, Robert; Puri, Anu; McCutchan, Francine E.; Birx, Deborah L.; Polonis, Victoria R.; Schengrund, Cara-Lynne

    2010-01-01

    Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3’-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC50s ranging from 0.1 – 7.4 µg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. PMID:20880566

  20. Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

    SciTech Connect

    Rosa Borges, Andrew; Wieczorek, Lindsay; Johnson, Benitra; Benesi, Alan J.; Brown, Bruce K.; Kensinger, Richard D.; Krebs, Fred C.; Wigdahl, Brian; Blumenthal, Robert; Puri, Anu; McCutchan, Francine E.; Birx, Deborah L.; Polonis, Victoria R.; Schengrund, Cara-Lynne

    2010-12-05

    Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC{sub 50}s ranging from 0.1 to 7.4 {mu}g/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. -- Research Highlights: {yields}Multivalent carbohydrates (MVCs) inhibited infection of PBMCs by HIV-1. {yields}MVCs inhibited infection by T cell line-adapted viruses. {yields}MVCs inhibited infection by primary isolates of HIV-1. {yields}MVCs inhibited Env-mediated membrane fusion.

  1. [HIV-1 subtype distribution determined by phylogenetic analysis of pol gene sequences and automated subtyping tools among HIV-1 isolates from the Aegian Region of Turkey].

    PubMed

    Uluer Biçeroğlu, Servet; Altuğlu, Imre; Nazli Zeka, Arzu; Gökengin, Deniz; Yazan Sertöz, Rüçhan

    2014-07-01

    Human immunodeficiency virus (HIV) exhibiting remarkable genetic variability, includes two genotypes namely HIV-1 (group M, N, O and P) and HIV-2 (group A-H). HIV-1 group M, which is mainly the cause of the AIDS pandemic, is divided into nine pure subtypes, more than 45 circulating recombinant forms (CRF) and numerous unique recombinant forms (URF). According to the documents of Turkish Government of Health, among a total of 6802 HIV-positive cases, 1096 of them were defined as AIDS as of June 2013 in Turkey. Although subtype B is the predominant subtype, recent studies indicate higher proportion of CRFs similar to their increasing role in the HIV pandemic. The aim of this study was to determine the subtype distribution of HIV-1 strains isolated from 70 patients (61 male, 9 female; age range: 16-73 yrs, mean age: 39.6 yrs) who presented to our institution between April 2008-June 2013. HIV-1 strains were subtyped by phylogenetic analysis of the pol gene region and commonly used automated subtyping tools namely, Stanford HIV db v6.2.0 and Rega v3.0. Pol sequences retrieved from the Los Alamos database and from GeneBank, were trimmed from full-length genomes. Phylogenetic analysis of the 1302 base pair of the pol gene region was performed using Mega v5.2 software. The sequences were aligned using Muscle and phylogenetic distances between sequences were estimated by using Kimura two-parameter model (transition/transversion ratio: 2.0). Tree topology was obtained using neighbour-joining method and bootstrap value was set at 1000. Sixty-one (87.1%) patients were antiretroviral treatment (ART)-naive and nine were on different ART regimens. The subtypes of the isolates according to phylogenetic analysis were found as follows; 31 (44.2%) subtype B, 24 (34.2%) CRF42_BF, 6 (8.5%) B/CRF02_AG recombinants, 5 (7.1%) sub-subtype A1, 1 (1.4%) sub-subtype F1, 1 (%1.4) CRF 25_cpx, 1 (1.4%) CRF02_AG and 1 (1.4%) CRF01_AE. Rega v3.0 subtyping tool produced five discrepant results (4 B

  2. Close phylogenetic relationship between Angolan and Romanian HIV-1 subtype F1 isolates

    PubMed Central

    Guimarães, Monick L; Vicente, Ana Carolina P; Otsuki, Koko; da Silva, Rosa Ferreira FC; Francisco, Moises; da Silva, Filomena Gomes; Serrano, Ducelina; Morgado, Mariza G; Bello, Gonzalo

    2009-01-01

    Background Here, we investigated the phylogenetic relationships of the HIV-1 subtype F1 circulating in Angola with subtype F1 strains sampled worldwide and reconstructed the evolutionary history of this subtype in Central Africa. Methods Forty-six HIV-1-positive samples were collected in Angola in 2006 and subtyped at the env-gp41 region. Partial env-gp120 and pol-RT sequences and near full-length genomes from those env-gp41 subtype F1 samples were further generated. Phylogenetic analyses of partial and full-length subtype F1 strains isolated worldwide were carried out. The onset date of the subtype F1 epidemic in Central Africa was estimated using a Bayesian Markov chain Monte Carlo approach. Results Nine Angolan samples were classified as subtype F1 based on the analysis of the env-gp41 region. All nine Angolan sequences were also classified as subtype F1 in both env-gp120 and pol-RT genomic regions, and near full-length genome analysis of four of these samples confirmed their classification as "pure" subtype F1. Phylogenetic analyses of subtype F1 strains isolated worldwide revealed that isolates from the Democratic Republic of Congo (DRC) were the earliest branching lineages within the subtype F1 phylogeny. Most strains from Angola segregated in a monophyletic group together with Romanian sequences; whereas South American F1 sequences emerged as an independent cluster. The origin of the subtype F1 epidemic in Central African was estimated at 1958 (1934–1971). Conclusion "Pure" subtype F1 strains are common in Angola and seem to be the result of a single founder event. Subtype F1 sequences from Angola are closely related to those described in Romania, and only distantly related to the subtype F1 lineage circulating in South America. Original diversification of subtype F1 probably occurred within the DRC around the late 1950s. PMID:19386115

  3. Inhibition of replication of primary HIV-1 isolates in huPBL-NOD/Scid mice by antibodies from HIV-1 infected patients.

    PubMed

    Steyaert, Sophia; Heyndrickx, Leo; Verhoye, Lieven; Vermoesen, Tine; Donners, Helen; Fransen, Katrien; Van Wanzeele, Filip; Vandergucht, Beatrijs; Vanham, Guido; Leroux-Roels, Geert; Vanlandschoot, Peter

    2007-08-01

    Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model.

  4. Novel two-round phenotypic assay for protease inhibitor susceptibility testing of recombinant and primary HIV-1 isolates.

    PubMed

    Puertas, Maria C; Buzón, Maria J; Ballestero, Mònica; Van Den Eede, Peter; Clotet, Bonaventura; Prado, Julia G; Martinez-Picado, Javier

    2012-12-01

    Antiretroviral drug susceptibility tests facilitate therapeutic management of HIV-1-infected patients. Although genotyping systems are affordable, inaccuracy in the interpretation of complex mutational patterns may limit their usefulness. Currently available HIV-1 phenotypic assays are based on the generation of recombinant viruses in which the specific viral gene of interest, derived from a patient plasma sample, is cloned into a susceptible genetic viral backbone prior to in vitro drug susceptibility evaluation. Nevertheless, in the case of protease inhibitors, not only are mutations in the HIV-1 protease-coding region involved in resistance, but the role of Gag in drug susceptibility has also recently been reported. In order to avoid the inherent limitations resulting from partial cloning of the viral genome, we designed and evaluated a new experimental strategy to test the in vitro susceptibility of primary viral isolates to protease inhibitors. Our protocol, which is based on a two-round infection protocol using the reporter TZM-bl cell line, showed a good correlation with genotypic resistance prediction and with the Antivirogram phenotypic assay, in both protease-recombinant viruses and primary viral isolates. The protocol is suitable for any HIV-1 subtype and enables rapid in-house measurement of protease inhibitor susceptibility, thus making it possible to evaluate the concomitant effects of both patient-derived gag and protease-coding regions.

  5. Genetic analysis and natural polymorphisms in HIV-1 gp41 isolates from Maputo City, Mozambique.

    PubMed

    Ismael, Nália; Bila, Dulce; Mariani, Diana; Vubil, Adolfo; Mabunda, Nedio; Abreu, Celina; Jani, Ilesh; Tanuri, Amilcar

    2014-06-01

    Enfuvirtide was the first fusion inhibitor approved by the Food and Drug Administration (FDA) in 2003 for HIV-1 infection in treatment-experienced patient. It is the first approved antiviral agent to attack the HIV life cycle in its early stages. For HIV fusion to occur, the HR1 and HR2 domains in the gp41 region need to interact. Enfuvirtide is a synthetic peptide that corresponds to 36 amino acids of the HR2, which competitively binds to HR1 inhibiting the interaction with the HR2 domain thus preventing fusogenic conformation and inhibiting viral entry into host cells. Resistance to enfuvirtide is conferred by mutations occurring in the HR1 region involving residues 36-45. Mozambique, a sub-Saharan country, with an HIV prevalence of 11.5%, provides first line and second line antiretroviral therapy (ART)-based treatment. In poor resource settings such as Mozambique the lack of adequate infrastructures, the high costs of viral load tests, and the availability of salvage treatment have hindered the intended objective of monitoring HIV treatment, suggesting an important concern regarding the development of drug resistance. The general aim of this study was to evaluate naturally occurring polymorphisms and resistance-associated mutations in the gp41 region of HIV-1 isolates from Mozambique. The study included 78 patients naive to ARV treatment and 28 patients failing first line regimen recruited from Centro de Saúde Alto-Maé situated in Maputo. The gp41 gene from 103 patients was sequenced and resistance-associated mutations for enfuvirtide were screened. Subtype analysis revealed that 96% of the sequences were classified as subtype C, 2% as subtype G, 1% as subtype A1, and the other 1% as a mosaic form composed of A1/C. No enfuvirtide resistance-associated mutations in HR1 of gp41 were detected. The major polymorphisms in the HR1 were N42S, L54M, A67T, and V72I. This study suggests that this new class of antiviral drug may be effective as a salvage therapy in

  6. Genetic Analysis and Natural Polymorphisms in HIV-1 gp41 Isolates from Maputo City, Mozambique

    PubMed Central

    Ismael, Nália; Bila, Dulce; Mariani, Diana; Vubil, Adolfo; Mabunda, Nedio; Abreu, Celina; Jani, Ilesh

    2014-01-01

    Abstract Enfuvirtide was the first fusion inhibitor approved by the Food and Drug Administration (FDA) in 2003 for HIV-1 infection in treatment-experienced patient. It is the first approved antiviral agent to attack the HIV life cycle in its early stages. For HIV fusion to occur, the HR1 and HR2 domains in the gp41 region need to interact. Enfuvirtide is a synthetic peptide that corresponds to 36 amino acids of the HR2, which competitively binds to HR1 inhibiting the interaction with the HR2 domain thus preventing fusogenic conformation and inhibiting viral entry into host cells. Resistance to enfuvirtide is conferred by mutations occurring in the HR1 region involving residues 36–45. Mozambique, a sub-Saharan country, with an HIV prevalence of 11.5%, provides first line and second line antiretroviral therapy (ART)-based treatment. In poor resource settings such as Mozambique the lack of adequate infrastructures, the high costs of viral load tests, and the availability of salvage treatment have hindered the intended objective of monitoring HIV treatment, suggesting an important concern regarding the development of drug resistance. The general aim of this study was to evaluate naturally occurring polymorphisms and resistance-associated mutations in the gp41 region of HIV-1 isolates from Mozambique. The study included 78 patients naive to ARV treatment and 28 patients failing first line regimen recruited from Centro de Saúde Alto-Maé situated in Maputo. The gp41 gene from 103 patients was sequenced and resistance-associated mutations for enfuvirtide were screened. Subtype analysis revealed that 96% of the sequences were classified as subtype C, 2% as subtype G, 1% as subtype A1, and the other 1% as a mosaic form composed of A1/C. No enfuvirtide resistance-associated mutations in HR1 of gp41 were detected. The major polymorphisms in the HR1 were N42S, L54M, A67T, and V72I. This study suggests that this new class of antiviral drug may be effective as a salvage

  7. Functional stability of unliganded envelope glycoprotein spikes among isolates of human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Agrawal, Nitish; Leaman, Daniel P; Rowcliffe, Eric; Kinkead, Heather; Nohria, Raman; Akagi, Junya; Bauer, Katherine; Du, Sean X; Whalen, Robert G; Burton, Dennis R; Zwick, Michael B

    2011-01-01

    The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T(90) values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T(90) (p = 0.029), though two 'outliers' were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1(ADA) was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1(JR-CSF). Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.

  8. Functional Stability of Unliganded Envelope Glycoprotein Spikes among Isolates of Human Immunodeficiency Virus Type 1 (HIV-1)

    PubMed Central

    Kinkead, Heather; Nohria, Raman; Akagi, Junya; Bauer, Katherine; Du, Sean X.; Whalen, Robert G.; Burton, Dennis R.; Zwick, Michael B.

    2011-01-01

    The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T90 (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines. PMID:21738637

  9. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    SciTech Connect

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-08-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

  10. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    PubMed Central

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-01-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO 140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo. PMID:18519143

  11. Entry inhibitor-based microbicides are active in vitro against HIV-1 isolates from multiple genetic subtypes

    SciTech Connect

    Ketas, Thomas J.; Schader, Susan M.; Zurita, Juan; Teo, Esther; Polonis, Victoria; Lu Min; Klasse, Per Johan; Moore, John P. . E-mail: jpm2003@med.cornell.edu

    2007-08-01

    Inhibitors of viral entry are under consideration as topical microbicides to prevent HIV-1 sexual transmission. Small molecules targeting HIV-1 gp120 (BMS-378806) or CCR5 (CMPD167), and a peptide fusion inhibitor (C52L), each blocks vaginal infection of macaques by a SHIV. A microbicide, however, must be active against multiple HIV-1 variants. We therefore tested BMS-C (a BMS-378806 derivative), CMPD167, C52L and the CXCR4 ligand AMD3465, alone and in combination, against 25 primary R5, 12 X4 and 7 R5X4 isolates from subtypes A-G. At high concentrations (0.1-1 {mu}M), the replication of most R5 isolates in human donor lymphocytes was inhibited by > 90%. At lower concentrations, double and triple combinations were more effective than individual inhibitors. Similar results were obtained with X4 viruses when AMD3465 was substituted for CMPD167. The R5X4 viruses were inhibited by combining AMD3465 with CMPD167, or by the coreceptor-independent compounds. Thus, combining entry inhibitors may improve microbicide effectiveness.

  12. Neutralization resistant HIV-1 primary isolates from antiretroviral naïve chronically infected children in India.

    PubMed

    Makhdoomi, Muzamil Ashraf; Singh, Deepti; Nair Pananghat, Ambili; Lodha, Rakesh; Kabra, Sushil Kumar; Luthra, Kalpana

    2016-12-01

    Anti-HIV-1 broadly neutralizing antibodies (bnAbs) have been extensively tested against pesudoviruses of diverse strains. We generated and characterized HIV-1 primary isolates from antiretroviral naïve infected Indian children, and determined their susceptibility to known NAbs. All the 8 isolates belonged to subtype-C and were R5 tropic. Majority of these viruses were resistant to neutralization by NAbs, suggesting that the bnAbs, known to efficiently neutralize pseudoviruses (adult and pediatric) of different strains, are less effective against pediatric primary isolates. Interestingly, AIIMS_329 isolate displayed high susceptibility to neutralization by PG9 and PG16bnAbs, with IC50 titer of 1.3 and 0.97μg/ml, suggesting exposure of this epitope on this virus. All isolates except AIIMS_506 were neutralized by contemporaneous plasma antibodies. Our findings suggest that primary isolates, due to close resemblance to viruses in natural infection, should be used to evaluate NAbs as effective vaccine candidates in both children and adults. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. 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.

  14. In vitro anti-HIV-1 activities of kaempferol and kaempferol-7-O-glucoside isolated from Securigera securidaca.

    PubMed

    Behbahani, M; Sayedipour, S; Pourazar, A; Shanehsazzadeh, M

    2014-01-01

    Previously, we reported that the kaempferol and kaempferol-7-O-glucoside isolated from Securigera securidaca showed potent anti-HSV activity. In the present study the anti-HIV-1 activities of kaempferol and kaempferol-7-O-glucoside are investigated at different concentrations (100, 50, 25 and 10 μg/ml) using HIV-1 p24 Antigen kit. Real-time Polymerase chain reaction (RT-PCR) assay was also used for quantification of full range of virus load observed in treated and untreated cells. According to the results of RT- PCR, tested compounds at a concentration of 100 μg/ml exerted potent inhibitory effect. Time of drug addition experiments demonstrated that these compounds exerted their inhibitory effects on the early stage of HIV infection. The results also showed potent anti-HIV-1 reverse transcriptase activity. Antiviral activity of kaempferol-7-O-glucoside was more pronounced than that of kaempferol. These findings demonstrate that kaempferol-7-O-glucoside could be considered as a new potential drug candidate for the treatment of HIV infection which requires further assessments.

  15. Isolation and characterization of HIV-1 envelope glycoprotein specific B cell from immortalized human naïve B cell library.

    PubMed

    Sun, Zehua; Lu, Shiqiang; Yang, Zheng; Li, Jingjing; Zhang, Meiyun

    2017-01-10

    With the recent development of single B cell cloning techniques, an increasing number of HIV-1-specific broadly neutralizing antibodies (bNAbs) have been isolated since 2009. However, knowledge regarding HIV-1-specific B cells in vivo is limited. In this study, an HIV-1-specific B cell line has been established using healthy PBMC donors by the highly efficient EBV transformation method to generate immortalized human naïve B cell libraries. The enrichment of HIV-1 envelope-specific B cells was observed after four rounds of cell panning with the HIV-1 envelope glycoprotein. An HIV-1 envelope-specific stable B cell line (LCL-P4) was generated. Although this cell line acquired a lymphoblastic phenotype, no expression was observed for activation-induced cytidine deaminase (AID), an enzyme responsible for initiating somatic hypermutation and class switch recombination in B cells. This study describes a method that enables fast isolation of HIV-1-specific B cells, and this approach may extend to isolating other B cell-specific antigens for further experiments.

  16. Similar Replicative Fitness Is Shared by the Subtype B and Unique BF Recombinant HIV-1 Isolates that Dominate the Epidemic in Argentina

    PubMed Central

    Rubio, Andrea E.; Abraha, Awet; Carpenter, Crystal A.; Troyer, Ryan M.; Reyes-Rodríguez, Ángel L.; Salomon, Horacio; Arts, Eric J.; Tebit, Denis M.

    2014-01-01

    The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (n = 9, 32.1%), C (n = 1, 3.6%), and CRFs (n = 18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous “CRF” B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events. PMID:24727861

  17. The replicative restriction of lymphocytotropic isolates of HIV-1 in macrophages is overcome by TGF-beta.

    PubMed

    Lazdins, J K; Klimkait, T; Woods-Cook, K; Walker, M; Alteri, E; Cox, D; Cerletti, N; Shipman, R; Bilbe, G; McMaster, G

    1992-04-01

    In vitro exposure of human blood monocyte-derived macrophages to T-cell tropic human immunodeficiency virus (HIV) isolates fails to establish a productive viral infection. Several studies have shown that such preferential HIV-1 replication in T cells or in mononuclear phagocytes (HIV tropism) may be determined by distinct viral characteristics. In the present study it was demonstrated that transforming growth factor-beta (TGF-beta), a factor known to be produced by platelets, macrophages, and other cells present at a wound site, can act as a mediator in overcoming the lymphocytotropic restriction of several well-characterized viral isolates of HIV-1 (i.e., LAV, Z84, pLAI, NY5). Macrophages infected with these isolates show cytopathic changes comparable to those seen upon infection with the monocytotropic isolate ADA. To achieve this effect with TGF-beta, the factor must be present after the infection period. The emerging virus retains its original cellular tropism. Based on these observations the authors propose a role for TGF-beta in the establishment and progression of HIV infection and disease.

  18. High prevalence of secondary resistance mutations in Venezuelan HIV-1 isolates.

    PubMed

    Dieudonne, Mariacarolina; Garzaro, Domingo; Torres, Jaime; Naranjo, Laura; Suárez, José Antonio; Castro, Julio; Martínez, Nahir; Castro, Erika; Berrueta, Lisbeth; Salmen, Siham; Devesa, Marisol; Rangel, Héctor R; Pujol, Flor Helene

    2006-03-01

    The genetic variability was studied in HIV-1 from Venezuelan patients with and without treatment, in order to evaluate the presence of polymorphisms and drug resistance mutations. Proviral DNA from peripheral blood mononuclear cells or viral RNA from plasma was extracted from the blood of 30 patients. Two regions from the polymerase gene, protease (Pr) and reverse transcriptase (RT) and one genomic fragment from the envelope (Env) gene were amplified and sequenced. All HIV-1 samples analyzed were classified as subtype B, without evidence of recombination. Although no primary protease mutations were detected, a high frequency of secondary mutations (86%, 19/22), associated to restoration of viral replicative fitness, was observed in strains circulating both in treated and non-treated patients. Resistance mutations to nucleoside RT inhibitors (NRTI) and non-nucleoside RT inhibitors (NNRTI) were detected in 35% (6/17) and 12% (2/17) of the viruses circulating in treated patients, respectively. Resistance mutations were also present in the virus infecting one antiretroviral naive individual (7.7%), suggesting that local screening for resistant mutation in naive patient might be important to minimize therapy failure. Future studies are warranted to assess the role of secondary mutation in the success of viral infection.

  19. Exploiting the Anti-HIV-1 Activity of Acyclovir: Suppression of Primary and Drug-Resistant HIV Isolates and Potentiation of the Activity by Ribavirin

    PubMed Central

    Vanpouille, Christophe; Lisco, Andrea; Introini, Andrea; Grivel, Jean-Charles; Munawwar, Arshi; Merbah, Melanie; Schinazi, Raymond F.; Derudas, Marco; McGuigan, Christopher; Balzarini, Jan

    2012-01-01

    Multiple clinical trials have demonstrated that herpes simplex virus 2 (HSV-2) suppressive therapy using acyclovir (ACV) or valacyclovir in HIV-1/HSV-2-infected persons increased the patient's survival and decreased the HIV-1 load. It has been shown that the incorporation of ACV-monophosphate into the nascent DNA chain instead of dGMP results in the termination of viral DNA elongation and directly inhibits laboratory strains of HIV-1. We evaluated here the anti-HIV activity of ACV against primary HIV-1 isolates of different clades and coreceptor specificity and against viral isolates resistant to currently used drugs, including zidovudine, lamivudine, nevirapine, a combination of nucleoside reverse transcriptase inhibitors (NRTIs), a fusion inhibitor, and two protease inhibitors. We found that, at clinically relevant concentrations, ACV inhibits the replication of these isolates in human tissues infected ex vivo. Moreover, addition of ribavirin, an antiviral capable of depleting the pool of intracellular dGTP, potentiated the ACV-mediated HIV-1 suppression. These data warrant further clinical investigations of the benefits of using inexpensive and safe ACV alone or in combination with other drugs against HIV-1, especially to complement or delay highly active antiretroviral therapy (HAART) initiation in low-resource settings. PMID:22314523

  20. Evolution of HIV-1 isolates that use a novel Vif-independent mechanism to resist restriction by human APOBEC3G.

    PubMed

    Haché, Guylaine; Shindo, Keisuke; Albin, John S; Harris, Reuben S

    2008-06-03

    The human APOBEC3G protein restricts the replication of Vif-deficient HIV-1 by deaminating nascent viral cDNA cytosines to uracils, leading to viral genomic strand G-to-A hypermutations. However, the HIV-1 Vif protein triggers APOBEC3G degradation, which helps to explain why this innate defense does not protect patients. The APOBEC3G-Vif interaction is a promising therapeutic target, but the benefit of the enabling of HIV-1 restriction in patients is unlikely to be known until Vif antagonists are developed. As a necessary prelude to such studies, cell-based HIV-1 evolution experiments were done to find out whether APOBEC3G can provide a long-term block to Vif-deficient virus replication and, if so, whether HIV-1 variants that resist restriction would emerge. APOBEC3G-expressing T cells were infected with Vif-deficient HIV-1. Virus infectivity was suppressed in 45/48 cultures for more than five weeks, but replication was eventually detected in three cultures. Virus-growth characteristics and sequencing demonstrated that these isolates were still Vif-deficient and that in fact, these viruses had acquired a promoter mutation and a Vpr null mutation. Resistance occurred by a novel tolerance mechanism in which the resistant viruses packaged less APOBEC3G and accumulated fewer hypermutations. These data support the development of antiretrovirals that antagonize Vif and thereby enable endogenous APOBEC3G to suppress HIV-1 replication.

  1. Rescue of HIV-1 Release by Targeting Widely Divergent NEDD4-Type Ubiquitin Ligases and Isolated Catalytic HECT Domains to Gag

    PubMed Central

    Weiss, Eric R.; Popova, Elena; Yamanaka, Hikaru; Kim, Hyung Cheol; Huibregtse, Jon M.; Göttlinger, Heinrich

    2010-01-01

    Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release. PMID:20862313

  2. Rescue of HIV-1 release by targeting widely divergent NEDD4-type ubiquitin ligases and isolated catalytic HECT domains to Gag.

    PubMed

    Weiss, Eric R; Popova, Elena; Yamanaka, Hikaru; Kim, Hyung Cheol; Huibregtse, Jon M; Göttlinger, Heinrich

    2010-09-16

    Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release.

  3. CD4 and MHC class I down-modulation activities of nef alleles from brain- and lymphoid tissue-derived primary HIV-1 isolates

    PubMed Central

    Gray, Lachlan R.; Gabuzda, Dana; Cowley, Daniel; Ellett, Anne; Chiavaroli, Lisa; Wesselingh, Steven L.; Churchill, Melissa J.; Gorry, Paul R.

    2015-01-01

    HIV-1 nef undergoes adaptive evolution in the CNS, reflecting altered requirements for HIV-1 replication in macrophages/microglia and brain-specific immune selection pressures. The role of Nef in HIV-1 neurotropism and the pathogenesis of HIV-associated dementia (HAD) is unclear. In this study, we characterized 82 nef alleles cloned from brain, CSF, spinal cord and blood/lymphoid tissue-derived HIV-1 isolates from 7 subjects with HAD. CNS isolate-derived nef alleles were genetically compartmentalized and had reduced sequence diversity compared to those from lymphoid tissue isolates. Defective nef alleles predominated in a brain-derived isolate from one of the 7 subjects (MACS2-br). The ability of Nef to down-modulate CD4 and MHC class 1 (MHC-1) was generally conserved among nef alleles from both CNS and lymphoid tissues. However, the potency of CD4 and MHC-1 down-modulation was variable, which was associated with sequence alterations known to influence these Nef functions. These results suggest that CD4 and MHC-1 down-modulation are highly conserved functions among nef alleles from CNS- and lymphoid tissue-derived HIV-1 isolates that may contribute to viral replication and escape from immune surveillance in the CNS. PMID:21165790

  4. Clade C HIV-1 isolates circulating in Southern Africa exhibit a greater frequency of dicysteine motif-containing Tat variants than those in Southeast Asia and cause increased neurovirulence

    PubMed Central

    2013-01-01

    Background HIV-1 Clade C (Subtype C; HIV-1C) is responsible for greater than 50% of infections worldwide. Unlike clade B HIV-1 (Subtype B; HIV-1B), which is known to cause HIV associated dementia (HAD) in approximately 15% to 30% of the infected individuals, HIV-1C has been linked with lower prevalence of HAD (0 to 6%) in India and Ethiopia. However, recent studies report a higher prevalence of HAD in South Africa, Zambia and Botswana, where HIV-1C infections predominate. Therefore, we examined whether Southern African HIV-1C is genetically distinct and investigated its neurovirulence. HIV-1 Tat protein is a viral determinant of neurocognitive dysfunction. Therefore, we focused our study on the variations seen in tat gene and its contribution to HIV associated neuropathogenesis. Results A phylogenetic analysis of tat sequences of Southern African (South Africa and Zambia) HIV isolates with those from the geographically distant Southeast Asian (India and Bangladesh) isolates revealed that Southern African tat sequences are distinct from Southeast Asian isolates. The proportion of HIV − 1C variants with an intact dicysteine motif in Tat protein (C30C31) was significantly higher in the Southern African countries compared to Southeast Asia and broadly paralleled the high incidence of HAD in these countries. Neuropathogenic potential of a Southern African HIV-1C isolate (from Zambia; HIV-1C1084i), a HIV-1C isolate (HIV-1IndieC1) from Southeast Asia and a HIV-1B isolate (HIV-1ADA) from the US were tested using in vitro assays to measure neurovirulence and a SCID mouse HIV encephalitis model to measure cognitive deficits. In vitro assays revealed that the Southern African isolate, HIV-1C1084i exhibited increased monocyte chemotaxis and greater neurotoxicity compared to Southeast Asian HIV-1C. In neurocognitive tests, SCID mice injected with MDM infected with Southern African HIV-1C1084i showed greater cognitive dysfunction similar to HIV-1B but much higher than

  5. In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease.

    PubMed

    Partaledis, J A; Yamaguchi, K; Tisdale, M; Blair, E E; Falcione, C; Maschera, B; Myers, R E; Pazhanisamy, S; Futer, O; Cullinan, A B

    1995-09-01

    Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.

  6. In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease.

    PubMed Central

    Partaledis, J A; Yamaguchi, K; Tisdale, M; Blair, E E; Falcione, C; Maschera, B; Myers, R E; Pazhanisamy, S; Futer, O; Cullinan, A B

    1995-01-01

    Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially. PMID:7636964

  7. In–Depth Characterization of Viral Isolates from Plasma and Cells Compared with Plasma Circulating Quasispecies in Early HIV-1 Infection

    PubMed Central

    Erkizia, Itziar; Pino, Maria; Pou, Christian; Paredes, Roger; Clotet, Bonaventura; Martinez-Picado, Javier; Prado, Julia G.

    2012-01-01

    Background The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies. Methodology/Principal Findings We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA. Conclusion This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and

  8. Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

    NASA Technical Reports Server (NTRS)

    Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

    2005-01-01

    CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

  9. Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

    NASA Technical Reports Server (NTRS)

    Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

    2005-01-01

    CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

  10. Evolution of CCR5 Antagonist Resistance in an HIV-1 Subtype C Clinical Isolate

    PubMed Central

    Henrich, Timothy J.; Tsibris, Athe M.N.; Lewine, Nicolas R.P.; Konstantinidis, Ioannis; Leopold, Kay E.; Sagar, Manish; Kuritzkes, Daniel R.

    2011-01-01

    Objectives We previously reported vicriviroc (VCV) resistance in an HIV-infected subject and used deep sequencing and clonal analyses to track the evolution of V3 sequence forms over 28 weeks of therapy. Here, we test the contribution of gp120 mutations to CCR5 antagonist resistance and investigate why certain minority V3 variants emerged as the dominant species under drug pressure. Methods 19 site-directed HIV-1 mutants were generated that contained gp120 VCV-resistance mutations. Viral sensitivities to VCV, maraviroc, TAK-779 and HGS004 were determined. Results Three patterns of susceptibilities were observed: sigmoid inhibition curves with IC50s similar to pre-treatment virus (07J-week 0 [W0]), single mutants with decreased IC50s compared to 07J-W0, and mutants that contained ≥5 of 7 VCV-resistance mutations with flattened inhibition curves and decreased or negative percent maximal inhibition. Substitutions such as S306P, which sensitized virus to CCR5 antagonists when present as single mutations, were not detected in the baseline virus population but were necessary for maximal resistance when incorporated into V3 backbones that included pre-existing VCV resistance mutations. Conclusion CCR5 antagonist resistance was reproduced only when a majority of V3 mutations were present. Minority V3 loop variants may serve as a scaffold upon which additional mutations lead to complete VCV resistance. PMID:20856130

  11. Infection of human and non-human cells by a highly fusogenic primary CD4-independent HIV-1 isolate with a truncated envelope cytoplasmic tail

    SciTech Connect

    Saha, Kunal . E-mail: sahak@pediatrics.ohio-state.edu; Yan Hui; Nelson, Julie A.E.; Zerhouni-Layachi, Bouchra

    2005-06-20

    Truncation of the envelope cytoplasmic tail has enabled FIV, SIV, and some laboratory HIV-1 strains to acquire broader cellular tropism and enhanced fusogenicity. Here we have characterized a primary CD4-independent HIV-1 isolate (92UG046-T8) with a truncated cytoplasmic tail that was able to infect and induce syncytia in primary lymphocytes from human, chimpanzee, and monkey, as well as CD4-negative cell lines from human and monkey. Increased syncytia were also noticeable with 293 cells expressing the cloned envelope from the 92UG046-T8 isolate suggesting envelope-mediated cellular fusion. Except pooled serum from HIV-1-infected individuals, monoclonal anti-envelope antibodies or antibodies/antagonists against CD4, CXCR4, and CCR5 were not able to prevent infection by the 92UG046-T8 isolate. This is the first report showing a primary HIV-1 variant with truncated cytoplasmic tail which is highly fusogenic and can infect a broad range of cells from human and non-human origins. In vivo evolution of similar HIV-1 mutants may have important implications in AIDS pathogenesis.

  12. 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.

  13. The search for a T cell line for testing novel antiviral strategies against HIV-1 isolates of diverse receptor tropism and subtype origin.

    PubMed

    Herrera-Carrillo, Elena; Paxton, William A; Berkhout, Ben

    2014-07-01

    The world-wide HIV epidemic is characterized by increasing genetic diversity with multiple viral subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Antiretroviral drug design and basic virology studies have largely focused on HIV-1 subtype B. There have been few direct comparisons by subtype, perhaps due to the lack of uniform and standardized culture systems for the in vitro propagation of diverse HIV-1 subtypes. Although peripheral blood mononuclear cells (PBMCs) are major targets and reservoirs of HIV, PBMCs culturing is relatively difficult and not always reproducible. In addition, long-term experiments cannot be performed because PBMCs are short-lived cells. We faced these problems during the in vitro testing of an experimental RNA interference (RNAi) based gene therapy. Therefore, many T cell lines that support HIV-1 infection were tested and compared for replication of HIV-1 isolates, including viruses that use different receptors and diverse subtypes. The PM1 T cell line was comparable to PBMCs for culturing of any of the HIV-1 strains and subtypes. The advantage of PM1 cells in long-term cultures for testing the safety and efficacy of an RNAi-based gene therapy was demonstrated. PM1 may thus provide a valuable research tool for studying new anti-HIV therapies. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; De Spiegelaere, Ward

    2014-01-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. PMID:25502524

  15. Modulation of NKG2D-mediated cytotoxic functions of natural killer cells by viral protein R from HIV-1 primary isolates.

    PubMed

    Pham, Tram N Q; Richard, Jonathan; Gerard, Francine C A; Power, Christopher; Cohen, Éric A

    2011-12-01

    HIV-1 viral protein R (Vpr) from laboratory-adapted virus strains activates the DNA damage/stress sensor ATR kinase and induces cell cycle arrest at the G(2)/M phase through a process that requires Vpr to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex. Activation of this DNA damage/stress checkpoint in G(2) by Vpr was shown to modulate NKG2D-dependent NK cell effector functions via enhancing expression of NKG2D ligands, notably ULBP2. However, it is unknown whether Vpr from HIV-1 primary isolates (groups M, N, O, and P) could modulate NKG2D-mediated cytotoxic functions of NK cells. Here, we report that Vpr from most HIV-1 primary isolates can upregulate ULBP2 expression and induce NKG2D-dependent NK cell killing. Importantly, these activities were always accompanied by an active G(2) cell cycle arrest function. Interestingly, Vpr variants from group P and a clade D isolate of group M were defective at enhancing NKG2D-mediated NK cell lysis owing to their inability to augment ULBP2 expression. However, distinct mechanisms were responsible for their failure to do so. While Vpr from group P was deficient in its ability to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex, the Vpr variant from group D was unable to properly localize to the nucleus, underlining the importance of these biological properties in Vpr function. In conclusion, the ability of Vpr from HIV-1 primary isolates to regulate NK cell effector function underscores the importance of this HIV-1 accessory protein in the modulation of the host's innate immune responses.

  16. Prothymosin α Variants Isolated From CD8+ T Cells and Cervicovaginal Fluid Suppress HIV-1 Replication Through Type I Interferon Induction

    PubMed Central

    Teixeira, Avelino; Yen, Benjamin; Gusella, Gabriele Luca; Thomas, Albert G.; Mullen, Michael P.; Aberg, Judith; Chen, Xintong; Hoshida, Yujin; van Bakel, Harm; Schadt, Eric; Basler, Christopher F.; García-Sastre, Adolfo; Mosoian, Arevik

    2015-01-01

    Soluble factors from CD8+ T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8+ T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8+ T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance. PMID:25404520

  17. Prothymosin α variants isolated from CD8+ T cells and cervicovaginal fluid suppress HIV-1 replication through type I interferon induction.

    PubMed

    Teixeira, Avelino; Yen, Benjamin; Gusella, Gabriele Luca; Thomas, Albert G; Mullen, Michael P; Aberg, Judith; Chen, Xintong; Hoshida, Yujin; van Bakel, Harm; Schadt, Eric; Basler, Christopher F; García-Sastre, Adolfo; Mosoian, Arevik

    2015-05-01

    Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance.

  18. Comparative evaluation of trimeric envelope glycoproteins derived from subtype C and B HIV-1 R5 isolates

    SciTech Connect

    Srivastava, Indresh K. Kan, Elaine; Sun Yide; Sharma, Victoria A.; Cisto, Jimna; Burke, Brian; Lian Ying; Hilt, Susan; Biron, Zohar; Hartog, Karin; Stamatatos, Leonidas; Cheng, R. Holland; Ulmer, Jeffrey B.; Barnett, Susan W.

    2008-03-15

    We previously reported that an envelope (Env) glycoprotein immunogen (o-gp140{delta}V2SF162) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162 partially protected vaccinated rhesus macaques against pathogenic SHIV{sub SF162P4} virus. Extending our studies to subtype C isolate TV1, we have purified o-gp140{delta}V2TV1 (subtype C {delta}V2 trimer) to homogeneity, performed glycosylation analysis, and determined its ability to bind CD4, as well as a panel of well-characterized neutralizing monoclonal antibodies (mAb). In general, critical epitopes are preserved on the subtype C {delta}V2 trimer; however, we did not observe significant binding for the b12 mAb. The molecular mass of subtype C {delta}V2 trimer was found to be 450 kDa, and the hydrodynamic radius was found to be 10.87 nm. Our data suggest that subtype C {delta}V2 trimer binds to CD4 with an affinity comparable to o-gp140{delta}V2SF162 (subtype B {delta}V2 trimer). Using isothermal titration calorimetric (ITC) analysis, we demonstrated that all three CD4 binding sites (CD4-BS) in both subtype C and B trimers are exposed and accessible. However, compared to subtype B trimer, the three CD4-BS in subtype C trimer have different affinities for CD4, suggesting a cooperativity of CD4 binding in subtype C trimer but not in subtype B trimer. Negative staining electron microscopy of the subtype C {delta}V2 trimer has demonstrated that it is in fact a trimer. These results highlight the importance of studying subtype C Env, and also of developing appropriate subtype C-specific reagents that may be used for better immunological characterization of subtype C Env for developing an AIDS vaccine.

  19. Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

    PubMed

    Singh, Deepali; Boeras, Ioana; Singh, Gatikrushna; Boris-Lawrie, Kathleen

    2016-01-01

    All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.

  20. HLA-B*57 Elite Suppressor and Chronic Progressor HIV-1 Isolates Replicate Vigorously and Cause CD4+ T Cell Depletion in Humanized BLT Mice

    PubMed Central

    Salgado, Maria; Swanson, Michael D.; Pohlmeyer, Christopher W.; Buckheit, Robert W.; Wu, Jin; Archin, Nancie M.; Williams, Thomas M.; Margolis, David M.; Siliciano, Robert F.

