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Sample records for hiv-1 tat dependent

  1. Interaction of the phospholipid scramblase 1 with HIV-1 Tat results in the repression of Tat-dependent transcription

    SciTech Connect

    Kusano, Shuichi Eizuru, Yoshito

    2013-04-19

    Highlights: •PLSCR1 specifically interacted with HIV-1 Tat in vitro and in vivo. •PLSCR1 repressed Tat-dependent transactivation of the HIV-1 LTR. •Suppression of PLSCR1 expression enhanced the levels of HIV-1 transcripts. •PLSCR1 reduced the nuclear localization of Tat. -- Abstract: Human phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)-stimulated gene and possesses an IFN-mediated antiviral function. We show here that PLSCR1 directly interacts with human immunodeficiency virus type-1 (HIV-1) Tat. This interaction occurs both in vitro and in vivo through amino acids 160–250 of PLSCR1. Overexpression of PLSCR1 efficiently represses the Tat-dependent transactivation of the HIV-1 long terminal repeat (LTR) and reduces the nuclear translocation of Tat. In addition, shRNA-mediated suppression of endogenous PLSCR1 expression enhances the levels of gag mRNA in an HIV-1-infected T-cell line. These findings indicate that PLSCR1 negatively regulates the Tat-dependent transactivation of the HIV-1 LTR during HIV-1 infection.

  2. Morphine Tolerance and Physical Dependence Are Altered in Conditional HIV-1 Tat Transgenic Mice

    PubMed Central

    Stevens, David L.; Khan, Fayez A.; Scoggins, Krista L.; Enga, Rachel M.; Beardsley, Patrick M.; Knapp, Pamela E.; Dewey, William L.

    2016-01-01

    Despite considerable evidence that chronic opiate use selectively affects the pathophysiologic consequences of human immunodeficiency virus type 1 (HIV-1) infection in the nervous system, few studies have examined whether neuro-acquired immune deficiency syndrome (neuroAIDS) might intrinsically alter the pharmacologic responses to chronic opiate exposure. This is an important matter because HIV-1 and opiate abuse are interrelated epidemics, and HIV-1 patients are often prescribed opiates as a treatment of HIV-1–related neuropathic pain. Tolerance and physical dependence are inevitable consequences of frequent and repeated administration of morphine. In the present study, mice expressing HIV-1 Tat in a doxycycline (DOX)–inducible manner [Tat(+)], their Tat(−) controls, and control C57BL/6 mice were chronically exposed to placebo or 75-mg morphine pellets to explore the effects of Tat induction on morphine tolerance and dependence. Antinociceptive tolerance and locomotor activity tolerance were assessed using tail-flick and locomotor activity assays, respectively, and physical dependence was measured with the platform-jumping assay and recording of other withdrawal signs. We found that Tat(+) mice treated with DOX [Tat(+)/DOX] developed an increased tolerance in the tail-flick assay compared with control Tat(−)/DOX and/or C57/DOX mice. Equivalent tolerance was developed in all mice when assessed by locomotor activity. Further, Tat(+)/DOX mice expressed reduced levels of physical dependence to chronic morphine exposure after a 1-mg/kg naloxone challenge compared with control Tat(−)/DOX and/or C57/DOX mice. Assuming the results seen in Tat transgenic mice can be generalized to neuroAIDS, our findings suggest that HIV-1-infected individuals may display heightened analgesic tolerance to similar doses of opiates compared with uninfected individuals and show fewer symptoms of physical dependence. PMID:26542403

  3. Morphine Tolerance and Physical Dependence Are Altered in Conditional HIV-1 Tat Transgenic Mice.

    PubMed

    Fitting, Sylvia; Stevens, David L; Khan, Fayez A; Scoggins, Krista L; Enga, Rachel M; Beardsley, Patrick M; Knapp, Pamela E; Dewey, William L; Hauser, Kurt F