    2014-01-01

    ABSTRACT Elite controllers or suppressors (ES) are HIV-1-infected patients who maintain undetectable viral loads without antiretroviral therapy. The mechanism of control remains unclear, but the HLA-B*57 allele is overrepresented in cohorts of these patients. However, many HLA-B*57 patients develop progressive disease, and some studies have suggested that infection with defective viruses may be the cause of the lack of high levels of virus replication and disease progression in ES. We therefore performed a comprehensive comparative in vivo and in vitro characterization of viruses isolated from well-defined ES. For this purpose, we first performed full-genome sequence analysis and in vitro fitness assays on replication-competent isolates from HLA-B*57 ES and HLA-B*57 chronic progressors (CPs). Under our experimental conditions, we found that isolates from ES and CPs can replicate in vitro. However, since inherently these assays involve the use of unnaturally in vitro-activated cells, we also investigated the replication competence and pathogenic potential of these HIV isolates in vivo using humanized BLT mice. The results from these analyses demonstrate that virus isolates from ES are fully replication competent in vivo and can induce peripheral and systemic CD4 T cell depletion. These results provide the first direct in vivo evidence that viral fitness does not likely determine clinical outcome in HLA-B*57 patients and that elite suppressors can control replication-competent, fully pathogenic viruses. A better understanding of the immunological bases of viral suppression in ES will serve to inform novel approaches to preventive and therapeutic HIV vaccine design. IMPORTANCE Elite suppressors are HIV-1-infected patients who have undetectable levels of viremia despite not being on antiviral drugs. One of the most fundamental questions about this phenomenon involves the mechanism of control. To address this question, we isolated virus from elite suppressors and from

  1. HLA-B*57 elite suppressor and chronic progressor HIV-1 isolates replicate vigorously and cause CD4+ T cell depletion in humanized BLT mice.

    PubMed

    Salgado, Maria; Swanson, Michael D; Pohlmeyer, Christopher W; Buckheit, Robert W; Wu, Jin; Archin, Nancie M; Williams, Thomas M; Margolis, David M; Siliciano, Robert F; Garcia, J Victor; Blankson, Joel N

    2014-03-01

    Elite controllers or suppressors (ES) are HIV-1-infected patients who maintain undetectable viral loads without antiretroviral therapy. The mechanism of control remains unclear, but the HLA-B*57 allele is overrepresented in cohorts of these patients. However, many HLA-B*57 patients develop progressive disease, and some studies have suggested that infection with defective viruses may be the cause of the lack of high levels of virus replication and disease progression in ES. We therefore performed a comprehensive comparative in vivo and in vitro characterization of viruses isolated from well-defined ES. For this purpose, we first performed full-genome sequence analysis and in vitro fitness assays on replication-competent isolates from HLA-B*57 ES and HLA-B*57 chronic progressors (CPs). Under our experimental conditions, we found that isolates from ES and CPs can replicate in vitro. However, since inherently these assays involve the use of unnaturally in vitro-activated cells, we also investigated the replication competence and pathogenic potential of these HIV isolates in vivo using humanized BLT mice. The results from these analyses demonstrate that virus isolates from ES are fully replication competent in vivo and can induce peripheral and systemic CD4 T cell depletion. These results provide the first direct in vivo evidence that viral fitness does not likely determine clinical outcome in HLA-B*57 patients and that elite suppressors can control replication-competent, fully pathogenic viruses. A better understanding of the immunological bases of viral suppression in ES will serve to inform novel approaches to preventive and therapeutic HIV vaccine design. Elite suppressors are HIV-1-infected patients who have undetectable levels of viremia despite not being on antiviral drugs. One of the most fundamental questions about this phenomenon involves the mechanism of control. To address this question, we isolated virus from elite suppressors and from HIV-1-infected

  2. Evaluation of anti-HIV-1 activity of a new iridoid glycoside isolated from Avicenna marina, in vitro.

    PubMed

    Behbahani, Mandana

    2014-11-01

    This study was carried out to check the efficacy of methanol seed extract of Avicenna marina and its column chromatographic fractions on Peripheral Blood Mono nuclear Cells (PBMCs) toxicity and HIV-1 replication. The anti-HIV-1 activities of crude methanol extract and its fractions were performed by use of real-time polymerase chain reaction (PCR) assay and HIV-1 p24 antigen kit. A time of drug addiction approach was also done to identify target of anti-HIV compound. The activity of the extracts on CD4, CD3, CD19 and CD45 expression in lymphocytes population was performed by use of flow cytometry. The most active anti-HIV agent was detected by spectroscopic analysis as 2'-O-(4-methoxycinnamoyl) mussaenosidic acid. The apparent effective concentrations for 50% virus replication (EC50) of methanol extract and iridoid glycoside were 45 and 0.1 μg/ml respectively. The iridoid glycoside also did not have any observable effect on the proportion of CD4, CD3, CD19 and CD45 cells or on the intensity of their expressions on PBMCs. In addition, the expression level of C-C chemokine receptor type 5 (CCR5) and chemokine receptor type 4 (CXCR4) on CD4(+) T cells were decreased in cells treated with this iridoid glycoside. The reduction of these two HIV coreceptors and the result of time of addition study demonstrated that this iridoid glycoside restricts HIV-1 replication on the early stage of HIV infection. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Seroprevalence and molecular epidemiology of HTLV-1 isolates from HIV-1 co-infected women in Feira de Santana, Bahia, Brazil.

    PubMed

    de Almeida Rego, Filipe Ferreira; Mota-Miranda, Aline; de Souza Santos, Edson; Galvão-Castro, Bernardo; Alcantara, Luiz Carlos

    2010-12-01

    HTLV-1/HIV-1 co-infection is associated with severe clinical manifestations, marked immunodeficiency, and opportunistic pathogenic infections, as well as risk behavior. Salvador, the capital of the State of Bahia, Brazil, has the highest HTLV-1 prevalence (1.74%) found in Brazil. Few studies exist which describe this co-infection found in Salvador and its surrounding areas, much less investigate how these viruses circulate or assess the relationship between them. To describe the epidemiological and molecular features of HTLV in HIV co-infected women. To investigate the prevalence of HTLV/HIV co-infection in surrounding areas, as well as the molecular epidemiology of HTLV, a cross sectional study was carried out involving 107 women infected with HIV-1 from the STD/HIV/AIDS Reference Center located in the neighboring City of Feira de Santana. Patient samples were submitted to ELISA, and HTLV infection was confirmed using Western Blot and Polymerase Chain Reaction (PCR). Phylogenetic analysis using Neighbor-Joining (NJ) and Maximum Likelihood (ML) was performed on HTLV LTR sequences in order to gain further insights about molecular epidemiology and the origins of this virus in Bahia. Four out of five reactive samples were confirmed to be infected with HTLV-1, and one with HTLV-2. The seroprevalence of HTLV among HIV-1 co-infected women was 4.7%. Phylogenetic analysis of the LTR region from four HTLV-1 sequences showed that all isolates were clustered into the main Latin American group within the Transcontinental subgroup of the Cosmopolitan subtype. The HTLV-2 sequence was classified as the HTLV-2c subtype. It was also observed that four HTLV/HIV-1 co-infected women exhibited risk behavior with two having parenteral exposure, while another two were sex workers. This article describes the characteristics of co-infected patients. This co-infection is known to be severe and further studies should be conducted to confirm the suggestion that HTLV-1 is spreading from

  4. UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate.

    PubMed

    Foucault, Marine; Mayol, Katia; Receveur-Bréchot, Véronique; Bussat, Marie-Claire; Klinguer-Hamour, Christine; Verrier, Bernard; Beck, Alain; Haser, Richard; Gouet, Patrice; Guillon, Christophe

    2010-05-01

    The 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt-Tat(133) or STLA-Tat(133) underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat(133) proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function.

  5. Comparative clonal analysis of human immunodeficiency virus type 1 (HIV- 1)-specific CD4+ and CD8+ cytolytic T lymphocytes isolated from seronegative humans immunized with candidate HIV-1 vaccines

    PubMed Central

    1992-01-01

    The lysis of infected host cells by virus-specific cytolytic T lymphocytes (CTL) is an important factor in host resistance to viral infection. An optimal vaccine against human immunodeficiency virus type 1 (HIV-1) would elicit virus-specific CTL as well as neutralizing antibodies. The induction by a vaccine of HIV-1-specific CD8+ CTL in humans has not been previously reported. In this study, CTL responses were evaluated in HIV-1-seronegative human volunteers participating in a phase I acquired immune deficiency syndrome (AIDS) vaccine trial involving a novel vaccine regimen. Volunteers received an initial immunization with a live recombinant vaccinia virus vector carrying the HIV-1 env gene and a subsequent boost with purified env protein. An exceptionally strong env-specific CTL response was detected in one of two vaccine recipients, while modest but significant env-specific CTL activity was present in the second vaccinee. Cloning of the responding CTL gave both CD4+ and CD8+ env-specific CTL clones, permitting a detailed comparison of critical functional properties of these two types of CTL. In particular, the potential antiviral effects of these CTL were evaluated in an in vitro system involving HIV-1 infection of cultures of normal autologous CD4+ lymphoblasts. At extremely low effector-to-target ratios, vaccine-induced CD8+ CTL clones lysed productively infected cells present within these cultures. When tested for lytic activity against target cells expressing the HIV-1 env gene, CD8+ CTL were 3-10-fold more active on a per cell basis than CD4+ CTL. However, when tested against autologous CD4+ lymphoblasts acutely infected with HIV-1, CD4+ clones lysed a much higher fraction of the target cell population than did CD8+ CTL. CD4+ CTL were shown to recognize not only the infected cells within these acutely infected cultures but also noninfected CD4+ T cells that had passively taken up gp120 shed from infected cells and/or free virions. These results were

  6. HIV-1 replication.

    PubMed

    Freed, E O

    2001-11-01

    surface (SU) Env glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41. gp120 contains the determinants that interact with receptor and coreceptor, while gp41 not only anchors the gp120/gp41 complex in the membrane (Fig. 2), but also contains domains that are critical for catalyzing the membrane fusion reaction between viral and host lipid bilayers during virus entry. Comparison of env sequences from a large number of virus isolates revealed that gp120 is organized into five conserved regions (C1-C5) and five highly variable domains (V1-V5). The variable regions tend to be located in disulfide-linked loops. gp41 is composed of three major domains: the ectodomain (which contains determinants essential for membrane fusion), the transmembrane anchor sequence, and the cytoplasmic tail. In addition to the gag, pol, and env genes, HIV-1 also encodes a number of regulatory and accessory proteins. Tat is critical for transcription from the HIV-1 LTR and Rev plays a major [figure: see text] role in the transport of viral RNAs from the nucleus to the cytoplasm. Vpu, Vif, Vpr and Nef have been termed "accessory" or "auxiliary" proteins to reflect the fact that they are not uniformly required for virus replication. The functions of these very interesting proteins will be discussed in more detail at the end of this chapter. HIV replication proceeds in a series of events that can be divided into two overall phases: "early" and "late" (Fig. 3). Although some events occur in a concerted or simultaneous fashion, the replication cycle can be viewed most simply as proceeding in an ordered, step-wise manner. In this chapter, each step in virus replication will be considered; additional information can be obtained from the more detailed reviews and primary references that are cited.

  7. HIV-1 Prevention for HIV-1 Serodiscordant Couples

    PubMed Central

    Curran, Kathryn; Baeten, Jared M.; Coates, Thomas J.; Kurth, Ann; Mugo, Nelly R.

    2013-01-01

    A substantial proportion of HIV-1-infected individuals in sub-Saharan Africa are in stable relationships with HIV-1-uninfected partners, and HIV-1 serodiscordant couples thus represent an important target population for HIV-1 prevention. Couple-based HIV-1 testing and counseling facilitates identification of HIV-1 serodiscordant couples, counseling about risk reduction, and referrals to HIV-1 treatment, reproductive health services, and support services. Maximizing HIV-1 prevention for HIV-1 serodiscordant couples requires a combination of strategies, including counseling about condoms, sexual risk, fertility, contraception, and the clinical and prevention benefits of antiretroviral therapy (ART) for the HIV-1-infected partner; provision of clinical care and ART for the HIV-1-infected partner; antenatal care and services to prevent mother to child transmission for HIV-1- infected pregnant women; male circumcision for HIV-1-uninfected men; and, pending guidelines and demonstration projects, oral pre-exposure prophylaxis (PrEP) for HIV-1-uninfected partners. PMID:22415473

  8. HIV-1 envelope glycoprotein

    DOEpatents

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  9. 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…

  10. The activity of the integrase inhibitor dolutegravir against HIV-1 variants isolated from raltegravir-treated adults.

    PubMed

    Underwood, Mark R; Johns, Brian A; Sato, Akihiko; Martin, Jeffrey N; Deeks, Steven G; Fujiwara, Tamio

    2012-11-01

    Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates. Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen. Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87), while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9-19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H, G140S + Q148R, T97A + Y143R, and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively. Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir.

  11. The Activity of the Integrase Inhibitor Dolutegravir Against HIV-1 Variants Isolated From Raltegravir-Treated Adults

    PubMed Central

    Underwood, Mark R.; Johns, Brian A.; Sato, Akihiko; Martin, Jeffrey N.; Deeks, Steven G.; Fujiwara, Tamio

    2013-01-01

    Background Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates. Methods Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen. Results Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87) while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9–19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H; G140S + Q148R; T97A + Y143R; and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively. Conclusions Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir. PMID:22878423

  12. Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop

    PubMed Central

    Qin, Yali; Shi, Heliang; Banasik, Marisa; Lin, Feng; Rohl, Kari; LaBranche, Celia; Montefiori, David C.; Cho, Michael W.

    2015-01-01

    We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs) against Human Immunodeficiency Virus type-1 (HIV-1) in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs) ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR), followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37) exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem. PMID:26039641

  13. Viral Determinants of HIV-1 Macrophage Tropism

    PubMed Central

    Duncan, Christopher J. A.; Sattentau, Quentin J.

    2011-01-01

    Macrophages are important target cells for HIV-1 infection that play significant roles in the maintenance of viral reservoirs and other aspects of pathogenesis. Understanding the determinants of HIV-1 tropism for macrophages will inform HIV-1 control and eradication strategies. Tropism for macrophages is both qualitative (infection or not) and quantitative (replication capacity). For example many R5 HIV-1 isolates cannot infect macrophages, but for those that can the macrophage replication capacity can vary by up to 1000-fold. Some X4 viruses are also capable of replication in macrophages, indicating that cellular tropism is partially independent of co-receptor preference. Preliminary data obtained with a small number of transmitted/founder viruses indicate inefficient macrophage infection, whereas isolates from later in disease are more frequently tropic for macrophages. Thus tropism may evolve over time, and more macrophage tropic viruses may be implicated in the pathogenesis of advanced HIV-1 infection. Compartmentalization of macrophage-tropic brain-derived envelope glycoproteins (Envs), and non-macrophage tropic non-neural tissue-derived Envs points to adaptation of HIV-1 quasi-species in distinct tissue microenvironments. Mutations within and adjacent to the Env-CD4 binding site have been identified that determine macrophage tropism at the entry level, but post-entry molecular determinants of macrophage replication capacity involving HIV-1 accessory proteins need further definition. PMID:22163344

  14. 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.

  15. HIV-1 vaccine antibody induction against a variable region of HIV-1: a possible link to protective immunity?

    PubMed

    Bauer, Gerhard

    2013-05-01

    Evaluation of: Liao H, Bonsignori M, Alam M et al. Vaccine induction of antibodies against a structurally heterogeneous site of immune pressure within HIV-1 envelope protein variable regions 1 and 2. Immunity 38, 176-186 (2013). In 2009, results from the Phase III HIV-1 vaccine clinical trial RV144 applying a prime/boost regimen with a canarypox vaccine vector ALVAC-HIV plus the AIDSVAX B/E subunit envelope vaccine conducted in Thailand were reported. The priming canarypox vector carried the HIV-1 vaccine genes gp120 linked to the transmembrane-anchoring portion of subtype B gp41, HIV-1 Gag and protease; the boosting vaccine was composed of clades B and E of HIV-1 gp120. A 31.2% vaccine efficacy could be seen in this trial, an encouraging result in HIV-1 vaccine research that had been previously plagued with little clinical efficacy. In this paper, results from tests of four monoclonal antibodies isolated from RV144 vaccinees are reported. The antibodies recognize a certain HIV-1 envelope residue (169), neutralize laboratory-adapted HIV-1 strains and mediate killing of CD4(+) cells infected with HIV-1 laboratory isolates. Crystal structure analysis suggests that the recognized HIV-1 envelope epitope can exist in different conformations. It is thought that the immune pressure elicited by the monoclonal antibodies targets a HIV-1 envelope region with variable sequence structure.

  16. HIV-1 vaccines

    PubMed Central

    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. PMID:24637946

  17. HIV-1 Reverse Transcription

    PubMed Central

    Hu, Wei-Shau; Hughes, Stephen H.

    2012-01-01

    Reverse transcription and integration are the defining features of the Retroviridae; the common name “retrovirus” derives from the fact that these viruses use a virally encoded enzyme, reverse transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral factors that can affect reverse transcription, and discusses fidelity and recombination, two processes in which reverse transcription plays an important role. In keeping with the theme of the collection, the emphasis is on HIV-1 and HIV-1 RT. PMID:23028129

  18. Molecular epidemiology of HIV-1 in Madrid.

    PubMed

    Rojas, J M; Dopazo, J; Nájera, I; Sánchez-Palomino, S; Olivares, I; Martin, M J; Bernal, A; García Saiz, A; Nájera, R; López-Galíndez, C

    1994-03-01

    Thirteen HIV-1 isolates from patients of different risk groups in Madrid (Spain) have been analyzed at the genetic level. Two distinct lineages of subtype B have been detected among the HIV-1 circulating in this area: one was related to SF-2/RF strains, whereas the other consists of a more heterogeneous group related to reference strain III-B. Variants of each lineage appeared to circulate preferentially within a risk group: III-B among intravenous drug users, and RF/SF-2 among male homosexuals.

  19. [A new unique HIV-1 recombinant form detected in Belarus].

    PubMed

    Eremin, V F; Gasich, E L; Sosinovich, S V

    2012-01-01

    Republican Research-and-Practical Center for Epidemiology and Microbiology, Ministry of Health of Belarus, Minsk The paper presents data on the molecular genetic characteristics of a new HIV-1 recombinant form. The study has shown that the virus is referred to as HIV-1 subtype B in terms of the gag gene and HIV-1 subtype A in terms of the pol and env genes. At the same time the new isolate is closer, in terms of the gag gene, to the HIV-1 DQ207943 strain isolated in Georgia, in terms of the pol gene, to the HIV-1 AF413987.1 strain isolated in Ukraine and, in terms of the env gene to the HIV-1 AY500393 strain isolated in Russia. Thus, the described new HIV-1 recombinant form has the following structure: BgagApolAenv. The gag, pol, and env gene sequences from the new unique HIV-1 recombinant form have been registered in the international database EMBL/Genbank/DDBJ under accession numbers FR775442.1, FN995656.1, and FR775443.1.

  20. Imaging HIV-1 Genomic DNA from Entry through Productive Infection.

    PubMed

    Stultz, Ryan D; Cenker, Jennifer J; McDonald, David

    2017-05-01

    In order to track the fate of HIV-1 particles from early entry events through productive infection, we developed a method to visualize HIV-1 DNA reverse transcription complexes by the incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) into nascent viral DNA during cellular entry. Monocyte-derived macrophages were chosen as natural targets of HIV-1, as they do not divide and therefore do not incorporate EdU into chromosomal DNA, which would obscure the detection of intranuclear HIV-1 genomes. Using this approach, we observed distinct EdU puncta in the cytoplasm of infected cells within 12 h postinfection and subsequent accumulation of puncta in the nucleus, which remained stable through 5 days. The depletion of the restriction factor SAMHD1 resulted in a markedly increased number of EdU puncta, allowing efficient quantification of HIV-1 reverse transcription events. Analysis of HIV-1 isolates bearing defined mutations in the capsid protein revealed differences in their cytoplasmic and nuclear accumulation, and data from quantitative PCR analysis closely recapitulated the EdU results. RNA fluorescence in situ hybridization identified actively transcribing, EdU-labeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at serine 175, was recruited to RNA-positive HIV-1 DNA, providing a means to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this system allows stable labeling and monitoring of HIV genomic DNA within infected cells during cytoplasmic transit, nuclear import, and mRNA synthesis.IMPORTANCE The fates of HIV-1 reverse transcription products within infected cells are not well understood. Although previous imaging approaches identified HIV-1 intermediates during early stages of infection, few have connected these events with the later stages that ultimately lead to proviral transcription and the

  1. HIV-1 protease and HIV-1 integrase inhibitory substances from Eclipta prostrata.

    PubMed

    Tewtrakul, Supinya; Subhadhirasakul, Sanan; Cheenpracha, Sarot; Karalai, Chatchanok

    2007-11-01

    The bioassay-guided fractionation for anti-HIV-1 integrase activity led to the isolation of six compounds from the whole plant extract of Eclipta prostrata extract. They were identified as 5-hydroxymethyl-(2,2':5',2'')-terthienyl tiglate (1), 5-hydroxymethyl-(2,2':5',2'')-terthienyl agelate (2), 5-hydroxymethyl-(2,2':5',2'')-terthienyl acetate (3), ecliptal (4), orobol (5) and wedelolactone (6). Of these, compound 6 showed the highest activity against HIV-1 integrase (IN) with an IC50 value of 4.0+/-0.2 microm, followed by compound 5 (IC50=8.1+/-0.5 microm), whereas the four terthiophene compounds (1-4) were inactive (IC50>100 microm). Regarding HIV-1 protease (PR) inhibitory activity, compound 1 exhibited appreciable activity against HIV-1 PR with an IC50 of 58.3+/-0.8 microm, followed by compound 4 (IC50=83.3+/-1.6 microm) and compound 3 (IC50=93.7+/-0.8 microm), while compounds 2, 5 and 6 were inactive against HIV-1 PR (IC50>100 microm). This is the first report of anti-HIV-1 IN activities for wedelolactone (6), a coumarin derivative, and orobol (5), an isoflavone derivative. This study supports the use of E. prostrata in AIDS patients, which is in accord with its traditional use by Thai traditional doctors for curing blood related diseases. Copyright (c) 2007 John Wiley & Sons, Ltd.

  2. 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.

  3. 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

  4. Chimeric Antigen Receptor T Cells Guided by the Single-Chain Fv of a Broadly Neutralizing Antibody Specifically and Effectively Eradicate Virus Reactivated from Latency in CD4+ T Lymphocytes Isolated from HIV-1-Infected Individuals Receiving Suppressive Combined Antiretroviral Therapy.

    PubMed

    Liu, Bingfeng; Zou, Fan; Lu, Lijuan; Chen, Cancan; He, Dalian; Zhang, Xu; Tang, Xiaoping; Liu, Chao; Li, Linghua; Zhang, Hui

    2016-11-01

    Despite the advent of combined antiretroviral therapy (cART), the persistence of viral reservoirs remains a major barrier to curing human immunodeficiency virus type 1 (HIV-1) infection. Recently, the shock and kill strategy, by which such reservoirs are eradicated following reactivation of latent HIV-1 by latency-reversing agents (LRAs), has been extensively practiced. It is important to reestablish virus-specific and reliable immune surveillance to eradicate the reactivated virus-harboring cells. In this report, we attempted to reach this goal by using newly developed chimeric antigen receptor (CAR)-T cell technology. To generate anti-HIV-1 CAR-T cells, we connected the single-chain variable fragment of the broadly neutralizing HIV-1-specific antibody VRC01 to a third-generation CAR moiety as the extracellular and intracellular domains and subsequently transduced this into primary CD8(+) T lymphocytes. We demonstrated that the resulting VC-CAR-T cells induced T cell-mediated cytolysis of cells expressing HIV-1 Env proteins and significantly inhibited HIV-1 rebound after removal of antiviral inhibitors in a viral infectivity model in cell culture that mimics the termination of the cART in the clinic. Importantly, the VC-CAR-T cells also effectively induced the cytolysis of LRA-reactivated HIV-1-infected CD4(+) T lymphocytes isolated from infected individuals receiving suppressive cART. Our data demonstrate that the special features of genetically engineered CAR-T cells make them a particularly suitable candidate for therapeutic application in efforts to reach a functional HIV cure. The presence of latently infected cells remains a key obstacle to the development of a functional HIV-1 cure. Reactivation of dormant viruses is possible with latency-reversing agents, but the effectiveness of these compounds and the subsequent immune response require optimization if the eradication of HIV-1-infected cells is to be achieved. Here, we describe the use of a chimeric

  5. Isolation of a Monoclonal Antibody That Targets the Alpha-2 Helix of gp120 and Represents the Initial Autologous Neutralizing-Antibody Response in an HIV-1 Subtype C-Infected Individual ▿

    PubMed Central

    Gray, Elin S.; Moody, M. Anthony; Wibmer, Constantinos Kurt; Chen, Xi; Marshall, Dawn; Amos, Joshua; Moore, Penny L.; Foulger, Andrew; Yu, Jae-Sung; Lambson, Bronwen; Abdool Karim, Salim; Whitesides, John; Tomaras, Georgia D.; Haynes, Barton F.; Morris, Lynn; Liao, Hua-Xin

    2011-01-01

    The C3-V4 region is a major target of autologous neutralizing antibodies in HIV-1 subtype C infection. We previously identified a Center for AIDS Program of Research in South Africa (CAPRISA) participant, CAP88, who developed a potent neutralizing-antibody response within 3 months of infection that targeted an epitope in the C3 region of the HIV-1 envelope (P. L. Moore et al., PLoS Pathog. 5:e1000598, 2009). Here we showed that these type-specific antibodies could be adsorbed using recombinant gp120 from the transmitted/founder virus from CAP88 but not by gp120 made from other isolates. Furthermore, this activity could be depleted using a chimeric gp120 protein that contained only the C3 region from the CAP88 viral envelope engrafted onto the unrelated CAP63 viral envelope (called 63-88C3). On the basis of this, a differential sorting of memory B cells was performed using gp120s made from 63-88C3 and CAP63 labeled with different fluorochromes as positive and negative probes, respectively. This strategy resulted in the isolation of a highly specific monoclonal antibody (MAb), called CAP88-CH06, that neutralized the CAP88 transmitted/founder virus and viruses from acute infection but was unable to neutralize CAP88 viruses isolated at 6 and 12 months postinfection. The latter viruses contained 2 amino acid changes in the alpha-2 helix of C3 that mediated escape from this MAb. One of these changes involved the introduction of an N-linked glycan at position 339 that occluded the epitope, while the other mutation (either E343K or E350K) was a charge change. Our data validate the use of differential sorting to isolate a MAb targeting a specific epitope in the envelope glycoprotein and provided insights into the mechanisms of autologous neutralization escape. PMID:21613396

  6. 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

  7. Hyperthermia stimulates HIV-1 replication.

    PubMed

    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.

  8. 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

  9. 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.

  10. Characterization of a dual-tropic human immunodeficiency virus (HIV-1) strain derived from the prototypical X4 isolate HXBc2.

    PubMed

    Xiang, Shi-hua; Pacheco, Beatriz; Bowder, Dane; Yuan, Wen; Sodroski, Joseph

    2013-03-30

    Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed CCR5 utilization with retention of the ability to use CXCR4. We also found that the stable association of gp120 with the trimeric envelope glycoprotein complex in R5X4 viruses, as in X4 viruses, is less sensitive to V3 loop changes than gp120-trimer association in R5 viruses.

  11. Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs

    PubMed Central

    2011-01-01

    Background Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection. Results Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved

  12. An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

    PubMed Central

    Bonsignori, Mattia; Wiehe, Kevin; Grimm, Sebastian K.; Lynch, Rebecca; Yang, Guang; Kozink, Daniel M.; Perrin, Florence; Cooper, Abby J.; Hwang, Kwan-Ki; Chen, Xi; Liu, Mengfei; McKee, Krisha; Parks, Robert J.; Eudailey, Joshua; Wang, Minyue; Clowse, Megan; Criscione-Schreiber, Lisa G.; Moody, M. Anthony; Ackerman, Margaret E.; Boyd, Scott D.; Gao, Feng; Kelsoe, Garnett; Verkoczy, Laurent; Tomaras, Georgia D.; Liao, Hua-Xin; Kepler, Thomas B.; Montefiori, David C.; Mascola, John R.; Haynes, Barton F.

    2014-01-01

    Broadly HIV-1–neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1–infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1–infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient’s plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells. PMID:24614107

  13. 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.

  14. 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.

  15. 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.

  16. Trichomonas vaginalis-Induced Epithelial Monolayer Disruption and Human Immunodeficiency Virus Type 1 (HIV-1) Replication: Implications for the Sexual Transmission of HIV-1

    PubMed Central

    Guenthner, Patricia C.; Secor, W. Evan; Dezzutti, Charlene S.

    2005-01-01

    The objective of this study was to evaluate potential mechanisms of Trichomonas vaginalis involvement in human immunodeficiency virus type 1 (HIV-1) transmission. Polarized monolayer integrity of primary cervical and prostate epithelial cells or cell lines cultured with T. vaginalis was measured by monitoring transepithelium resistance. The effect of T. vaginalis isolates on HIV-1 passage through polarized epithelial cell monolayers was evaluated for HIV-1 p24gag in the basolateral supernatants. Coincubation with T. vaginalis isolates induced disruption of monolayer integrity and resulted in passage of virus to the basolateral side of the monolayer. Furthermore, there was isolate variability in which two isolates induced greater monolayer damage and increased HIV-1 passage than did the other two isolates. Coincubation of T. vaginalis isolates with acutely HIV-1-infected peripheral blood mononuclear cells enhanced HIV-1 replication. This enhancement was associated with cellular proliferation and activation, as well as with tumor necrosis factor alpha production. In contrast to the monolayer disruption, the effect of T. vaginalis on HIV-1 replication was not isolate dependent. Thus, two mechanisms have been identified that could contribute to the epidemiologic association of trichomoniasis with the sexual transmission of HIV-1. (i) T. vaginalis disruption of urogenital epithelial monolayers could facilitate passage of HIV-1 to underlying layers. (ii) Activation of local immune cells by T. vaginalis in the presence of infectious HIV-1 might lead to increased viral replication. Collectively, these data suggest the need for more vigilant efforts in the diagnosis and treatment of T. vaginalis in women and men, especially in countries with a high prevalence of HIV-1. PMID:15972505

  17. Trichomonas vaginalis-induced epithelial monolayer disruption and human immunodeficiency virus type 1 (HIV-1) replication: implications for the sexual transmission of HIV-1.

    PubMed

    Guenthner, Patricia C; Secor, W Evan; Dezzutti, Charlene S

    2005-07-01

    The objective of this study was to evaluate potential mechanisms of Trichomonas vaginalis involvement in human immunodeficiency virus type 1 (HIV-1) transmission. Polarized monolayer integrity of primary cervical and prostate epithelial cells or cell lines cultured with T. vaginalis was measured by monitoring transepithelium resistance. The effect of T. vaginalis isolates on HIV-1 passage through polarized epithelial cell monolayers was evaluated for HIV-1 p24gag in the basolateral supernatants. Coincubation with T. vaginalis isolates induced disruption of monolayer integrity and resulted in passage of virus to the basolateral side of the monolayer. Furthermore, there was isolate variability in which two isolates induced greater monolayer damage and increased HIV-1 passage than did the other two isolates. Coincubation of T. vaginalis isolates with acutely HIV-1-infected peripheral blood mononuclear cells enhanced HIV-1 replication. This enhancement was associated with cellular proliferation and activation, as well as with tumor necrosis factor alpha production. In contrast to the monolayer disruption, the effect of T. vaginalis on HIV-1 replication was not isolate dependent. Thus, two mechanisms have been identified that could contribute to the epidemiologic association of trichomoniasis with the sexual transmission of HIV-1. (i) T. vaginalis disruption of urogenital epithelial monolayers could facilitate passage of HIV-1 to underlying layers. (ii) Activation of local immune cells by T. vaginalis in the presence of infectious HIV-1 might lead to increased viral replication. Collectively, these data suggest the need for more vigilant efforts in the diagnosis and treatment of T. vaginalis in women and men, especially in countries with a high prevalence of HIV-1.

  18. 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

  19. 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.

  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. Chimpanzee Reservoirs of Pandemic and Nonpandemic HIV-1

    PubMed Central

    Keele, Brandon F.; Van Heuverswyn, Fran; Li, Yingying; Bailes, Elizabeth; Takehisa, Jun; Santiago, Mario L.; Bibollet-Ruche, Frederic; Chen, Yalu; Wain, Louise V.; Liegeois, Florian; Loul, Severin; Ngole, Eitel Mpoudi; Bienvenue, Yanga; Delaporte, Eric; Brookfield, John F. Y.; Sharp, Paul M.; Shaw, George M.; Peeters, Martine; Hahn, Beatrice H.

    2008-01-01

    Human immunodeficiency virus type 1 (HIV-1), the cause of human acquired immunodeficiency syndrome (AIDS), is a zoonotic infection of staggering proportions and social impact. Yet uncertainty persists regarding its natural reservoir. The virus most closely related to HIV-1 is a simian immunodeficiency virus (SIV) thus far identified only in captive members of the chimpanzee subspecies Pan troglodytes troglodytes. Here we report the detection of SIVcpz antibodies and nucleic acids in fecal samples from wild-living P. t. troglodytes apes in southern Cameroon, where prevalence rates in some communities reached 29 to 35%. By sequence analysis of endemic SIVcpz strains, we could trace the origins of pandemic (group M) and nonpandemic (group N) HIV-1 to distinct, geographically isolated chimpanzee communities. These findings establish P. t. troglodytes as a natural reservoir of HIV-1. PMID:16728595

  2. Chimpanzee reservoirs of pandemic and nonpandemic HIV-1.

    PubMed

    Keele, Brandon F; Van Heuverswyn, Fran; Li, Yingying; Bailes, Elizabeth; Takehisa, Jun; Santiago, Mario L; Bibollet-Ruche, Frederic; Chen, Yalu; Wain, Louise V; Liegeois, Florian; Loul, Severin; Ngole, Eitel Mpoudi; Bienvenue, Yanga; Delaporte, Eric; Brookfield, John F Y; Sharp, Paul M; Shaw, George M; Peeters, Martine; Hahn, Beatrice H

    2006-07-28

    Human immunodeficiency virus type 1 (HIV-1), the cause of human acquired immunodeficiency syndrome (AIDS), is a zoonotic infection of staggering proportions and social impact. Yet uncertainty persists regarding its natural reservoir. The virus most closely related to HIV-1 is a simian immunodeficiency virus (SIV) thus far identified only in captive members of the chimpanzee subspecies Pan troglodytes troglodytes. Here we report the detection of SIVcpz antibodies and nucleic acids in fecal samples from wild-living P. t. troglodytes apes in southern Cameroon, where prevalence rates in some communities reached 29 to 35%. By sequence analysis of endemic SIVcpz strains, we could trace the origins of pandemic (group M) and nonpandemic (group N) HIV-1 to distinct, geographically isolated chimpanzee communities. These findings establish P. t. troglodytes as a natural reservoir of HIV-1.

  3. A multifaceted analysis of HIV-1 protease multidrug resistance phenotypes

    PubMed Central

    2011-01-01

    Background Great strides have been made in the effective treatment of HIV-1 with the development of second-generation protease inhibitors (PIs) that are effective against historically multi-PI-resistant HIV-1 variants. Nevertheless, mutation patterns that confer decreasing susceptibility to available PIs continue to arise within the population. Understanding the phenotypic and genotypic patterns responsible for multi-PI resistance is necessary for developing PIs that are active against clinically-relevant PI-resistant HIV-1 variants. Results In this work, we use globally optimal integer programming-based clustering techniques to elucidate multi-PI phenotypic resistance patterns using a data set of 398 HIV-1 protease sequences that have each been phenotyped for susceptibility toward the nine clinically-approved HIV-1 PIs. We validate the information content of the clusters by evaluating their ability to predict the level of decreased susceptibility to each of the available PIs using a cross validation procedure. We demonstrate the finding that as a result of phenotypic cross resistance, the considered clinical HIV-1 protease isolates are confined to ~6% or less of the clinically-relevant phenotypic space. Clustering and feature selection methods are used to find representative sequences and mutations for major resistance phenotypes to elucidate their genotypic signatures. We show that phenotypic similarity does not imply genotypic similarity, that different PI-resistance mutation patterns can give rise to HIV-1 isolates with similar phenotypic profiles. Conclusion Rather than characterizing HIV-1 susceptibility toward each PI individually, our study offers a unique perspective on the phenomenon of PI class resistance by uncovering major multidrug-resistant phenotypic patterns and their often diverse genotypic determinants, providing a methodology that can be applied to understand clinically-relevant phenotypic patterns to aid in the design of novel inhibitors that

  4. High levels of divergent HIV-1 quasispecies in patients with neurological opportunistic infections in China.

    PubMed

    Zhang, Yulin; Wei, Feili; Liang, Qi; Ding, Wei; Qiao, Luxin; Song, Fengli; Liu, Lifeng; Yang, Sufang; Jin, Ronghua; Gu, Jianhua; Li, Ning; Chen, Dexi

    2013-08-01

    Despite the fact that the survival of people infected with human immunodeficiency virus (HIV) has improved worldwide because of the increasingly powerful and highly active antiretroviral therapy, opportunistic infections (OIs) of the central nervous system (CNS) remain a serious burden. HIV-1 is capable of entering the CNS through infected peripheral monocytes, but its effect on OIs of CNS remains unclear. In this study, we investigated the characteristics of HIV-1 in acquired immunodeficiency syndrome (AIDS) patients with CNS OIs. A total of 24 patients with CNS OIs and 16 non-CNS OIs (control) cases were selected. These AIDS patients were infected with HIV-1 by paid blood donors in China. HIV-1 loads in plasma and cerebrospinal fluid (CSF) were detected using RT-PCR, and the C2-V5 region of HIV-1 envelope gene was amplified from viral quasispecies isolated from CSF using nested PCR. The CSF HIV-1 load of CNS OIs was higher than that of non-CNS OIs, but plasma HIV-1 load of CNS OIs was not higher than that of non-CNS OIs. The nucleotide sequence of C2-V5 region of the HIV-1 quasispecies isolated from the CSF of CNS OIs had a high diversity, and the HIV-1 quasispecies isolated from the CSF of CNS OIs revealed R5 tropism as 11/25 charge rule. These results suggest that high levels of divergent HIV-1 quasispecies in the CNS probably contribute to opportunistic infections.

  5. Specific sequences commonly found in the V3 domain of HIV-1 subtype C isolates affect the overall conformation of native Env and induce a neutralization-resistant phenotype independent of V1/V2 masking.

    PubMed

    Salomon, Aidy; Krachmarov, Chavdar; Lai, Zhong; Honnen, William; Zingman, Barry S; Sarlo, Julie; Gorny, Miroslaw K; Zolla-Pazner, Susan; Robinson, James E; Pinter, Abraham

    2014-01-05

    Primary HIV-1 isolates are relatively resistant to neutralization by antibodies commonly induced after infection or vaccination. This is generally attributed to masking of sensitive epitopes by the V1/V2 domain and/or glycans situated at various positions in Env. Here we identified a novel masking effect mediated by subtype C-specific V3 sequences that contributes to the V1/V2-independent and glycan-independent neutralization resistance of chimeric and primary Envs to antibodies directed against multiple neutralization domains. Positions at several conserved charged and hydrophobic sites in the V3 crown and stem were also shown to affect neutralization phenotype. These results indicated that substitutions typically present in subtype C and related V3 sequences influence the overall conformation of native Env in a way that occludes multiple neutralization targets located both within and outside of the V3 domain, and may reflect an alternative mechanism for neutralization resistance that is particularly active in subtype C and related isolates.

  6. 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

  7. Tracing HIV-1 transmission: envelope traits of HIV-1 transmitter and recipient pairs.

    PubMed

    Oberle, Corinna S; Joos, Beda; Rusert, Peter; Campbell, Nottania K; Beauparlant, David; Kuster, Herbert; Weber, Jacqueline; Schenkel, Corinne D; Scherrer, Alexandra U; Magnus, Carsten; Kouyos, Roger; Rieder, Philip; Niederöst, Barbara; Braun, Dominique L; Pavlovic, Jovan; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Trkola, Alexandra; Metzner, Karin J; Günthard, Huldrych F

    2016-09-05

    Mucosal HIV-1 transmission predominantly results in a single transmitted/founder (T/F) virus establishing infection in the new host despite the generally high genetic diversity of the transmitter virus population. To what extent HIV-1 transmission is a stochastic process or driven by selective forces that allow T/F viruses best to overcome bottlenecks in transmission has not been conclusively resolved. Building on prior investigations that suggest HIV-1 envelope (Env) features to contribute in the selection process during transmission, we compared phenotypic virus characteristics of nine HIV-1 subtype B transmission pairs, six men who have sex with men and three male-to-female transmission pairs. All recipients were identified early in acute infection and harbored based on extensive sequencing analysis a single T/F virus allowing a controlled analysis of virus properties in matched transmission pairs. Recipient and transmitter viruses from the closest time point to transmission showed no signs of selection for specific Env modifications such as variable loop length and glycosylation. Recipient viruses were resistant to circulating plasma antibodies of the transmitter and also showed no altered sensitivity to a large panel of entry inhibitors and neutralizing antibodies. The recipient virus did not consistently differ from the transmitter virus in terms of entry kinetics, cell-cell transmission and replicative capacity in primary cells. Our paired analysis revealed a higher sensitivity of several recipient virus isolates to interferon-α (IFNα) which suggests that resistance to IFNα cannot be a general driving force in T/F establishment. With the exception of increased IFNα sensitivity, none of the phenotypic virus properties we investigated clearly distinguished T/F viruses from their matched transmitter viruses supporting the notion that at least in subtype B infection HIV-1 transmission is to a considerable extent stochastic.

  8. Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase.

    PubMed

    Paskaleva, Elena E; Lin, Xudong; Duus, Karen; McSharry, James J; Veille, Jean-Claude L; Thornber, Carol; Liu, Yanze; Lee, David Yu-Wei; Canki, Mario

    2008-01-15

    Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 mug/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 mug. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 mug/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.

  9. Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase

    PubMed Central

    Paskaleva, Elena E; Lin, Xudong; Duus, Karen; McSharry, James J; Veille, Jean-Claude L; Thornber, Carol; Liu, Yanze; Lee, David Yu-Wei; Canki, Mario

    2008-01-01

    Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 μg/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 μg. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 μg/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development. PMID:18197976

  10. Structured antiretroviral treatment interruptions in chronically HIV-1-infected subjects

    PubMed Central

    Ortiz, Gabriel M.; Wellons, Melissa; Brancato, Jason; Vo, Ha T. T.; Zinn, Rebekah L.; Clarkson, Daniel E.; Van Loon, Katherine; Bonhoeffer, Sebastian; Miralles, G. Diego; Montefiori, David; Bartlett, John A.; Nixon, Douglas F.

    2001-01-01

    The risks and benefits of structured treatment interruption (STI) in HIV-1-infected subjects are not fully understood. A pilot study was performed to compare STI with continuous highly active antiretroviral therapy (HAART) in chronic HIV-1-infected subjects with HIV-1 plasma RNA levels (VL) <400 copies per ml and CD4+ T cells >400 per μl. CD4+ T cells, VL, HIV-1-specific neutralizing antibodies, and IFN-γ-producing HIV-1-specific CD8+ and CD4+ T cells were measured in all subjects. STIs of 1-month duration separated by 1 month of HAART, before a final 3-month STI, resulted in augmented CD8+ T cell responses in all eight STI subjects (P = 0.003), maintained while on HAART up to 22 weeks after STI, and augmented neutralization titers to autologous HIV-1 isolate in one of eight subjects. However, significant decline of CD4+ T cell count from pre-STI level, and VL rebound to pre-HAART baseline, occurred during STI (P = 0.001 and 0.34, respectively). CD4+ T cell counts were regained on return to HAART. Control subjects (n = 4) maintained VL <400 copies per ml and stable CD4+ T cell counts, and showed no enhancement of antiviral CD8+ T cell responses. Despite increases in antiviral immunity, no control of VL was observed. Future studies of STI should proceed with caution. PMID:11687611

  11. Developing strategies for HIV-1 eradication

    PubMed Central

    Durand, Christine M.; Blankson, Joel N.; Siliciano, Robert F.

    2014-01-01

    Highly active antiretroviral therapy (HAART) suppresses HIV-1 replication, transforming the outlook for infected patients. However, reservoirs of replication-competent forms of the virus persist during HAART, and when treatment is stopped, high rates of HIV-1 replication return. Recent insights into HIV-1 latency, as well as a report that HIV-1 infection was eradicated in one individual, have renewed interest in finding a cure for HIV-1 infection. Strategies for HIV-1 eradication include gene therapy and hematopoietic stem cell transplantation, stimulating host immunity to control HIV-1 replication, and targeting latent HIV-1 in resting memory CD4+ T cells. Future efforts should aim to provide better understanding of how to reconstitute the CD4+ T cell compartment with genetically engineered cells, exert immune control over HIV-1 replication, and identify and eliminate all viral reservoirs. PMID:22867874

  12. Vaccination Against Hepatitis B Virus (HBV) in HIV-1-Infected Patients With Isolated Anti-HBV Core Antibody: The ANRS HB EP03 CISOVAC Prospective Study.

    PubMed

    Piroth, Lionel; Launay, Odile; Michel, Marie-Louise; Bourredjem, Abderrahmane; Miailhes, Patrick; Ajana, Faiza; Chirouze, Catherine; Zucman, David; Wendling, Marie-Josee; Nazzal, Dani; Carrat, Fabrice; Rey, David; Binquet, Christine

    2016-06-01

    Although an isolated anti-hepatitis B virus (HBV) core antibody (anti-HBc) serological profile is frequent in human immunodeficiency virus (HIV)-infected patients, data on HBV vaccination in these patients are scarce. A prospective multicenter study was conducted to assess the immunogenicity of HBV vaccination in 54 patients with an isolated anti-HBc profile and undetectable HIV load. They were vaccinated with 1 dose (20 µg) of recombinant HBV vaccine. Those with an anti-HBV surface antibody (anti-HBs) level of <10 mIU/mL 4 weeks after vaccination received 3 additional double doses (40 µg) at weeks 5, 9, and 24. At week 4, 25 patients (46%) were responders. Only the ratio of CD4(+) T cells to CD8(+) T cells was associated with this response in multivariate analysis (odds ratio for +0.1, 1.32; 95% confidence interval, 1.07-1.63; P = .008). At week 28 and month 18, 58% of these patients (14 of 24) and 50% (10 of 20), respectively, maintained anti-HBs level of ≥10 mIU/mL.Among nonresponding patients at week 4, who received further vaccinations, 89% (24 of 27) and 81% (21 of 26) had an anti-HBs level of ≥10 mIU/mL at week 28 and month 18, respectively. The preS2-specific interferon γ T-cell response increased between week 0 and week 28 in patients who finally responded to reinforced vaccination (P = .03). All of the patients with an isolated anti-HBc profile who did not have an anti-HBs titer of >100 mIU/mL 4 weeks after a single recall dose of HBV vaccine should be further vaccinated with a reinforced triple double-dose scheme. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  13. 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

  14. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages

    PubMed Central

    Kishore, Shivendra; Jaskiewicz, Lukasz; Hall, Jonathan; Günthard, Huldrych F.; Beerenwinkel, Niko; Metzner, Karin J.

    2015-01-01

    Background MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM). Methods The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs. Results and Conclusion PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages. PMID:26226348

  15. Potent Intratype Neutralizing Activity Distinguishes Human Immunodeficiency Virus Type 2 (HIV-2) from HIV-1

    PubMed Central

    Özkaya Şahin, Gülşen; Holmgren, Birgitta; da Silva, Zacarias; Nielsen, Jens; Nowroozalizadeh, Salma; Esbjörnsson, Joakim; Månsson, Fredrik; Andersson, Sören; Norrgren, Hans; Aaby, Peter

    2012-01-01

    HIV-2 has a lower pathogenicity and transmission rate than HIV-1. Neutralizing antibodies could be contributing to these observations. Here we explored side by side the potency and breadth of intratype and intertype neutralizing activity (NAc) in plasma of 20 HIV-1-, 20 HIV-2-, and 11 dually HIV-1/2 (HIV-D)-seropositive individuals from Guinea-Bissau, West Africa. Panels of primary isolates, five HIV-1 and five HIV-2 isolates, were tested in a plaque reduction assay using U87.CD4-CCR5 cells as targets. Intratype NAc in HIV-2 plasma was found to be considerably more potent and also broader than intratype NAc in HIV-1 plasma. This indicates that HIV-2-infected individuals display potent type-specific neutralizing antibodies, whereas such strong type-specific antibodies are absent in HIV-1 infection. Furthermore, the potency of intratype NAc was positively associated with the viral load of HIV-1 but not HIV-2, suggesting that NAc in HIV-1 infection is more antigen stimulation dependent than in HIV-2 infection, where plasma viral loads typically are at least 10-fold lower than in HIV-1 infection. Intertype NAc of both HIV-1 and HIV-2 infections was, instead, of low potency. HIV-D subjects had NAc to HIV-2 with similar high potency as singly HIV-2-infected individuals, whereas neutralization of HIV-1 remained poor, indicating that the difference in NAc between HIV-1 and HIV-2 infections depends on the virus itself. We suggest that immunogenicity and/or antigenicity, meaning the neutralization phenotype, of HIV-2 is distinct from that of HIV-1 and that HIV-2 may display structures that favor triggering of potent neutralizing antibody responses. PMID:22072782

  16. Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women.

    PubMed

    Shen, Ruizhong; Achenbach, Jenna; Shen, Yue; Palaia, Jana; Rahkola, Jeremy T; Nick, Heidi J; Smythies, Lesley E; McConnell, Michelle; Fowler, Mary G; Smith, Phillip D; Janoff, Edward N

    2015-01-01

    Breast milk is a vehicle of infection and source of protection in post-natal mother-to-child HIV-1 transmission (MTCT). Understanding the mechanism by which breast milk limits vertical transmission will provide critical insight into the design of preventive and therapeutic approaches to interrupt HIV-1 mucosal transmission. However, characterization of the inhibitory activity of breast milk in human intestinal mucosa, the portal of entry in postnatal MTCT, has been constrained by the limited availability of primary mucosal target cells and tissues to recapitulate mucosal transmission ex vivo. Here, we characterized the impact of skimmed breast milk, breast milk antibodies (Igs) and non-Ig components from HIV-1-infected Ugandan women on the major events of HIV-1 mucosal transmission using primary human intestinal cells and tissues. HIV-1-specific IgG antibodies and non-Ig components in breast milk inhibited the uptake of Ugandan HIV-1 isolates by primary human intestinal epithelial cells, viral replication in and transport of HIV-1- bearing dendritic cells through the human intestinal mucosa. Breast milk HIV-1-specific IgG and IgA, as well as innate factors, blocked the uptake and transport of HIV-1 through intestinal mucosa. Thus, breast milk components have distinct and complementary effects in reducing HIV-1 uptake, transport through and replication in the intestinal mucosa and, therefore, likely contribute to preventing postnatal HIV-1 transmission. Our data suggests that a successful preventive or therapeutic approach would require multiple immune factors acting at multiple steps in the HIV-1 mucosal transmission process.

  17. Bioorthogonal mimetics of palmitoyl-CoA and myristoyl-CoA and their subsequent isolation by click chemistry and characterization by mass spectrometry reveal novel acylated host-proteins modified by HIV-1 infection.

    PubMed

    Colquhoun, David R; Lyashkov, Alexey E; Ubaida Mohien, Ceereena; Aquino, Veronica N; Bullock, Brandon T; Dinglasan, Rhoel R; Agnew, Brian J; Graham, David R M

    2015-06-01

    Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems.

  18. 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.

  19. Depression does not influence basal ganglia-mediated psychomotor speed in HIV-1 infection.

    PubMed

    von Giesen, H J; Bäcker, R; Hefter, H; Arendt, G

    2001-01-01

    The authors examined the effects of depressive mood (Hamilton Rating Scale for Depression [Ham-D]) on basal ganglia-mediated psychomotor speed (motor test battery) in 202 HIV-1 seropositive homosexual males with no prior history of antiretroviral treatment. HIV-1 seropositive patients showed a significant slowing of most rapid alternating movements (MRAM) and significantly prolonged contraction times (CT) compared with 66 HIV-1 seronegative male control subjects. Factor analysis of Ham-D scores isolated a factor containing the items depressed mood, suicide, and psychic and somatic anxiety. This factor did not correlate with MRAM or CT. Depression and psychomotor speed are independent in HIV-1infection.

  20. Phylodynamics of the HIV-1 epidemic in Cuba.

    PubMed

    Delatorre, Edson; Bello, Gonzalo

    2013-01-01

    Previous studies have shown that the HIV-1 epidemic in Cuba displayed a complex molecular epidemiologic profile with circulation of several subtypes and circulating recombinant forms (CRF); but the evolutionary and population history of those viral variants remains unknown. HIV-1 pol sequences of the most prevalent Cuban lineages (subtypes B, C and G, CRF18_cpx, CRF19_cpx, and CRFs20/23/24_BG) isolated between 1999 and 2011 were analyzed. Maximum-likelihood analyses revealed multiple introductions of subtype B (n≥66), subtype C (n≥10), subtype G (n≥8) and CRF18_cpx (n≥2) viruses in Cuba. The bulk of HIV-1 infections in this country, however, was caused by dissemination of a few founder strains probably introduced from North America/Europe (clades B(CU-I) and B(CU-II)), east Africa (clade C(CU-I)) and central Africa (clades G(CU), CRF18(CU) and CRF19(CU)), or locally generated (clades CRFs20/23/24_BG). Bayesian-coalescent analyses show that the major HIV-1 founder strains were introduced into Cuba during 1985-1995; whereas the CRFs_BG strains emerged in the second half of the 1990s. Most HIV-1 Cuban clades appear to have experienced an initial period of fast exponential spread during the 1990s and early 2000s, followed by a more recent decline in growth rate. The median initial growth rate of HIV-1 Cuban clades ranged from 0.4 year⁻¹ to 1.6 year⁻¹. Thus, the HIV-1 epidemic in Cuba has been a result of the successful introduction of a few viral strains that began to circulate at a rather late time of the AIDS pandemic, but then were rapidly disseminated through local transmission networks.

  1. 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.

  2. Preclinical Development of the Green Tea Catechin, Epigallocatechin Gallate, as an HIV-1 Therapy

    PubMed Central

    Nance, Christina L.; Siwak, Edward B.; Shearer, William T.

    2009-01-01

    Background Previously, we presented evidence that at physiologic concentrations the green tea catechin, epigallocatechin gallate (EGCG), inhibited attachment of gp120 to the CD4 molecule on T cells, but the downstream effects of EGCG upon HIV-1 infectivity were not determined. Objective To evaluate the inhibition of HIV-1 infectivity by EGCG and begin preclinical development of EGCG as a possible therapy. Methods Peripheral blood mononuclear cells, CD4+ T cells, and macrophages were isolated from blood of HIV-1 uninfected donors. HIV-1 infectivity was assessed by an HIV-1 p24 enzyme linked immunoassay. Cell survival was assessed by cell viability by trypan blue exclusion assay; cell growth by thymidine incorporation; and apoptosis by flow cytometric analysis of Annexin-V binding. Results EGCG inhibited HIV-1 infectivity on human CD4+ T cells and macrophages in a dose-dependent manner. At a physiologic concentration of 6μM, EGCG significantly inhibited HIV-1 p24 antigen production across a broad spectrum of both HIV-1 clinical isolates and laboratory-adapted subtypes [B (p<0.001), C, D, and G (p<0.01)]. The specificity of the EGCG-induced inhibition was substantiated by the failure of EGCG derivatives lacking galloyl and/or pyrogallol side groups to alter HIV-1 p24 levels. EGCG-induced inhibition of HV-1 infectivity was not due to cytotoxicity, cell growth inhibition, nor apoptosis. Conclusion We conclude that by preventing the attachment of HIV-1-gp120 to the CD4 molecule, EGCG inhibits HIV-1 infectivity. As this inhibition can be achieved at physiologic concentrations, the natural anti-HIV agent, EGCG, is a candidate as an alternative therapy in HIV-1 therapy. PMID:19203663

  3. HIV-1 Ribonuclease H Inhibitory Phenolic Glycosides from Eugenia hyemalis

    PubMed Central

    Bokesch, Heidi R.; Wamiru, Antony; Le Grice, Stuart F. J.; Beutler, John A.; McKee, Tawnya C.; McMahon, James B.

    2008-01-01

    Three new galloyl arbutins, hyemalosides A–C (1–3), along with nine known compounds were isolated from the evergreen tree Eugenia hyemalis. The structures of compounds 1–3 were determined by analysis of NMR and MS data. Compounds 1–3 inhibited HIV-1 RNase H in vitro with IC50 values of 1.46, >18, and 1.19 μM, respectively. However, in a XTT-based cell viability assay using the human T-cell line CEM-SS infected with HIV-1RT, none of the compounds inhibited the cytopathic effect of HIV-1 infection at the highest dose tested (20 μg/mL). PMID:18763827

  4. Suppression of HIV-1 replication by microRNA effectors

    PubMed Central

    Chable-Bessia, Christine; Meziane, Oussama; Latreille, Daniel; Triboulet, Robinson; Zamborlini, Alessia; Wagschal, Alexandre; Jacquet, Jean-Marc; Reynes, Jacques; Levy, Yves; Saib, Ali; Bennasser, Yamina; Benkirane, Monsef

    2009-01-01

    The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART. PMID:19272132

  5. Cocaine enhances HIV-1-induced CD4(+) T-cell apoptosis: implications in disease progression in cocaine-abusing HIV-1 patients.

    PubMed

    Pandhare, Jui; Addai, Amma B; Mantri, Chinmay K; Hager, Cynthia; Smith, Rita M; Barnett, Louis; Villalta, Fernando; Kalams, Spyros A; Dash, Chandravanu

    2014-04-01

    Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers.

  6. Rare HIV-1 Subtype J Genomes and a New H/U/CRF02_AG Recombinant Genome Suggests an Ancient Origin of HIV-1 in Angola.

    PubMed

    Bártolo, Inês; Calado, Rita; Borrego, Pedro; Leitner, Thomas; Taveira, Nuno

    2016-08-01

    Angola has an extremely diverse HIV-1 epidemic fueled in part by the frequent interchange of people with the Democratic Republic of Congo (DRC) and Republic of Congo (RC). Characterization of HIV-1 strains circulating in Angola should help to better understand the origin of HIV-1 subtypes and recombinant forms and their transmission dynamics. In this study we characterize the first near full-length HIV-1 genomic sequences from HIV-1 infected individuals from Angola. Samples were obtained in 1993 from three HIV-1 infected patients living in Cabinda, Angola. Near full-length genomic sequences were obtained from virus isolates. Maximum likelihood phylogenetic tree inference and analyses of potential recombination patterns were performed to evaluate the sequence classifications and origins. Phylogenetic and recombination analyses revealed that one virus was a pure subtype J, another mostly subtype J with a small uncertain region, and the final virus was classified as a H/U/CRF02_AG recombinant. Consistent with their epidemiological data, the subtype J sequences were more closely related to each other than to other J sequences previously published. Based on the env gene, taxa from Angola occur throughout the global subtype J phylogeny. HIV-1 subtypes J and H are present in Angola at low levels since at least 1993. Low transmission efficiency and/or high recombination potential may explain their limited epidemic success in Angola and worldwide. The high diversity of rare subtypes in Angola suggests that Angola was part of the early establishment of the HIV-1 pandemic.

  7. In Vitro Reactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors

    PubMed Central

    Banga, Riddhima; Procopio, Francesco Andrea; Cavassini, Matthias

    2015-01-01

    ABSTRACT The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects. IMPORTANCE The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an

  8. Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.

    PubMed

    Permar, Sallie R; Fong, Youyi; Vandergrift, Nathan; Fouda, Genevieve G; Gilbert, Peter; Parks, Robert; Jaeger, Frederick H; Pollara, Justin; Martelli, Amanda; Liebl, Brooke E; Lloyd, Krissey; Yates, Nicole L; Overman, R Glenn; Shen, Xiaoying; Whitaker, Kaylan; Chen, Haiyan; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Marshall, Dawn J; Whitesides, John F; Gurley, Thaddeus C; Von Holle, Tarra; Martinez, David R; Cai, Fangping; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Louzao, Raul; Wilkes, Samantha; Datta, Saheli; Sarzotti-Kelsoe, Marcella; Liao, Hua-Xin; Ferrari, Guido; Alam, S Munir; Montefiori, David C; Denny, Thomas N; Moody, M Anthony; Tomaras, Georgia D; Gao, Feng; Haynes, Barton F

    2015-07-01

    Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1-transmitting mothers and 165 propensity score-matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1-infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3-specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.

  9. Maternal HIV-1 envelope–specific antibody responses and reduced risk of perinatal transmission

    PubMed Central

    Permar, Sallie R.; Fong, Youyi; Vandergrift, Nathan; Fouda, Genevieve G.; Gilbert, Peter; Parks, Robert; Jaeger, Frederick H.; Pollara, Justin; Martelli, Amanda; Liebl, Brooke E.; Lloyd, Krissey; Yates, Nicole L.; Overman, R. Glenn; Shen, Xiaoying; Whitaker, Kaylan; Chen, Haiyan; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Marshall, Dawn J.; Whitesides, John F.; Gurley, Thaddeus C.; Von Holle, Tarra; Martinez, David R.; Cai, Fangping; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Louzao, Raul; Wilkes, Samantha; Datta, Saheli; Sarzotti-Kelsoe, Marcella; Liao, Hua-Xin; Ferrari, Guido; Alam, S. Munir; Montefiori, David C.; Denny, Thomas N.; Moody, M. Anthony; Tomaras, Georgia D.; Gao, Feng; Haynes, Barton F.

    2015-01-01

    Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1–transmitting mothers and 165 propensity score–matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1–infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3–specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT. PMID:26053661

  10. HIV-1 Group O Genotypes and Phenotypes: Relationship to Fitness and Susceptibility to Antiretroviral Drugs

    PubMed Central

    Patel, Hamish; Ratcliff, Annette; Alessandri, Elodie; Liu, Joseph; Carpenter, Crystal; Plantier, Jean-Christophe

    2016-01-01

    Abstract Despite only 30,000 group O HIV-1 infections, a similar genetic diversity is observed among the O subgroups H (head) and T (tail) (previously described as subtypes A, B) as in the 9 group M subtypes (A–K). Group O isolates bearing a cysteine at reverse transcriptase (RT) position 181, predominantly the H strains are intrinsically resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, their susceptibility to newer antiretroviral drugs such as etravirine, maraviroc, raltegravir (RAL), and elvitegravir (EVG) remains relatively unknown. We tested a large collection of HIV-1 group O strains for their susceptibility to four classes of antiretroviral drugs namely nucleoside RT, non-nucleoside RT, integrase, and entry inhibitors knowing in advance the intrinsic resistance to NNRTIs. Drug target regions were sequenced to determine various polymorphisms and were phylogenetically analyzed. Replication kinetics and fitness assays were performed in U87-CD4+CCR5 and CXCR4 cells and peripheral blood mononuclear cells. With all antiretroviral drugs, group O HIV-1 showed higher variability in IC50 values than group M HIV-1. The mean IC50 values for entry and nucleoside reverse transcriptase inhibitor (NRTI) were similar for group O and M HIV-1 isolates. Despite similar susceptibility to maraviroc, the various phenotypic algorithms failed to predict CXCR4 usage based on the V3 Env sequences of group O HIV-1 isolates. Decreased sensitivity of group O HIV-1 to integrase or NNRTIs had no relation to replicative fitness. Group O HIV-1 isolates were 10-fold less sensitive to EVG inhibition than group M HIV-1. These findings suggest that in regions where HIV-1 group O is endemic, first line treatment regimens combining two NRTIs with RAL may provide more sustained virologic responses than the standard regimens involving an NNRTI or protease inhibitors. PMID:26861573

  11. Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

    PubMed

    Sacha, C R; Vandergrift, N; Jeffries, T L; McGuire, E; Fouda, G G; Liebl, B; Marshall, D J; Gurley, T C; Stiegel, L; Whitesides, J F; Friedman, J; Badiabo, A; Foulger, A; Yates, N L; Tomaras, G D; Kepler, T B; Liao, H X; Haynes, B F; Moody, M A; Permar, S R

    2015-03-01

    A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

  12. Isolation and anti-HIV-1 integrase activity of lentzeosides A-F from extremotolerant lentzea sp. H45, a strain isolated from a high-altitude Atacama Desert soil.

    PubMed

    Wichner, Dominik; Idris, Hamidah; Houssen, Wael E; McEwan, Andrew R; Bull, Alan T; Asenjo, Juan A; Goodfellow, Michael; Jaspars, Marcel; Ebel, Rainer; Rateb, Mostafa E

    2017-04-01

    The extremotolerant isolate H45 was one of several actinomycetes isolated from a high-altitude Atacama Desert soil collected in northwest Chile. The isolate was identified as a new Lentzea sp. using a combination of chemotaxonomic, morphological and phylogenetic properties. Large scale fermentation of the strain in two different media followed by chromatographic purification led to the isolation of six new diene and monoene glycosides named lentzeosides A-F, together with the known compound (Z)-3-hexenyl glucoside. The structures of the new compounds were confirmed by HRESIMS and NMR analyses. Compounds 1-6 displayed moderate inhibitory activity against HIV integrase.

  13. Antiviral Breadth and Combination Potential of Peptide Triazole HIV-1 Entry Inhibitors

    PubMed Central

    McFadden, Karyn; Fletcher, Patricia; Rossi, Fiorella; Kantharaju; Umashankara, Muddagowda; Pirrone, Vanessa; Rajagopal, Srivats; Gopi, Hosahudya; Krebs, Fred C.; Martin-Garcia, Julio; Shattock, Robin J.

    2012-01-01

    The first stage of human immunodeficiency virus type 1 (HIV-1) infection involves the fusion of viral and host cellular membranes mediated by viral envelope glycoprotein gp120. Inhibitors that specifically target gp120 are gaining increased attention as therapeutics or preventatives to prevent the spread of HIV-1. One promising new group of inhibitors is the peptide triazoles, which bind to gp120 and simultaneously block its interaction with both CD4 and the coreceptor. In this study, we assessed the most potent peptide triazole, HNG-156, for inhibitory breadth, cytotoxicity, and efficacy, both alone and in combination with other antiviral compounds, against HIV-1. HNG-156 inhibited a panel of 16 subtype B and C isolates of HIV-1 in a single-round infection assay. Inhibition of cell infection by replication-competent clinical isolates of HIV-1 was also observed with HNG-156. We found that HNG-156 had a greater than predicted effect when combined with several other entry inhibitors or the reverse transcriptase inhibitor tenofovir. Overall, we find that HNG-156 is noncytotoxic, has a broad inhibition profile, and provides a positive combination with several inhibitors of the HIV-1 life cycle. These results support the pursuit of efficacy and toxicity analyses in more advanced cell and animal models to develop peptide triazole family inhibitors of HIV-1 into antagonists of HIV-1 infection. PMID:22083481

  14. Ex vivo production of autologous whole inactivated HIV-1 for clinical use in therapeutic vaccines.

    PubMed

    Gil, Cristina; Climent, Núria; García, Felipe; Hurtado, Carmen; Nieto-Márquez, Sara; León, Agathe; García, M Teresa; Rovira, Cristina; Miralles, Laia; Dalmau, Judith; Pumarola, Tomás; Almela, Manel; Martinez-Picado, Javier; Lifson, Jeffrey D; Zamora, Laura; Miró, José M; Brander, Christian; Clotet, Bonaventura; Gallart, Teresa; Gatell, José M

    2011-08-05

    This study provides a detailed description and characterization of the preparation of individualized lots of autologous heat inactivated HIV-1 virions used as immunogen in a clinical trial designed to test an autologous dendritic-cell-based therapeutic HIV-1 vaccine (Clinical Trial DCV-2, NCT00402142). For each participant, ex vivo isolation and expansion of primary virus were performed by co-culturing CD4-enriched PBMCs from the HIV-1-infected patient with PBMC from HIV-seronegative unrelated healthy volunteer donors. The viral supernatants were heat-inactivated and concentrated to obtain 1 mL of autologous immunogen, which was used to load autologous dendritic cells of each patient. High sequence homology was found between the inactivated virus immunogen and the HIV-1 circulating in plasma at the time of HIV-1 isolation. Immunogens contained up to 10⁹ HIV-1 RNA copies/mL showed considerably reduced infectivity after heat inactivation (median of 5.6 log₁₀), and were free of specified adventitious agents. The production of individualized lots of immunogen based on autologous inactivated HIV-1 virus fulfilling clinical-grade good manufacturing practice proved to be feasible, consistent with predetermined specifications, and safe for use in a clinical trial designed to test autologous dendritic cell-based therapeutic HIV-1 vaccine.

  15. Shedding of HIV-1 subtype E in semen and cervico-vaginal fluid.

    PubMed

    Sutthent, R; Chaisilwattana, P; Roongpisuthipong, A; Wirachsilp, P; Samrangsarp, K; Chaiyakul, P; Puthavathana, P; Wasi, C

    1997-06-01

    The uneven expansion of HIV-1 subtypes in each transmitted group raises the possibility that some viruses have less/more potential by qualitative/quantitative for heterosexual transmission compared to others. In Thailand, HIV-1 subtype E is mainly spread via heterosexual route and accounts for about 95 per cent of the infected cases. To determine whether high sexual infectivity of HIV-1 subtype E is due to the presence of a virus in genital fluid, we conducted a study to characterize shedding of HIV-1 in seminal and cervico-vaginal fluids of 30 HIV-1 subtype E infected Thai couples by PCR and virus isolation methods. All subjects had no HIV-associated diseases and other sexually transmitted diseases. HIV-1 subtype E DNA was detected in 22/30 (77.33%) of cervico-vaginal and also 22/30 (77.33%) of seminal fluid samples. The isolation rate of HIV-1 from semen and cervico-vaginal secretion was 36.67 per cent and 16.67 per cent, respectively. Number of HIV-1 subtype E DNA copies in the blood is reversely correlated with the number of blood CD4+ T cells, while that in genital fluid was not related to CD4+ T cell count. An increase in shedding of HIV- DNA subtype E in female genital tract compared to other HIV subtypes reported by other investigators might be one reason to explain the rapid spread of subtype E by heterosexual transmission in Thailand.

  16. 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.

  17. Willingness of Kenyan HIV-1 serodiscordant couples to use antiretroviral based HIV-1 prevention strategies

    PubMed Central

    Heffron, Renee; Ngure, Kenneth; Mugo, Nelly; Celum, Connie; Kurth, Ann; Curran, Kathryn; Baeten, Jared M.

    2012-01-01

    Introduction Antiretroviral treatment (ART) and pre-exposure prophylaxis (PrEP) have demonstrated efficacy as new HIV-1 prevention approaches for HIV-1 serodiscordant couples. Methods Among Kenyan HIV-1 serodiscordant heterosexual couples participating in a clinical trial of PrEP, we conducted a cross-sectional study and used descriptive statistical methods to explore couples' willingness to use antiretrovirals for HIV-1 prevention. The study was conducted prior to July 2011, when studies among heterosexual populations reported that ART and PrEP reduced HIV-1 risk. Results For 181 couples in which the HIV-1 infected partner had a CD4 count ≥350 cells/μL and had not yet initiated ART (and thus did not qualify for ART under Kenyan guidelines), 60.2% of HIV-1 infected partners (69.4% of men and 57.9% of women) were willing to use early ART (at CD4 ≥350 cells/μL) for HIV-1 prevention. Among HIV-1 uninfected partners, 92.7% (93.8% of men and 86.1% of women) reported willingness to use PrEP. When given a hypothetical choice of early ART or PrEP for HIV-1 prevention, 52.5% of HIV-1 infected participants would prefer to initiate ART early and 56.9% of HIV-1 uninfected participants would prefer to use PrEP. Conclusions Nearly 40% of Kenyan HIV-1 infected individuals in known HIV-1 serodiscordant partnerships reported reservations about early ART initiation for HIV-1 prevention. PrEP interest in this PrEP-experienced population was high. Strategies to achieve high uptake and sustained adherence to ART and PrEP for HIV-1 prevention in HIV-1 serodiscordant couples will require responding to couples' preferences for prevention strategies. PMID:22595872

  18. Identification of dual-tropic HIV-1 using evolved neural networks.

    PubMed

    Fogel, Gary B; Lamers, Susanna L; Liu, Enoch S; Salemi, Marco; McGrath, Michael S

    2015-11-01

    Blocking the binding of the envelope HIV-1 protein to immune cells is a popular concept for development of anti-HIV therapeutics. R5 HIV-1 binds CCR5, X4 HIV-1 binds CXCR4, and dual-tropic HIV-1 can bind either coreceptor for cellular entry. R5 viruses are associated with early infection and over time can evolve to X4 viruses that are associated with immune failure. Dual-tropic HIV-1 is less studied; however, it represents functional antigenic intermediates during the transition of R5 to X4 viruses. Viral tropism is linked partly to the HIV-1 envelope V3 domain, where the amino acid sequence helps dictate the receptor a particular virus will target; however, using V3 sequence information to identify dual-tropic HIV-1 isolates has remained difficult. Our goal in this study was to elucidate features of dual-tropic HIV-1 isolates that assist in the biological understanding of dual-tropism and develop an approach for their detection. Over 1559 HIV-1 subtype B sequences with known tropisms were analyzed. Each sequence was represented by 73 structural, biochemical and regional features. These features were provided to an evolved neural network classifier and evaluated using balanced and unbalanced data sets. The study resolved R5X4 viruses from R5 with an accuracy of 81.8% and from X4 with an accuracy of 78.8%. The approach also identified a set of V3 features (hydrophobicity, structural and polarity) that are associated with tropism transitions. The ability to distinguish R5X4 isolates will improve computational tropism decisions for R5 vs. X4 and assist in HIV-1 research and drug development efforts.

  19. 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.

  20. Cytopathic Mechanisms of HIV-1

    PubMed Central

    Costin, Joshua M

    2007-01-01

    The human immunodeficiency virus type 1 (HIV-1) has been intensely investigated since its discovery in 1983 as the cause of acquired immune deficiency syndrome (AIDS). With relatively few proteins made by the virus, it is able to accomplish many tasks, with each protein serving multiple functions. The Envelope glycoprotein, composed of the two noncovalently linked subunits, SU (surface glycoprotein) and TM (transmembrane glycoprotein) is largely responsible for host cell recognition and entry respectively. While the roles of the N-terminal residues of TM is well established as a fusion pore and anchor for Env into cell membranes, the role of the C-terminus of the protein is not well understood and is fiercely debated. This review gathers information on TM in an attempt to shed some light on the functional regions of this protein. PMID:17945027

  1. Cytopathic mechanisms of HIV-1.

    PubMed

    Costin, Joshua M

    2007-10-18

    The human immunodeficiency virus type 1 (HIV-1) has been intensely investigated since its discovery in 1983 as the cause of acquired immune deficiency syndrome (AIDS). With relatively few proteins made by the virus, it is able to accomplish many tasks, with each protein serving multiple functions. The Envelope glycoprotein, composed of the two noncovalently linked subunits, SU (surface glycoprotein) and TM (transmembrane glycoprotein) is largely responsible for host cell recognition and entry respectively. While the roles of the N-terminal residues of TM is well established as a fusion pore and anchor for Env into cell membranes, the role of the C-terminus of the protein is not well understood and is fiercely debated. This review gathers information on TM in an attempt to shed some light on the functional regions of this protein.

  2. Breast milk transmission of HIV-1.

    PubMed

    Nduati, R; John, G

    1995-12-01

    Breast milk provides infants and children immunologic, nutritional, and child spacing benefits. Yet it also transmits some viruses, for example, HIV-1. The World Health Organization recommends that, in conditions with poor access to breast milk substitutes, HIV-positive women should still breast feed due to the nutritional and infectious risk of artificial feeding. It appears that breast fed infants experience a slower progression of AIDS and death. Vertical transmission of HIV-1 may occur during pregnancy, at delivery, or through breast milk. The HIV-1 transmission rate via breast milk from acutely infected women is estimated to be 29-36%. A meta-analysis of case reports and small case series of women with chronic HIV-1 infection indicated a breast feeding transmission rate of 14%. Studies suggest that the likelihood of HIV-1 transmission via breast milk increases as duration of breast feeding increases. Infants with detectable HIV-1 DNA tend to have mothers whose absolute CD4 counts are less than 400 and have severe vitamin A deficiency. Breast milk has HIV-1 specific immunoglobulins (IgG, IgA, and IgM). It appears that HIV-1 elicits a local immune response. Breast milk of HIV-1 positive mothers with non-infected children tends to still have IgM and IgA until 18 months. Potential risk factors for breast milk transmission of HIV-1 include cracked nipples and mastitis in the mother; oral thrush, malnutrition, inflammation of the lips, and mucosal compromise in the infant; and vigorous suction of the neonate and use of the wrong equipment for suctioning. Inhibiting factors of HIV-1 in breast milk are bovine and human lactoferrin and a membrane associated protein that attaches to the CD4 receptor and thus prevents attachment of the HIV antigen gp120 to the CD4 receptor on T-cells.

  3. 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

  4. B-cell-lineage immunogen design in vaccine development with HIV-1 as a case study.

    PubMed

    Haynes, Barton F; Kelsoe, Garnett; Harrison, Stephen C; Kepler, Thomas B

    2012-05-07

    Failure of immunization with the HIV-1 envelope to induce broadly neutralizing antibodies against conserved epitopes is a major barrier to producing a preventive HIV-1 vaccine. Broadly neutralizing monoclonal antibodies (BnAbs) from those subjects who do produce them after years of chronic HIV-1 infection have one or more unusual characteristics, including polyreactivity for host antigens, extensive somatic hypermutation and long, variable heavy-chain third complementarity-determining regions, factors that may limit their expression by host immunoregulatory mechanisms. The isolation of BnAbs from HIV-1-infected subjects and the use of computationally derived clonal lineages as templates provide a new path for HIV-1 vaccine immunogen design. This approach, which should be applicable to many infectious agents, holds promise for the construction of vaccines that can drive B cells along rare but desirable maturation pathways.

  5. Evidence of at least two introductions of HIV-1 in the Amerindian Warao population from Venezuela.

    PubMed

    Rangel, Héctor R; Maes, Mailis; Villalba, Julian; Sulbarán, Yoneira; de Waard, Jacobus H; Bello, Gonzalo; Pujol, Flor H

    2012-01-01

    The Venezuelan Amerindians were, until recently, free of human immunodeficiency virus (HIV) infection. However, in 2007, HIV-1 infection was detected for the first time in the Warao Amerindian population living in the Eastern part of Venezuela, in the delta of the Orinoco river. The aim of this study was to analyze the genetic diversity of the HIV-1 circulating in this population. The pol genomic region was sequenced for 16 HIV-1 isolates and for some of them, sequences from env, vif and nef genomic regions were obtained. All HIV-1 isolates were classified as subtype B, with exception of one that was classified as subtype C. The 15 subtype B isolates exhibited a high degree of genetic similarity and formed a highly supported monophyletic cluster in each genomic region analyzed. Evolutionary analyses of the pol genomic region indicated that the date of the most recent common ancestor of the Waraos subtype B clade dates back to the late 1990s. At least two independent introductions of HIV-1 have occurred in the Warao Amerindians from Venezuela. The HIV-1 subtype B was successfully established and got disseminated in the community, while no evidence of local dissemination of the HIV-1 subtype C was detected in this study. These results warrant further surveys to evaluate the burden of this disease, which can be particularly devastating in this Amerindian population, with a high prevalence of tuberculosis, hepatitis B, among other infectious diseases, and with limited access to primary health care.