    2016-01-01

    Despite considerable evidence that chronic opiate use selectively affects the pathophysiologic consequences of human immunodeficiency virus type 1 (HIV-1) infection in the nervous system, few studies have examined whether neuro-acquired immune deficiency syndrome (neuroAIDS) might intrinsically alter the pharmacologic responses to chronic opiate exposure. This is an important matter because HIV-1 and opiate abuse are interrelated epidemics, and HIV-1 patients are often prescribed opiates as a treatment of HIV-1-related neuropathic pain. Tolerance and physical dependence are inevitable consequences of frequent and repeated administration of morphine. In the present study, mice expressing HIV-1 Tat in a doxycycline (DOX)-inducible manner [Tat(+)], their Tat(-) controls, and control C57BL/6 mice were chronically exposed to placebo or 75-mg morphine pellets to explore the effects of Tat induction on morphine tolerance and dependence. Antinociceptive tolerance and locomotor activity tolerance were assessed using tail-flick and locomotor activity assays, respectively, and physical dependence was measured with the platform-jumping assay and recording of other withdrawal signs. We found that Tat(+) mice treated with DOX [Tat(+)/DOX] developed an increased tolerance in the tail-flick assay compared with control Tat(-)/DOX and/or C57/DOX mice. Equivalent tolerance was developed in all mice when assessed by locomotor activity. Further, Tat(+)/DOX mice expressed reduced levels of physical dependence to chronic morphine exposure after a 1-mg/kg naloxone challenge compared with control Tat(-)/DOX and/or C57/DOX mice. Assuming the results seen in Tat transgenic mice can be generalized to neuroAIDS, our findings suggest that HIV-1-infected individuals may display heightened analgesic tolerance to similar doses of opiates compared with uninfected individuals and show fewer symptoms of physical dependence. Copyright © 2015 by The American Society for Pharmacology and Experimental

  4. Thioredoxin reductase-1 negatively regulates HIV-1 transactivating protein Tat-dependent transcription in human macrophages.

    PubMed

    Kalantari, Parisa; Narayan, Vivek; Natarajan, Sathish K; Muralidhar, Kambadur; Gandhi, Ujjawal H; Vunta, Hema; Henderson, Andrew J; Prabhu, K Sandeep

    2008-11-28

    Epidemiological studies suggest a correlation between severity of acquired immunodeficiency syndrome (AIDS) and selenium deficiency, indicating a protective role for this anti-oxidant during HIV infection. Here we demonstrate that thioredoxin reductase-1 (TR1), a selenium-containing pyridine nucleotide-disulfide oxidoreductase that reduces protein disulfides to free thiols, negatively regulates the activity of the HIV-1 encoded transcriptional activator, Tat, in human macrophages. We used a small interfering RNA approach as well as a high affinity substrate of TR1, ebselen, to demonstrate that Tat-dependent transcription and HIV-1 replication were significantly increased in human macrophages when TR1 activity was reduced. The increase in HIV-1 replication in TR1 small interfering RNA-treated cells was independent of the redox-sensitive transcription factor, NF-kappaB. These studies indicate that TR-1 acts as a negative regulator of Tat-dependent transcription. Furthermore, in vitro biochemical assays with recombinant Tat protein confirmed that TR1 targets two disulfide bonds within the Cys-rich motif required for efficient HIV-1 transactivation. Increasing TR1 expression along with other selenoproteins by supplementing with selenium suggests a potential inexpensive adjuvant therapy for HIV/AIDS patients.

  5. Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon

    PubMed Central

    López-Huertas, M. R.; Callejas, S.; Abia, D.; Mateos, E.; Dopazo, A.; Alcamí, J.; Coiras, M.

    2010-01-01

    The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1–72aa) is sufficient for viral transcript elongation and second exon (73–101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-κB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells. PMID:20139419

  6. Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon.

    PubMed

    López-Huertas, M R; Callejas, S; Abia, D; Mateos, E; Dopazo, A; Alcamí, J; Coiras, M

    2010-06-01

    The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.

  7. Mechanism of HIV-1 Tat RNA translation and its activation by the Tat protein

    PubMed Central

    Charnay, Nicolas; Ivanyi-Nagy, Roland; Soto-Rifo, Ricardo; Ohlmann, Théophile; López-Lastra, Marcelo; Darlix, Jean-Luc