  6. Characterizing HIV-1 Splicing by Using Next-Generation Sequencing.

    PubMed

    Emery, Ann; Zhou, Shuntai; Pollom, Elizabeth; Swanstrom, Ronald

    2017-03-15

    Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of

  7. Epitope-vaccine strategy against HIV-1: today and tomorrow.

    PubMed

    Liu, Zuqiang; Xiao, Yi; Chen, Ying-Hua

    2003-01-01

    Vaccines play important roles in preventing infectious diseases caused by different pathogens. However, some pathogens such as HIV-1 challenge current vaccine strategy. Poor immunogenicity and the high mutation rate of HIV-1 make great difficulties in inducing potent immune responses strong enough to prevent infection via vaccination. Epitope-vaccine, which could intensively enhance predefined epitope-specific immune responses, was suggested as a new strategy against HIV-1 and HIV-1 mutation. Epitope-vaccines afford powerful approaches to elicit potent, broad and complete immune protection against not only primary homologous viral isolates but also heterologous viral mutants. Although most studies are still preliminary now, epitope-vaccine as a novel strategy against the AIDS epidemic has great developmental potential. To trigger T-cell-dependent IgG antibody responses and improve affinities of the epitope-specific antibodies, approaches such as recombinant multi-epitope-vaccination and prime-boosting vaccination were suggested. Cellular immune responses, especially CTL responses, could also be elicited and enhanced in addition to humoral immune responses. Developed epitope-vaccines activating both arms of the immune system would benefit prevention and immunotherapy not only against HIV but also other chronic infections.

  8. Quantifying Ongoing HIV-1 Exposure in HIV-1–Serodiscordant Couples to Identify Individuals With Potential Host Resistance to HIV-1

    PubMed Central

    Mackelprang, Romel D.; Baeten, Jared M.; Donnell, Deborah; Celum, Connie; Farquhar, Carey; de Bruyn, Guy; Essex, Max; McElrath, M. Juliana; Nakku-Joloba, Edith; Lingappa, Jairam R.

    2012-01-01

    Background. Immunogenetic correlates of resistance to HIV-1 in HIV-1–exposed seronegative (HESN) individuals with consistently high exposure may inform HIV-1 prevention strategies. We developed a novel approach for quantifying HIV-1 exposure to identify individuals remaining HIV-1 uninfected despite persistent high exposure. Methods. We used longitudinal predictors of HIV-1 transmission in HIV-1 serodiscordant couples to score HIV-1 exposure and define HESN clusters with persistently high, low, and decreasing risk trajectories. The model was validated in an independent cohort of serodiscordant couples. We describe a statistical tool that can be applied to other HESN cohorts to identify individuals with high exposure to HIV-1. Results. HIV-1 exposure was best quantified by frequency of unprotected sex with, plasma HIV-1 RNA levels among, and presence of genital ulcer disease among HIV-1–infected partners and by age, pregnancy status, herpes simplex virus 2 serostatus, and male circumcision status among HESN participants. Overall, 14% of HESN individuals persistently had high HIV-1 exposure and exhibited a declining incidence of HIV-1 infection over time. Conclusions. A minority of HESN individuals from HIV-1–discordant couples had persistent high HIV-1 exposure over time. Decreasing incidence of infection in this group suggests these individuals were selected for resistance to HIV-1 and may be most appropriate for identifying biological correlates of natural host resistance to HIV-1 infection. PMID:22926009

  9. Contribution of Epidemiological Predictors in Unraveling the Phylogeographic History of HIV-1 Subtype C in Brazil

    PubMed Central

    Vrancken, Bram; Maletich Junqueira, Dennis; de Medeiros, Rúbia Marília; Suchard, Marc A.; Lemey, Philippe; Esteves de Matos Almeida, Sabrina; Pinto, Aguinaldo Roberto

    2015-01-01

    ABSTRACT The phylogeographic history of the Brazilian HIV-1 subtype C (HIV-1C) epidemic is still unclear. Previous studies have mainly focused on the capital cities of Brazilian federal states, and the fact that HIV-1C infections increase at a higher rate than subtype B infections in Brazil calls for a better understanding of the process of spatial spread. A comprehensive sequence data set sampled across 22 Brazilian locations was assembled and analyzed. A Bayesian phylogeographic generalized linear model approach was used to reconstruct the spatiotemporal history of HIV-1C in Brazil, considering several potential explanatory predictors of the viral diffusion process. Analyses were performed on several subsampled data sets in order to mitigate potential sample biases. We reveal a central role for the city of Porto Alegre, the capital of the southernmost state, in the Brazilian HIV-1C epidemic (HIV-1C_BR), and the northward expansion of HIV-1C_BR could be linked to source populations with higher HIV-1 burdens and larger proportions of HIV-1C infections. The results presented here bring new insights to the continuing discussion about the HIV-1C epidemic in Brazil and raise an alternative hypothesis for its spatiotemporal history. The current work also highlights how sampling bias can confound phylogeographic analyses and demonstrates the importance of incorporating external information to protect against this. IMPORTANCE Subtype C is responsible for the largest HIV infection burden worldwide, but our understanding of its transmission dynamics remains incomplete. Brazil witnessed a relatively recent introduction of HIV-1C compared to HIV-1B, but it swiftly spread throughout the south, where it now circulates as the dominant variant. The northward spread has been comparatively slow, and HIV-1B still prevails in that region. While epidemiological data and viral genetic analyses have both independently shed light on the dynamics of spread in isolation, their combination

  10. Contribution of Epidemiological Predictors in Unraveling the Phylogeographic History of HIV-1 Subtype C in Brazil.

    PubMed

    Gräf, Tiago; Vrancken, Bram; Maletich Junqueira, Dennis; de Medeiros, Rúbia Marília; Suchard, Marc A; Lemey, Philippe; Esteves de Matos Almeida, Sabrina; Pinto, Aguinaldo Roberto

    2015-12-01

    The phylogeographic history of the Brazilian HIV-1 subtype C (HIV-1C) epidemic is still unclear. Previous studies have mainly focused on the capital cities of Brazilian federal states, and the fact that HIV-1C infections increase at a higher rate than subtype B infections in Brazil calls for a better understanding of the process of spatial spread. A comprehensive sequence data set sampled across 22 Brazilian locations was assembled and analyzed. A Bayesian phylogeographic generalized linear model approach was used to reconstruct the spatiotemporal history of HIV-1C in Brazil, considering several potential explanatory predictors of the viral diffusion process. Analyses were performed on several subsampled data sets in order to mitigate potential sample biases. We reveal a central role for the city of Porto Alegre, the capital of the southernmost state, in the Brazilian HIV-1C epidemic (HIV-1C_BR), and the northward expansion of HIV-1C_BR could be linked to source populations with higher HIV-1 burdens and larger proportions of HIV-1C infections. The results presented here bring new insights to the continuing discussion about the HIV-1C epidemic in Brazil and raise an alternative hypothesis for its spatiotemporal history. The current work also highlights how sampling bias can confound phylogeographic analyses and demonstrates the importance of incorporating external information to protect against this. Subtype C is responsible for the largest HIV infection burden worldwide, but our understanding of its transmission dynamics remains incomplete. Brazil witnessed a relatively recent introduction of HIV-1C compared to HIV-1B, but it swiftly spread throughout the south, where it now circulates as the dominant variant. The northward spread has been comparatively slow, and HIV-1B still prevails in that region. While epidemiological data and viral genetic analyses have both independently shed light on the dynamics of spread in isolation, their combination has not yet been

  11. HIV-1 epidemic in Warao Amerindians from Venezuela: spatial phylodynamics and epidemiological patterns.

    PubMed

    Villalba, Julian A; Bello, Gonzalo; Maes, Mailis; Sulbaran, Yoneira F; Garzaro, Domingo; Loureiro, Carmen L; Rangel, Hector R; de Waard, Jacobus H; Pujol, Flor H

    2013-07-17

    We previously reported HIV-1 infection in Warao Amerindians from Venezuela. The aim of this study was to evaluate the extent and the dynamic of HIV-1 dissemination in eight Warao communities. HIV-1 infection was evaluated in 576 Warao Amerindians from the Orinoco Delta. Partial HIV-1 pol sequences were analyzed to reconstruct the spatiotemporal and demographic dynamics of the epidemic. HIV-1 antibodies were present in 9.55% of Warao Amerindians, ranging from 0 to 22%. A significantly higher prevalence was found in men (15.6%) compared with women (2.6%), reaching up to 35% in men from one community. All but one isolates were classified as subtype B. Warao's HIV-1 subtype-B epidemic resulted from a single viral introduction at around the early 2000s. After an initial phase of slow growth, the subtype B started to spread at a fast rate (0.8/year) following two major routes of migration within the communities. A dramatic high prevalence was documented in almost all the communities of Warao Amerindians from the Orinoco Delta tested for HIV-1 infection. This epidemic resulted from the dissemination of a single HIV-1 subtype B founder strain introduced about 10 years ago and its size is probably doubling every year, creating a situation that can be devastating for this vulnerable Amerindian group.

  12. Replication potentials of HIV-1/HSIV in PBMCs from northern pig-tailed macaque (Macaca leonina).

    PubMed

    Lei, Ai-Hua; Zhang, Gao-Hong; Tian, Ren-Rong; Zhu, Jia-Wu; Zheng, Hong-Yi; Pang, Wei; Zheng, Yong-Tang

    2014-05-01

    The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 (HIV-1), which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-1NL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of vif-substituted HIV-1 (HSIV) in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-1SV chimeras, two HIV-1NL4-3-derived viruses encoding the viral infectivity factor (Vif) protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS.

  13. Replication potentials of HIV-1/HSIV in PBMCs from northern pigtailed macaque (Macaca leonina)

    PubMed Central

    LEI, Ai-Hua; ZHANG, Gao-Hong; TIAN, Ren-Rong; ZHU, Jia-Wu; ZHENG, Hong-Yi; PANG, Wei; ZHENG, Yong-Tang

    2014-01-01

    The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 (HIV-1), which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-1NL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of vif-substituted HIV-1 (HSIV) in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-1SV chimeras, two HIV-1NL4-3-derived viruses encoding the viral infectivity factor (Vif) protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS. PMID:24866489

  14. Cocaine Enhances HIV-1–Induced CD4+ T-Cell Apoptosis

    PubMed Central

    Pandhare, Jui; Addai, Amma B.; Mantri, Chinmay K.; Hager, Cynthia; Smith, Rita M.; Barnett, Louis; Villalta, Fernando; Kalams, Spyros A.; Dash, Chandravanu

    2015-01-01

    Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1–associated CD4+ T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4+ T cells from HIV-1–negative and HIV-1–positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4+ T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4+ T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4+ T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4+ T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1–infected drug abusers. PMID:24486327

  15. 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.

  16. 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.

  17. Uneven Genetic Robustness of HIV-1 Integrase

    PubMed Central

    Rihn, Suzannah J.; Hughes, Joseph; Wilson, Sam J.

    2014-01-01

    ABSTRACT Genetic robustness (tolerance of mutation) may be a naturally selected property in some viruses, because it should enhance adaptability. Robustness should be especially beneficial to viruses like HIV-1 that exhibit high mutation rates and exist in immunologically hostile environments. Surprisingly, however, the HIV-1 capsid protein (CA) exhibits extreme fragility. To determine whether fragility is a general property of HIV-1 proteins, we created a large library of random, single-amino-acid mutants in HIV-1 integrase (IN), covering >40% of amino acid positions. Despite similar degrees of sequence variation in naturally occurring IN and CA sequences, we found that HIV-1 IN was significantly more robust than CA, with random nonsilent IN mutations only half as likely to cause lethal defects. Interestingly, IN and CA were similar in that a subset of mutations with high in vitro fitness were rare in natural populations. IN mutations of this type were more likely to occur in the buried interior of the modeled HIV-1 intasome, suggesting that even very subtle fitness effects suppress variation in natural HIV-1 populations. Lethal mutations, in particular those that perturbed particle production, proteolytic processing, and particle-associated IN levels, were strikingly localized at specific IN subunit interfaces. This observation strongly suggests that binding interactions between particular IN subunits regulate proteolysis during HIV-1 virion morphogenesis. Overall, use of the IN mutant library in conjunction with structural models demonstrates the overall robustness of IN and highlights particular regions of vulnerability that may be targeted in therapeutic interventions. IMPORTANCE The HIV-1 integrase (IN) protein is responsible for the integration of the viral genome into the host cell chromosome. To measure the capacity of IN to maintain function in the face of mutation, and to probe structure/function relationships, we created a library of random single

  18. Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Naïve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

    PubMed Central

    Vigil, Karen J.; Vey, Elana; Arduino, Roberto C.; Kimata, Jason T.

    2012-01-01

    Background Xenotropic murine leukemia virus-related virus (XMRV) has been found in the prostatic tissue of prostate cancer patients and in the blood of chronic fatigue syndrome patients. However, numerous studies have found little to no trace of XMRV in different human cohorts. Based on evidence suggesting common transmission routes between XMRV and HIV-1, HIV-1 infected individuals may represent a high-risk group for XMRV infection and spread. Methodology/Principal Findings DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of 179 HIV-1 infected treatment naïve patients, 86 of which were coinfected with HCV, and 54 healthy blood donors. DNA was screened for XMRV provirus with two sensitive, published PCR assays targeting XMRV gag and env and one sensitive, published nested PCR assay targeting env. Detection of XMRV was confirmed by DNA sequencing. One of the 179 HIV-1 infected patients tested positive for gag by non-nested PCR whereas the two other assays did not detect XMRV in any specimen. All healthy blood donors were negative for XMRV proviral sequences. Sera from 23 HIV-1 infected patients (15 HCV+) and 12 healthy donors were screened for the presence of XMRV-reactive antibodies by Western blot. Thirteen sera (57%) from HIV-1+ patients and 6 sera (50%) from healthy donors showed reactivity to XMRV-infected cell lysate. Conclusions/Significance The virtual absence of XMRV in PBMCs suggests that XMRV is not associated with HIV-1 infected or HIV-1/HCV coinfected patients, or blood donors. Although we noted isolated incidents of serum reactivity to XMRV, we are unable to verify the antibodies as XMRV specific. PMID:22348082

  19. Correlation of Naturally Occurring HIV-1 Resistance to DEB025 with Capsid Amino Acid Polymorphisms

    PubMed Central

    Gallay, Philippe A.; Ptak, Roger G.; Bobardt, Michael D.; Dumont, Jean-Maurice; Vuagniaux, Grégoire; Rosenwirth, Brigitte

    2013-01-01

    DEB025 (alisporivir) is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV). It binds to cyclophilin A (CypA) and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA) region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96), as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent. PMID:23524389

  20. 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.

  1. Lessons from HIV-1 vaccine efficacy trials.

    PubMed

    Excler, Jean-Louis; Michael, Nelson L

    2016-11-01

    Only four HIV-1 vaccine concepts have been tested in six efficacy trials with no product licensed to date. Several scientific and programmatic lessons can be learned from these studies generating new hypotheses and guiding future steps. RV144 [ALVAC-HIV (canarypox vector) and AIDSVAX B/E (bivalent gp120 HIV-1 subtype B and CRF01_AE)] remains the only efficacy trial that demonstrated a modest vaccine efficacy, which led to the identification of immune correlates of risk. Progress on subtype-specific, ALVAC (canarypox vector) and gp120 vaccine prime-boost approaches has been slow, but we are finally close to the launch of an efficacy study in Africa in 2016. The quest of a globally effective HIV-1 vaccine has led to the development of new approaches. Efficacy studies of combinations of Adenovirus type 26 (Ad26)/Modified Vaccinia Ankara (MVA)/gp140 vaccines with mosaic designs will enter efficacy studies mid-2017 and cytomegalovirus (CMV)-vectored vaccines begin Phase I studies at the same time. Future HIV-1 vaccine efficacy trials face practical challenges as effective nonvaccine prevention programs are projected to decrease HIV-1 incidence. An HIV-1 vaccine is urgently needed. Increased industry involvement, mobilization of resources, expansion of a robust pipeline of new concepts, and robust preclinical challenge studies will be essential to accelerate efficacy testing of next generation HIV-1 vaccine candidates.

  2. Unexplained diarrhoea in HIV-1 infected individuals

    PubMed Central

    2014-01-01

    Background Gastrointestinal symptoms, in particular diarrhoea, are common in non-treated HIV-1 infected individuals. Although various enteric pathogens have been implicated, the aetiology of diarrhoea remains unexplained in a large proportion of HIV-1 infected patients. Our aim is to identify the cause of diarrhoea for patients that remain negative in routine diagnostics. Methods In this study stool samples of 196 HIV-1 infected persons, including 29 persons with diarrhoea, were examined for enteropathogens and HIV-1. A search for unknown and unexpected viruses was performed using virus discovery cDNA-AFLP combined with Roche-454 sequencing (VIDISCA-454). Results HIV-1 RNA was detected in stool of 19 patients with diarrhoea (66%) compared to 75 patients (45%) without diarrhoea. In 19 of the 29 diarrhoea cases a known enteropathogen could be identified (66%). Next to these known causative agents, a range of recently identified viruses was identified via VIDISCA-454: cosavirus, Aichi virus, human gyrovirus, and non-A non-B hepatitis virus. Moreover, a novel virus was detected which was named immunodeficiency-associated stool virus (IASvirus). However, PCR based screening for these viruses showed that none of these novel viruses was associated with diarrhoea. Notably, among the 34% enteropathogen-negative cases, HIV-1 RNA shedding in stool was more frequently observed (80%) compared to enteropathogen-positive cases (47%), indicating that HIV-1 itself is the most likely candidate to be involved in diarrhoea. Conclusion Unexplained diarrhoea in HIV-1 infected patients is probably not caused by recently described or previously unknown pathogens, but it is more likely that HIV-1 itself plays a role in intestinal mucosal abnormalities which leads to diarrhoea. PMID:24410947

  3. Disulfiram reactivates latent HIV-1 expression through depletion of the phosphatase and tensin homolog.

    PubMed

    Doyon, Geneviève; Zerbato, Jennifer; Mellors, John W; Sluis-Cremer, Nicolas

    2013-01-14

    Disulfiram (DSF), an inhibitor of acetaldehyde dehydrogenase that is used for the treatment of alcoholism, was shown to reactivate latent HIV-1 expression in a primary cell model of virus latency and is currently being assessed in a clinical trial for its potential to deplete the latent HIV-1 reservoir in patients on combination antiretroviral therapy. The mechanism by which DSF reactivates latent HIV-1 expression, however, is not known and was the focus of this study. The impact of DSF treatment on HIV-1 latency was assessed in the ACH2, J89GFP and U1 cell line models of HIV-1 latency and in resting CD4 T cells isolated from HIV-negative donors. DSF reactivated latent HIV-1 expression in the U1 cell line, but not in the J89GFP or ACH2 cell lines. Interestingly, we found that DSF significantly reduced phosphatase and tensin homolog (PTEN) protein levels in U1 cells and in resting CD4 T cells from HIV-negative donors. Decreased PTEN resulted in increased phosphorylation of protein kinase B (Akt) and activation of the Akt signaling pathway. Consistent with these finding, pharmacological inhibitors of Akt and nuclear factor-kappaB (NF-κB) block the latent HIV-1-reactivating activity of DSF. Furthermore, we show that HIV-1 expression in the U1 cell line could be activated by a small molecule inhibitor of PTEN or by siRNA knockdown of PTEN expression. Neither the J89GFP nor ACH2 cells express PTEN, explaining the lack of DSF effect on HIV-1 expression in both these cell lines. DSF reactivates latent HIV-1 expression via the Akt signaling pathway through depletion of PTEN.

  4. Blocking HIV-1 entry by a gp120 surface binding inhibitor

    PubMed Central

    Tsou, Lun K.; Chen, Chin-Ho; Dutschman, Ginger E.

    2012-01-01

    We report the mode of action of a proteomimetic compound that binds to the exterior surface of gp120 and blocks HIV-1 entry into cells. Using a one cycle time-of-addition study and antibody competition binding studies, we have determined that the compound blocks HIV-1 entry through modulation of key protein-protein interactions mediated by gp120. The compound exhibits anti-HIV-1 replication activities against several pseudotype viruses derived from primary isolates and the resistant strains isolated from existing drug candidates with equal potency. Together, these data provide evidence that the proteomimetic compound represents a novel class of HIV-1 viral entry inhibitor that functions through protein surface recognition in analogy to an antibody. PMID:22487177

  5. Neuroinflammation and Behavior in HIV-1 Transgenic Rats Exposed to Chronic Adolescent Stress

    PubMed Central

    Rowson, Sydney A.; Harrell, Constance S.; Bekhbat, Mandakh; Gangavelli, Apoorva; Wu, Matthew J.; Kelly, Sean D.; Reddy, Renuka; Neigh, Gretchen N.

    2016-01-01

    Highly active antiretroviral therapy (HAART) has improved prognosis for people living with HIV (PLWH) and dramatically reduced the incidence of AIDS. However, even when viral load is controlled, PLWH develop psychiatric and neurological disorders more frequently than those living without HIV. Adolescents with HIV are particularly susceptible to the development of psychiatric illnesses and neurocognitive impairments. While both psychiatric and neurocognitive disorders have been found to be exacerbated by stress, the extent to which chronic stress and HIV-1 viral proteins interact to impact behavior and relevant neuroinflammatory processes is unknown. Determination of the individual contributions of stress and HIV to neuropsychiatric disorders is heavily confounded in humans. In order to isolate the influence of HIV-1 proteins and chronic stress on behavior and neuroinflammation, we employed the HIV-1 transgenic (Tg) rat model, which expresses HIV-1 proteins with a gag and pol deletion, allowing for viral protein expression without viral replication. This Tg line has been characterized as a model of HAART-controlled HIV-1 infection due to the lack of viral replication but continued presence of HIV-1 proteins. We exposed male and female adolescent HIV-1 Tg rats to a mixed-modality chronic stress paradigm consisting of isolation, social defeat and restraint, and assessed behavior, cerebral vascularization, and neuroinflammatory endpoints. Stress, sex, and presence of the HIV-1 transgene impacted weight gain in adolescent rats. Female HIV-1 Tg rats showed decreases in central tendency during the light cycle in the open field regardless of stress exposure. Both male and female HIV-1 Tg rats exhibited decreased investigative behavior in the novel object recognition task, but no memory impairments. Adolescent stress had no effect on the tested behaviors. Microglia in female HIV-1 Tg rats exhibited a hyper-ramified structure, and gene expression of complement factor B was

  6. 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

  7. Neuroinflammation and Behavior in HIV-1 Transgenic Rats Exposed to Chronic Adolescent Stress.

    PubMed

    Rowson, Sydney A; Harrell, Constance S; Bekhbat, Mandakh; Gangavelli, Apoorva; Wu, Matthew J; Kelly, Sean D; Reddy, Renuka; Neigh, Gretchen N

    2016-01-01

    Highly active antiretroviral therapy (HAART) has improved prognosis for people living with HIV (PLWH) and dramatically reduced the incidence of AIDS. However, even when viral load is controlled, PLWH develop psychiatric and neurological disorders more frequently than those living without HIV. Adolescents with HIV are particularly susceptible to the development of psychiatric illnesses and neurocognitive impairments. While both psychiatric and neurocognitive disorders have been found to be exacerbated by stress, the extent to which chronic stress and HIV-1 viral proteins interact to impact behavior and relevant neuroinflammatory processes is unknown. Determination of the individual contributions of stress and HIV to neuropsychiatric disorders is heavily confounded in humans. In order to isolate the influence of HIV-1 proteins and chronic stress on behavior and neuroinflammation, we employed the HIV-1 transgenic (Tg) rat model, which expresses HIV-1 proteins with a gag and pol deletion, allowing for viral protein expression without viral replication. This Tg line has been characterized as a model of HAART-controlled HIV-1 infection due to the lack of viral replication but continued presence of HIV-1 proteins. We exposed male and female adolescent HIV-1 Tg rats to a mixed-modality chronic stress paradigm consisting of isolation, social defeat and restraint, and assessed behavior, cerebral vascularization, and neuroinflammatory endpoints. Stress, sex, and presence of the HIV-1 transgene impacted weight gain in adolescent rats. Female HIV-1 Tg rats showed decreases in central tendency during the light cycle in the open field regardless of stress exposure. Both male and female HIV-1 Tg rats exhibited decreased investigative behavior in the novel object recognition task, but no memory impairments. Adolescent stress had no effect on the tested behaviors. Microglia in female HIV-1 Tg rats exhibited a hyper-ramified structure, and gene expression of complement factor B was

  8. 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.

  9. The global spread of HIV-1 subtype B epidemic.

    PubMed

    Magiorkinis, Gkikas; Angelis, Konstantinos; Mamais, Ioannis; Katzourakis, Aris; Hatzakis, Angelos; Albert, Jan; Lawyer, Glenn; Hamouda, Osamah; Struck, Daniel; Vercauteren, Jurgen; Wensing, Annemarie; Alexiev, Ivailo; Åsjö, Birgitta; Balotta, Claudia; Gomes, Perpétua; Camacho, Ricardo J; Coughlan, Suzie; Griskevicius, Algirdas; Grossman, Zehava; Horban, Anders; Kostrikis, Leondios G; Lepej, Snjezana J; Liitsola, Kirsi; Linka, Marek; Nielsen, Claus; Otelea, Dan; Paredes, Roger; Poljak, Mario; Puchhammer-Stöckl, Elizabeth; Schmit, Jean Claude; Sönnerborg, Anders; Staneková, Danica; Stanojevic, Maja; Stylianou, Dora C; Boucher, Charles A B; Nikolopoulos, Georgios; Vasylyeva, Tetyana; Friedman, Samuel R; van de Vijver, David; Angarano, Gioacchino; Chaix, Marie-Laure; de Luca, Andrea; Korn, Klaus; Loveday, Clive; Soriano, Vincent; Yerly, Sabine; Zazzi, Mauricio; Vandamme, Anne-Mieke; Paraskevis, Dimitrios

    2016-12-01

    Human immunodeficiency virus type 1 (HIV-1) was discovered in the early 1980s when the virus had already established a pandemic. For at least three decades the epidemic in the Western World has been dominated by subtype B infections, as part of a sub-epidemic that traveled from Africa through Haiti to United States. However, the pattern of the subsequent spread still remains poorly understood. Here we analyze a large dataset of globally representative HIV-1 subtype B strains to map their spread around the world over the last 50years and describe significant spread patterns. We show that subtype B travelled from North America to Western Europe in different occasions, while Central/Eastern Europe remained isolated for the most part of the early epidemic. Looking with more detail in European countries we see that the United Kingdom, France and Switzerland exchanged viral isolates with non-European countries than with European ones. The observed pattern is likely to mirror geopolitical landmarks in the post-World War II era, namely the rise and the fall of the Iron Curtain and the European colonialism. In conclusion, HIV-1 spread through specific migration routes which are consistent with geopolitical factors that affected human activities during the last 50years, such as migration, tourism and trade. Our findings support the argument that epidemic control policies should be global and incorporate political and socioeconomic factors.

  10. Host factors dictate control of viral replication in two HIV-1 controller/chronic progressor transmission pairs.

    PubMed

    Buckheit, Robert W; Allen, Tracy G; Alme, Angela; Salgado, Maria; O'Connell, Karen A; Huculak, Sarah; Falade-Nwulia, Oluwaseun; Williams, Thomas M; Gallant, Joel E; Siliciano, Robert F; Blankson, Joel N

    2012-03-06

    Viremic controllers and elite controllers/suppressors maintain control over HIV-1 replication. Some studies have suggested that control is a result of infection with a defective viral strain, while others suggested host immune factors have a key role. Here we document two HIV-1 transmission pairs: one consisting of a patient with progressive disease and an individual who became an elite suppressor, and the second consisting of a patient with progressive disease and a viremic controller. In contrast to another elite suppressor transmission pair, virus isolated from all patients was fully competent. These data suggest that some viremic controllers and elite suppressors are infected with HIV-1 isolates that replicate vigorously in vitro and are able to cause progressive disease in vivo. These data suggest that host factors have a dominant role in the control of HIV-1 infection, thus it may be possible to control fully pathogenic HIV-1 isolates with therapeutic vaccination.

  11. Host Factors Dictate Control of Viral Replication in two HIV-1 Controller/Chronic Progressor Transmission Pairs

    PubMed Central

    Buckheit, Robert W.; Allen, Tracy G.; Alme, Angela; Salgado, Maria; O’Connell, Karen A.; Huculak, Sarah; Falade-Nwulia, Oluwaseun; Williams, Thomas M.; Gallant, Joel E.; Siliciano, Robert F.; Blankson, Joel N.

    2012-01-01

    Viremic controllers (VC) and elite controllers/suppressors (ES) maintain control over HIV-1 replication. Some studies suggested that control is a result of infection with a defective viral strain while others suggested host immune factors play a key role. Here we document two HIV-1 transmission pairs: one consisting of a patient with progressive disease and an individual who became an ES, and the second consisting of a patient with progressive disease and a VC. In contrast to another ES transmission pair, virus isolated from all patients was fully replication competent. These data suggests that some VC and ES are infected with HIV-1 isolates that replicate vigorously in vitro and are able to cause progressive disease in vivo. These data suggest that host factors play a dominant role in control of HIV-1 infection, thus it may be possible to control fully pathogenic HIV-1 isolates with therapeutic vaccination. PMID:22395607

  12. Women's barriers to HIV-1 testing and disclosure: challenges for HIV-1 voluntary counselling and testing.

    PubMed

    Maman, S; Mbwambo, J; Hogan, N M; Kilonzo, G P; Sweat, M

    2001-10-01

    In view of the ever-increasing HIV/AIDS epidemic in sub-Saharan Africa, the expansion of HIV-1 voluntary counselling and testing (VCT) as an integral part of prevention strategies and medical research is both a reality and an urgent need. As the availability of HIV-1 VCT grows two limitations need to be addressed, namely: low rates of HIV-1 serostatus disclosure to sexual partners and negative outcomes of serostatus disclosure. Results from a study among men, women and couples at an HIV-1 VCT clinic in Dar es Salaam, Tanzania are presented. The individual, relational and environmental factors that influence the decision to test for HIV-1 and to share test results with partners are described. The most salient barriers to HIV-1 testing and serostatus disclosure described by women include fear of partners' reaction, decision-making and communication patterns between partners, and partners' attitudes towards HIV-1 testing. Perception of personal risk for HIV-1 is the major factor driving women to overcome barriers to HIV-1 testing. The implications of findings for the promotion of HIV-1 VCT programmes, the implementation of partner notification policies and the development of post-test support services are discussed.

  13. Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection

    PubMed Central

    Hanauske-Abel, Hartmut M.; Saxena, Deepti; Palumbo, Paul E.; Hanauske, Axel-Rainer; Luchessi, Augusto D.; Cambiaghi, Tavane D.; Hoque, Mainul; Spino, Michael; Gandolfi, Darlene D'Alliessi; Heller, Debra S.; Singh, Sukhwinder; Park, Myung Hee; Cracchiolo, Bernadette M.; Tricta, Fernando; Connelly, John; Popowicz, Anthony M.; Cone, Richard A.; Holland, Bart; Pe’ery, Tsafi; Mathews, Michael B.

    2013-01-01

    HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal

  14. Human Mucosal Mast Cells Capture HIV-1 and Mediate Viral trans-Infection of CD4+ T Cells

    PubMed Central

    Jiang, Ai-Ping; Jiang, Jin-Feng; Wei, Ji-Fu; Guo, Ming-Gao; Qin, Yan; Guo, Qian-Qian; Ma, Li; Liu, Bao-Chi; Wang, Xiaolei; Veazey, Ronald S.

    2015-01-01

    ABSTRACT The gastrointestinal mucosa is the primary site where human immunodeficiency virus type 1 (HIV-1) invades, amplifies, and becomes persistently established, and cell-to-cell transmission of HIV-1 plays a pivotal role in mucosal viral dissemination. Mast cells are widely distributed in the gastrointestinal tract and are early targets for invasive pathogens, and they have been shown to have increased density in the genital mucosa in HIV-infected women. Intestinal mast cells express numerous pathogen-associated molecular patterns (PAMPs) and have been shown to combat various viral, parasitic, and bacterial infections. However, the role of mast cells in HIV-1 infection is poorly defined. In this study, we investigated their potential contributions to HIV-1 transmission. Mast cells isolated from gut mucosal tissues were found to express a variety of HIV-1 attachment factors (HAFs), such as DC-SIGN, heparan sulfate proteoglycan (HSPG), and α4β7 integrin, which mediate capture of HIV-1 on the cell surface. Intriguingly, following coculture with CD4+ T cells, mast cell surface-bound viruses were efficiently transferred to target T cells. Prior blocking with anti-HAF antibody or mannan before coculture impaired viral trans-infection. Cell-cell conjunctions formed between mast cells and T cells, to which viral particles were recruited, and these were required for efficient cell-to-cell HIV-1 transmission. Our results reveal a potential function of gut mucosal mast cells in HIV-1 dissemination in tissues. Strategies aimed at preventing viral capture and transfer mediated by mast cells could be beneficial in combating primary HIV-1 infection. IMPORTANCE In this study, we demonstrate the role of human mast cells isolated from mucosal tissues in mediating HIV-1 trans-infection of CD4+ T cells. This finding facilitates our understanding of HIV-1 mucosal infection and will benefit the development of strategies to combat primary HIV-1 dissemination. PMID:26719250

  15. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Improved quantification of HIV-1-infected CD4+ T cells using an optimised method of intracellular HIV-1 gag p24 antigen detection.

    PubMed

    Yang, Hongbing; Yorke, Elisabeth; Hancock, Gemma; Clutton, Genevieve; Sande, Nellia; Angus, Brian; Smyth, Redmond; Mak, Johnson; Dorrell, Lucy

    2013-05-31

    The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro strongly correlates with virus control in vivo. Post-hoc evaluations of HIV-1 vaccine candidates suggest that this immunological parameter is a promising benchmark of vaccine efficacy. Large-scale analysis of CD8+ T cell antiviral activity requires a rapid, robust and economical assay for accurate quantification of HIV-1 infection in primary CD4+ T cells. Detection of intracellular HIV-1 p24 antigen (p24 Ag) by flow cytometry is one such method but it is thought to be less sensitive and quantitative than p24 Ag ELISA. We report that fixation and permeabilisation of HIV-infected cells using paraformaldehyde/50% methanol/Nonidet P-40 instead of a conventional paraformaldehyde/saponin-based protocol improved their detection across multiplicities of infection (MOI) ranging from 10(-2) to 8×10(-5), and by nearly two-fold (p<0.001) at the optimal MOI tested (10(-2)). The frequency of infected cells was strongly correlated with p24 Ag release during culture, thus validating its use as a measure of productive infection. We were also able to quantify infection with a panel of HIV-1 isolates representing the major clades. The protocol described here is rapid and cost-effective compared with ELISA and thus could be a useful component of immune monitoring of HIV-1 vaccines and interventions to reduce viral reservoirs.

  17. Substance abuse, HIV-1 and hepatitis

    PubMed Central

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

    2013-01-01

    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. PMID:22973853

  18. 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.

  19. Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

    PubMed Central

    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. PMID:22754323

  20. Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response.

    PubMed

    Shcherbakov, D N; Bakulina, A Y; Karpenko, L I; Ilyichev, A A

    2015-01-01

    The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins.

  1. How HIV-1 entry mechanism and broadly neutralizing antibodies guide structure-based vaccine design.

    PubMed

    Pancera, Marie; Changela, Anita; Kwong, Peter D

    2017-05-01

    An HIV-1 vaccine that elicits broadly neutralizing antibodies (bNAbs) remains to be developed. Here, we review how knowledge of bNAbs and HIV-1 entry mechanism is guiding the structure-based design of vaccine immunogens and immunization regimens. Isolation of bNAbs from HIV-1-infected donors has led to an unprecedented understanding of the sites of vulnerability that these antibodies target on the HIV-1 envelope (Env) as well as of the immunological pathways that these antibody lineages follow to develop broad and potent neutralization. Sites of vulnerability, however, reside in the context of diverse Env conformations required for HIV-1 entry, including a prefusion-closed state, a single-CD4-bound intermediate, a three-CD4-bound intermediate, a prehairpin intermediate and postfusion states, and it is not always clear which structural state optimally presents a particular site of vulnerability in the vaccine context. Furthermore, detailed knowledge of immunological pathways has led to debate among vaccine developers as to how much of the natural antibody-developmental pathway immunogens should mimic, ranging from only the recognized epitope to multiple antigens from the antibody-virus coevolution process. A plethora of information on bNAbs is guiding HIV-1-vaccine development. We highlight consideration of the appropriate structural context from the HIV-1-entry mechanism and extraordinary progress with replicating template B-cell ontogenies.

  2. 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.

  3. Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response

    PubMed Central

    Shcherbakov, D. N.; Bakulina, A. Y.; Karpenko, L. I.; Ilyichev, A. A.

    2015-01-01

    The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins. PMID:26798488

  4. Anti-HIV-1 activity of eight monofloral Iranian honey types.

    PubMed

    Behbahani, Mandana

    2014-01-01

    Monofloral Iranian honeys from eight floral sources were analyzed to determine their anti-HIV-1 activities as well as their effects on lymphocyte proliferation. The Peripheral Blood Mononuclear Cells (PBMCs) used in this study were prepared from five healthy volunteers who were seronegative for HIV, HCV, HBV and TB. The anti-HIV-1 activity of eight different honeys was performed by quantitative polymerase chain reaction (PCR) assay and high pure viral nucleic acid kit. The results demonstrated that monofloral honeys from Petro selinum sativum, Nigella sativa, Citrus sinensis, Zataria multiflora, Citrus aurantium and Zizyphus mauritiana flowers had potent anti-HIV-1 activity with half maximal effective concentration (EC50) values of 37.5, 88, 70, 88, 105 and 5 µg/ml respectively. However, monofloral Iranian honeys from Astragalus gummifer and Chamaemelum nobile flowers had weak anti-HIV-1 activity. The frequency and intensity of CD4 expression on PBMCs increased in the presence of all honey types. CD19 marker were also increased after the treatment with monofloral honeys from Z. multiflora and N. sativa. The anti-HIV-1 agent in monofloral honeys from P. sativum, N. sativa, Z. multiflora and Z. mauritiana flowers was detected by spectroscopic analysis as methylglyoxal. Time of drug addition studies demonstrated that the inhibitory effect of methylglyoxal is higher on the late stage of HIV-1 infection. The result demonstrated that methylglyoxal isolated from monofloral honey types is a good candidate for preclinical evaluation of anti-HIV-1 therapies.

  5. Sex differences in HIV-1-mediated immunopathology.

    PubMed

    Ziegler, Susanne; Altfeld, Marcus

    2016-03-01

    The article reviews our current knowledge regarding the role of sex and sex hormones in regulating innate immune responses to viral infections, which may account for the described sex differences in immunity to HIV-1. Prominent sex differences exist in various infectious and autoimmune diseases. Biological mechanisms underlying these differences include the modulation of immunological pathways by sex hormones and gene dosage effects of immunomodulatory genes encoded by the X chromosome. During HIV-1 infections, women have been shown to present with lower viral load levels in primary infection, although their progression to AIDS is faster in comparison with men when accounting for viral load levels in chronic infection. HIV-1-infected women furthermore tend to have higher levels of immune activation and interferon-stimulated gene expression in comparison with men for the same viral load, which has been associated to innate sensing of HIV-1 by Toll-like receptor 7 and the consequent interferon-α production by plasmacytoid dendritic cells. Improvement in understanding the mechanisms associated with sex differences in HIV-1-mediated immunopathology will be critical to take sex differences into consideration when designing experimental and clinical studies in HIV-1-infected populations.

  6. 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

  7. 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.