    2009-01-01

    Background The human immunodeficiency virus type 1 (HIV-1) Tat protein is a major viral transactivator required for HIV-1 replication. In the nucleus Tat greatly stimulates the synthesis of full-length transcripts from the HIV-1 promoter by causing efficient transcriptional elongation. Tat induces elongation by directly interacting with the bulge of the transactivation response (TAR) RNA, a hairpin-loop located at the 5'-end of all nascent viral transcripts, and by recruiting cellular transcriptional co-activators. In the cytoplasm, Tat is thought to act as a translational activator of HIV-1 mRNAs. Thus, Tat plays a central role in the regulation of HIV-1 gene expression both at the level of mRNA and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of Tat can be made early on in HIV-1 infected cells to sustain its own synthesis. To address this issue we studied translation of the Tat mRNA in vitro and in human cells using recombinant monocistronic and dicistronic RNAs containing the 5' untranslated region (5'-UTR) of Tat RNA. Results This study shows that the Tat mRNA can be efficiently translated both in vitro and in cells. Furthermore, our data suggest that translation initiation from the Tat mRNA probably occurs by a internal ribosome entry site (IRES) mechanism. Finally, we show that Tat protein can strongly stimulate translation from its cognate mRNA in a TAR dependent fashion. Conclusion These results indicate that Tat mRNA translation is efficient and benefits from a feedback stimulation by the Tat protein. This translational control mechanism would ensure that minute amounts of Tat mRNA are sufficient to generate enough Tat protein required to stimulate HIV-1 replication. PMID:19671151

  8. Tat is required for efficient HIV-1 reverse transcription.

    PubMed Central

    Harrich, D; Ulich, C; García-Martínez, L F; Gaynor, R B

    1997-01-01

    The ability of human immunodeficiency virus-1 (HIV-1) to undergo efficient reverse transcription is dependent on a number of parameters. These include the binding of the tRNA(3)(Lys) to the HIV-1 primer binding site and the subsequent interaction with the heterodimeric reverse transcriptase. Recently, we demonstrated that TAR RNA was also necessary for efficient HIV-1 reverse transcription. Given the fact that the Tat protein is involved in the activation of HIV-1 gene expression in conjunction with TAR, we wished to determine whether Tat might also be involved in the control of HIV-1 reverse transcription. HIV-1 virions deleted in the tat gene were unable to initiate reverse transcription efficiently upon infection of peripheral blood mononuclear cells (PBMCs). This defect was not due to decreased amounts of genomic RNA, reverse transcriptase or other HIV-1 proteins which were incorporated into the virion. Following transfection of wild-type but not mutant tat genes into cell lines producing HIV-1 lacking tat, the virions produced could be complemented for defects in reverse transcription upon subsequent infection of PBMCs. In contrast, the defect in reverse transcription seen with HIV-1 lacking the tat gene could not be complemented when the target cells rather than the producer cells contained tat. Viruses lacking tat were also defective in endogenous assays of reverse transcription, although these viruses contained similar levels of reverse transcriptase. These results indicate that the Tat protein, in addition to regulating the level of gene expression, is also important for efficient HIV-1 reverse transcription. PMID:9135139

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

    PubMed Central

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

    2016-01-01

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

  10. DDX3 RNA helicase is required for HIV-1 Tat function.

    PubMed

    Yasuda-Inoue, Mariko; Kuroki, Misao; Ariumi, Yasuo

    2013-11-22

    Host RNA helicase has been involved in human immunodeficiency virus type 1 (HIV-1) replication, since HIV-1 does not encode an RNA helicase. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether DDX RNA helicases modulate the HIV-1 Tat function. In this study, we demonstrate, for the first time, that DDX3 is required for the HIV-1 Tat function. Notably, DDX3 colocalized and interacted with HIV-1 Tat in cytoplasmic foci. Indeed, DDX3 localized in the cytoplasmic foci P-bodies or stress granules under stress condition after the treatment with arsenite. Importantly, only DDX3 enhanced the Tat function, while various distinct DEAD-box RNA helicases including DDX1, DDX3, DDX5, DDX17, DDX21, and DDX56, stimulated the HIV-1 Rev-dependent RNA export function, indicating a specific role of DDX3 in Tat function. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be required for the Tat function as well as the colocalization with Tat. Furthermore, the combination of DDX3 with other distinct DDX RNA helicases cooperated to stimulate the Rev but not Tat function. Thus, DDX3 seems to interact with the HIV-1 Tat and facilitate the Tat function. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Analysis of HIV-1 Tat effects in Xenopus laevis embryos.

    PubMed

    Venanzi, S; Destrée, O H; Gigliani, F; Battaglia, P A

    1998-01-01

    Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a 'loss of function' mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.

  12. 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

  13. Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat.

    PubMed

    Nukuzuma, Souichi; Kameoka, Masanori; Sugiura, Shigeki; Nakamichi, Kazuo; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu

    2009-11-01

    Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter. In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.

  14. USP7 deubiquitinase controls HIV-1 production by stabilizing Tat protein.

    PubMed

    Ali, Amjad; Raja, Rameez; Farooqui, Sabihur Rahman; Ahmad, Shaista; Banerjea, Akhil C

    2017-05-04

    Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  15. The effects of HIV-1 regulatory TAT protein expression on brain reward function, response to psychostimulants and delay-dependent memory in mice.

    PubMed

    Kesby, James P; Markou, Athina; Semenova, Svetlana

    2016-10-01

    Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Molecular mechanisms in the dramatic enhancement of HIV-1 Tat transduction by cationic liposomes

    PubMed Central

    Li, Guan-Han; Li, Wenxue; Mumper, Russell J.; Nath, Avindra

    2012-01-01

    Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein possesses a unique membrane-transduction property. Interestingly, Tat transduction could be dramatically increased 1000-fold based on LTR-transactivation assay when complexed with cationic liposomes (lipo-Tat), compared with Tat alone. Therefore, underlining mechanisms were explored further. Microscopy and flow cytometry showed that this effect was associated with enhanced membrane binding, large particle formation (1–2 μm) and increased intracellular uptake of Tat fluorescent proteins. Using pharmacological assays and immune colocalizations, it was found that lipid raft-dependent endocytosis and macropinocytosis were major pathways involved in lipo-Tat uptake, and actin-filaments played a major role in intracellular trafficking of lipo-Tat to the nucleus. Furthermore, we found that the Tat hydrophobic domain (aa 36–47) mediated formation of two positively charged molecules into lipo-Tat complexes via hydrophobic bonds, based on LTR-transactivation inhibition assay. Thus, the hydrophobic domain may play an important role in Tat protein uptake and be useful for intracellular delivery of biomacromolecules if coupled together with Tat basic peptide, a cell-penetrating peptide.—Li, G.-H., Li, W., Mumper, R. J., Nath, A. Molecular mechanisms in the dramatic enhancement of HIV-1 Tat transduction by cationic liposomes. PMID:22447980

  17. HIV-1 Tat and Cocaine Impair Survival of Cultured Primary Neuronal Cells via a Mitochondrial Pathway.

    PubMed

    De Simone, Francesca Isabella; Darbinian, Nune; Amini, Shohreh; Muniswamy, Madesh; White, Martyn K; Elrod, John W; Datta, Prasun K; Langford, Dianne; Khalili, Kamel

    2016-06-01

    Addictive stimulant drugs, such as cocaine, are known to increase the risk of exposure to HIV-1 infection and hence predispose towards the development of AIDS. Previous findings suggested that the combined effect of chronic cocaine administration and HIV-1 infection enhances cell death. Neuronal survival is highly dependent on the health of mitochondria providing a rationale for assessing mitochondrial integrity and functionality following cocaine treatment, either alone or in combination with the HIV-1 viral protein Tat, by monitoring ATP release and mitochondrial membrane potential (ΔΨm). Our results indicate that exposing human and rat primary hippocampal neurons to cocaine and HIV-1 Tat synergistically decreased both mitochondrial membrane potential and ATP production. Additionally, since previous studies suggested HIV-1 infection alters autophagy in the CNS, we investigated how HIV-1 Tat and cocaine affect autophagy in neurons. The results indicated that Tat induces an increase in LC3-II levels and the formation of Parkin-ring-like structures surrounding damaged mitochondria, indicating the possible involvement of the Parkin/PINK1/DJ-1 (PPD) complex in neuronal degeneration. The importance of mitochondrial damage is also indicated by reductions in mitochondrial membrane potential and ATP content induced by HIV-1 Tat and cocaine.

  18. Latent HIV-1 Can Be Reactivated by Cellular Superinfection in a Tat-Dependent Manner, Which Can Lead to the Emergence of Multidrug-Resistant Recombinant Viruses

    PubMed Central

    Donahue, Daniel A.; Bastarache, Sophie M.; Sloan, Richard D.

    2013-01-01

    The HIV-1 latent reservoir represents an important source of genetic diversity that could contribute to viral evolution and multidrug resistance following latent virus reactivation. This could occur by superinfection of a latently infected cell. We asked whether latent viruses might be reactivated when their host cells are superinfected, and if so, whether they could contribute to the generation of recombinant viruses. Using populations of latently infected Jurkat cells, we found that latent viruses were efficiently reactivated upon superinfection. Pathways leading to latent virus reactivation via superinfection might include gp120-CD4/CXCR4-induced signaling, modulation of the cellular environment by Nef, and/or the activity of Tat produced upon superinfection. Using a range of antiviral compounds and genetic approaches, we show that gp120 and Nef are not required for latent virus reactivation by superinfection, but this process depends on production of functional Tat by the superinfecting virus. In a primary cell model of latency in unstimulated CD4 T cells, superinfection also led to latent virus reactivation. Drug-resistant latent viruses were also reactivated following superinfection in Jurkat cells and were able to undergo recombination with the superinfecting virus. Under drug-selective pressure, this generated multidrug-resistant recombinants that were identified by unique restriction digestion band patterns and by population-level sequencing. During conditions of poor drug adherence, treatment interruption or treatment failure, or in drug-impermeable sanctuary sites, reactivation of latent viruses by superinfection or other means could provide for the emergence or spread of replicatively fit viruses in the face of strong selective pressures. PMID:23804632