  8. 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

  9. Kynurenine pathway inhibition reduces neurotoxicity of HIV-1-infected macrophages.

    PubMed

    Kerr, S J; Armati, P J; Pemberton, L A; Smythe, G; Tattam, B; Brew, B J

    1997-12-01

    The AIDS dementia complex (ADC) is a consequence of excessive immune activation driven at least in part by systemic HIV infection and probably brain infection. Quinolinic acid (QUIN) is a neurotoxic tryptophan metabolite produced by macrophages in response to stimulation with cytokines or infection with HIV-1. Consequently it has been implicated in ADC pathogenesis. However, macrophages infected with HIV-1 synthesize numerous neurotoxic substances. Therefore we conducted experiments using human fetal brain tissue to determine the relative importance of QUIN as a neurotoxin in ADC. Human macrophages were infected with HIV-1 in vitro using a viral isolate from a demented patient. 6-Chloro-D-tryptophan, an inhibitor of QUIN biosynthesis, was added to half the macrophage cultures to block formation of QUIN. Supernatants containing QUIN (SQpos) or in which QUIN biosynthesis had been inhibited (SQneg) were then added to human fetal brain aggregate cultures. Toxicity was evaluated using lactate dehydrogenase efflux, trypan blue exclusion, immunohistochemistry, image analysis, and electron microscopy. Each technique showed a reduction of toxicity in SQneg-treated cultures. These studies confirm the significance of QUIN as a neurotoxin in ADC and suggest that neuroprotective strategies may have a place in the treatment of this disease.

  10. Prolonged Antiretroviral Therapy Preserves HIV-1-Specific CD8 T Cells with Stem Cell-Like Properties

    PubMed Central

    Vigano, Selena; Negron, Jordi; Ouyang, Zhengyu; Rosenberg, Eric S.; Walker, Bruce D.; Lichterfeld, Mathias

    2015-01-01

    physically identified and isolated in humans, mice, and nonhuman primates. Here, we investigated whether cellular immune responses against HIV-1 include such T memory stem cells. Our data show that HIV-1-specific CD8 T memory stem cells are detectable during all stages of HIV-1 infection but occur most visibly at times of prolonged viral antigen suppression by antiretroviral combination therapy. These cells may therefore be particularly relevant for designing antiviral immune defense strategies against the residual reservoir of HIV-1-infected cells that persists despite treatment and leads to viral rebound upon treatment discontinuation. PMID:25995260

  11. Sialoadhesin Expressed on IFN-Induced Monocytes Binds HIV-1 and Enhances Infectivity

    PubMed Central

    Rempel, Hans; Calosing, Cyrus; Sun, Bing; Pulliam, Lynn

    2008-01-01

    Background HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1), a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases. Methodology/Principal Findings We analyzed sialoadhesin expression on CD14+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-α and interferon-γ but not tumor necrosis factor-α. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection. Conclusions/Significance Increased sialoadhesin expression on CD14+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells. PMID:18414664

  12. HIV-1 decreases Nrf2/ARE activity and phagocytic function in alveolar macrophages.

    PubMed

    Staitieh, Bashar S; Ding, Lingmei; Neveu, Wendy A; Spearman, Paul; Guidot, David M; Fan, Xian

    2017-08-01

    Respiratory complications occur frequently in individuals living with human immunodeficiency-1 virus (HIV) infection, and there is evidence that HIV-related oxidative stress impairs alveolar macrophage immune function. We hypothesized that nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master transcription factor that activates the antioxidant response element (ARE) and regulates antioxidant defenses, has an important role in alveolar macrophage (AMs) immune dysfunction in individuals with HIV infections. To test that hypothesis, we analyzed human monocyte-derived macrophages (MDMs) that were either infected with HIV-1 or were exposed to the HIV-related proteins gp120 and Tat ex vivo and determined that either stress affected the expression of Nrf2 and the Nrf2-ARE-dependent genes for NAD(P)H dehydrogenase, quinone 1 (NQO1) and glutamate-cysteine ligase, catalytic subunit (GCLC). We then determined that the expression of Nrf2, NQO1, and GCLC was significantly decreased in primary AMs isolated from HIV-1 transgenic rats. In parallel, treating a rat macrophage cell line (NR8383 cells) with the HIV-related proteins gp120 or Tat similarly decreased the gene and protein expression of Nrf2, NQO1, and GCLC. Further, phagocytic function was decreased in both human MDMs infected with HIV-1 and primary AMs from HIV-1 transgenic rats. Importantly, treating HIV-1-infected human MDMs or AMs from HIV-1 transgenic rats with sulforaphane (SFN, an Nrf2 activator) significantly improved their phagocytic function. The salutary effects of SFN were abrogated by silencing RNA to Nrf2 in wild-type rat macrophages. Our findings demonstrate that HIV-1 infection and exposure to HIV-1-related proteins inhibit Nrf2-ARE activity in the AMs and impair their phagocytic function. Treatments targeted at increasing Nrf2-ARE activity could, therefore, enhance lung innate immunity in people living with HIV-1. © Society for Leukocyte Biology.

  13. Interleukin-7 Facilitates HIV-1 Transmission to Cervico-Vaginal Tissue ex vivo

    PubMed Central

    Introini, Andrea; Vanpouille, Christophe; Lisco, Andrea; Grivel, Jean-Charles; Margolis, Leonid

    2013-01-01

    The majority of HIV-1 infections in women occur through vaginal intercourse, in which virus-containing semen is deposited on the cervico-vaginal mucosa. Semen is more than a mere carrier of HIV-1, since it contains many biological factors, in particular cytokines, that may affect HIV-1 transmission. The concentration of interleukin (IL)-7, one of the most prominent cytokines in semen of healthy individuals, is further increased in semen of HIV-1-infected men. Here, we investigated the potential role of IL-7 in HIV-1 vaginal transmission in an ex vivo system of human cervico-vaginal tissue. We simulated an in vivo situation by depositing HIV-1 on cervico-vaginal tissue in combination with IL-7 at concentrations comparable with those measured in semen of HIV-1-infected individuals. We found that IL-7 significantly enhanced virus replication in ex vivo infected cervico-vaginal tissue. Similarly, we observed an enhancement of HIV-1 replication in lymphoid tissue explants. Analysis of T cells isolated from infected tissues showed that IL-7 reduced CD4+ T cell depletion preventing apoptosis, as shown by the decrease in the number of cells expressing the apoptotic marker APO2.7 and the increase in the expression of the anti-apoptotic protein B-cell lymphoma (Bcl)-2. Also, IL-7 increased the fraction of cycling CD4+ T cells, as evidenced by staining for the nuclear factor Ki-67. High levels of seminal IL-7 in vivo may be relevant to the survival of the founder pool of HIV-1-infected cells in the cervico-vaginal mucosa at the initial stage of infection, promoting local expansion and dissemination of HIV infection. PMID:23408885

  14. Evaluation of Cervical Mucosa in Transmission Bottleneck during Acute HIV-1 Infection Using a Cervical Tissue-Based Organ Culture

    PubMed Central

    Shen, Chengli; Ding, Ming; Ratner, Deena; Montelaro, Ronald C.; Chen, Yue; Gupta, Phalguni

    2012-01-01

    Background Although there are different strains of HIV-1 in a chronically infected individual, only one or limited virus strains are successfully transmitted to a new individual. The reason for this “transmission bottleneck” is as yet unknown. Methodology/Principal Findings A human cervical explant model was used to measure HIV-1 transmission efficiency of viral strains from chronic infections, and transmitter/founder variants. We also evaluated the genetic characteristics of HIV-1 variants in the inoculums compared to those transmitted across the cervical mucosa. Eight different HIV-1 isolates were used in this study, six chronic isolates and two transmitter/founder viruses. The transmission efficiency of the chronic and transmitter/founder virus isolates and the viral diversity of chronic isolates before and after viral transmission were assessed. The results indicate that transmitter/founder viruses did not display higher transmission efficiency than chronic HIV-1 isolates. Furthermore, no evidence for a difference in diversity was found between the inoculums and transmitted virus strains. Phylogenetic analysis indicated that the sequences of variants in the inoculums and those present in transmitted virus intermingled irrespective of co-receptor usage. In addition, the inoculum and transmitted variants had a similar pairwise distance distribution. Conclusion There was no selection of a single or limited number of viral variants during HIV-1 transmission across the cervical mucosa in the organ culture model, indicating that the cervical mucosa alone may not produce the transmission bottleneck of HIV-1 infection observed in vivo. PMID:22412886

  15. 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

  16. In vitro anti-HIV-1 activity of fucoidan from Sargassum swartzii.

    PubMed

    Dinesh, Subramaniam; Menon, Thangam; Hanna, Luke E; Suresh, V; Sathuvan, M; Manikannan, M

    2016-01-01

    Sargassum swartzii, a marine brown algae with wide range of biological properties belongs to the family Sargassaceae. Bioactive fucoidan fractions (CFF, FF1 and FF2) were isolated from S. swartzii and characterized by linear gradient anion-exchange chromatography and FT-IR. The characterized fucoidan fractions contained mainly sugars, sulfate and uronic acid. In the present study, anti-HIV-1 property of the fucoidan fractions was investigated. Fraction FF2 was found to exhibit significant anti-HIV-1 activity at concentrations of 1.56 and 6.25 μg/ml as observed by >50% reduction in HIV-1 p24 antigen levels and reverse transcriptase activity. Fucoidan fractions have no cytotoxic effects on PBMCs at the concentration range of 1.56-1000 μg/ml. These results suggest that fucoidan fractions could have inhibitory activity against HIV and has potential as an anti-HIV-1 agent.

  17. Relative resistance of HIV-1 founder viruses to control by interferon-alpha

    PubMed Central

    2013-01-01

    Background Following mucosal human immunodeficiency virus type 1 (HIV-1) transmission, type 1 interferons (IFNs) are rapidly induced at sites of initial virus replication in the mucosa and draining lymph nodes. However, the role played by IFN-stimulated antiviral activity in restricting HIV-1 replication during the initial stages of infection is not clear. We hypothesized that if type 1 IFNs exert selective pressure on HIV-1 replication in the earliest stages of infection, the founder viruses that succeed in establishing systemic infection would be more IFN-resistant than viruses replicating during chronic infection, when type 1 IFNs are produced at much lower levels. To address this hypothesis, the relative resistance of virus isolates derived from HIV-1-infected individuals during acute and chronic infection to control by type 1 IFNs was analysed. Results The replication of plasma virus isolates generated from subjects acutely infected with HIV-1 and molecularly cloned founder HIV-1 strains could be reduced but not fully suppressed by type 1 IFNs in vitro. The mean IC50 value for IFNα2 (22 U/ml) was lower than that for IFNβ (346 U/ml), although at maximally-inhibitory concentrations both IFN subtypes inhibited virus replication to similar extents. Individual virus isolates exhibited differential susceptibility to inhibition by IFNα2 and IFNβ, likely reflecting variation in resistance to differentially up-regulated IFN-stimulated genes. Virus isolates from subjects acutely infected with HIV-1 were significantly more resistant to in vitro control by IFNα than virus isolates generated from the same individuals during chronic, asymptomatic infection. Viral IFN resistance declined rapidly after the acute phase of infection: in five subjects, viruses derived from six-month consensus molecular clones were significantly more sensitive to the antiviral effects of IFNs than the corresponding founder viruses. Conclusions The establishment of systemic HIV-1 infection by

  18. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.

    PubMed

    Liao, Hua-Xin; Lynch, Rebecca; Zhou, Tongqing; Gao, Feng; Alam, S Munir; Boyd, Scott D; Fire, Andrew Z; Roskin, Krishna M; Schramm, Chaim A; Zhang, Zhenhai; Zhu, Jiang; Shapiro, Lawrence; Mullikin, James C; Gnanakaran, S; Hraber, Peter; Wiehe, Kevin; Kelsoe, Garnett; Yang, Guang; Xia, Shi-Mao; Montefiori, David C; Parks, Robert; Lloyd, Krissey E; Scearce, Richard M; Soderberg, Kelly A; Cohen, Myron; Kamanga, Gift; Louder, Mark K; Tran, Lillian M; Chen, Yue; Cai, Fangping; Chen, Sheri; Moquin, Stephanie; Du, Xiulian; Joyce, M Gordon; Srivatsan, Sanjay; Zhang, Baoshan; Zheng, Anqi; Shaw, George M; Hahn, Beatrice H; Kepler, Thomas B; Korber, Bette T M; Kwong, Peter D; Mascola, John R; Haynes, Barton F

    2013-04-25

    Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

  19. HIV-1 Subtype C Reverse Transcriptase and Protease Genotypes in Zimbabwean Patients Failing Antiretroviral Therapy

    PubMed Central

    KANTOR, RAMI; ZIJENAH, LYNN S.; SHAFER, ROBERT W.; MUTETWA, SOLOMON; JOHNSTON, ELIZABETH; LLOYD, ROBERT; VON LIEVEN, ANDREA; ISRAELSKI, DENNIS; KATZENSTEIN, DAVID A.

    2008-01-01

    HIV-1 drug resistance mutations have been identified and characterized mostly in subtype B HIV-1 infection. The extent to which antiretroviral drugs select for drug resistance mutations in non-subtype B HIV-1 is not known. We obtained HIV-1 reverse transcriptase (RT) and protease sequences from 21 Zimbabwean patients failing antiretroviral drug therapy. We compared these sequences with 56 published RT and protease subtype C sequences from untreated patients, 990 RT and 1140 protease subtype B sequences from treated patients, and 340 RT and 907 protease subtype B sequences from untreated patients and identified four mutation categories of subtype C HIV-1. Seventeen of the 21 patients (81%) had known drug resistance mutations. Mutations at 15 RT and 11 protease positions were more common in subtype C isolates than in subtype B isolates. HIV-1 subtype C-infected individuals receiving antiretroviral therapy develop many of the known subtype B drug resistance mutations. Comparison of subtype C RT and protease sequences with a large database of subtype B sequences identified subtype C-specific polymorphisms and candidate drug resistance mutations. PMID:12512512

  20. Bone marrow plasma cells are a primary source of serum HIV-1-specific antibodies in chronically infected individuals.

    PubMed

    Montezuma-Rusca, Jairo M; Moir, Susan; Kardava, Lela; Buckner, Clarisa M; Louie, Aaron; Kim, Leo J Y; Santich, Brian H; Wang, Wei; Fankuchen, Olivia R; Diaz, Gabriella; Daub, Janine R; Rosenzweig, Sergio D; Chun, Tae-Wook; Li, Yuxing; Braylan, Raul C; Calvo, Katherine R; Fauci, Anthony S

    2015-03-15

    Several potent and broadly neutralizing Abs to HIV-1 have been isolated recently from peripheral blood B cells of infected individuals, based on prescreening of Ab activity in the serum. However, little is known regarding the cells that make the Abs that circulate in the blood. Accordingly, we investigated the most likely source, the bone marrow, of chronically HIV-1-infected individuals who were not receiving antiretroviral therapy. Increased frequencies of plasma cells, as well as B cell precursors, namely preB-I and preB-II, and decreased frequencies of mature B cells were observed in bone marrow aspirates of these individuals compared with HIV-negative counterparts. Increased frequencies of bone marrow plasma cells are consistent with known hallmarks of HIV-1 infection, namely hypergammaglobulinemia and increased frequencies of peripheral blood plasmablasts. Levels of HIV-1 envelope (Env)-binding and HIV-1-neutralizing Abs were measured in serum, and corresponding frequencies of Ab-secreting or Env-binding cells were measured in the blood (plasmablasts and memory B cells) and in the bone marrow (plasma cells). A strong correlation was observed between serum HIV-1-specific Abs and Env-specific bone marrow-derived plasma cells, but not circulating plasmablasts or memory B cells. These findings demonstrate that, despite HIV-1-induced phenotypic and functional B cell dysregulation in the peripheral blood and secondary lymphoid tissues, bone marrow plasma cells remain a primary source for circulating HIV-1-specific Abs in HIV-1-infected individuals.

  1. Regulatory T Cells Expanded from HIV-1-Infected Individuals Maintain Phenotype, TCR Repertoire and Suppressive Capacity

    PubMed Central

    Angin, Mathieu; Klarenbeek, Paul L.; King, Melanie; Sharma, Siddhartha M.; Moodley, Eshia S.; Rezai, Ashley; Piechocka-Trocha, Alicja; Toth, Ildiko; Chan, Andrew T.; Goulder, Philip J.; Ndung'u, Thumbi; Kwon, Douglas S.; Addo, Marylyn M.

    2014-01-01

    While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4+ Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection. PMID:24498287

  2. 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

  3. 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

  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. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Acyclovir and Transmission of HIV-1 from Persons Infected with HIV-1 and HSV-2

    PubMed Central

    Celum, Connie; Wald, Anna; Lingappa, Jairam R.; Magaret, Amalia S.; Wang, Richard S.; Mugo, Nelly; Mujugira, Andrew; Baeten, Jared M.; Mullins, James I.; Hughes, James P.; Bukusi, Elizabeth A.; Cohen, Craig R.; Katabira, Elly; Ronald, Allan; Kiarie, James; Farquhar, Carey; Stewart, Grace John; Makhema, Joseph; Essex, Myron; Were, Edwin; Fife, Kenneth H.; de Bruyn, Guy; Gray, Glenda E.; McIntyre, James A.; Manongi, Rachel; Kapiga, Saidi; Coetzee, David; Allen, Susan; Inambao, Mubiana; Kayitenkore, Kayitesi; Karita, Etienne; Kanweka, William; Delany, Sinead; Rees, Helen; Vwalika, Bellington; Stevens, Wendy; Campbell, Mary S.; Thomas, Katherine K.; Coombs, Robert W.; Morrow, Rhoda; Whittington, William L.H.; McElrath, M. Juliana; Barnes, Linda; Ridzon, Renee; Corey, Lawrence

    2010-01-01

    BACKGROUND Most persons who are infected with human immunodeficiency virus type 1 (HIV-1) are also infected with herpes simplex virus type 2 (HSV-2), which is frequently reactivated and is associated with increased plasma and genital levels of HIV-1. Therapy to suppress HSV-2 reduces the frequency of reactivation of HSV-2 as well as HIV-1 levels, suggesting that suppression of HSV-2 may reduce the risk of transmission of HIV-1. METHODS We conducted a randomized, placebo-controlled trial of suppressive therapy for HSV-2 (acyclovir at a dose of 400 mg orally twice daily) in couples in which only one of the partners was seropositive for HIV-1 (CD4 count, ≥250 cells per cubic millimeter) and that partner was also infected with HSV-2 and was not taking antiretroviral therapy at the time of enrollment. The primary end point was transmission of HIV-1 to the partner who was not initially infected with HIV-1; linkage of transmissions was assessed by means of genetic sequencing of viruses. RESULTS A total of 3408 couples were enrolled at 14 sites in Africa. Of the partners who were infected with HIV-1, 68% were women, and the baseline median CD4 count was 462 cells per cubic millimeter. Of 132 HIV-1 seroconversions that occurred after randomization (an incidence of 2.7 per 100 person-years), 84 were linked within couples by viral sequencing: 41 in the acyclovir group and 43 in the placebo group (hazard ratio with acyclovir, 0.92, 95% confidence interval [CI], 0.60 to 1.41; P = 0.69). Suppression with acyclovir reduced the mean plasma concentration of HIV-1 by 0.25 log10 copies per milliliter (95% CI, 0.22 to 0.29; P<0.001) and the occurrence of HSV-2–positive genital ulcers by 73% (risk ratio, 0.27; 95% CI, 0.20 to 0.36; P<0.001). A total of 92% of the partners infected with HIV-1 and 84% of the partners not infected with HIV-1 remained in the study for 24 months. The level of adherence to the dispensed study drug was 96%. No serious adverse events related to acyclovir

  11. Acyclovir and transmission of HIV-1 from persons infected with HIV-1 and HSV-2.

    PubMed

    Celum, C; Wald, A; Lingappa, J R; Magaret, A S; Wang, R S; Mugo, N; Mujugira, A; Baeten, J M; Mullins, J I; Hughes, J P; Bukusi, E A; Cohen, C R; Katabira, E; Ronald, A; Kiarie, J; Farquhar, C; Stewart, G J; Makhema, J; Essex, M; Were, E; Fife, K H; de Bruyn, G; Gray, G E; McIntyre, J A; Manongi, R; Kapiga, S; Coetzee, D; Allen, S; Inambao, M; Kayitenkore, K; Karita, E; Kanweka, W; Delany, S; Rees, H; Vwalika, B; Stevens, W; Campbell, M S; Thomas, K K; Coombs, R W; Morrow, R; Whittington, W L H; McElrath, M J; Barnes, L; Ridzon, R; Corey, L

    2010-02-04

    Most persons who are infected with human immunodeficiency virus type 1 (HIV-1) are also infected with herpes simplex virus type 2 (HSV-2), which is frequently reactivated and is associated with increased plasma and genital levels of HIV-1. Therapy to suppress HSV-2 reduces the frequency of reactivation of HSV-2 as well as HIV-1 levels, suggesting that suppression of HSV-2 may reduce the risk of transmission of HIV-1. We conducted a randomized, placebo-controlled trial of suppressive therapy for HSV-2 (acyclovir at a dose of 400 mg orally twice daily) in couples in which only one of the partners was seropositive for HIV-1 (CD4 count, > or = 250 cells per cubic millimeter) and that partner was also infected with HSV-2 and was not taking antiretroviral therapy at the time of enrollment. The primary end point was transmission of HIV-1 to the partner who was not initially infected with HIV-1; linkage of transmissions was assessed by means of genetic sequencing of viruses. A total of 3408 couples were enrolled at 14 sites in Africa. Of the partners who were infected with HIV-1, 68% were women, and the baseline median CD4 count was 462 cells per cubic millimeter. Of 132 HIV-1 seroconversions that occurred after randomization (an incidence of 2.7 per 100 person-years), 84 were linked within couples by viral sequencing: 41 in the acyclovir group and 43 in the placebo group (hazard ratio with acyclovir, 0.92, 95% confidence interval [CI], 0.60 to 1.41; P=0.69). Suppression with acyclovir reduced the mean plasma concentration of HIV-1 by 0.25 log(10) copies per milliliter (95% CI, 0.22 to 0.29; P<0.001) and the occurrence of HSV-2-positive genital ulcers by 73% (risk ratio, 0.27; 95% CI, 0.20 to 0.36; P<0.001). A total of 92% of the partners infected with HIV-1 and 84% of the partners not infected with HIV-1 remained in the study for 24 months. The level of adherence to the dispensed study drug was 96%. No serious adverse events related to acyclovir were observed. Daily

  12. Platelets and erythrocyte-bound platelets bind infectious HIV-1 in plasma of chronically infected patients.

    PubMed

    Beck, Zoltan; Jagodzinski, Linda L; Eller, Michael A; Thelian, Doris; Matyas, Gary R; Kunz, Anjali N; Alving, Carl R

    2013-01-01

    Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC) either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC), platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture) from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a) platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b) platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV-1; (c) DC

  13. Impairment of HIV-1 cDNA synthesis by DBR1 knockdown.

    PubMed

    Galvis, Alvaro E; Fisher, Hugh E; Nitta, Takayuki; Fan, Hung; Camerini, David

    2014-06-01

    Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different

  14. Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

    PubMed Central

    Chen, Xi; Munshaw, Supriya; Zhang, Ruijun; Marshall, Dawn J.; Vandergrift, Nathan; Whitesides, John F.; Lu, Xiaozhi; Yu, Jae-Sung; Hwang, Kwan-Ki; Gao, Feng; Markowitz, Martin; Heath, Sonya L.; Bar, Katharine J.; Goepfert, Paul A.; Montefiori, David C.; Shaw, George C.; Alam, S. Munir; Margolis, David M.; Denny, Thomas N.; Boyd, Scott D.; Marshal, Eleanor; Egholm, Michael; Simen, Birgitte B.; Hanczaruk, Bozena; Fire, Andrew Z.; Voss, Gerald; Kelsoe, Garnett; Tomaras, Georgia D.; Moody, M. Anthony; Kepler, Thomas B.

    2011-01-01

    The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens. PMID:21987658

  15. Comparative Evaluation of HIV-1 Neutralization in External Secretions and Sera of HIV-1-Infected Women.

    PubMed

    Wei, Qing; Moldoveanu, Zina; Huang, Wen-Qiang; Alexander, Rashada C; Goepfert, Paul A; Mestecky, Jiri

    2012-01-01

    Although human immunodeficiency virus type 1 (HIV-1)-specific antibodies are detectable in external secretions by ELISA and western blot (WB), the presence of HIV-1 neutralizing antibodies is difficult to evaluate due to the low levels of immunoglobulins (Ig) and the presence of humoral factors of innate immunity. The objective of this study was to determine virus neutralization activity and the relative contribution of HIV-1-specific antibodies of various isotypes to virus neutralization in serum/plasma samples, cervicovaginal lavages (CVL), and rectal lavages (RL). Serum/plasma, CVL, and RL samples were examined by ELISA, WB and HIV-1 neutralization assays. Selected samples were Ig depleted and analyzed for virus neutralization. IgG specific for three HIV-1 ENV antigens was detected in all serum/plasma samples, while IgA to at least one ENV glycoprotein was found at the low levels in 95% samples. Serum/plasma samples had the ability to neutralize at least one of three clade B and two clade C viruses. The neutralizing titers were reduced significantly or became undetectable after IgG removal. In corresponding CVL and RL, HIV-1 ENV-specific IgG antibodies were readily detected compared to IgA. Furthermore, IgG in CVL had greater ability than IgA to reduce virus infectivity. The difference in HIV-1 neutralization before and after Ig depletion was not observed in RL, implying that innate humoral factors were involved in anti-HIV-1 activity. Results demonstrate that HIV-1-specific neutralizing antibodies are almost exclusively of the IgG isotype in serum/plasma and CVL samples. HIV-1-specific binding antibodies detected in RL are not responsible for neutralization activity, suggesting that the antibody-mediated virus neutralization in external secretions should be verified by means of a selective depletion of Ig.

  16. HIV-1 Reservoirs During Suppressive Therapy

    PubMed Central

    Barton, Kirston; Winckelmann, Anni; Palmer, Sarah

    2016-01-01

    The introduction of antiretroviral therapy (ART) 20 years ago has dramatically reduced morbidity and mortality associated with HIV-1. Initially there was hope that ART would be curative, but it quickly became clear that even though ART was able to restore CD4+ T cell counts and suppress viral loads below levels of detection, discontinuation of treatment resulted in a rapid rebound of infection. This is due to persistence of a small reservoir of latently infected cells with a long half-life, which necessitates life-long ART. Over the past few years, significant progress has been made in defining and characterizing the latent reservoir of HIV-1, and here we review how understanding the latent reservoir during suppressive therapy will lead to significant advances in curative approaches for HIV-1. PMID:26875617

  17. 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

  18. The ESCRT pathway and HIV-1 budding.

    PubMed

    Usami, Yoshiko; Popov, Sergei; Popova, Elena; Inoue, Michio; Weissenhorn, Winfried; G Göttlinger, Heinrich

    2009-02-01

    HIV-1 Gag engages components of the ESCRT (endosomal sorting complex required for transport) pathway via so-called L (late-assembly) domains to promote virus budding. Specifically, the PTAP (Pro-Thr-Ala-Pro)-type primary L domain of HIV-1 recruits ESCRT-I by binding to Tsg101 (tumour susceptibility gene 101), and an auxiliary LYPX(n)L (Leu-Tyr-Pro-Xaa(n)-Leu)-type L domain recruits the ESCRT-III-binding partner Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X]. The structurally related CHMPs (charged multivesicular body proteins), which form ESCRT-III, are kept in an inactive state through intramolecular interactions, and become potent inhibitors of HIV-1 budding upon removal of an autoinhibitory region. In the absence of the primary L domain, HIV-1 budding is strongly impaired, but can be efficiently rescued through the overexpression of Alix. This effect of Alix depends on its ability to interact with CHMP4, suggesting that it is the recruitment of CHMPs that ultimately drives virus release. Surprisingly, HIV-1 budding defects can also be efficiently corrected by overexpressing Nedd (neural-precursor-cell-expressed developmentally down-regulated) 4-2s, a member of a family of ubiquitin ligases previously implicated in the function of PPXY (Pro-Pro-Xaa-Tyr)-type L domains, which are absent from HIV-1. At least under certain circumstances, Nedd4-2s stimulates the activity of PTAP-type L domains, raising the possibility that the ubiquitin ligase regulates the activity of ESCRT-I.

  19. Analysis of HIV-1 intersubtype recombination breakpoints suggests region with high pairing probability may be a more fundamental factor than sequence similarity affecting HIV-1 recombination.

    PubMed

    Jia, Lei; Li, Lin; Gui, Tao; Liu, Siyang; Li, Hanping; Han, Jingwan; Guo, Wei; Liu, Yongjian; Li, Jingyun

    2016-09-21

    With increasing data on HIV-1, a more relevant molecular model describing mechanism details of HIV-1 genetic recombination usually requires upgrades. Currently an incomplete structural understanding of the copy choice mechanism along with several other issues in the field that lack elucidation led us to perform an analysis of the correlation between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarity to further explore structural mechanisms. Near full length sequences of URFs from Asia, Europe, and Africa (one sequence/patient), and representative sequences of worldwide CRFs were retrieved from the Los Alamos HIV database. Their recombination patterns were analyzed by jpHMM in detail. Then the relationships between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarities were investigated. Pearson correlation test showed that all URF groups and the CRF group exhibit the same breakpoint distribution pattern. Additionally, the Wilcoxon two-sample test indicated a significant and inexplicable limitation of recombination in regions with high pairing probability. These regions have been found to be strongly conserved across distinct biological states (i.e., strong intersubtype similarity), and genetic similarity has been determined to be a very important factor promoting recombination. Thus, the results revealed an unexpected disagreement between intersubtype similarity and breakpoint distribution, which were further confirmed by genetic similarity analysis. Our analysis reveals a critical conflict between results from natural HIV-1 isolates and those from HIV-1-based assay vectors in which genetic similarity has been shown to be a very critical factor promoting recombination. These results indicate the region with high-pairing probabilities may be a more fundamental factor affecting HIV-1 recombination than sequence similarity in natural HIV-1 infections. Our

  20. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  1. 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.

  2. Evidence of at Least Two Introductions of HIV-1 in the Amerindian Warao Population from Venezuela

    PubMed Central

    Rangel, Héctor R.; Maes, Mailis; Villalba, Julian; Sulbarán, Yoneira; de Waard, Jacobus H.; Bello, Gonzalo; Pujol, Flor H.

    2012-01-01

    Background The Venezuelan Amerindians were, until recently, free of human immunodeficiency virus (HIV) infection. However, in 2007, HIV-1 infection was detected for the first time in the Warao Amerindian population living in the Eastern part of Venezuela, in the delta of the Orinoco river. The aim of this study was to analyze the genetic diversity of the HIV-1 circulating in this population. Methodology/Principal Findings The pol genomic region was sequenced for 16 HIV-1 isolates and for some of them, sequences from env, vif and nef genomic regions were obtained. All HIV-1 isolates were classified as subtype B, with exception of one that was classified as subtype C. The 15 subtype B isolates exhibited a high degree of genetic similarity and formed a highly supported monophyletic cluster in each genomic region analyzed. Evolutionary analyses of the pol genomic region indicated that the date of the most recent common ancestor of the Waraos subtype B clade dates back to the late 1990s. Conclusions/Significance At least two independent introductions of HIV-1 have occurred in the Warao Amerindians from Venezuela. The HIV-1 subtype B was successfully established and got disseminated in the community, while no evidence of local dissemination of the HIV-1 subtype C was detected in this study. These results warrant further surveys to evaluate the burden of this disease, which can be particularly devastating in this Amerindian population, with a high prevalence of tuberculosis, hepatitis B, among other infectious diseases, and with limited access to primary health care. PMID:22808212

  3. Potent Inhibitor of Drug-Resistant HIV-1 Strains Identified from the Medicinal Plant Justicia gendarussa.

    PubMed

    Zhang, Hong-Jie; Rumschlag-Booms, Emily; Guan, Yi-Fu; Wang, Dong-Ying; Liu, Kang-Lun; Li, Wan-Fei; Nguyen, Van H; Cuong, Nguyen M; Soejarto, Djaja D; Fong, Harry H S; Rong, Lijun

    2017-06-23

    Justicia gendarussa, a medicinal plant collected in Vietnam, was identified as a potent anti-HIV-1 active lead from the evaluation of over 4500 plant extracts. Bioassay-guided separation of the extracts of the stems and roots of this plant led to the isolation of an anti-HIV arylnaphthalene lignan (ANL) glycoside, patentiflorin A (1). Evaluation of the compound against both the M- and T-tropic HIV-1 isolates showed it to possess a significantly higher inhibition effect than the clinically used anti-HIV drug AZT. Patentiflorin A and two congeners were synthesized, de novo, as an efficient strategy for resupply as well as for further structural modification of the anti-HIV ANL glycosides in the search for drug leads. Subsequently, it was determined that the presence of a quinovopyranosyloxy group in the structure is likely essential to retain the high degree of anti-HIV activity of this type of compounds. Patentiflorin A was further investigated against the HIV-1 gene expression of the R/U5 and U5/gag transcripts, and the data showed that the compound acts as a potential inhibitor of HIV-1 reverse transcription. Importantly, the compound displayed potent inhibitory activity against drug-resistant HIV-1 isolates of both the nucleotide analogue (AZT) and non-nucleotide analogue (nevaripine). Thus, the ANL glycosides have the potential to be developed as novel anti-HIV drugs.

  4. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. An outbreak of HIV-1 BC recombinants in Southern Italy.

    PubMed

    Monno, Laura; Brindicci, Gaetano; Lai, Alessia; Punzi, Grazia; Altamura, Maurantonio; Simonetti, Francesco Roberto; Ladisa, Nicoletta; Saracino, Annalisa; Balotta, Claudia; Angarano, Gioacchino

    2012-12-01

    In Western Europe, a previously subtype B HIV-1 restricted area, BC recombinants have been rarely reported. To describe an outbreak of HIV-1 BC recombinants in southern Italy. We analyzed pol (protease/reverse transcriptase) sequences from 135 newly diagnosed HIV-1-infected patients during the years 2009-2011. For phylogenetic relationships, sequences were aligned to the most recent reference data set from the Los Alamos database using BioEdit (version 7.1.3). The resulting alignment was analyzed with the Phylip package (version 3.67) building a neighbor-joining tree based on the Kimura two-parameter substitution model. The reliability of the tree topology was assessed through bootstrapping using 1000 replicates. The recombination pattern was characterized using SimPlot 3.5.1 and SplitsTree 4. At phylogenetic analysis, 22 (16.2%) isolates whose sequences were not unequivocally assigned to a pure subtype or known CRF, formed a distinct monophyletic clade (100% of bootstrap value). For these isolates, the recombination analysis identified a BC mosaic pattern with two breakpoints at positions 2778±5 and 3162±8 (HXB2 numbering) which differed from those of known BC CRFs. All patients from whom these sequences were derived were highly educated youth Italians, 91% males and 82% MSM. Sequences of pol integrase, gp120 and gp41 from these same patients were classified as C subtype. This outbreak which further reflects the increasing heterogeneity of HIV epidemic in our country is the first report of an Italian outbreak of a BC recombinant, possibly a novel candidate CRF. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines

    PubMed Central

    Liao, Hua-Xin; Alam, S. Munir; Scearce, Richard M.; Plonk, M. Kelly; Kozink, Daniel M.; Drinker, Mark S.; Zhang, Ruijun; Xia, Shi-Mao; Sutherland, Laura L.; Tomaras, Georgia D.; Giles, Ian P.; Kappes, John C.; Ochsenbauer-Jambor, Christina; Edmonds, Tara G.; Soares, Melina; Barbero, Gustavo; Forthal, Donald N.; Landucci, Gary; Chang, Connie; King, Steven W.; Kavlie, Anita; Denny, Thomas N.; Hwang, Kwan-Ki; Chen, Pojen P.; Thorpe, Philip E.; Montefiori, David C.

    2010-01-01

    Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes. PMID:20368576

  7. A genotypic HIV-1 proviral DNA coreceptor tropism assay: characterization in viremic subjects

    PubMed Central

    2014-01-01

    Background HIV-1 coreceptor tropism testing is used to evaluate eligibility for CCR5 antagonist therapy. However, HIV-1 RNA-based tests are not suitable for virologically suppressed patients, therefore the use of proviral DNA tropism testing has been investigated. We describe a novel proviral DNA-based genotypic tropism assay and compare its performance to that of a sensitive HIV-1 RNA-based genotypic test. Methods Tropism was determined using HIV-1 plasma RNA and proviral DNA from 42 paired samples from patients with plasma viral loads ≥1000 HIV-1 RNA copies/mL. Proviral DNA sample types included whole blood, separated peripheral blood mononuclear cells resuspended in phosphate-buffered saline and peripheral blood mononuclear cells resuspended in spun plasma. The HIV-1 envelope V3 region was PCR-amplified, sequenced in triplicate, and analyzed for tropism with the geno2pheno algorithm using a 10% false-positive rate (FPR). Results Amplicons were obtained from proviral DNA and plasma RNA in 41/42 samples. Tropism predictions were highly concordant (93%–98%) between proviral DNA and plasma RNA, regardless of the proviral DNA isolation method. Non-R5 proviral DNA results were obtained for 100% of patients with detectable non-R5 plasma HIV-1 RNA results. Geno2pheno FPRs for proviral DNA and plasma RNA were highly correlated (Spearman rho = 0.86). Conclusions Our findings demonstrate that proviral DNA tropism determinations from whole blood or peripheral blood mononuclear cells were highly concordant with plasma HIV-1 RNA tropism determinations. This assay may be useful for screening virologically suppressed patients for CCR5-antagonist eligibility and for research purposes. PMID:24904682

  8. Limited HIV-1 Reactivation in Resting CD4+ T cells from Aviremic Patients under Protease Inhibitors

    PubMed Central

    Kumar, Amit; Abbas, Wasim; Bouchat, Sophie; Gatot, Jean-Stéphane; Pasquereau, Sébastien; Kabeya, Kabamba; Clumeck, Nathan; De Wit, Stéphane; Van Lint, Carine; Herbein, Georges

    2016-01-01

    A latent viral reservoir that resides in resting CD4+ T cells represents a major barrier for eradication of HIV infection. We test here the impact of HIV protease inhibitor (PI) based combination anti-retroviral therapy (cART) over nonnucleoside reverse transcriptase inhibitor (NNRTI)-based cART on HIV-1 reactivation and integration in resting CD4+ T cells. This is a prospective cohort study of patients with chronic HIV-1 infection treated with conventional cART with an undetectable viremia. We performed a seven-year study of 47 patients with chronic HIV-infection treated with cART regimens and with undetectable plasma HIV-1 RNA levels for at least 1 year. Of these 47 patients treated with cART, 24 were treated with a PI-based regimen and 23 with a NNRTI-based regimen as their most recent treatment for more than one year. We evaluated the HIV-1 reservoir using reactivation assay and integrated HIV-1 DNA, respectively, in resting CD4+ T cells. Resting CD4+ T cells isolated from PI-treated patients compared to NNRTI-treated patients showed a limited HIV-1 reactivation upon T-cell stimulation (p = 0·024) and a lower level of HIV-1 integration (p = 0·024). Our study indicates that PI-based cART could be more efficient than NNRTI-based cART for limiting HIV-1 reactivation in aviremic chronically infected patients. PMID:27922055

  9. In vitro anti-HIV-1 activity of salicylidene acylhydrazide compounds

    PubMed Central

    Forthal, Donald N.; Phan, Tran B.; Slepenkin, Anatoly V.; Landucci, Gary; Chu, Hencelyn; Elofsson, Mikael; Peterson, Ellena

    2012-01-01

    Introduction Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides. Methods Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle. Results We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step. Conclusions We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide. PMID:22819150

  10. Viral linkage in HIV-1 seroconverters and their partners in an HIV-1 prevention clinical trial.

    PubMed

    Campbell, Mary S; Mullins, James I; Hughes, James P; Celum, Connie; Wong, Kim G; Raugi, Dana N; Sorensen, Stefanie; Stoddard, Julia N; Zhao, Hong; Deng, Wenjie; Kahle, Erin; Panteleeff, Dana; Baeten, Jared M; McCutchan, Francine E; Albert, Jan; Leitner, Thomas; Wald, Anna; Corey, Lawrence; Lingappa, Jairam R

    2011-03-02

    Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #NCT00194519) was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners. We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners' sequences and a Bayesian posterior probability of ≥ 50%. Adjudicators classified each seroconversion, finding 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%). Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters. 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

  11. Distribution of HIV-1 Infection in Different T Lymphocyte Subsets: Antiretroviral Therapy-Naïve vs. Experienced Patients

    PubMed Central

    Perez, Raul; Gibson, Sonia; Lopez, Pablo; Koenig, Ellen; De Castro, Marisol

    2011-01-01

    Abstract Memory CD4 T cells are the primary targets of HIV-1 infection, which then subsequently spreads to other T lymphocyte subsets. Antiretroviral therapy (ART) alters the pattern of HIV-1 distribution. Blood samples were collected from ART-naïve or -experienced HIV-1 patients, and the memory and naïve subsets of CD4+ and CD8+ T lymphocytes, respectively, were isolated by cell sorting. DNA was extracted and the HIV-1 env C2/V3 region PCR amplified. Amplicons were cloned and sequenced, and genetic relatedness among different HIV-1 compartments was determined by the phylogenetic analysis of clonal sequences. The viral V3 sequence of HIV-1 in each compartment was analyzed by using webPSSM to determine CCR5 or CXCR4 coreceptor binding property of the virus. The direction of viral migration among involved compartments was determined by using the MacClade program. In ART-naïve patients, HIV-1 was generally confined to the memory CD4 T (mT4) cell compartment, even though in a few cases, naïve CD4 T (nT4) cells were also infected. When this occurred, the HIV-1 gene migrated from nT4 to mT4. In contrast, HIV-1 was detected in nT4 and mT4 as well as in the memory CD8 T (mT8) compartments of ART-experienced patients. However, no clear pattern of directional HIV-1 gene flow among the compartments could be determined because of the small sample size. All HIV-1–infected T cell compartments housed the virus that used either CCR5 or CXCR4 as the coreceptor. PMID:21054214

  12. HIV-1 Continues To Replicate and Evolve in Patients with Natural Control of HIV Infection ▿ †

    PubMed Central

    Mens, Helene; Kearney, Mary; Wiegand, Ann; Shao, Wei; Schønning, Kristian; Gerstoft, Jan; Obel, Niels; Maldarelli, Frank; Mellors, John W.; Benfield, Thomas; Coffin, John M.