  19. Latent HIV-1 can be reactivated by cellular superinfection in a Tat-dependent manner, which can lead to the emergence of multidrug-resistant recombinant viruses.

    PubMed

    Donahue, Daniel A; Bastarache, Sophie M; Sloan, Richard D; Wainberg, Mark A

    2013-09-01

    The HIV-1 latent reservoir represents an important source of genetic diversity that could contribute to viral evolution and multidrug resistance following latent virus reactivation. This could occur by superinfection of a latently infected cell. We asked whether latent viruses might be reactivated when their host cells are superinfected, and if so, whether they could contribute to the generation of recombinant viruses. Using populations of latently infected Jurkat cells, we found that latent viruses were efficiently reactivated upon superinfection. Pathways leading to latent virus reactivation via superinfection might include gp120-CD4/CXCR4-induced signaling, modulation of the cellular environment by Nef, and/or the activity of Tat produced upon superinfection. Using a range of antiviral compounds and genetic approaches, we show that gp120 and Nef are not required for latent virus reactivation by superinfection, but this process depends on production of functional Tat by the superinfecting virus. In a primary cell model of latency in unstimulated CD4 T cells, superinfection also led to latent virus reactivation. Drug-resistant latent viruses were also reactivated following superinfection in Jurkat cells and were able to undergo recombination with the superinfecting virus. Under drug-selective pressure, this generated multidrug-resistant recombinants that were identified by unique restriction digestion band patterns and by population-level sequencing. During conditions of poor drug adherence, treatment interruption or treatment failure, or in drug-impermeable sanctuary sites, reactivation of latent viruses by superinfection or other means could provide for the emergence or spread of replicatively fit viruses in the face of strong selective pressures.

  20. Intersubtype Genetic Variation of HIV-1 Tat Exon 1.

    PubMed

    Roy, Chandra Nath; Khandaker, Irona; Oshitani, Hitoshi

    2015-06-01

    HIV-1 Tat is a regulatory protein that plays a pivotal role in viral transcription and replication. Our study aims to investigate the genetic variation of Tat exon 1 in all subtypes of HIV-1: A, B, C, D, F, G, H, J, and K. We performed phylogenetic, mutation, and selection pressure analyses on a total of 1,179 sequences of different subtypes of HIV-1 Tat obtained from the Los Alamos National Laboratory (LANL). The mean nucleotide divergences (%) among the analyzed sequences of subtypes A, B, C, D, F, G, H, J, and K were 88, 89, 90, 88, 86, 89, 88, 97, and 97, respectively. We revealed that subtype B evolved relatively faster than other subtypes. The second and fifth domains were found comparatively more variable among all subtypes. Site-by-site tests of positive selection revealed that several positions in all subtypes were under significant positive selection. Positively selected sites were found in the acidic domain at positions 3, 4, and 19, in the cysteine-rich domains at positions 24, 29, 32, and 36, in the core domain at position 40, and in the basic domain for the rest of the positions for all subtypes. Positions 58 and 68 in the basic domain were positively selected in subtypes A, B, C and B, C, F, respectively. We also observed high variability within positively selected sites in amino acid positions. Our study findings on HIV-1 Tat genetic variability may contribute to a better understanding of HIV-1 evolution as well as to the development of effective Tat-targeted therapeutics and vaccines.

  1. HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

    PubMed

    Vivarini, Áislan de Carvalho; Pereira, Renata de Meirelles Santos; Barreto-de-Souza, Victor; Temerozo, Jairo Ramos; Soares, Deivid C; Saraiva, Elvira M; Saliba, Alessandra Mattos; Bou-Habib, Dumith Chequer; Lopes, Ulisses Gazos

    2015-11-26

    HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

  2. HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR

    PubMed Central

    Vivarini, Áislan de Carvalho; Santos Pereira, Renata de Meirelles; Barreto-de-Souza, Victor; Temerozo, Jairo Ramos; Soares, Deivid C.; Saraiva, Elvira M.; Saliba, Alessandra Mattos; Bou-Habib, Dumith Chequer; Lopes, Ulisses Gazos

    2015-01-01

    HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49–57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling. PMID:26608746

  3. Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors.

    PubMed

    Kehn-Hall, Kylene; Guendel, Irene; Carpio, Lawrence; Skaltsounis, Leandros; Meijer, Laurent; Al-Harthi, Lena; Steiner, Joseph P; Nath, Avindra; Kutsch, Olaf; Kashanchi, Fatah

    2011-06-20

    The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC(50) of 40nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC(50) of 4nM in primary macrophages and 0.5nM in astrocytes infected with HIV-1. 6BIOder displayed an IC(50) value of 0.03nM through in vitro GSK-3β kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors

    PubMed Central

    Kehn-Hall, Kylene; Guendel, Irene; Carpio, Lawrence; Skaltsounis, Leandros; Meijer, Laurent; Al-Harthi, Lena; Steiner, Joseph P.; Nath, Avindra; Kutsch, Olaf; Kashanchi, Fatah

    2013-01-01

    The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC50 of 40 nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC50 of 4 nM in primary macrophages and 0.5 nM in astrocytes infected with HIV-1. 6BIOder displayed an IC50 value of 0.03 nM through in vitro GSK-3β kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity. PMID:21514616

  5. Differential immune mechanism to HIV-1 Tat variants and its regulation by AEA [corrected].

    PubMed

    Krishnan, Gopinath; Chatterjee, Nivedita

    2015-05-06

    In the retina, Müller glia is a dominant player of immune response. The HIV-1 transactivator viral protein (Tat) induces production of several neurotoxic cytokines in retinal cells. We show that HIV-1 clades Tat B and C act differentially on Müller glia, which is reflected in apoptosis, activation of cell death pathway components and pro-inflammatory cytokines. The harsher immune-mediated pathology of Tat B, as opposed to milder effects of Tat C, manifests at several signal transduction pathways, notably, MAPK, STAT, SOCS, the NFκB signalosome, and TTP. In activated cells, anandamide (AEA), acting as an immune-modulator, suppresses Tat B effect through MKP-1 but Tat C action via MEK-1. AEA lowers nuclear NF-κB and TAB2 for both variants while elevating IRAK1BP1 in activated Müller glia. Müller glia exposed to Tat shows enhanced PBMC attachment. Tat-induced increase in leukocyte adhesion to Müller cells can be mitigated by AEA, involving both CB receptors. This study identifies multiple signalling components that drive immune-mediated pathology and contribute to disease severity in HIV clades. We show that the protective effects of AEA occur at various stages in cytokine generation and are clade-dependant.

  6. HIV-1 Tat interaction with RNA polymerase II C-terminal domain (CTD) and a dynamic association with CDK2 induce CTD phosphorylation and transcription from HIV-1 promoter.

    PubMed

    Deng, Longwen; Ammosova, Tatyana; Pumfery, Anne; Kashanchi, Fatah; Nekhai, Sergei

    2002-09-13

    Human immunodeficiency virus, type 1 (HIV-1), Tat protein activates viral gene expression through promoting transcriptional elongation by RNA polymerase II (RNAPII). In this process Tat enhances phosphorylation of the C-terminal domain (CTD) of RNAPII by activating cell cycle-dependent kinases (CDKs) associated with general transcription factors of the promoter complex, specifically CDK7 and CDK9. We reported a Tat-associated T-cell-derived kinase, which contained CDK2. Here, we provide further evidence that CDK2 is involved in Tat-mediated CTD phosphorylation and in HIV-1 transcription in vitro. Tat-mediated CTD phosphorylation by CDK2 required cysteine 22 in the activation domain of Tat and amino acids 42-72 of Tat. CDK2 phosphorylated Tat itself, apparently by forming dynamic contacts with amino acids 15-24 and 36-49 of Tat. Also, amino acids 24-36 and 45-72 of Tat interacted with CTD. CDK2 associated with RNAPII and was found in elongation complexes assembled on HIV-1 long-terminal repeat template. Recombinant CDK2/cyclin E stimulated Tat-dependent HIV-1 transcription in reconstituted transcription assay. Immunodepletion of CDK2/cyclin E in HeLa nuclear extract blocked Tat-dependent transcription. We suggest that CDK2 is part of a transcription complex that is required for Tat-dependent transcription and that interaction of Tat with CTD and a dynamic association of Tat with CDK2/cyclin E stimulated CTD phosphorylation by CDK2.