    2010-01-01

    Elucidating mechanisms leading to the natural control of HIV-1 infection is of great importance for vaccine design and for understanding viral pathogenesis. Rare HIV-1-infected individuals, termed HIV-1 controllers, have plasma HIV-1 RNA levels below the limit of detection by standard clinical assays (<50 to 75 copies/ml) without antiretroviral therapy. Although several recent studies have documented persistent low-grade viremia in HIV-1 controllers at a level not significantly different from that in HIV-1-infected individuals undergoing treatment with combination antiretroviral therapy (cART), it is unclear if plasma viruses are undergoing full cycles of replication in vivo or if the infection of new cells is completely blocked by host immune mechanisms. We studied a cohort of 21 HIV-1 controllers with a median level of viremia below 1 copy/ml, followed for a median of 11 years. Less than half of the cohort carried known protective HLA types (B*57/27). By isolating HIV-1 RNA from large volumes of plasma, we amplified single genome sequences of both pro-rt and env longitudinally. This study is the first to document that HIV-1 pro-rt and env evolve in this patient group, albeit at rates somewhat lower than in HIV-1 noncontrollers, in HLA B*57/27-positive, as well as HLA B*57/27-negative, individuals. Viral diversity and adaptive events associated with immune escape were found to be restricted in HIV-1 controllers, suggesting that replication occurs in the face of less overall immune selection. PMID:20926564

  13. National survey of prevalent HIV strains: limited genetic variation of Korean HIV-1 clade B within the population of Korean men who have sex with men.

    PubMed

    Kim, Gab Jung; Nam, Jeong-Gu; Shin, Bo Gyeong; Kee, Mee Kyeong; Kim, Eun-Jin; Lee, Joo-Shil; Kim, Sung Soon

    2008-06-01

    The evolution of HIV is the result of an explosive combination of factors-a high rate of mutation, replication dynamics, frequent recombination, and natural selection. To understand the evolution of the distinctive Korean HIV-1 B clade, we investigated the characteristics of the genetic variation of the HIV-1 subtype B env gene within the group of Korean men who have sex with men (MSM). From 1985 to 2005, 700 HIV-1-infected Koreans were sequenced at the V1 to V5 region of the HIV-1 env gene. In the phylogenetic analysis, 560 isolates were identified as HIV-1 subtype B, and 489 of the 560 isolates were HIV-1 Korean clade B. Based on epidemiologic investigation, 249 of 700 HIV-1-infected patients were HIV-1 subtype B-infected MSM. Interestingly, the proportion of the GPGS motif in MSM infected by Koreans was 1.6 times higher than in MSM infected by foreigners, and the genetic expansions of diversity and divergence for HIV-1 subtype B in Korean MSM were 2.1% and 2.5%, respectively. This was much lower than those observed in other countries. Therefore, our findings imply that the HIV strains in this group were closely related. This result may be helpful for understanding the evolution of the distinct HIV-1 Korean B clade.

  14. 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

  15. 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

  16. Enteric viruses in HIV-1 seropositive and HIV-1 seronegative children with diarrheal diseases in Brazil

    PubMed Central

    Rocha, Monica Simões; Fumian, Tulio Machado; Maranhão, Adriana Gonçalves; de Assis, Rosane Maria; Xavier, Maria da Penha Trindade Pinheiro; Rocha, Myrna Santos; Miagostovich, Marize Pereira; Leite, José Paulo Gagliardi; Volotão, Eduardo de Mello

    2017-01-01

    Diarrheal diseases (DD) have distinct etiological profiles in immune-deficient and immune-competent patients. This study compares detection rates, genotype distribution and viral loads of different enteric viral agents in HIV-1 seropositive (n = 200) and HIV-1 seronegative (n = 125) children hospitalized with DD in Rio de Janeiro, Brazil. Except for group A rotavirus (RVA), which were detected through enzyme immunoassay, the other enteric viruses (norovirus [NoV], astrovirus [HAstV], adenovirus [HAdV] and bocavirus [HBoV]) were detected through PCR or RT-PCR. A quantitative PCR was performed for RVA, NoV, HAstV, HAdV and HBoV. Infections with NoV (19% vs. 9.6%; p<0.001), HBoV (14% vs. 7.2%; p = 0.042) and HAdV (30.5% vs. 14.4%; p<0.001) were significantly more frequent among HIV-1 seropositive children. RVA was significantly less frequent among HIV-1 seropositive patients (6.5% vs. 20%; p<0.001). Similarly, frequency of infection with HAstV was lower among HIV-1 seropositive children (5.5% vs. 12.8%; p = 0.018). Among HIV-1 seropositive children 33 (16.5%) had co-infections, including three enteric viruses, such as NoV, HBoV and HAdV (n = 2) and NoV, HAstV and HAdV (n = 2). The frequency of infection with more than one virus was 17 (13.6%) in the HIV-1 negative group, triple infection (NoV + HAstV + HBoV) being observed in only one patient. The median viral load of HAstV in feces was significantly higher among HIV-1 positive children compared to HIV-1 negative children. Concerning children infected with RVA, NoV, HBoV and HAdV, no statistically significant differences were observed in the medians of viral loads in feces, comparing HIV-1 seropositive and HIV-1 seronegative children. Similar detection rates were observed for RVA, HAstV and HAdV, whilst NoV and HBoV were significantly more prevalent among children with CD4+ T lymphocyte count below 200 cells/mm3. Enteric viruses should be considered an important cause of DD in HIV-1 seropositive children, along with

  17. 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

  18. 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.

  19. Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

    PubMed Central

    2010-01-01

    Background Two HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans et al., JAIDS 2008, 47:69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection. Results Biological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the tat gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus. Conclusions These preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate. PMID:20646276

  20. Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Moldt, Brian; Le, Khoa; Robinson, James E.; Burton, Dennis R.

    2016-01-01

    ABSTRACT Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO. ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. IMPORTANCE This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. PMID:27122574

  1. High prevalence of diverse forms of HIV-1 intersubtype recombinants in Central Myanmar: geographical hot spot of extensive recombination.

    PubMed

    Takebe, Yutaka; Motomura, Kazushi; Tatsumi, Masashi; Lwin, Hla Htut; Zaw, Myint; Kusagawa, Shigeru

    2003-09-26

    To investigate the molecular epidemiology and genetic structure of HIV-1s causing the epidemic in Central Myanmar and to explore the genesis of HIV epidemic in this area. A molecular epidemiological investigation was conducted in 1999-2000 in the city of Mandalay among high-risk populations and the structural features of circulating HIV-1s were analyzed. HIV-1 genotypes of 59 specimens were screened based on gag (p17) and env (C2/V3) regions. Near full-length nucleotide sequences of HIV-1 isolates with subtype discordance were determined and their recombinant structures were characterized. Three lineages of HIV-1 strains, including CRF01_AE (27, 45.8%), subtype B' (Thailand variant of subtype B) (15, 25.4%) and subtype C (8, 13.6%), were distributed in Mandalay, while substantial portions (9, 15.3%) of specimens showed various patterns of subtype discordance in different regions of HIV-1 genomes. The study on six HIV-1 isolates with subtype discordance revealed that they were highly diverse types of unique recombinant forms (URFs) comprised of various combinations of three circulating subtypes. One URF was a particularly complex mosaic that contained 13 recombination breakpoints between three HIV-1 subtypes. Approximately half of recombinants showed 'pseudotype' virion structures, in which the external portions of envelope glycoproteins were exchanged with different lineages of HIV-1 strains, suggesting the potential selective advantage of 'pseudotype' viruses over parental strains. The study revealed the unique geographical hot spot in Central Myanmar where extensive recombination events appeared to be taking place continually. This reflects the presence of highly exposed individuals and social networks of HIV-1 transmission.

  2. Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies.

    PubMed

    von Bredow, Benjamin; Arias, Juan F; Heyer, Lisa N; Moldt, Brian; Le, Khoa; Robinson, James E; Zolla-Pazner, Susan; Burton, Dennis R; Evans, David T

    2016-07-01

    Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Reactivation Kinetics of HIV-1 and Susceptibility of Reactivated Latently Infected CD4+ T Cells to HIV-1-Specific CD8+ T Cells

    PubMed Central

    Walker-Sperling, Victoria E. K.; Cohen, Valerie J.; Tarwater, Patrick M.

    2015-01-01

    ABSTRACT The “shock and kill” model of human immunodeficiency virus type 1 (HIV-1) eradication involves the induction of transcription of HIV-1 genes in latently infected CD4+ T cells, followed by the elimination of these infected CD4+ T cells by CD8+ T cells or other effector cells. CD8+ T cells may also be needed to control the spread of new infection if residual infected cells are present at the time combination antiretroviral therapy (cART) is discontinued. In order to determine the time frame needed for CD8+ T cells to effectively prevent the spread of HIV-1 infection, we examined the kinetics of HIV transcription and virus release in latently infected cells reactivated ex vivo. Isolated resting, primary CD4+ T cells from HIV-positive (HIV+) subjects on suppressive regimens were found to upregulate cell-associated HIV-1 mRNA within 1 h of stimulation and produce extracellular virus as early as 6 h poststimulation. In spite of the rapid kinetics of virus production, we show that CD8+ T cells from 2 out of 4 viremic controllers were capable of effectively eliminating reactivated autologous CD4+ cells that upregulate cell-associated HIV-1 mRNA. The results have implications for devising strategies to prevent rebound viremia due to reactivation of rare latently infected cells that persist after potentially curative therapy. IMPORTANCE A prominent HIV-1 cure strategy termed “shock and kill” involves the induction of HIV-1 transcription in latently infected CD4+ T cells with the goal of elimination of these cells by either the cytotoxic T lymphocyte response or other immune cell subsets. However, the cytotoxic T cell response may also be required after curative treatment if residual latently infected cells remain. The kinetics of HIV-1 reactivation indicate rapid upregulation of cell-associated HIV-1 mRNA and a 5-h window between transcription and virus release. Thus, HIV-specific CD8+ T cell responses likely have a very short time frame to eliminate

  4. 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.

  5. 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.

  6. [Mutation frequencies in HIV-1 subtype-A genome in regions containing efficient RNAi targets].

    PubMed

    Kravatsky, Y V; Chechetkin, V R; Fedoseeva, D M; Gorbacheva, M A; Kretova, O V; Tchurikov, N A

    2016-01-01

    The development of gene-therapy technology using RNAi for AIDS/HIV-1 treatment is a prospective alternative to traditional anti-retroviral therapy. RNAi targets could be selected in HIV-1 transcripts and in CCR5 mRNA. Previously, we experimentally selected a number of efficient siRNAs that target HIV-1 RNAs. The viral genome mutates frequently, and RNAi strength is very sensitive, even for a single mismatches. That is why it is important to study nucleotide sequences of targets in clinical isolates of HIV-1. In the present study, we analyzed mutations in 6 of about 300-bp regions containing RNAi targets from HIV-1 subtype A isolates in Russia. Estimates of the mean frequencies of mutations in the targets were obtained and the frequencies of mutations in the different codon positions were compared. The frequencies of mutations in the vicinity of the targets and directly within the targets were also compared and have been shown to be approximately the same. The frequencies of indels in the chosen regions have been assessed. Their frequencies have proved to be two to three orders of magnitude less compared to that for mutations.

  7. Dendritic cell-mediated HIV-1 infection of T-cells demonstrates a direct relationship to plasma viral RNA levels

    PubMed Central

    Arora, Reetakshi; Bull, Lara; Siwak, Edward B.; Thippeshappa, Rajesh; Arduino, Roberto C.; Kimata, Jason T.

    2011-01-01

    Objective To examine the relationship between infectivity of HIV-1 variants in dendritic cell-mediated in trans infection of T-cells and plasma viral RNA levels in infected subjects. Methods HIV-1 was isolated from PBMCs of chronically infected individuals, typed for coreceptor usage, and viral replication was examined in monocyte derived-dendritic cells-peripheral blood lymphocytes (DC-PBL) co-cultures. The rate of p24 antigen production during the logarithmic phase of viral replication was determined by ELISA. Additionally, nef variants were cloned and expressed in trans with a HIV-luciferase vector and CCR5-tropic HIV-1 envelope, and infectivity was measured in DC-mediated capture-transfer assays. Results Replication capacity of HIV-1 viral CCR5-tropic isolates in DC-PBL co-cultures was linearly associated with the plasma viral RNA levels in a cohort of HIV-1 infected individuals exhibiting an inverse relationship between plasma viral RNA and CD4 cell count. Furthermore, infectivity activity of Nef variants in context of DC-mediated enhanced infection of T-cells also showed a linear relationship to plasma viral RNA levels. Conclusion These results illustrate that replication capacity of HIV-1 in DC-T-cell cultures is a significant predictor of plasma viral RNA level. The data suggest that adaptation of HIV-1 to DC interactions with T-cells influences the level of viral replication in the host. PMID:20386455

  8. 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.

  9. Affinity purification of HIV-1 and HIV-2 proteases from recombinant E. coli strains using pepstatin-agarose.

    PubMed

    Rittenhouse, J; Turon, M C; Helfrich, R J; Albrecht, K S; Weigl, D; Simmer, R L; Mordini, F; Erickson, J; Kohlbrenner, W E

    1990-08-31

    A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E. coli constructs that were developed for the expression of these enzymes. HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates. A Mono S ionic exchange step following pepstatin-agarose chromatography was sufficient to bring the HIV-1 protease to homogeneity. Purification of either enzyme can be completed in several days yielding homogeneous preparations suitable for crystallization and other physical characterization.

  10. 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.

  11. 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.

  12. Primary Human Mammary Epithelial Cells Endocytose HIV-1 and Facilitate Viral Infection of CD4+ T Lymphocytes ▿

    PubMed Central

    Dorosko, Stephanie M.; Connor, Ruth I.

    2010-01-01

    The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4+ cells throughout the breast-feeding period, it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4+ cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4+ target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-, CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription and integration and productive viral infection. MEC were also tested for the ability to transfer HIV to CD4+ target cells and to activate resting CD4+ T cells. Our results demonstrate that MEC express HIV-1 receptor proteins CD4, CCR5, CXCR4, and galactosyl ceramide (GalCer). While no evidence for direct infection of MEC was found, HIV-1 virions were observed in MEC endosomal compartments. Coculture of HIV-exposed MEC resulted in productive infection of activated CD4+ T cells. In addition, MEC secretions increased HIV-1 replication and proliferation of infected target cells. Overall, our results indicate that MEC are capable of endosomal uptake of HIV-1 and can facilitate virus infection and replication in CD4+ target cells. These findings suggest that MEC may serve as a viral reservoir for HIV-1 and may enhance infection of CD4+ T lymphocytes in vivo. PMID:20702626

  13. Biochemical and Biologic Characterization of Exosomes and Microvesicles as Facilitators of HIV-1 Infection in Macrophages1

    PubMed Central

    Kadiu, Irena; Narayanasamy, Prabagaran; Dash, Prasanta K.; Zhang, Wei; Gendelman, Howard E.

    2013-01-01

    Exosomes and microvesicles are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Lipidomic, proteomic and cell biologic approaches uncovered processes by which the human immunodeficiency virus type-1 (HIV-1) can use exosomes and MV to facilitate its dissemination. Macrophage MV and exosomes were isolated by immunoaffinity and sucrose cushion centrifugation and characterized by morphologic, biochemical and molecular assays. HIV-1 was “entrapped” in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV and exosomes facilitated viral infection that was affected by a range of cell surface receptors and adhesion proteins. HIV-1 readily completed its life cycle in human monocyte-derived macrophages but not in CD4 negative cells. The data support a significant role for exosomes as facilitators of viral infection. PMID:22711894

  14. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

    PubMed

    Stantchev, Tzanko S; Paciga, Mark; Lankford, Carla R; Schwartzkopff, Franziska; Broder, Christopher C; Clouse, Kathleen A

    2012-12-03

    The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences

  15. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

    PubMed Central

    2012-01-01

    Background The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/ reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. Results We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets - primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Conclusions Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an

  16. 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.

  17. HIV-1 LTR subtype and perinatal transmission.

    PubMed

    Blackard, J T; Renjifo, B; Fawzi, W; Hertzmark, E; Msamanga, G; Mwakagile, D; Hunter, D; Spiegelman, D; Sharghi, N; Kagoma, C; Essex, M

    2001-09-01

    Multiple subtypes of HIV-1 have been identified; however, there is little data on the relative transmissibility of viruses belonging to different subtypes. A matched case-control study addressed whether viruses with different long terminal repeat (LTR) subtypes were transmitted equally from mother to infant. The LTR subtype was determined for 45 matched cases and controls who participated in a clinical trial in Tanzania. HIV-1 subtypes A, C, and D and intersubtype recombinant sequences were identified. Exact matched logistic regression analysis showed that viruses containing subtype A or intersubtype recombinant LTRs were 3.2 and 4.8 times more likely to be transmitted from mother to infant than viruses with subtype D LTRs. Viruses containing subtype C LTRs were 6.1 times more likely to be transmitted than those with subtype D LTRs. These differences in transmission were independent of maternal CD4 at enrollment. Thus, it appears that HIV-1 subtype may be associated with differing rates of perinatal transmission in Tanzania. Copyright 2001 Academic Press.

  18. 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.

  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. Punica granatum (Pomegranate) juice provides an HIV-1 entry inhibitor and candidate topical microbicide

    PubMed Central

    Neurath, A Robert; Strick, Nathan; Li, Yun-Yao; Debnath, Asim K

    2004-01-01

    Background For ≈ 24 years the AIDS pandemic has claimed ≈ 30 million lives, causing ≈ 14,000 new HIV-1 infections daily worldwide in 2003. About 80% of infections occur by heterosexual transmission. In the absence of vaccines, topical microbicides, expected to block virus transmission, offer hope for controlling the pandemic. Antiretroviral chemotherapeutics have decreased AIDS mortality in industrialized countries, but only minimally in developing countries. To prevent an analogous dichotomy, microbicides should be: acceptable; accessible; affordable; and accelerative in transition from development to marketing. Already marketed pharmaceutical excipients or foods, with established safety records and adequate anti-HIV-1 activity, may provide this option. Methods Fruit juices were screened for inhibitory activity against HIV-1 IIIB using CD4 and CXCR4 as cell receptors. The best juice was tested for inhibition of: (1) infection by HIV-1 BaL, utilizing CCR5 as the cellular coreceptor; and (2) binding of gp120 IIIB and gp120 BaL, respectively, to CXCR4 and CCR5. To remove most colored juice components, the adsorption of the effective ingredient(s) to dispersible excipients and other foods was investigated. A selected complex was assayed for inhibition of infection by primary HIV-1 isolates. Results HIV-1 entry inhibitors from pomegranate juice adsorb onto corn starch. The resulting complex blocks virus binding to CD4 and CXCR4/CCR5 and inhibits infection by primary virus clades A to G and group O. Conclusion These results suggest the possibility of producing an anti-HIV-1 microbicide from inexpensive, widely available sources, whose safety has been established throughout centuries, provided that its quality is adequately standardized and monitored. PMID:15485580

  1. Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets

    PubMed Central

    Phogat, S.; Wyatt, R. T.; Hedestam, G. B. Karlsson

    2008-01-01

    Phogat S, Wyatt RT, Karlsson Hedestam GB (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; Department of Microbiology Tumor and Cell Biology, Karolinska Institutet, Stockholm; and the Swedish Institute for Infectious Disease Control, Solna, Sweden). Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets (Review). Vaccine-induced antibodies that interfere with viral entry are the protective correlate of most existing prophylactic vaccines. However, for highly variable viruses such as HIV-1, the ability to elicit broadly neutralizing antibody responses through vaccination has proven to be extremely difficult. The major targets for HIV-1 neutralizing antibodies are the viral envelope glycoprotein trimers on the surface of the virus that mediate receptor binding and entry. HIV-1 has evolved many mechanisms on the surface of envelope glyco-proteins to evade antibody-mediated neutralization, including the masking of conserved regions by glycan, quaternary protein interactions and the presence of immunodominant variable elements. The primary challenge in the development of an HIV-1 vaccine that elicits broadly neutralizing antibodies therefore lies in the design of suitable envelope glycoprotein immunogens that circumvent these barriers. Here, we describe neutralizing determinants on the viral envelope glyco-proteins that are defined by their function in receptor binding or by rare neutralizing antibodies isolated from HIV-infected individuals. We also describe the nonvariable cellular receptors involved in the HIV-1 entry process, or other cellular proteins, and ongoing studies to determine if antibodies against these proteins have efficacy as therapeutic reagents or, in some cases, as vaccine targets to interfere with HIV-1 entry. PMID:17598813

  2. Anti-HIV-1 Activity of Eight Monofloral Iranian Honey Types

    PubMed Central

    Behbahani, Mandana

    2014-01-01

    Monofloral Iranian honeys from eight floral sources were analyzed to determine their anti-HIV-1 activities as well as their effects on lymphocyte proliferation. The Peripheral Blood Mononuclear Cells (PBMCs) used in this study were prepared from five healthy volunteers who were seronegative for HIV, HCV, HBV and TB. The anti-HIV-1 activity of eight different honeys was performed by quantitative polymerase chain reaction (PCR) assay and high pure viral nucleic acid kit. The results demonstrated that monofloral honeys from Petro selinum sativum, Nigella sativa, Citrus sinensis, Zataria multiflora, Citrus aurantium and Zizyphus mauritiana flowers had potent anti-HIV-1 activity with half maximal effective concentration (EC50) values of 37.5, 88, 70, 88, 105 and 5 µg/ml respectively. However, monofloral Iranian honeys from Astragalus gummifer and Chamaemelum nobile flowers had weak anti-HIV-1 activity. The frequency and intensity of CD4 expression on PBMCs increased in the presence of all honey types. CD19 marker were also increased after the treatment with monofloral honeys from Z.multiflora and N. sativa. The anti-HIV-1 agent in monofloral honeys from P.sativum, N. sativa, Z. multiflora and Z. mauritiana flowers was detected by spectroscopic analysis as methylglyoxal. Time of drug addition studies demonstrated that the inhibitory effect of methylglyoxal is higher on the late stage of HIV-1 infection. The result demonstrated that methylglyoxal isolated from monofloral honey types is a good candidate for preclinical evaluation of anti-HIV-1 therapies. PMID:25333699

  3. Prevalence of HIV-1 resistant strains in recent seroconverters.

    PubMed

    Balotta, C; Berlusconi, A; Pan, A; Violin, M; Riva, C; Gori, A; Corvasce, S; Mazzucchelli, R; Facchi, G; Velleca, R; Senese, D; Dehò, L; Galli, M; Rusconi, S; Moroni, M

    2000-01-01

    Twenty-nine HIV-1 recently infected subjects were retrospectively studied to investigate both the prevalence of nucleoside reverse transcriptase inhibitors (NRTI)-related mutations at primary infection and the proportion of naturally occurring mutations in protease inhibitor (PI)-naive patients. Neither HIV-1 plasma viremia nor CD4 absolute count at baseline could distinguish patients with NRTI pre-existing mutations from those with wild-type virus. An increasing proportion of ZDV-related mutations was observed over time with an overall frequency of 20.7% in the study period. Only 1 out of 6 patients (16.7%) with ZDV-related mutations showed a phenotypically ZDV resistant isolate. A striking proportion of polymorphic changes was present in the protease region of pol gene in newly infected individuals. As many as 80% of seroconverters presented at least one naturally occurring substitution. Some PI-associated substitutions, thought to be compensatory in protease enzymatic function, could confer intermediate to high PI-resistance. Their role following PI administration remains to be elucidated. Our data suggest that the choice of drugs should be oriented by both genotypic and phenotypic evaluations to tailor individual regimens in seroconverters.

  4. Sequence determinants of breakpoint location during HIV-1 intersubtype recombination.

    PubMed

    Baird, Heather A; Galetto, Román; Gao, Yong; Simon-Loriere, Etienne; Abreha, Measho; Archer, John; Fan, Jun; Robertson, David L; Arts, Eric J; Negroni, Matteo

    2006-01-01

    Retroviral recombination results from strand switching, during reverse transcription, between the two copies of genomic RNA present in the virus. We analysed recombination in part of the envelope gene, between HIV-1 subtype A and D strains. After a single infection cycle, breakpoints clustered in regions corresponding to the constant portions of Env. With some exceptions, a similar distribution was observed after multiple infection cycles, and among recombinant sequences in the HIV Sequence Database. We compared the experimental data with computer simulations made using a program that only allows recombination to occur whenever an identical base is present in the aligned parental RNAs. Experimental recombination was more frequent than expected on the basis of simulated recombination when, in a region spanning 40 nt from the 5' border of a breakpoint, no more than two discordant bases between the parental RNAs were present. When these requirements were not fulfilled, breakpoints were distributed randomly along the RNA, closer to the distribution predicted by computer simulation. A significant preference for recombination was also observed for regions containing homopolymeric stretches. These results define, for the first time, local sequence determinants for recombination between divergent HIV-1 isolates.

  5. Sequence determinants of breakpoint location during HIV-1 intersubtype recombination

    PubMed Central

    Baird, Heather A.; Galetto, Román; Gao, Yong; Simon-Loriere, Etienne; Abreha, Measho; Archer, John; Fan, Jun; Robertson, David L.; Arts, Eric J.; Negroni, Matteo

    2006-01-01

    Retroviral recombination results from strand switching, during reverse transcription, between the two copies of genomic RNA present in the virus. We analysed recombination in part of the envelope gene, between HIV-1 subtype A and D strains. After a single infection cycle, breakpoints clustered in regions corresponding to the constant portions of Env. With some exceptions, a similar distribution was observed after multiple infection cycles, and among recombinant sequences in the HIV Sequence Database. We compared the experimental data with computer simulations made using a program that only allows recombination to occur whenever an identical base is present in the aligned parental RNAs. Experimental recombination was more frequent than expected on the basis of simulated recombination when, in a region spanning 40 nt from the 5′ border of a breakpoint, no more than two discordant bases between the parental RNAs were present. When these requirements were not fulfilled, breakpoints were distributed randomly along the RNA, closer to the distribution predicted by computer simulation. A significant preference for recombination was also observed for regions containing homopolymeric stretches. These results define, for the first time, local sequence determinants for recombination between divergent HIV-1 isolates. PMID:17003055

  6. Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses.

    PubMed

    Swanson, Priscilla; Huang, Shihai; Abravaya, Klara; de Mendoza, Carmen; Soriano, Vincent; Devare, Sushil G; Hackett, John

    2007-04-01

    Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. Suppression of HIV-1 Infectivity by Human Glioma Cells

    PubMed Central

    Hoque, Sheikh Ariful; Tanaka, Atsushi; Islam, Salequl; Ahsan, Gias Uddin; Jinno-Oue, Atsushi

    2016-01-01

    Abstract 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. PMID:26650729

  12. 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

  13. 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.

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

    PubMed

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

    2015-01-01

    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. Copyright © 2014. Published by Elsevier B.V.

  15. Quantification of the Epitope Diversity of HIV-1-Specific Binding Antibodies by Peptide Microarrays for Global HIV-1 Vaccine Development

    PubMed Central

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

    2014-01-01

    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 6,564 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. PMID:25445329

  16. 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

  17. Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

    SciTech Connect

    Wu, Xueling; Zhou, Tongqing; Zhu, Jiang; Zhang, Baoshan; Georgiev, Ivelin; Wang, Charlene; Chen, Xuejun; Longo, Nancy S.; Louder, Mark; McKee, Krisha; O’Dell, Sijy; Perfetto, Stephen; Schmidt, Stephen D.; Shi, Wei; Wu, Lan; Yang, Yongping; Yang, Zhi-Yong; Yang, Zhongjia; Zhang, Zhenhai; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Doria-Rose, Nicole A.; Connors, Mark; Mullikin, James C.; Nabel, Gary J.; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D.; Mascola, John R.

    2013-03-04

    Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

  18. Modulation of the proteome of peripheral blood mononuclear cells from HIV-1 infected patients by drugs of abuse

    PubMed Central

    Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikunar; Nair, Bindukumar; Sykes, Donald E.; Agosto-Mujica, Arnadri; Hsiao, Chiu Bin; Schwartz, Stanley A.

    2010-01-01

    We used proteomic analyses to assess how drug abuse modulates immunologic responses to infections with the human immunodeficiency virus type 1 (HIV-1). Two dimensional (2D) difference gel electrophoresis was utilized to determine changes in the proteome of peripheral blood mononuclear cells (PBMC) isolated from HIV-1 positive donors that occurred after treatment with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins. We further isolated specific subpopulations of PBMC to determine which subpopulations were selectively affected by treatment with drugs of abuse. Monocytes, B cells and T cells were positively or negatively selected from PBMC isolated from HIV-1 positive donors. Our results demonstrate that cocaine and methamphetamine modulate gene expression primarily in monocytes and T cells, the primary targets of HIV-1 infection. Proteomic data were validated with quantitative, real-time PCR. These studies elucidate the molecular mechanisms underlying the effects of drugs of abuse on HIV-1 infections. Several functionally relevant classes of proteins were identified as potential mediators of HIV-1 pathogenesis and disease progression associated with drug abuse. PMID:19543960

  19. Homozygous delta 32 deletion of the CCR-5 chemokine receptor gene in an HIV-1-infected patient.

    PubMed

    Balotta, C; Bagnarelli, P; Violin, M; Ridolfo, A L; Zhou, D; Berlusconi, A; Corvasce, S; Corbellino, M; Clementi, M; Clerici, M; Moroni, M; Galli, M

    1997-08-01

    Recent research has found that entry of non-syncytium-inducing (NSI), monocyte-macrophage-tropic HIV-1 isolates requires binding to both CD4 and CCR5 receptors, and that delta 32/delta 32 homozygous individuals are protected against infection. To analyse the polymorphism of CCR-5 gene in HIV-1-infected and uninfected subjects. CCR-5 sequences were amplified by polymerase chain reaction (PCR) from DNA of peripheral blood mononuclear cells. Samples from 152 HIV-1-infected subjects and 122 uninfected controls were tested for the detection of the 32 base-pair deletion. HIV-1 phenotype was determined by viral isolation and MT-2 evaluation. The wild-type/delta 32 heterozygous and delta 32/delta 32 homozygous conditions were represented in 10.7 and 0.8% of healthy controls and in 9.8 and 0.7% of HIV-1-infected subjects, respectively. Of note, the delta 32/delta 32 deletion of the CCR-5 gene was detected by PCR and sequencing confirmed in a patient with progressive infection harbouring a clade B virus with SI phenotype. delta 32/delta 32 homozygosity for the CCR-5 gene does not confer absolute protection against HIV-1 infection, suggesting that either macrophage-tropic viral strains could use coreceptors other than CCR-5 or infect independently of the presence of a functional CCR-5 coreceptor. Alternatively, primary infection sustained by T-cell-tropic isolates, although exceptional, may occur.

  20. Antibody 10-1074 suppresses viremia in HIV-1-infected individuals.

    PubMed

    Caskey, Marina; Schoofs, Till; Gruell, Henning; Settler, Allison; Karagounis, Theodora; Kreider, Edward F; Murrell, Ben; Pfeifer, Nico; Nogueira, Lilian; Oliveira, Thiago Y; Learn, Gerald H; Cohen, Yehuda Z; Lehmann, Clara; Gillor, Daniel; Shimeliovich, Irina; Unson-O'Brien, Cecilia; Weiland, Daniela; Robles, Alexander; Kümmerle, Tim; Wyen, Christoph; Levin, Rebeka; Witmer-Pack, Maggi; Eren, Kemal; Ignacio, Caroline; Kiss, Szilard; West, Anthony P; Mouquet, Hugo; Zingman, Barry S; Gulick, Roy M; Keler, Tibor; Bjorkman, Pamela J; Seaman, Michael S; Hahn, Beatrice H; Fätkenheuer, Gerd; Schlesinger, Sarah J; Nussenzweig, Michel C; Klein, Florian

    2017-02-01

    Monoclonal antibody 10-1074 targets the V3 glycan supersite on the HIV-1 envelope (Env) protein. It is among the most potent anti-HIV-1 neutralizing antibodies isolated so far. Here we report on its safety and activity in 33 individuals who received a single intravenous infusion of the antibody. 10-1074 was well tolerated and had a half-life of 24.0 d in participants without HIV-1 infection and 12.8 d in individuals with HIV-1 infection. Thirteen individuals with viremia received the highest dose of 30 mg/kg 10-1074. Eleven of these participants were 10-1074-sensitive and showed a rapid decline in viremia by a mean of 1.52 log10 copies/ml. Virologic analysis revealed the emergence of multiple independent 10-1074-resistant viruses in the first weeks after infusion. Emerging escape variants were generally resistant to the related V3-specific antibody PGT121, but remained sensitive to antibodies targeting nonoverlapping epitopes, such as the anti-CD4-binding-site antibodies 3BNC117 and VRC01. The results demonstrate the safety and activity of 10-1074 in humans and support the idea that antibodies targeting the V3 glycan supersite might be useful for the treatment and prevention of HIV-1 infection.

  1. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner.

    PubMed

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé; Emiliani, Stéphane; Gallois-Montbrun, Sarah

    2016-12-20

    Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.

  2. DC-SIGN plays a stronger role than DCIR in mediating HIV-1 capture and transfer.

    PubMed

    Jin, Wei; Li, Chang; Du, Tao; Hu, Kai; Huang, Xin; Hu, Qinxue

    2014-06-01

    The C-type lectin receptors (CLRs) expressed on dendritic cells (DCs), in particular DC-SIGN and DCIR, likely play an important role in HIV-1 early infection. Here, we systematically compared the capture and transfer capability of DC-SIGN and DCIR using a wide range of HIV-1 isolates. Our results indicated that DC-SIGN plays a stronger role than DCIR in DC-mediated HIV-1 capture and transfer. This was further strengthened by the data from transient and stable transfectants, showing that DC-SIGN had better capability, compared with DCIR in HIV-1 capture and transfer. Following constructing and analyzing a series of soluble DC-SIGN and DCIR truncates and chimeras, we demonstrated that the neck domain, but not the CRD, renders DC-SIGN higher binding affinity to gp120 likely via the formation of tetramerization. Our findings provide insights into CLR-mediated HIV-1 capture and transfer, highlighting potential targets for intervention strategies against gp120-CLR interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Genetic and structural analyses of affinity maturation in the humoral response to HIV-1.

    PubMed

    Kepler, Thomas B; Wiehe, Kevin

    2017-01-01

    Most broadly neutralizing antibodies (BNAbs) elicited in response to HIV-1 infection are extraordinarily mutated. One goal of HIV-1 vaccine development is to induce antibodies that are similar to the most potent and broad BNAbs isolated from infected subjects. The most effective BNAbs have very high mutation frequencies, indicative of the long periods of continual activation necessary to acquire the BNAb phenotype through affinity maturation. Understanding the mutational patterns that define the maturation pathways in BNAb development is critical to vaccine design efforts to recapitulate through vaccination the successful routes to neutralization breadth and potency that have occurred in natural infection. Studying the mutational changes that occur during affinity maturation, however, requires accurate partitioning of sequence data into B-cell clones and identification of the starting point of a B-cell clonal lineage, the initial V(D)J rearrangement. Here, we describe the statistical framework we have used to perform these tasks. Through the recent advancement of these and similar computational methods, many HIV-1 ancestral antibodies have been inferred, synthesized and their structures determined. This has allowed, for the first time, the investigation of the structural mechanisms underlying the affinity maturation process in HIV-1 antibody development. Here, we review what has been learned from this atomic-level structural characterization of affinity maturation in HIV-1 antibodies and the implications for vaccine design.

  4. Influence of mutation and recombination on HIV-1 in vitro fitness recovery.

    PubMed

    Arenas, Miguel; Lorenzo-Redondo, Ramon; Lopez-Galindez, Cecilio

    2016-01-01

    The understanding of the evolutionary processes underlying HIV-1 fitness recovery is fundamental for HIV-1 pathogenesis, antiretroviral treatment and vaccine design. It is known that HIV-1 can present very high mutation and recombination rates, however the specific contribution of these evolutionary forces in the "in vitro" viral fitness recovery has not been simultaneously quantified. To this aim, we analyzed substitution, recombination and molecular adaptation rates in a variety of HIV-1 biological clones derived from a viral isolate after severe population bottlenecks and a number of large population cell culture passages. These clones presented an overall but uneven fitness gain, mean of 3-fold, respect to the initial passage values. We found a significant relationship between the fitness increase and the appearance and fixation of mutations. In addition, these fixed mutations presented molecular signatures of positive selection through the accumulation of non-synonymous substitutions. Interestingly, viral recombination correlated with fitness recovery in most of studied viral quasispecies. The genetic diversity generated by these evolutionary processes was positively correlated with the viral fitness. We conclude that HIV-1 fitness recovery can be derived from the genetic heterogeneity generated through both mutation and recombination, and under diversifying molecular adaptation. The findings also suggest nonrandom evolutionary pathways for in vitro fitness recovery.

  5. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner

    PubMed Central

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé

    2017-01-01

    Abstract Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway. PMID:28003477

  6. A global approach to HIV-1 vaccine development

    PubMed Central

    Stephenson, Kathryn E; Barouch, Dan H

    2013-01-01

    Summary A global human immunodeficiency virus-1 (HIV-1) vaccine will have to elicit immune responses capable of providing protection against a tremendous diversity of HIV-1 variants. In this review, we first describe the current state of the HIV-1 vaccine field, outlining the immune responses that are desired in a global HIV-1 vaccine. In particular, we emphasize the likely importance of Env-specific neutralizing and non-neutralizing antibodies for protection against HIV-1 acquisition and the likely importance of effector Gag-specific T lymphocytes for virologic control. We then highlight four strategies for developing a global HIV-1 vaccine. The first approach is to design specific vaccines for each geographic region that include antigens tailor-made to match local circulating HIV-1 strains. The second approach is to design a vaccine that will elicit Env-specific antibodies capable of broadly neutralizing all HIV-1 subtypes. The third approach is to design a vaccine that will elicit cellular immune responses that are focused on highly conserved HIV-1 sequences. The fourth approach is to design a vaccine to elicit highly diverse HIV-1-specific responses. Finally, we emphasize the importance of conducting clinical efficacy trials as the only way to determine which strategies will provide optimal protection against HIV-1 in humans. PMID:23772627

  7. Semi-synthesis of oxygenated dolabellane diterpenes with highly in vitro anti-HIV-1 activity.

    PubMed

    Pardo-Vargas, Alonso; Ramos, Freddy A; Cirne-Santos, Claudio Cesar; Stephens, Paulo Roberto; Paixão, Izabel Christina Palmer; Teixeira, Valeria Laneuville; Castellanos, Leonardo

    2014-09-15

    Research on dolabellane diterpenes of brown algae Dictyota spp. has shown that these diterpenoids have strong anti-HIV-1 activity, but there are not data about antiviral activity of dolabellane diterpenes isolated from octocorals, which are antipodes of those isolated from the brown algae. Dolabellanes 13-keto-1(R),11(S)-dolabella-3(E),7(E),12(18)-triene (1) and β-Araneosene (2) were isolated from the Caribbean octocoral Eunicea laciniata, and both showed low anti-HIV-1 activity and low toxicity. Since it was shown that oxygenated dolabellanes from algae have better anti-HIV-1 activity, in this work some derivatives of the main dolabellane of E. laciniata1 were obtained by epoxidation (3), epoxide opening (4), and allylic oxidation (5). The derivatives showed significant improvement in the anti-HIV-1potency (100-fold), being compounds 3 and 5 the most active ones. Their high antiviral activities, along with their low cytotoxicity, make them promissory antiviral compounds; and it is worth noting that the absolute configuration at the ring junction in the dolabellane skeleton does not seem to be determinant in the antiviral potency of these diterpeneoids.

  8. Genotypic characterization of human immunodeficiency virus type 1 in Greece. Multicentre Study on HIV-1 Heterogeneity.

    PubMed

    Nasioulas, G; Paraskevis, D; Paparizos, V; Lazanas, M; Karafoulidou, A; Hatzakis, A

    1998-05-20

    The HIV-1 subtype distribution in 83 HIV-1-seropositive individuals living in Greece was investigated by using the heteroduplex mobility assay (HMA), DNA sequencing, and phylogenetic analysis. The results revealed that partial HIV-1 gp120 sequences from 71 (86%) patients were subtype B, 5 (6%) were subtype A, 4 were subtype D (5%), 2 (2%) were subtype C, and 1 (1%) was subtype I. The subtype I isolate was documented in an intravenous drug user. A high prevalence (90-100%) of B isolates among intravenous drug users, hemophiliacs, and homosexual men was observed, in contrast to heterosexuals, among whom non-B subtypes seemed to be common (42.9%, p < 0.001). Among the Greek population subtype B is the most frequent (94%), in contrast to the high prevalence (57%) of non-B isolates found in emigrants living in Greece (p < 0.001). A heterosexual transmission case of subtype D in a Greek individual not traveling abroad was also documented. The broad HIV-1 diversity in Greece may be explained by population movements, such as migration and traveling.

  9. Clinical significance and characterization of AZT-resistant strains of HIV-1

    PubMed Central

    Wainberg, Mark A; Rooke, Ronald; Tremblay, Michel; Li, XuGuang; Parniak, Michael A; Gao, Qing; Yao, Xiao-Jian; Tsoukas, Chris; Montaner, JSG; Fanning, M; Ruedy, J

    1991-01-01

    A number of laboratories have now independently confirmed that zidovudine (AZT)-resistant strains of human immunodeficiency virus type 1 (HIV-1) may be isolated from patients undergoing prolonged therapy with this drug. In certain instances, such drug-resistant viral isolates have been obtained from patients with clinical acquired immune deficiency syndrome (aids), while in others, isolation of drug-resistant strains has been achieved in the case of HIV seropositive, asymptomatic subjects. Most of the evidence points to a series of mutations within the polymerase gene of HIV-1, which encodes viral reverse transcriptase, as being responsible for development of the drug-resistant phenotype. It further appears that over 50% of patients treated with AZT for periods longer than six months are likely to yield drug-resistant strains of HIV-1 in their circulation. Furthermore, the development of drug resistance soon after initiation of AZT therapy may potentially be correlated with the likelihood of AZT treatment failure. In several instances, cross resistance has been observed between AZT and other nucleosides being considered for potential therapy of HIV-1-associated disease. PMID:22451746

  10. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

    PubMed

    Jeffries, T L; Sacha, C R; Pollara, J; Himes, J; Jaeger, F H; Dennison, S M; McGuire, E; Kunz, E; Eudailey, J A; Trama, A M; LaBranche, C; Fouda, G G; Wiehe, K; Montefiori, D C; Haynes, B F; Liao, H-X; Ferrari, G; Alam, S M; Moody, M A; Permar, S R

    2016-03-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

  11. Near Full-Length Identification of a Novel HIV-1 CRF01_AE/B/C Recombinant in Northern Myanmar.

    PubMed

    Zhou, Yan-Heng; Chen, Xin; Liang, Yue-Bo; Pang, Wei; Qin, Wei-Hong; Zhang, Chiyu; Zheng, Yong-Tang

    2015-08-01

    The Myanmar-China border appears to be the "hot spot" region for the occurrence of HIV-1 recombination. The majority of the previous analyses of HIV-1 recombination were based on partial genomic sequences, which obviously cannot reflect the reality of the genetic diversity of HIV-1 in this area well. Here, we present a near full-length characterization of a novel HIV-1 CRF01_AE/B/C recombinant isolated from a long-distance truck driver in Northern Myanmar. It is the first description of a near full-length genomic sequence in Myanmar since 2003, and might be one of the most complicated HIV-1 chimeras ever detected in Myanmar, containing four CRF01_AE, six B segments, and five C segments separated by 14 breakpoints throughout its genome. The discovery and characterization of this new CRF01_AE/B/C recombinant indicate that intersubtype recombination is ongoing in Myanmar, continuously generating new forms of HIV-1. More work based on near full-length sequence analyses is urgently needed to better understand the genetic diversity of HIV-1 in these regions.

  12. Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment

    PubMed Central

    Yuan, Jinyun; Wang, Jianbin; Crain, Karen; Fearns, Colleen; Kim, Kenneth A; Hua, Kevin L; Gregory, Philip D; Holmes, Michael C; Torbett, Bruce E

    2012-01-01

    HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4+ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4+ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4+ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4+ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4+ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4+ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4+ T cells during HIV-1 infection. PMID:22273578

  13. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells

    PubMed Central

    Jeffries, Thomas L; Sacha, CR; Pollara, Justin; Himes, Jon; Jaeger, Frederick H; Dennison, S Moses; McGuire, Erin; Kunz, Erika; Eudailey, Joshua A; Trama, Ashley M; LaBranche, Celia; Fouda, Genevieve G; Wiehe, Kevin; Montefiori, David C; Haynes, Barton F; Liao, Hua-Xin; Ferrari, Guido; Alam, S Munir; Moody, M Anthony; Permar, Sallie R

    2015-01-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants due to beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 Envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. Interestingly, we also identified divergent patterns of colostrum Env-specific B cell lineage evolution with respect to cross-reactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk IgG repertoire. Maternal vaccine strategies to specifically target this breast milk B cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants. PMID:26242599

  14. Short Communication: Neutralizing Antibodies in HIV-1-Infected Brazilian Individuals

    PubMed Central

    Morgado, Mariza Gonçalvez; Côrtes, Fernanda Heloise; Guimarães, Monick Lindermeyer; Mendonça-Lima, Leila; Pilotto, Jose Henrique; Grinsztejn, Beatriz; Veloso, Valdiléa Gonçalves; Bongertz, Vera

    2013-01-01

    Abstract Tests for the detection of the humoral immune response to HIV-1 have to be standardized and established, demanding regional efforts. For this purpose the neutralizing antibody (NAb) assay for HIV-1 in TZM-bl cells was introduced in Brazil. Twenty plasma samples from HIV-1-infected individuals were assayed: 10 progressors and 10 long-term nonprogressors. These were tested against eight env-pseudotyped viruses (psVs) in the TZM-bl NAb assay and against HIV-1 strain HTLV/IIIB (HIV-1 IIIB) in primary lymphocytes. Forty-four percent of the samples showed neutralizing titers for psVs and 55% for HIV-1 IIIB. Plasma from progressors showed a broader neutralization and a higher potency. The introduction of these reference reagents encourages the participation of Brazil in future comparative assessments of anti-HIV-1 antibodies. PMID:23145941

  15. Improving Neutralization Potency and Breadth by Combining Broadly Reactive HIV-1 Antibodies Targeting Major Neutralization Epitopes

    PubMed Central

    Kong, Rui; Louder, Mark K.; Wagh, Kshitij; Bailer, Robert T.; deCamp, Allan; Greene, Kelli; Gao, Hongmei; Taft, Justin D.; Gazumyan, Anna; Liu, Cassie; Nussenzweig, Michel C.; Korber, Bette

    2014-01-01

    ABSTRACT The isolation of broadly neutralizing HIV-1 monoclonal antibodies (MAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of MAbs for prevention and treatment of HIV-1 infection. Since many of these MAbs have been isolated in the last few years, the potency and breadth of MAb combinations have not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double-, triple-, and quadruple-MAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite, and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All MAb combinations showed substantially improved neutralization breadth compared to the corresponding single MAbs, while the neutralization potency of individual MAbs was maintained. At a 50% inhibitory concentration (IC50) cutoff of 1 μg/ml per antibody, double-MAb combinations neutralized 89 to 98% of viruses, and triple combinations neutralized 98 to 100%. Overall, the improvement of neutralization breadth was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some MAb combinations, whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan MAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 MAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment. IMPORTANCE Over the last 5 years, numerous broadly reactive HIV-1-neutralizing MAbs have been isolated from B cells of HIV-1-infected donors. Each of these MAbs binds to one of the major vulnerable sites (epitopes) on the surface of the viral envelope glycoprotein. Since antibodies to distinct viral epitopes

  16. Improving neutralization potency and breadth by combining broadly reactive HIV-1 antibodies targeting major neutralization epitopes.

    PubMed

    Kong, Rui; Louder, Mark K; Wagh, Kshitij; Bailer, Robert T; deCamp, Allan; Greene, Kelli; Gao, Hongmei; Taft, Justin D; Gazumyan, Anna; Liu, Cassie; Nussenzweig, Michel C; Korber, Bette; Montefiori, David C; Mascola, John R

    2015-03-01

    The isolation of broadly neutralizing HIV-1 monoclonal antibodies (MAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of MAbs for prevention and treatment of HIV-1 infection. Since many of these MAbs have been isolated in the last few years, the potency and breadth of MAb combinations have not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double-, triple-, and quadruple-MAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite, and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All MAb combinations showed substantially improved neutralization breadth compared to the corresponding single MAbs, while the neutralization potency of individual MAbs was maintained. At a 50% inhibitory concentration (IC50) cutoff of 1 μg/ml per antibody, double-MAb combinations neutralized 89 to 98% of viruses, and triple combinations neutralized 98 to 100%. Overall, the improvement of neutralization breadth was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some MAb combinations, whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan MAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 MAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment. Over the last 5 years, numerous broadly reactive HIV-1-neutralizing MAbs have been isolated from B cells of HIV-1-infected donors. Each of these MAbs binds to one of the major vulnerable sites (epitopes) on the surface of the viral envelope glycoprotein. Since antibodies to distinct viral epitopes could theoretically

  17. 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.

  18. Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method

    PubMed Central

    Kim, Eun-Young; Stanton, Jennifer; Korber, Bette TM; Krebs, Kendall; Bogdan, Derek; Kunstman, Kevin; Wu, Samuel; Phair, John P; Mirkin, Chad A

    2010-01-01

    Background Detection of HIV-1 in patients is limited by the sensitivity and selectivity of available tests. The nanotechnology-based bio-barcode-amplification method offers an innovative approach to detect specific HIV-1 antigens from diverse HIV-1 subtypes. We evaluated the efficacy of this protein-detection method in detecting HIV-1 in men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). Methods The method relies on magnetic microparticles with antibodies that specifically bind the HIV-1 p24 Gag protein and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the microparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes (hundreds per target) were identified by a nanoparticle-based detection method that does not rely on PCR. Results Of 112 plasma samples from HIV-1-infected subjects, 111 were positive for HIV-1 p24 Gag protein (range: 0.11–71.5 ng/ml of plasma) by the bio-barcode-amplification method. HIV-1 p24 Gag protein was detected in only 23 out of 112 men by the conventional ELISA. A total of 34 uninfected subjects were negative by both tests. Thus, the specificity of the bio-barcode-amplification method was 100% and the sensitivity 99%. The bio-barcode-amplification method detected HIV-1 p24 Gag protein in plasma from all study subjects with less than 200 CD4+ T cells/μl of plasma (100%) and 19 out of 20 (95%) HIV-1-infected men who had less than 50 copies/ml of plasma of HIV-1 RNA. In a separate group of 60 diverse international isolates, representative of clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG, the bio-barcode-amplification method identified the presence of virus correctly. Conclusions The bio-barcode-amplification method was superior to the conventional ELISA assay for the detection of HIV-1 p24 Gag protein in plasma

  19. Human Cytosolic Extracts Stabilize the HIV-1 Core

    PubMed Central

    Fricke, Thomas; Brandariz-Nuñez, Alberto; Wang, Xiaozhao; Smith, Amos B.

    2013-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects on HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure the stability of in vitro-assembled HIV-1 CA-NC complexes. The assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core. Interestingly, stabilization of in vitro-assembled HIV-1 CA-NC complexes is not due solely to macromolecular crowding, suggesting the presence of specific cellular factors that stabilize the HIV-1 core. By using our novel assay, we measured the abilities of different drugs, such as PF74, CAP-1, IXN-053, cyclosporine, Bi2 (also known as BI-2), and the peptide CAI, to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes. Interestingly, we found that PF74 and Bi2 strongly stabilized HIV-1 CA-NC complexes. On the other hand, the peptide CAI destabilized HIV-1 CA-NC complexes. We also found that purified cyclophilin A destabilizes in vitro-assembled HIV-1 CA-NC complexes in the presence of cellular extracts in a cyclosporine-sensitive manner. In agreement with previous observations using the fate-of-the-capsid assay, we also demonstrated the ability of recombinant CPSF6 to stabilize HIV-1 CA-NC complexes. Overall, our findings suggested that cellular extracts specifically stabilize the HIV-1 core. We believe that our assay can be a powerful tool to assess HIV-1 core stability in vitro. PMID:23885082

  20. 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

  1. Productive Replication and Evolution of HIV-1 in Ferret Cells

    PubMed Central

    Fadel, Hind J.; Saenz, Dyana T.; Guevara, Rebekah; von Messling, Veronika; Peretz, Mary

    2012-01-01

    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

  2. Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization

    PubMed Central

    Asokan, M.; Rudicell, R. S.; Louder, M.; McKee, K.; O'Dell, S.; Stewart-Jones, G.; Wang, K.; Xu, L.; Chen, X.; Choe, M.; Chuang, G.; Georgiev, I. S.; Joyce, M. G.; Kirys, T.; Ko, S.; Pegu, A.; Shi, W.; Todd, J. P.; Yang, Z.; Bailer, R. T.; Rao, S.; Kwong, P. D.; Nabel, G. J.

    2015-01-01

    ABSTRACT The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 μg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different

  3. De novo Generation of Cells within Human Nurse Macrophages and Consequences following HIV-1 Infection

    PubMed Central

    Gartner, Suzanne

    2012-01-01

    Nurse cells are defined as those that provide for the development of other cells. We report here, that in vitro, human monocyte-derived macrophages can behave as nurse cells with functional capabilities that include de novo generation of CD4+ T-lymphocytes and a previously unknown small cell with monocytoid characteristics. We named these novel cells “self-renewing monocytoid cells” (SRMC), because they could develop into nurse macrophages that produced another generation of SRMC. SRMC were not detectable in blood. Their transition to nurse behavior was characterized by expression of CD10, a marker of thymic epithelium and bone marrow stroma, typically absent on macrophages. Bromodeoxyuridine labeling and immunostaining for cdc6 expression confirmed DNA synthesis within nurse macrophages. T-cell excision circles were detected in macrophages, along with expression of pre-T-cell receptor alpha and recombination activating gene 1, suggesting that genetic recombination events associated with generation of the T-cell receptor were occurring in these cells. SRMC expressed CCR5, the coreceptor for R5 HIV-1 isolates, and were highly susceptible to HIV-1 entry leading to productive infection. While expressing HIV-1, SRMC could differentiate into nurse macrophages that produced another generation of HIV-1-expressing SRMC. The infected nurse macrophage/SRMC cycle could continue in vitro for multiple generations, suggesting it might represent a mechanism whereby HIV-1 can maintain persistence in vivo. HIV-1 infection of nurse macrophages led to a decline in CD4+ T-cell production. There was severe, preferential loss of the CCR5+ CD4+ T-cell subpopulation. Confocal microscopy revealed individual HIV-1-expressing nurse macrophages simultaneously producing both HIV-1-expressing SRMC and non-expressing CD3+ cells, suggesting that nurse macrophages might be a source of latently infected CD4+ T-cells. Real-time PCR experiments confirmed this by demonstrating 10-fold more HIV-1

  4. An Integrated Overview of HIV-1 Latency

    PubMed Central

    Ruelas, Debbie S.; Greene, Warner C.

    2015-01-01

    Despite significant advances in our understanding of HIV, a cure has not been realized for the more than 34 million infected with this virus. HIV is incurable because infected individuals harbor cells where the HIV provirus is integrated into the host’s DNA but is not actively replicating and thus is not inhibited by antiviral drugs. Similarly, these latent viruses are not detected by the immune system. In this review, we discuss HIV-1 latency and the mechanisms that allow this pathogenic retrovirus to hide and persist by exploiting the cellular vehicles of immunological memory. PMID:24243012

  5. 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

  6. Prospective study of correlates of vaginal Lactobacillus colonisation among high-risk HIV-1 seronegative women.

    PubMed

    Baeten, J M; Hassan, W M; Chohan, V; Richardson, B A; Mandaliya, K; Ndinya-Achola, J O; Jaoko, W; McClelland, R S

    2009-09-01

    Vaginal colonisation with Lactobacillus species is characteristic of normal vaginal ecology. The absence of vaginal lactobacilli, particularly hydrogen peroxide (H(2)O(2))-producing isolates, has been associated with symptomatic bacterial vaginosis (BV) and increased risk for HIV-1 acquisition. Identification of factors associated with vaginal Lactobacillus colonisation may suggest interventions to improve vaginal health. We conducted a prospective cohort study of correlates of vaginal Lactobacillus colonisation among Kenyan HIV-1 seronegative female sex workers. At monthly follow-up visits, vaginal Lactobacillus cultures were obtained. Generalised estimating equations were used to examine demographic, behavioural and medical correlates of Lactobacillus isolation, including isolation of H(2)O(2)-producing strains. Lactobacillus cultures were obtained from 1020 women who completed a total of 8896 follow-up visits. Vaginal washing, typically with water alone or with soap and water, was associated with an approximately 40% decreased likelihood of Lactobacillus isolation, including isolation of H(2)O(2)-producing strains. Recent antibiotic use, excluding metronidazole and treatments for vaginal candidiasis, reduced Lactobacillus isolation by approximately 30%. H(2)O(2)-producing lactobacilli were significantly less common among women with Trichomonas vaginalis infection and those who were seropositive for herpes simplex virus type 2. In contrast, H(2)O(2)-producing lactobacilli were significantly more common among women with concurrent vaginal candidiasis. Modifiable biological and behavioural factors are associated with Lactobacillus colonisation in African women. Our results suggest intervention strategies to improve vaginal health in women at high risk for HIV-1.

  7. Prospective study of correlates of vaginal Lactobacillus colonization among high-risk HIV-1 seronegative women

    PubMed Central

    Baeten, Jared M.; Hassan, Wisal M.; Chohan, Vrasha; Richardson, Barbra A.; Mandaliya, Kishorchandra; Ndinya-Achola, Jeckoniah O.; Jaoko, Walter; McClelland, R. Scott

    2009-01-01

    Objective Vaginal colonization with Lactobacillus species is characteristic of normal vaginal ecology. The absence of vaginal lactobacilli, particularly hydrogen peroxide (H2O2)-producing isolates, has been associated with symptomatic bacterial vaginosis (BV) and increased risk for HIV-1 acquisition. Identification of factors associated with vaginal Lactobacillus colonization may suggest interventions to improve vaginal health. Methods We conducted a prospective cohort study of correlates of vaginal Lactobacillus colonization among Kenyan HIV-1 seronegative female sex workers. At monthly follow-up visits, vaginal Lactobacillus cultures were obtained. Generalized estimating equations were used to examine demographic, behavioral, and medical correlates of Lactobacillus isolation, including isolation of H2O2-producing strains. Results Lactobacillus cultures were obtained from 1020 women who completed a total of 8896 follow-up visits. Vaginal washing, typically with water alone or with soap and water, was associated with an approximately 40% decreased likelihood of Lactobacillus isolation, including isolation of H2O2-producing strains. Recent antibiotic use, excluding metronidazole and treatments for vaginal candidiasis, reduced Lactobacillus isolation by ~30%. H2O2-producing lactobacilli were significantly less common among women with Trichomonas vaginalis infection and those who were seropositive for herpes simplex virus type 2. In contrast, H2O2-producing lactobacilli were significantly more common among women with concurrent vaginal candidiasis. Conclusions Modifiable biologic and behavioral factors are associated with Lactobacillus colonization in African women. Our results suggest intervention strategies to improve vaginal health in women at high risk for HIV-1. PMID:19329442

  8. 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.

  9. 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

  10. The impact of HIV-1 genetic diversity on the efficacy of a combinatorial RNAi-based gene therapy.

    PubMed

    Herrera-Carrillo, E; Berkhout, B

    2015-06-01

    A hurdle for human immunodeficiency virus (HIV-1) therapy is the genomic diversity of circulating viruses and the possibility that drug-resistant virus variants are selected. Although RNA interference (RNAi) is a powerful tool to stably inhibit HIV-1 replication by the expression of antiviral short hairpin RNAs (shRNAs) in transduced T cells, this approach is also vulnerable to pre-existing genetic variation and the development of viral resistance through mutation. To prevent viral escape, we proposed to combine multiple shRNAs against important regions of the HIV-1 RNA genome, which should ideally be conserved in all HIV-1 subtypes. The vulnerability of RNAi therapy to viral escape has been studied for a single subtype B strain, but it is unclear whether the antiviral shRNAs can inhibit diverse virus isolates and subtypes, including drug-resistant variants that could be present in treated patients. To determine the breadth of the RNAi gene therapy approach, we studied the susceptibility of HIV-1 subtypes A-E and drug-resistant variants. In addition, we monitored the evolution of HIV-1 escape variants. We demonstrate that the combinatorial RNAi therapy is highly effective against most isolates, supporting the future testing of this gene therapy in appropriate in vivo models.

  11. 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.

  12. HIV-1 imposes rigidity on blood and semen cytokine networks.

    PubMed

    Lisco, Andrea; Introini, Andrea; Munawwar, Arshi; Vanpouille, Christophe; Grivel, Jean-Charles; Blank, Paul; Singh, Sarman; Margolis, Leonid

    2012-12-01

    Although it is established that the levels of individual cytokines are altered by HIV-1 infection, the changes in cytokine interrelations that organize them into networks have been poorly studied. Here, we evaluated these networks in HIV-infected and HIV-uninfected individuals in fluid compartments that are critical for HIV-1 pathogenesis and transmission, namely blood and semen. In samples collected from therapy-naïve HIV-1-infected and HIV-1-uninfected individuals, we measured HIV-1-load, CD4 cell count, and levels of 21 cytokines using a multiplex bead assay. Cytokine networks in blood and semen were different for HIV-1-infected and HIV-1-uninfected individuals. In both compartments of HIV-1-infected individuals, the cytokine networks were more interlocked than in controls: HIV-1 infection resulted in the establishment of new correlations and in the strengthening of pre-existing correlations between different cytokines. In blood and semen of HIV-infected patients, there were, respectively, 68 and 72 statistically significant correlations between cytokines, while in uninfected individuals, there were 18 and 21 such correlations. HIV-1 infection reorganizes the cytokine networks, establishing new strong correlations between various cytokines and thus imposes a high rigidity on the cytokine network. This rigidity may reflect the impairment of the ability of the immune system to respond to microbial challenges. © 2012 John Wiley & Sons A/S.

  13. 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

  14. 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.

  15. Candida albicans Delays HIV-1 Replication in Macrophages

    PubMed Central

    Rodriguez Rodrigues, Christian; Remes Lenicov, Federico; Jancic, Carolina; Sabatté, Juan; Cabrini, Mercedes; Ceballos, Ana; Merlotti, Antonela; Gonzalez, Heidi; Ostrowski, Matías; Geffner, Jorge

    2013-01-01

    Macrophages are one of the most important HIV-1 target cells. Unlike CD4+ T cells, macrophages are resistant to the cytophatic effect of HIV-1. They are able to produce and harbor the virus for long periods acting as a viral reservoir. Candida albicans (CA) is a commensal fungus that colonizes the portals of HIV-1 entry, such as the vagina and the rectum, and becomes an aggressive pathogen in AIDS patients. In this study, we analyzed the ability of CA to modulate the course of HIV-1 infection in human monocyte-derived macrophages. We found that CA abrogated HIV-1 replication in macrophages when it was evaluated 7 days after virus inoculation. A similar inhibitory effect was observed in monocyte-derived dendritic cells. The analysis of the mechanisms responsible for the inhibition of HIV-1 production in macrophages revealed that CA efficiently sequesters HIV-1 particles avoiding its infectivity. Moreover, by acting on macrophages themselves, CA diminishes their permissibility to HIV-1 infection by reducing the expression of CD4, enhancing the production of the CCR5-interacting chemokines CCL3/MIP-1α, CCL4/MIP-1β, and CCL5/RANTES, and stimulating the production of interferon-α and the restriction factors APOBEC3G, APOBEC3F, and tetherin. Interestingly, abrogation of HIV-1 replication was overcome when the infection of macrophages was evaluated 2-3 weeks after virus inoculation. However, this reactivation of HIV-1 infection could be silenced by CA when added periodically to HIV-1-challenged macrophages. The induction of a silent HIV-1 infection in macrophages at the periphery, where cells are continuously confronted with CA, might help HIV-1 to evade the immune response and to promote resistance to antiretroviral therapy. PMID:24009706

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Novel Approaches to Inhibiting HIV-1 Replication

    PubMed Central

    Adamson, Catherine S.; Freed, Eric O.

    2009-01-01

    Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. PMID:19782103

  1. Rational Development of Radiopharmaceuticals for HIV-1

    PubMed Central

    Lau, Chuen-Yen; Maldarelli, Frank; Eckelman, William C; Neumann, Ronald D

    2014-01-01

    The global battle against HIV-1 would benefit from a sensitive and specific radiopharmaceutical to localize HIV-infected cells. Ideally, this probe would be able to identify latently infected host cells containing replication competent HIV sequences. Clinical and research applications would include assessment of reservoirs, informing clinical management by facilitating assessment of burden of infection in different compartments, monitoring disease progression and monitoring response to therapy. A “rational” development approach could facilitate efficient identification of an appropriate targeted radiopharmaceutical. Rational development starts with understanding characteristics of the disease that can be effectively targeted and then engineering radiopharmaceuticals to hone in on an appropriate target, which in the case of HIV-1 (HIV) might be an HIV-specific product on or in the host cell, a differentially expressed gene product, an integrated DNA sequence specific enzymatic activity, part of the inflammatory response, or a combination of these. This is different from the current approach that starts with a radiopharmaceutical for a target associated with a disease, mostly from autopsy studies, without a strong rationale for the potential to impact patient care. At present, no targeted therapies are available for HIV latency, although a number of approaches are under study. Here we discuss requirements for a radiopharmaceutical useful in strategies targeting persistently infected cells. The radiopharmaceutical for HIV should be developed based on HIV biology, studied in an animal model and then in humans, and ultimately used in clinical and research settings. PMID:24607432

  2. Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein

    PubMed Central

    Azarnezhad, Asaad; Sharifi, Zohreh; Seyedabadi, Rahmatollah; Hosseini, Arshad; Johari, Behrooz; Sobhani Fard, Mahsa

    2016-01-01

    Background: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli (E. coli) BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA. Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 μg/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA. Conclusion: Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests. PMID:27920885

  3. Origin and epidemiological history of HIV-1 CRF14_BG.

    PubMed

    Bártolo, Inês; Abecasis, Ana B; Borrego, Pedro; Barroso, Helena; McCutchan, Francine; Gomes, Perpétua; Camacho, Ricardo; Taveira, Nuno

    2011-01-01

    CRF14_BG isolates, originally found in Spain, are characterized by CXCR4 tropism and rapid disease progression. This study aimed to identify the origin of CRF14_BG and reconstruct its epidemiological history based on new isolates from Portugal. C2V3C3 env gene sequences were obtained from 62 samples collected in 1993-1998 from Portuguese HIV-1 patients. Full-length genomic sequences were obtained from three patients. Viral subtypes, diversity, divergence rate and positive selection were investigated by phylogenetic analysis. The molecular structure of the genomes was determined by bootscanning. A relaxed molecular clock model was used to date the origin of CRF14_BG. Geno2pheno was used to predict viral tropism. Subtype B was the most prevalent subtype (45 sequences; 73%) followed by CRF14_BG (8; 13%), G (4; 6%), F1 (2; 3%), C (2; 3%) and CRF02_AG (1; 2%). Three CRF14_BG sequences were derived from 1993 samples. Near full-length genomic sequences were strongly related to the CRF14_BG isolates from Spain. Genetic diversity of the Portuguese isolates was significantly higher than the Spanish isolates (0.044 vs 0.014, P<0.0001). The mean date of origin of the CRF14_BG cluster was estimated to be 1992 (range, 1989 and 1996) based on the subtype G genomic region and 1989 (range, 1984-1993) based on the subtype B genomic region. Most CRF14_BG strains (78.9%) were predicted to be CXCR4. Finally, up to five amino acids were under selective pressure in subtype B V3 loop whereas only one was found in the CRF14_BG cluster. CRF14_BG emerged in Portugal in the early 1990 s soon after the beginning of the HIV-1 epidemics, spread to Spain in late 1990 s as a consequence of IVDUs migration and then to the rest of Europe. CXCR4 tropism is a general characteristic of this CRF that may have been selected for by escape from neutralizing antibody response.

  4. Origin and Epidemiological History of HIV-1 CRF14_BG

    PubMed Central

    Bártolo, Inês; Abecasis, Ana B.; Borrego, Pedro; Barroso, Helena; McCutchan, Francine; Gomes, Perpétua; Camacho, Ricardo; Taveira, Nuno

    2011-01-01

    Background CRF14_BG isolates, originally found in Spain, are characterized by CXCR4 tropism and rapid disease progression. This study aimed to identify the origin of CRF14_BG and reconstruct its epidemiological history based on new isolates from Portugal. Methodology/Principal Findings C2V3C3 env gene sequences were obtained from 62 samples collected in 1993–1998 from Portuguese HIV-1 patients. Full-length genomic sequences were obtained from three patients. Viral subtypes, diversity, divergence rate and positive selection were investigated by phylogenetic analysis. The molecular structure of the genomes was determined by bootscanning. A relaxed molecular clock model was used to date the origin of CRF14_BG. Geno2pheno was used to predict viral tropism. Subtype B was the most prevalent subtype (45 sequences; 73%) followed by CRF14_BG (8; 13%), G (4; 6%), F1 (2; 3%), C (2; 3%) and CRF02_AG (1; 2%). Three CRF14_BG sequences were derived from 1993 samples. Near full-length genomic sequences were strongly related to the CRF14_BG isolates from Spain. Genetic diversity of the Portuguese isolates was significantly higher than the Spanish isolates (0.044 vs 0.014, P<0.0001). The mean date of origin of the CRF14_BG cluster was estimated to be 1992 (range, 1989 and 1996) based on the subtype G genomic region and 1989 (range, 1984–1993) based on the subtype B genomic region. Most CRF14_BG strains (78.9%) were predicted to be CXCR4. Finally, up to five amino acids were under selective pressure in subtype B V3 loop whereas only one was found in the CRF14_BG cluster. Conclusions CRF14_BG emerged in Portugal in the early 1990 s soon after the beginning of the HIV-1 epidemics, spread to Spain in late 1990 s as a consequence of IVDUs migration and then to the rest of Europe. CXCR4 tropism is a general characteristic of this CRF that may have been selected for by escape from neutralizing antibody response. PMID:21969855

  5. Identification of a small-molecule inhibitor of HIV-1 assembly that targets the phosphatidylinositol (4,5)-bisphosphate binding site of the HIV-1 matrix protein.

    PubMed

    Zentner, Isaac; Sierra, Luz-Jeannette; Fraser, Ayesha K; Maciunas, Lina; Mankowski, Marie K; Vinnik, Andrei; Fedichev, Peter; Ptak, Roger G; Martín-García, Julio; Cocklin, Simon

    2013-03-01

    The development of drug resistance remains a critical problem for current HIV-1 antiviral therapies, creating a need for new inhibitors of HIV-1 replication. We previously reported on a novel anti-HIV-1 compound, N(2)-(phenoxyacetyl)-N-[4-(1-piperidinylcarbonyl)benzyl]glycinamide (14), that binds to the highly conserved phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) binding pocket of the HIV-1 matrix (MA) protein. In this study, we re-evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV-1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2-(4-{[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]methyl})-1-piperazinyl)-N-(4-methylphenyl)acetamide (7), 3-(2-ethoxyphenyl)-5-[[4-(4-nitrophenyl)piperazin-1-yl]methyl]-1,2,4-oxadiazole (17), and N-[4-ethoxy-3-(1-piperidinylsulfonyl)phenyl]-2-(imidazo[2,1-b][1,3]thiazol-6-yl)acetamide (18), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV-1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P(2) for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P(2) binding site of MA decreased the antiviral effect of compound 7. Additionally, compound 7 displays a broadly neutralizing anti-HIV activity, with IC(50) values of 7.5-15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti-HIV-1 therapeutics.

  6. Hydroxytyrosol: a new class of microbicide displaying broad anti-HIV-1 activity

    PubMed Central

    Bedoya, Luis M.; Beltrán, Manuela; Obregón-Calderón, Patricia; García-Pérez, Javier; de la Torre, Humberto E.; González, Nuria; Pérez-Olmeda, Mayte; Auñón, David; Capa, Laura; Gómez-Acebo, Eduardo; Alcamí, José

    2016-01-01

    Objective: To investigate the toxicity and activity against HIV of 5-hydroxytyrosol as a potential microbicide. Design: The anti-HIV-1 activity of 5-hydroxytyrosol, a polyphenolic compound, was tested against wild-type HIV-1 and viral clones resistant to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and integrase inhibitors. In addition to its activity against founder viruses, different viral subtypes and potential synergy with tenofovir disoproxil fumarate, lamivudine and emtricitabine was also tested. 5-Hydroxytyrosol toxicity was evaluated in vivo in rabbit vaginal mucosa. Methods: We have cloned pol gene from drug-resistant HIV-1 isolated from infected patients and env gene from Fiebeg III/IV patients or A, C, D, E, F and G subtypes in the NL4.3-Ren backbone. 5-Hydroxytyrosol anti-HIV-1 activity was evaluated in infections of MT-2, U87-CCR5 or peripheral blood mononuclear cells preactivated with phytohemagglutinin + interleukin-2 with viruses obtained through 293T transfections. Inhibitory concentration 50% and cytotoxic concentration 50% were calculated. Synergy was analysed according to Chou and Talalay method. In-vivo toxicity was evaluated for 14 days in rabbit vaginal mucosa. Results: 5-Hydroxytyrosol inhibited HIV-1 infections of recombinant or wild-type viruses in all the target cells tested. Moreover, 5-hydroxytyrosol showed similar inhibitory concentration 50% values for infections with NRTIs, NNRTIs, protease inhibitors and INIs resistant viruses; founder viruses and all the subtypes tested. Combination of 5-hydroxytyrosol with tenofovir was found to be synergistic, whereas it was additive with lamivudine and emtricitabine. In-vivo toxicity of 5-hydroxytyrosol was very low even at the highest tested doses. Conclusion: 5-Hydroxytyrosol displayed a broad anti-HIV-1 activity in different cells systems in the absent of in-vivo toxicity, therefore supporting its

  7. Reviewing the History of HIV-1: Spread of Subtype B in the Americas

    PubMed Central

    Junqueira, Dennis Maletich; de Medeiros, Rúbia Marília; Matte, Maria Cristina Cotta; Araújo, Leonardo Augusto Luvison; Chies, Jose Artur Bogo; Ashton-Prolla, Patricia; Almeida, Sabrina Esteves de Matos

    2011-01-01

    The dispersal of HIV-1 subtype B (HIV-1B) is a reflection of the movement of human populations in response to social, political, and geographical issues. The initial dissemination of HIV-1B outside Africa seems to have included the passive involvement of human populations from the Caribbean in spreading the virus to the United States. However, the exact pathways taken during the establishment of the pandemic in the Americas remain unclear. Here, we propose a geographical scenario for the dissemination of HIV-1B in the Americas, based on phylogenetic and genetic statistical analyses of 313 available sequences of the pol gene from 27 countries. Maximum likelihood and Bayesian inference methods were used to explore the phylogenetic relationships between HIV-1B sequences, and molecular variance estimates were analyzed to infer the genetic structure of the viral population. We found that the initial dissemination and subsequent spread of subtype B in the Americas occurred via a single introduction event in the Caribbean around 1964 (1950–1967). Phylogenetic trees present evidence of several primary outbreaks in countries in South America, directly seeded by the Caribbean epidemic. Cuba is an exception insofar as its epidemic seems to have been introduced from South America. One clade comprising isolates from different countries emerged in the most-derived branches, reflecting the intense circulation of the virus throughout the American continents. Statistical analysis supports the genetic compartmentalization of the virus among the Americas, with a close relationship between the South American and Caribbean epidemics. These findings reflect the complex establishment of the HIV-1B pandemic and contribute to our understanding between the migration process of human populations and virus diffusion. PMID:22132104

  8. 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

  9. Evaluation of Hologic Aptima HIV-1 Quant Dx Assay on the Panther System on HIV Subtypes.

    PubMed

    Manak, Mark M; Hack, Holly R; Nair, Sangeetha V; Worlock, Andrew; Malia, Jennifer A; Peel, Sheila A; Jagodzinski, Linda L

    2016-10-01

    Quantitation of the HIV-1 viral load in plasma is the current standard of care for clinical monitoring of HIV-infected individuals undergoing antiretroviral therapy. This study evaluated the analytical and clinical performances of the Aptima HIV-1 Quant Dx assay (Hologic, San Diego, CA) for monitoring viral load by using 277 well-characterized subtype samples, including 171 cultured virus isolates and 106 plasma samples from 35 countries, representing all major HIV subtypes, recombinants, and circulating recombinant forms (CRFs) currently in circulation worldwide. Linearity of the Aptima assay was tested on each of 6 major HIV-1 subtypes (A, B, C, D, CRF01_AE, and CRF02_AG) and demonstrated an R(2) value of ≥0.996. The performance of the Aptima assay was also compared to those of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v.2 (CAP/CTM) and Abbott m2000 RealTime HIV-1 (RealTime) assays on all subtype samples. The Aptima assay values averaged 0.21 log higher than the CAP/CTM values and 0.30 log higher than the RealTime values, and the values were >0.4 log higher than CAP/CTM values for subtypes F and G and than RealTime values for subtypes C, F, and G and CRF02_AG. Two samples demonstrated results with >1-log differences from RealTime results. When the data were adjusted by the average difference, 94.9% and 87.0% of Aptima results fell within 0.5 log of the CAP/CTM and RealTime results, respectively. The linearity and accuracy of the Aptima assay in correctly quantitating all major HIV-1 subtypes, coupled with the completely automated format and high throughput of the Panther system, make this system well suited for reliable measurement of viral load in the clinical laboratory. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. A metal-induced conformational change and activation of HIV-1 integrase.