  7. Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury

    PubMed Central

    Stevens, Patrick R.; Gawryluk, Jeremy W.; Hui, Liang; Chen, Xuesong; Geiger, Jonathan D.

    2015-01-01

    HIV-1 infected individuals are living longer but experiencing a prevalence rate of over 50% for HIV-1 associated neurocognitive disorders (HAND) for which no effective treatment is available. Viral and cellular factors secreted by HIV-1 infected cells leads to neuronal injury and HIV-1 Tat continues to be implicated in the pathogenesis of HAND. Here we tested the hypothesis that creatine protected against HIV-1 Tat-induced neuronal injury by preventing mitochondrial bioenergetic crisis and/or redox catastrophe. Creatine blocked HIV-1 Tat1-72-induced increases in neuron cell death and synaptic area loss. Creatine protected against HIV-1 Tat-induced decreases in ATP. Creatine and creatine plus HIV-1 Tat increased cellular levels of creatine, and creatine plus HIV-1 Tat further decreased ratios of phosphocreatine to creatine observed with creatine or HIV-1 Tat treatments alone. Additionally, creatine protected against HIV-1 Tat-induced mitochondrial hypopolarization and HIV-1 Tat-induced mitochondrial permeability transition pore opening. Thus, creatine may be a useful adjunctive therapy against HAND. PMID:25613139

  8. HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription.

    PubMed Central

    Nekhai, Sergei; Zhou, Meisheng; Fernandez, Anne; Lane, William S; Lamb, Ned J C; Brady, John; Kumar, Ajit

    2002-01-01

    HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation. PMID:12049628

  9. Cannabinoids Occlude the HIV-1 Tat-Induced Decrease in GABAergic Neurotransmission in Prefrontal Cortex Slices.

    PubMed

    Xu, Changqing; Hermes, Douglas J; Mackie, Ken; Lichtman, Aron H; Ignatowska-Jankowska, Bogna M; Fitting, Sylvia

    2016-06-01

    In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is now considered a chronic disease that specifically targets the brain and causes HIV-1-associated neurocognitive disorders (HAND). Endocannabinoids exhibit neuroprotective and anti-inflammatory properties in several central nervous system (CNS) disease models, but their effects in HAND are poorly understood. To address this issue, whole-cell recordings were performed on young (14-24 day old) C57BL/6J mice. We investigated the actions of the synthetic cannabinoid WIN55,212-2 (1 μM) and the endocannabinoid N-arachidonoyl ethanolamine (anandamide; AEA, 1 μM) in the presence of HIV-1 Tat on GABAergic neurotransmission in mouse prefrontal cortex (PFC) slices. We found a Tat concentration-dependent (5-50 nM) decrease in the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid 1 receptor (CB1R) antagonist rimonabant (1 μM) and zero extracellular calcium prevented the significant Tat-induced decrease in mIPSCs. Further, bath-applied WIN55,212-2 or AEA by itself, significantly decreased the frequency, but not amplitude of mIPSCs and/or spontaneous IPSCs (sIPSCs), and occluded a further downregulation of IPSCs by Tat. Pretreatment with rimonabant but not the CB2R antagonist AM630 (1 μM) prevented the WIN55,212-2- and AEA-induced decrease in IPSCs frequency without any further Tat effect. Results indicated a Tat-induced decrease in GABAergic neurotransmission, which was occluded by cannabinoids via a CB1R-related mechanism. Understanding the relationship between Tat toxicity and endocannabinoid signaling has the potential to identify novel therapeutic interventions to benefit individuals suffering from HAND and other cognitive impairments.

  10. Cannabinoids occlude the HIV-1 Tat-induced decrease in GABAergic neurotransmission in prefrontal cortex slices

    PubMed Central

    Xu, Changqing; Hermes, Douglas J; Mackie, Ken; Lichtman, Aron H; Ignatowska-Jankowska, Bogna M; Fitting, Sylvia