    PubMed

    Asante-Appiah, E; Skalka, A M

    1997-06-27

    Retroviral integrases are composed of three independently folding domains whose organization relevant to one another is largely unknown. As an approach to understanding its structure, we have investigated the effect of the required metal cofactor(s), Mn2+ or Mg2+, on the conformation of human immunodeficiency virus type 1 (HIV-1) integrase (IN) using monoclonal antibodies (mAbs) that are specific for each of these three domains. Upon the addition of increasing concentrations of the divalent cations to immobilized HIV-1 IN in ELISA assays, binding of mAbs specific for either the C-terminal domain or for an epitope in the catalytic core domain was lost, whereas binding of an N terminus-specific mAb was unaffected. Size exclusion chromatography of a nonaggregating derivative of HIV-1 IN showed that the oligomeric state of the protein did not change under conditions in which recognition of the core and C terminus-specific mAbs was lost. Preincubation with Mn2+ increased the resistance of HIV-1 IN to proteolytic digestion and produced a digestion pattern that was significantly different from that observed with the apoprotein. A derivative that lacked the N-terminal domain, IN(50-288), exhibited the same metal-dependent changes observed with the full-length protein, whereas the isolated catalytic core domain IN(50-212) did not. From this we conclude that the metal-induced conformational change comprises a reorganization of the core and C-terminal domains. Preincubation with Mn2+ increased the specific activity of HIV-1 IN 5-fold. Enzymatic activity was inhibited by the conformation-sensitive C terminus-specific mAb, but this inhibition was reduced greatly if the enzyme was first preincubated with metal ions. Thus, it appears that apo-HIV-1 IN exists predominantly in an inactive conformation that is converted into a catalytically competent form upon the addition of metal ions.

  11. 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

  12. Evaluation of Hologic Aptima HIV-1 Quant Dx Assay on the Panther System on HIV Subtypes

    PubMed Central

    Hack, Holly R.; Nair, Sangeetha V.; Worlock, Andrew; Malia, Jennifer A.; Peel, Sheila A.; Jagodzinski, Linda L.

    2016-01-01

    Quantitation of the HIV-1 viral load in plasma is the current standard of care for clinical monitoring of HIV-infected individuals undergoing antiretroviral therapy. This study evaluated the analytical and clinical performances of the Aptima HIV-1 Quant Dx assay (Hologic, San Diego, CA) for monitoring viral load by using 277 well-characterized subtype samples, including 171 cultured virus isolates and 106 plasma samples from 35 countries, representing all major HIV subtypes, recombinants, and circulating recombinant forms (CRFs) currently in circulation worldwide. Linearity of the Aptima assay was tested on each of 6 major HIV-1 subtypes (A, B, C, D, CRF01_AE, and CRF02_AG) and demonstrated an R2 value of ≥0.996. The performance of the Aptima assay was also compared to those of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v.2 (CAP/CTM) and Abbott m2000 RealTime HIV-1 (RealTime) assays on all subtype samples. The Aptima assay values averaged 0.21 log higher than the CAP/CTM values and 0.30 log higher than the RealTime values, and the values were >0.4 log higher than CAP/CTM values for subtypes F and G and than RealTime values for subtypes C, F, and G and CRF02_AG. Two samples demonstrated results with >1-log differences from RealTime results. When the data were adjusted by the average difference, 94.9% and 87.0% of Aptima results fell within 0.5 log of the CAP/CTM and RealTime results, respectively. The linearity and accuracy of the Aptima assay in correctly quantitating all major HIV-1 subtypes, coupled with the completely automated format and high throughput of the Panther system, make this system well suited for reliable measurement of viral load in the clinical laboratory. PMID:27510829

  13. Reviewing the history of HIV-1: spread of subtype B in the Americas.

    PubMed

    Junqueira, Dennis Maletich; de Medeiros, Rúbia Marília; Matte, Maria Cristina Cotta; Araújo, Leonardo Augusto Luvison; Chies, Jose Artur Bogo; Ashton-Prolla, Patricia; Almeida, Sabrina Esteves de Matos

    2011-01-01

    The dispersal of HIV-1 subtype B (HIV-1B) is a reflection of the movement of human populations in response to social, political, and geographical issues. The initial dissemination of HIV-1B outside Africa seems to have included the passive involvement of human populations from the Caribbean in spreading the virus to the United States. However, the exact pathways taken during the establishment of the pandemic in the Americas remain unclear. Here, we propose a geographical scenario for the dissemination of HIV-1B in the Americas, based on phylogenetic and genetic statistical analyses of 313 available sequences of the pol gene from 27 countries. Maximum likelihood and bayesian inference methods were used to explore the phylogenetic relationships between HIV-1B sequences, and molecular variance estimates were analyzed to infer the genetic structure of the viral population. We found that the initial dissemination and subsequent spread of subtype B in the Americas occurred via a single introduction event in the Caribbean around 1964 (1950-1967). Phylogenetic trees present evidence of several primary outbreaks in countries in South America, directly seeded by the Caribbean epidemic. Cuba is an exception insofar as its epidemic seems to have been introduced from South America. One clade comprising isolates from different countries emerged in the most-derived branches, reflecting the intense circulation of the virus throughout the American continents. Statistical analysis supports the genetic compartmentalization of the virus among the Americas, with a close relationship between the South American and Caribbean epidemics. These findings reflect the complex establishment of the HIV-1B pandemic and contribute to our understanding between the migration process of human populations and virus diffusion.

  14. 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.

  15. Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1.

    PubMed

    Bunders, Madeleine J; van Hamme, John L; Jansen, Machiel H; Boer, Kees; Kootstra, Neeltje A; Kuijpers, Taco W

    2014-10-24

    Absolute numbers of lymphocytes are decreased in uninfected infants born to HIV-1-infected women (HIV-1-exposed). Although the exact mechanism is unknown, fetal exposure to maternal HIV-1-infection could prime the immune system and affect T cell trafficking. We compared the expression of chemokine receptors on cord blood CD4(+) T cells from HIV-1-exposed children and healthy controls. At baseline CD4(+) T cells had a largely naïve phenotype. However, stimulation with cytokines resulted in an upregulation of inflammatory response-related chemokine receptors on CD4(+) T cells, with HIV-1-exposed infants having a significantly higher frequency of CD4(+) T cells expressing, in particularly Th2 associated chemokine receptors (CCR3 p < 0.01, CCR8 p = 0.03). Numbers of naive CCR7(+) CD4(+) T cells were reduced (p = 0.01) in HIV-1-exposed infants. We further assessed whether the inflammatory phenotype was associated with susceptibility to HIV-1 and detected higher levels of p24 upon in in vitro infection of stimulated CD4(+) T cells of HIV-1-exposed infants. In summary, fetal exposure to HIV-1 primes the immune system in the infant leading to an enhanced immune activation and altered T cell homing, with potential ramifications regarding T cell responses and the acquisition of HIV-1 as an infant.

  16. Curdlan sulfate and HIV-1. I. In vitro inhibitory effects of curdlan sulfate on HIV-1 infection.

    PubMed

    Aoki, T; Kaneko, Y; Stefanski, M S; Nguyen, T; Ting, R C

    1991-04-01

    Action mechanisms of a newly synthesized polysaccharide, curdlan sulfate (CRDS), on human immunodeficiency virus type 1 (HIV-1) infection were investigated in vitro using syncytium formation microassay and p24 antigen capture enzyme-linked immunosorbent assay. These assays measured the titer of infectious virions and the amounts of HIV-1 core antigen p24 in soluble, intraviral, and intracellular forms. CRDS treatments were performed for 1 h at 37 degrees C. H9 cells pretreated with 0.1 to 100.0 micrograms/ml of CRDS appreciably inhibited HIV-1 infection. CRDS-treated HIV-1 virions were less able to infect H9 cells than untreated virions. The simultaneous treatment of H9 cells and HIV-1 virions with CRDS induced a significant inhibition of HIV-1 infection, resulting in the temporary disappearance of virions at the highest dose of CRDS. In contrast, CRDS treatment of newly HIV-1-infected H9 cells caused a significant decrease in the titer of infectious HIV-1 and the p24 amounts of all three forms, but no absolute elimination. Taken together, these results indicate that CRDS may block the binding of the HIV-1 envelope to the H9 cell surface, with emphasis on the high affinity of CRDS to the HIV-1 envelope.

  17. Innate immune sensing of HIV-1 by dendritic cells.

    PubMed

    Luban, Jeremy

    2012-10-18

    HIV-1-specific antibodies and CD8(+) cytotoxic T cells are detected in most HIV-1-infected people, yet HIV-1 infection is not eradicated. Contributing to the failure to mount a sterilizing immune response may be the inability of antigen-presenting dendritic cells (DCs) to sense HIV-1 during acute infection, and thus the inability to effectively prime naive, HIV-1-specific T cells. Recent findings related to DC-expressed innate immune factors including SAMHD1, TREX1, and TRIM5 provide a molecular basis for understanding why DCs fail to adequately sense invasion by this deadly pathogen and suggest experimental approaches to improve T cell priming to HIV-1 in prophylactic vaccination protocols. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. 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.

  19. Differential binding of the HIV-1 envelope to phosphatidylserine receptors.

    PubMed

    Gu, Linlin; Sims, Brian; Krendelchtchikov, Alexandre; Tabengwa, Edlue; Matthews, Qiana L

    2017-10-01

    Prior work has shown that the HIV-1 envelope of the human immunodeficiency virus (HIV) interacts directly with T-cell immunoglobulin mucin (TIM) family proteins. Herein, we demonstrate that HIV-1 envelope glycoproteins from varying HIV-1 clades bind differentially to TIM proteins and functionally similar proteins acting as phosphatidylserine (PtdSer) receptors. Using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology, we show that lysate containing HIV-1 envelope and recombinant HIV-1 envelope glycoproteins bind TIM-4 and advanced glycosylation end product-specific receptor (AGER). The complex binding of HIV-1 UG21 gp140 to TIM-4 or AGER suggests a biphasic interaction with these proteins. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  20. 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

  1. Human Erythrocytes Selectively Bind and Enrich Infectious HIV-1 Virions

    PubMed Central

    Beck, Zoltan; Brown, Bruce K.; Wieczorek, Lindsay; Peachman, Kristina K.; Matyas, Gary R.; Polonis, Victoria R.; Rao, Mangala; Alving, Carl R.

    2009-01-01

    Although CD4(+) cells represent the major target for HIV infection in blood, claims of complement-independent binding of HIV-1 to erythrocytes and the possible role of Duffy blood group antigen, have generated controversy. To examine the question of binding to erythrocytes, HIV-1 was incubated in vitro with erythrocytes from 30 healthy leukapheresis donors, and binding was determined by p24 analysis and adsorption of HIV-1 with reduction of infectivity for CD4(+) target cells. All of the cells, regardless of blood group type, bound HIV-1 p24. A typical preparation of erythrocytes bound <2.4% of the added p24, but erythrocytes selectively removed essentially all of the viral infectivity as determined by decreased infection of CD4(+) target cells; however, cell-associated HIV-1 was approximately 100-fold more efficient, via trans infection, than unadsorbed virus for infection of CD4(+) cells. All of the bound HIV-1 p24 was released by treatment of the cells with EDTA, and binding was optimized by adding Ca2+ and Mg2+ during the washing of erythrocytes containing bound HIV-1. Although the small number of contaminating leukocytes in the erythrocyte preparation also bound HIV-1 p24, there was no significant binding to CD4, and it thus appears that the binding occurred on leukocytes at non-CD4 sites. Furthermore, binding occurred to erythrocyte ghosts from which contaminating leukocytes had been previously removed. The results demonstrate that erythrocytes incubated in vitro with HIV-1 differentially adsorb all of the infectious HIV-1 virions (as opposed to non-infectious or degraded virions) in the absence of complement and independent of blood group, and binding is dependent on divalent cations. By analogy with HIV-1 bound to DC-SIGN on dendritic cells, erythrocyte-bound HIV-1 might comprise an important surface reservoir for trans infection of permissive cells. PMID:20011536

  2. The V3 Loop of HIV-1 Env Determines Viral Susceptibility to IFITM3 Impairment of Viral Infectivity.

    PubMed

    Wang, Yimeng; Pan, Qinghua; Ding, Shilei; Wang, Zhen; Yu, Jingyou; Finzi, Andrés; Liu, Shan-Lu; Liang, Chen

    2017-04-01

    Interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates, including transmitted founder viruses, is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, and the V3 loop sequence in particular, determines the extent of inhibition of viral infectivity by IFITM3. IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody than are IFITM3-resistant Env proteins. Together, data from our study suggest that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition.IMPORTANCE Results of our study have revealed the key features of the HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defense. A variety of viruses are inhibited by IFITMs at the virus entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3

  3. 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

  4. 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

  5. [Genetic subtyping of HIV-1 strains by heteroduplex mobility assay].

    PubMed

    Yu, H; Su, L; Shao, J J

    1997-08-01

    DNA fragments of HIV-1 env gene were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 24 HIV-1 infected individuals. The PCR products were separated by melting and annealing with denatured PCR product prepared from reference plasimd of representative subtypes. Heteroduplex were then formed between the single-stranded DNA from the two sources and were analysed on polyacrylamide gels. The results from heteroduplex mobility assay (HMA) were compared with HIV-1 subtype results determined by DNA sequencing. With advantages of high speed, low cost and high specificity, HMA is a reliable screening method for HIV-1 subtyping.

  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. TIM-family proteins inhibit HIV-1 release

    PubMed Central

    Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

    2014-01-01

    Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

  8. HIV-1 imposes rigidity on blood and semen cytokine networks

    PubMed Central

    LISCO, Andrea; INTROINI, Andrea; MUNAWWAR, Arshi; VANPOUILLE, Christope; GRIVEL, Jean-Charles; BLANK, Paul; SINGH, Sarman

    2012-01-01

    Problem Although it is established that the levels of individual cytokines are altered by HIV-1 infection, the changes in cytokine interrelations that organize them into networks have been poorly studied. Here, we evaluated these networks in HIV-infected and -uninfected individuals in fluid compartments that are critical for HIV-1 pathogenesis and transmission, namely blood and semen. Method of Study In samples collected from therapy-naïve HIV-1-infected and HIV-1-uninfected individuals, we measured HIV-1-load, CD4-cell-count, and levels of 21 cytokines using a multiplex bead-assay. Results Cytokine networks in blood and semen were different for HIV-1-infected and -uninfected individuals. In both compartments of HIV-1-infected individuals, the cytokine networks were more interlocked than in controls: HIV -1 infection results in the establishment of new correlations and the strengthening of pre-existing correlations between different cytokines. In blood and semen of HIV-infected patients there were, respectively, 68 and 72 statistically significant correlations between cytokines, while in uninfected individuals there were 18 and 21 such correlations. Conclusions HIV-1 infection reorganizes the cytokine networks, establishing new strong correlations between various cytokines and thus imposing a high rigidity on the cytokine network. This rigidity may reflect the impairment of the ability of the immune system to respond to microbial challenges. PMID:23006048

  9. Genetic Signatures of HIV-1 Envelope-mediated Bystander Apoptosis

    PubMed Central

    Joshi, Anjali; Lee, Raphael T. C.; Mohl, Jonathan; Sedano, Melina; Khong, Wei Xin; Ng, Oon Tek; Maurer-Stroh, Sebastian; Garg, Himanshu

    2014-01-01

    The envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4+ T cell loss. However, most of the studies testing this phenomenon have been conducted with laboratory-adapted HIV-1 isolates. This raises the question of whether primary Envs derived from HIV-infected patients are capable of inducing bystander apoptosis and whether specific Env signatures are associated with this phenomenon. We developed a high throughput assay to determine the bystander apoptosis inducing activity of a panel of primary Envs. We tested 38 different Envs for bystander apoptosis, virion infectivity, neutralizing antibody sensitivity, and putative N-linked glycosylation sites along with a comprehensive sequence analysis to determine if specific sequence signatures within the viral Env are associated with bystander apoptosis. Our studies show that primary Envs vary considerably in their bystander apoptosis-inducing potential, a phenomenon that correlates inversely with putative N-linked glycosylation sites and positively with virion infectivity. By use of a novel phylogenetic analysis that avoids subtype bias coupled with structural considerations, we found specific residues like Arg-476 and Asn-425 that were associated with differences in bystander apoptosis induction. A specific role of these residues was also confirmed experimentally. These data demonstrate for the first time the potential of primary R5 Envs to mediate bystander apoptosis in CD4+ T cells. Furthermore, we identify specific genetic signatures within the Env that may be associated with the bystander apoptosis-inducing phenotype. PMID:24265318

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. HIV-1 protease inhibitors in development.

    PubMed

    Rusconi, Stefano; La Seta Catamancio, Simona

    2002-03-01

    Several pharmaceutical companies have developed an increasing number of second generation protease inhibitors (PI) during the last few years. Many of these compounds have been in preclinical trials and some are now in clinical use. All drugs in this category have been designed to be well absorbed and overcome the crucial problem of cross-resistance within this class of compounds. Taking into account the rapid occurrence of PI cross-resistance, clinicians who are treating patients with the HIV-1 infection will need new active PIs in the near future. The clinical and antiviral efficacy of the new molecules versus the older PIs will be investigated through comparative trials that are likely to be completed over the next 12 months. These third-generation PIs currently in development will be the subject of our review.

  15. 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

  16. HIV-1 Strategies of Immune Evasion

    NASA Astrophysics Data System (ADS)

    Castiglione, F.; Bernaschi, M.

    We simulate the progression of the HIV-1 infection in untreated host organisms. The phenotype features of the virus are represented by the replication rate, the probability of activating the transcription, the mutation rate and the capacity to stimulate an immune response (the so-called immunogenicity). It is very difficult to study in-vivo or in-vitro how these characteristics of the virus influence the evolution of the disease. Therefore we resorted to simulations based on a computer model validated in previous studies. We observe, by means of computer experiments, that the virus continuously evolves under the selective pressure of an immune response whose effectiveness downgrades along with the disease progression. The results of the simulations show that immunogenicity is the most important factor in determining the rate of disease progression but, by itself, it is not sufficient to drive the disease to a conclusion in all cases.

  17. HIV-1 Envelope Proteins and V1/V2 Domain Scaffolds with Mannose-5 to Improve the Magnitude and Quality of Protective Antibody Responses to HIV-1*

    PubMed Central

    Morales, Javier F.; Morin, Trevor J.; Yu, Bin; Tatsuno, Gwen P.; O'Rourke, Sara M.; Theolis, Richard; Mesa, Kathryn A.; Berman, Phillip W.

    2014-01-01

    Two lines of investigation have highlighted the importance of antibodies to the V1/V2 domain of gp120 in providing protection from HIV-1 infection. First, the recent RV144 HIV-1 vaccine trial documented a correlation between non-neutralizing antibodies to the V2 domain and protection. Second, multiple broadly neutralizing monoclonal antibodies to the V1/V2 domain (e.g. PG9) have been isolated from rare infected individuals, termed elite neutralizers. Interestingly, the binding of both types of antibodies appears to depend on the same cluster of amino acids (positions 167–171) adjacent to the junction of the B and C strands of the four-stranded V1/V2 domain β-sheet structure. However, the broadly neutralizing mAb, PG9, additionally depends on mannose-5 glycans at positions 156 and 160 for binding. Because the gp120 vaccine immunogens used in previous HIV-1 vaccine trials were enriched for complex sialic acid-containing glycans, and lacked the high mannose structures required for the binding of PG9-like mAbs, we wondered if these immunogens could be improved by limiting glycosylation to mannose-5 glycans. Here, we describe the PG9 binding activity of monomeric gp120s from multiple strains of HIV-1 produced with mannose-5 glycans. We also describe the properties of glycopeptide scaffolds from the V1/V2 domain also expressed with mannose-5 glycans. The V1/V2 scaffold from the A244 isolate was able to bind the PG9, CH01, and CH03 mAbs with high affinity provided that the proper glycans were present. We further show that immunization with A244 V1/V2 fragments alone, or in a prime/boost regimen with gp120, enhanced the antibody response to sequences in the V1/V2 domain associated with protection in the RV144 trial. PMID:24872420

  18. 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

  19. Nanotherapeutics Using an HIV-1 Poly A and Transactivator of the HIV-1 LTR-(TAR-) Specific siRNA

    PubMed Central

    Mahajan, Supriya D.; Aalinkeel, Ravikumar; Reynolds, Jessica L.; Nair, Bindukumar; Sykes, Donald E.; Law, Wing-Cheung; Ding, Hong; Bergey, Earl J.; Prasad, Paras N.; Schwartz, Stanley A.

    2011-01-01

    HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. We used a well-validated siRNA (si510) which targets the poly A/TAR (transactivator of the HIV-1 LTR) site and suppresses viral replication. Nanotechnology holds much potential for impact in the field of HIV-1 therapeutics, and nanoparticles such as quantum rods (QRs) can be easily functionalized to incorporate siRNA forming stable nanoplexes that can be used for gene silencing. We evaluated the efficacy of the QR-si510 HIV-1 siRNA nanoplex in suppressing viral replication in the HIV-1-infected monocytic cell line THP-1 by measuring p24 antigen levels and gene expression levels of HIV-1 LTR. Our results suggest that the QR-si510 HIV-1 siRNA nanoplex is not only effective in delivering siRNA, but also in suppressing HIV-1 viral replication for a longer time period. HIV-1 nanotherapeutics can thus enhance systemic bioavailability and offer multifunctionality. PMID:21660279

  20. Diversity of HIV-1 RNA and DNA in breast milk from HIV-1-infected mothers.

    PubMed

    Becquart, Pierre; Courgnaud, Valerie; Willumsen, Juana; Van de Perre, Philippe

    2007-07-05

    We compared human immunodeficiency virus type 1 (HIV-1) RNA and DNA populations in the different fractions of breast milk (lactoserum, lipid layer, cell pellet) and between right and left breasts in four HIV-1-infected mothers by analyzing the hypervariable env C2-V5 region. Phylogenetic analyses of the viral quasispecies revealed that RNA populations and DNA populations were clearly distinct and that viral RNA sequences were similar in lipid layer and lactoserum in the milk of 3 out of 4 mothers. Comparison of viral DNA between milk from right and left breast showed a differential distribution of variants in three mothers. In contrast, RNA variants detected from milk of the two breasts were mixed in 3 out of 4 mothers. This study suggests that each mammary gland is subjected to microenvironmental pressure that may differ from the contralateral breast.

  1. Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration

    PubMed Central

    Kline, Erik R.; Kleinhenz, Dean J.; Liang, Bill; Dikalov, Sergey; Guidot, David M.; Hart, C. Michael; Jones, Dean P.; Sutliff, Roy L.

    2008-01-01

    Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation

  2. HIV-1 and Human PEG10 Frameshift Elements Are Functionally Distinct and Distinguished by Novel Small Molecule Modulators

    PubMed Central

    Sleebs, Brad E.; Lackovic, Kurt; Parisot, John P.; Moss, Rebecca M.; Crowe-McAuliffe, Caillan; Mathew, Suneeth F.; Edgar, Christina D.; Kleffmann, Torsten; Tate, Warren P.

    2015-01-01

    Frameshifting during translation of viral or in rare cases cellular mRNA results in the synthesis of proteins from two overlapping reading frames within the same mRNA. In HIV-1 the protease, reverse transcriptase, and integrase enzymes are in a second reading frame relative to the structural group-specific antigen (gag), and their synthesis is dependent upon frameshifting. This ensures that a strictly regulated ratio of structural proteins and enzymes, which is critical for HIV-1 replication and viral infectivity, is maintained during protein synthesis. The frameshift element in HIV-1 RNA is an attractive target for the development of a new class of anti HIV-1 drugs. However, a number of examples are now emerging of human genes using −1 frameshifting, such as PEG10 and CCR5. In this study we have compared the HIV-1 and PEG10 frameshift elements and shown they have distinct functional characteristics. Frameshifting occurs at several points within each element. Moreover, frameshift modulators that were isolated by high-throughput screening of a library of 114,000 lead-like compounds behaved differently with the PEG10 frameshift element. The most effective compounds affecting the HIV-1 element enhanced frameshifting by 2.5-fold at 10 μM in two different frameshift reporter assay systems. HIV-1 protease:gag protein ratio was affected by a similar amount in a specific assay of virally-infected cultured cell, but the modulation of frameshifting of the first-iteration compounds was not sufficient to show significant effects on viral infectivity. Importantly, two compounds did not affect frameshifting with the human PEG10 element, while one modestly inhibited rather than enhanced frameshifting at the human element. These studies indicate that frameshift elements have unique characteristics that may allow targeting of HIV-1 and of other viruses specifically for development of antiviral therapeutic molecules without effect on human genes like PEG10 that use the same

  3. Influence of sequence identity and unique breakpoints on the frequency of intersubtype HIV-1 recombination

    PubMed Central

    Baird, Heather A; Gao, Yong; Galetto, Román; Lalonde, Matthew; Anthony, Reshma M; Giacomoni, Véronique; Abreha, Measho; Destefano, Jeffrey J; Negroni, Matteo; Arts, Eric J

    2006-01-01

    Background HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population. Results Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies. Conclusion Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A

  4. 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

  5. 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.

  6. SEROPREVALENCE OF HTLV IN A POPULATION OF HIV1-INFECTED PATIENTS IN MIDWESTERN BRAZIL

    PubMed Central

    KOZLOWSKI, Aline Garcia; de MATOS, Márcia Alves Dias; CARNEIRO, Megmar Aparecida dos Santos; LOPES, Carmen Luci Rodrigues; TELES, Sheila Araújo; VICENTE, Carolina Paulo; MARTINS, Regina Maria Bringel

    2016-01-01

    SUMMARY Human T-cell lymphotropic virus (HTLV) may affect the clinical course of human immunodeficiency virus 1 (HIV1). Both infections are common in endemic areas because these viruses share similar routes of transmission. The aim of this study was to estimate the seroprevalence of HTLV1/2 in a population of HIV1-infected patients in the state of Goiás, Midwestern Brazil. Of the 505 studied patients, four (0.79%) were positive for anti-HTLV1/2 by enzyme-linked immunosorbent assay (ELISA), with HTLV1 infection confirmed by line immunoassay (LIA) and polymerase chain reaction (PCR) in all of the ELISA-positive samples. No cases of HTLV2 infection were observed. The prevalence of HTLV1/HIV1 coinfection was 0.79% (4/505; 95% CI: 0.25-2.16). All the coinfected patients reported sexual risk behaviors and only one reported intravenous drug use. Sequencing of the viral long terminal repeat (LTR) region and phylogenetic analysis revealed that the four HTLV1 isolates belonged to the Transcontinental a subgroup of the Cosmopolitan (1a) subtype, the most frequent subgroup detected in Brazil. This study shows a low prevalence of HTLV1/2 in HIV1-infected patients in Midwestern Brazil. PMID:27828621

  7. On the role of four small hairpins in the HIV-1 RNA genome

    PubMed Central

    Knoepfel, Stefanie A.; Berkhout, Ben

    2013-01-01

    An RNA secondary structure model for the complete HIV-1 genome has recently been published based on SHAPE technology. Several well-known RNA motifs such as TAR and RRE were confirmed and numerous new structured motifs were described that may play important roles in virus replication. The 9 kb viral RNA genome is densely packed with many RNA hairpin motifs and the collective fold may play an important role in HIV-1 biology. We initially focused on 16 RNA hairpin motifs scattered along the viral genome. We considered conservation of these structures, despite sequence variation among virus isolates, as a first indication for a significant function. Four relatively small hairpins exhibited considerable structural conservation and were selected for experimental validation in virus replication assays. Mutations were introduced into the HIV-1 RNA genome to destabilize individual RNA structures without affecting the protein-coding properties (silent codon changes). No major virus replication defects were scored, suggesting that these four hairpin structures do not play essential roles in HIV-1 replication. PMID:23535706

  8. The Genetic Diversity and Evolution of HIV-1 Subtype B Epidemic in Puerto Rico.

    PubMed

    López, Pablo; Rivera-Amill, Vanessa; Rodríguez, Nayra; Vargas, Freddie; Yamamura, Yasuhiro

    2015-12-23

    HIV-1 epidemics in Caribbean countries, including Puerto Rico, have been reported to be almost exclusively associated with the subtype B virus (HIV-1B). However, while HIV infections associated with other clades have been only sporadically reported, no organized data exist to accurately assess the prevalence of non-subtype B HIV-1 infection. We analyzed the nucleotide sequence data of the HIV pol gene associated with HIV isolates from Puerto Rican patients. The sequences (n = 945) were obtained from our "HIV Genotyping" test file, which has been generated over a period of 14 years (2001-2014). REGA subtyping tool found the following subtypes: B (90%), B-like (3%), B/D recombinant (6%), and D/B recombinant (0.6%). Though there were fewer cases, the following subtypes were also found (in the given proportions): A1B (0.3%), BF1 (0.2%), subtype A (01-AE) (0.1%), subtype A (A2) (0.1%), subtype F (12BF) (0.1%), CRF-39 BF-like (0.1%), and others (0.1%). Some of the recombinants were identified as early as 2001. Although the HIV epidemic in Puerto Rico is primarily associated with HIV-1B virus, our analysis uncovered the presence of other subtypes. There was no indication of subtype C, which has been predominantly associated with heterosexual transmission in other parts of the world.

  9. Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity.

    PubMed

    Bradley, Todd; Trama, Ashley; Tumba, Nancy; Gray, Elin; Lu, Xiaozhi; Madani, Navid; Jahanbakhsh, Fatemeh; Eaton, Amanda; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey E; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Barnett, Susan; Abdool-Karim, Salim S; Boyd, Scott D; Melillo, Bruno; Smith, Amos B; Sodroski, Joseph; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Moody, M Anthony; Montefiori, David; Santra, Sampa; Morris, Lynn; Haynes, Barton F

    2016-10-01

    Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

  10. Anti-human immunodeficiency virus type 1 (HIV-1) activity of lectins from Narcissus species.

    PubMed

    López, Susana; Armand-Ugon, Mercedes; Bastida, Jaume; Viladomat, Francesc; Esté, José A; Stewart, Derek; Codina, Carles

    2003-02-01

    Mannose-specific lectins (MSLs) were isolated from bulbs of fifteen wild Narcissus species growing in Spain and assayed for their HIV-1 infection inhibitory activity in MT-4 cells and compared to the Narcissus pseudonarcissus agglutinin (NPA), the commercially available MSL obtained from daffodils. Almost all the tested MSLs were found to be active, showing EC50 values (microg/mL) similar to that of NPA, with some being comparable to those obtained with dextran sulfate without significant cytotoxicity. However, on a molar basis almost all of the MSLs tested exhibited lower EC50 values than dextran sulfate whilst six MSLs had values lower than AZT. The most efficacious anti-HIV-1 activity was exhibited by the Narcissus tortifolious MSL, which was 10- (microg/mL) and 100- (molarity) fold more potent than dextran sulfate. Significantly, although this MSL was 15-fold less potent than AZT in terms of quantity (microg/mL), it was 68-fold more potent on a molar basis. The antiviral indices, a ratio of the concentrations that produce cytotoxicity and HIV-1 replication, were calculated and three of the MSLs, N. confusus, N. leonensis and N. tortifolius reported 1.5-, 2- and 8.5-fold greater AI values than dextran sulfate or AZT. Comparison of MSL haemagglutination activities (HAA) to their anti-HIV-1 activities showed that there was no significant correlation. It was suggested that this may be due to a dissociation between both activities as a consequence of multiple isolectin composition.

  11. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

    PubMed Central

    Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

    2015-01-01

    Background Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. Methods We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Results Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Conclusions Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents. PMID:26154172

  12. 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.

  13. On the role of four small hairpins in the HIV-1 RNA genome.

    PubMed

    Knoepfel, Stefanie A; Berkhout, Ben

    2013-04-01

    An RNA secondary structure model for the complete HIV-1 genome has recently been published based on SHAPE technology. Several well-known RNA motifs such as TAR and RRE were confirmed and numerous new structured motifs were described that may play important roles in virus replication. The 9 kb viral RNA genome is densely packed with many RNA hairpin motifs and the collective fold may play an important role in HIV-1 biology. We initially focused on 16 RNA hairpin motifs scattered along the viral genome. We considered conservation of these structures, despite sequence variation among virus isolates, as a first indication for a significant function. Four relatively small hairpins exhibited considerable structural conservation and were selected for experimental validation in virus replication assays. Mutations were introduced into the HIV-1 RNA genome to destabilize individual RNA structures without affecting the protein-coding properties (silent codon changes). No major virus replication defects were scored, suggesting that these four hairpin structures do not play essential roles in HIV-1 replication.

  14. The implications of viral reservoirs on the elite control of HIV-1 infection.

    PubMed

    Buckheit, Robert W; Salgado, Maria; Martins, Karen O; Blankson, Joel N

    2013-03-01

    The mechanisms by which a small percentage of HIV-1 infected individuals known as elite suppressors or controllers are able to control viral replication are not fully understood. Early cases of viremic control were attributed to infection with defective virus, but subsequent work has demonstrated that infection with a defective virus is not the exclusive cause of control. Replication-competent virus has been isolated from patients who control viral replication, and studies have demonstrated that evolution occurs in plasma virus but not in virus isolates from the latent reservoir. Additionally, transmission pair studies have demonstrated that patients infected with similar viruses can have dramatically different outcomes of infection. An increased understanding of the viral factors associated with control is important to understand the interplay between viral replication and host control, and has implications for the design of an effective therapeutic vaccine that can lead to a functional cure of HIV-1 infection.

  15. Computational Prediction of Broadly Neutralizing HIV-1 Antibody Epitopes from Neutralization Activity Data

    PubMed Central

    Ferguson, Andrew L.; Falkowska, Emilia; Walker, Laura M.; Seaman, Michael S.; Burton, Dennis R.; Chakraborty, Arup K.

    2013-01-01

    Broadly neutralizing monoclonal antibodies effective against the majority of circulating isolates of HIV-1 have been isolated from a small number of infected individuals. Definition of the conformational epitopes on the HIV spike to which these antibodies bind is of great value in defining targets for vaccine and drug design. Drawing on techniques from compressed sensing and information theory, we developed a computational methodology to predict key residues constituting the conformational epitopes on the viral spike from cross-clade neutralization activity data. Our approach does not require the availability of structural information for either the antibody or antigen. Predictions of the conformational epitopes of ten broadly neutralizing HIV-1 antibodies are shown to be in good agreement with new and existing experimental data. Our findings suggest that our approach offers a means to accelerate epitope identification for diverse pathogenic antigens. PMID:24312481

  16. A Novel Tricyclic Ligand-Containing Nonpeptidic HIV-1 Protease Inhibitor, GRL-0739, Effectively Inhibits the Replication of Multidrug-Resistant HIV-1 Variants and Has a Desirable Central Nervous System Penetration Property In Vitro

    PubMed Central

    Amano, Masayuki; Tojo, Yasushi; Salcedo-Gómez, Pedro Miguel; Parham, Garth L.; Nyalapatla, Prasanth R.; Das, Debananda; Ghosh, Arun K.

    2015-01-01

    We report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0019 to 0.0036 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC50], 21.0 μM). GRL-0739 blocked the infectivity and replication of HIV-1NL4-3 variants selected by concentrations of up to 5 μM ritonavir or atazanavir (EC50, 0.035 to 0.058 μM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2ROD variant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novel in vitro blood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles. PMID:25691652

  17. Complexity in human immunodeficiency virus type 1 (HIV-1) co-receptor usage: roles of CCR3 and CCR5 in HIV-1 infection of monocyte-derived macrophages and brain microglia.

    PubMed

    Agrawal, Lokesh; Maxwell, Christina R; Peters, Paul J; Clapham, Paul R; Liu, Sue M; Mackay, Charles R; Strayer, David S

    2009-03-01

    CCR3 has been implicated as a co-receptor for human immunodeficiency virus type 1 (HIV-1), particularly in brain microglia cells. We sought to clarify the comparative roles of CCR3 and CCR5 in the central nervous system (CNS) HIV-1 infection and the potential utility of CCR3 as a target for manipulation via gene transfer. To target CCR3, we developed a single-chain antibody (SFv) and an interfering RNA (RNAi), R3-526. Coding sequences for both were cloned into Tag-deleted SV40-dervied vectors, as these vectors transduce brain microglia and monocyte-derived macrophages (MDM) highly efficiently. These anti-CCR3 transgenes were compared to SFv-CCR5, an SFv against CCR5, and RNAi-R5, an RNAi that targets CCR5, for the ability to protect primary human brain microglia and MDM from infection with peripheral and neurotropic strains of HIV-1. Downregulation of CCR3 and CCR5 by these transgenes was independent from one another. Confocal microscopy showed that CCR3 and CCR5 co-localized at the plasma membrane with each other and with CD4. Targeting either CCR5 or CCR3 largely protected both microglia and MDM from infection by many strains of HIV-1. That is, some HIV-1 strains, isolated from either the CNS or periphery, required both CCR3 and CCR5 for optimal productive infection of microglia and MDM. Some HIV-1 strains were relatively purely CCR5-tropic. None was purely CCR3-tropic. Thus, some CNS-tropic strains of HIV-1 utilize CCR5 as a co-receptor but do not need CCR3, while for other isolates both CCR3 and CCR5 may be required.

  18. 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

  19. 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. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Impact of HIV-1 Backbone on Neutralization Sensitivity: Neutralization Profiles of Heterologous Envelope Glycoproteins Expressed in Native Subtype C and CRF01_AE Backbone

    PubMed Central

    Sanders-Buell, Eric; Wesberry, Maggie; Towle, Teresa; Pillis, Devin M.; Molnar, Sebastian; McLinden, Robert; Edmonds, Tara; Hirsch, Ivan; O’Connell, Robert; McCutchan, Francine E.; Montefiori, David C.; Ochsenbauer, Christina; Kappes, John C.; Kim, Jerome H.; Polonis, Victoria R.; Tovanabutra, Sodsai

    2013-01-01

    Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and –unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy. PMID:24312165

  6. Development of candidate reference reagent for HIV-1 RNA and comparison analysis for different HIV-1 RNA quantitative assay.

    PubMed

    Park, Borae G; Park, Ae Ja; Choi, Jee-Hye; Park, Jina; Kim, Sung Soon; Wang, Jin-Sook; Kee, Mee Kyung; Choi, Ju-yeon

    2011-09-01

    Human immunodeficiency virus type-1 (HIV-1) RNA viral load is a surrogate marker that is routinely used to determine indications for, and monitor the effectiveness of HIV-1 treatment. We developed three reagents for potential use in routine quality control of HIV-1 RNA quantitative assays. In this report, we compare the stability of these re-agents in storage and compare their performance in three different HIV-1 RNA quantitative assays. The candidate reagents were derived from readily available pre-existing reagents and examined for stability at different storage temperatures. They were compared in three commercially available HIV-1 RNA quantitative assays: the Cobas TaqMan HIV-1 Test (Cobas TaqMan), the RealTime HIV-1 Assay (Abbott RealTime), and the NucliSens EasyQ HIV-1 Assay v1.1 (NucliSens EasyQ). The candidate reagent derived from an HIV culture supernatant (candidate CS) was the most stable of the three candidates and showed g