    2016-01-01

    In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is now considered a chronic disease that specifically targets the brain and causes HIV-1-associated neurocognitive disorders (HAND). Endocannabinoids exhibit neuroprotective and anti-inflammatory properties in several central nervous system (CNS) disease models, but their effects in HAND are poorly understood. To address this issue, whole-cell recordings were performed on young (14 – 21 day old) C57BL/6J mice. We investigated the actions of the synthetic cannabinoid WIN55,212-2 (1 μM) and the endocannabinoid N-arachidonoyl ethanolamine (anandamide; AEA, 1 μM) in the presence of HIV-1 Tat on GABAergic neurotransmission in mouse prefrontal cortex (PFC) slices. We found a Tat concentration dependent (5 – 50 nM) decrease in the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid 1 receptor (CB1R) antagonist rimonabant (1 μM) and zero extracellular calcium prevented the significant Tat-induced decrease in mIPSCs. Further, bath-applied WIN55,212-2 or AEA by itself, significantly decreased the frequency, but not amplitude of mIPSCs and/or spontaneous IPSCs (sIPSCs), and occluded a further down-regulation of IPSCs by Tat. Pretreatment with rimonabant but not the CB2R antagonist AM630 (1 μM) prevented the WIN55,212-2- and AEA-induced decrease in IPSCs frequency without any further Tat effect. Results indicated a Tat-induced decrease in GABAergic neurotransmission, which was occluded by cannabinoids via a CB1R-related mechanism. Understanding the relationship between Tat toxicity and endocannabinoid signaling has the potential to identify novel therapeutic interventions to benefit individuals suffering from HAND and other cognitive impairments. PMID:26993829

  11. HIV-1 Tat regulates the expression of the dcw operon and stimulates the proliferation of bacteria.

    PubMed

    Wei, Jinsong; Zhang, Yumin; Knapp, Pamela E; Zhao, Tianyong

    2016-01-01

    Infections of pathogenic bacteria are very common in acquired immunodeficiency syndrome (AIDS) patients. However, the biological effects of HIV-1 Tat on bacteria are incompletely understood. In this study, HIV-1 Tat was expressed in Escherichia coli and Pseudomonas aeruginosa (PA01) to investigate its biological effects on bacteria. Bacterial cells expressing either HIV-1 Tat1-86 (Tat1-86) or HIV-1 Tat1-72 (Tat1-72) grow significantly faster than those with either only an empty vector or an unrelated control (GFP or Rluc). Supplementation of purified HIV-1 Tat1-86 or Tat1-101 protein into bacterial culture medium stimulated the growth of both E. coli and PA01. The expression profile of certain cell division-associated genes, such as those in the division cell wall (dcw) operon (ftsA, ftsQ, ftsW and ftsZ), yafO and zipA, was altered in HIV-1 Tat1-86 expressing E. coli BL21(DE3). Furthermore, the expression of firefly luciferase (Fluc) reporter gene, when engineered for control by the dcw promoter and terminator, was enhanced by HIV-1 Tat in E. coli, confirming that HIV-1 Tat transcriptionally regulates the expression of the dcw operon. The finding that HIV-1 Tat stimulates bacterial growth whether it is produced intracellularly or applied extracellularly may have relevance for HIV patients who are highly susceptible to opportunistic bacterial infections. Contents category: Viruses -Retroviruses. The GenBank accession number for the sequence of HIV-1 Tat1-86 is AF324439.1.

  12. Activation of Egr-1 expression in astrocytes by HIV-1 Tat: new insights into astrocyte-mediated Tat neurotoxicity.

    PubMed

    Fan, Yan; Zou, Wei; Green, Linden A; Kim, Byung Oh; He, Johnny J

    2011-03-01

    Human immunodeficiency virus type 1 (HIV-1) Tat plays an important role in HIV-associated neuropathogenesis; the underlying mechanisms are still evolving. We have recently shown that HIV-1 Tat induces expression of glial fibrillary acidic protein (GFAP), a characteristic of HIV-1 infection of the central nervous system. We have also shown that the Tat-induced GFAP expression in astrocytes is regulated by p300 and that deletion of the early growth response 1 (Egr-1) cis-transacting element within the p300 promoter abolishes Tat-induced GFAP expression. In this study, we further examined the relationship between Tat and Egr-1 in astrocytes. We found increased Egr-1 protein expression in Tat-expressing human astrocytoma cells and mouse primary astrocytes. Using the Egr-1 promoter-driven firefly luciferase reporter gene assay and the site-directed mutagenesis, we demonstrated that Tat increased Egr-1 expression by transactivating the Egr-1 promoter and involving specific serum response elements within the promoter. Consistent with these data, we showed that Tat transactivation of the Egr-1 promoter was abrogated when astrocytes were cultured in serum-reduced media. Taken together, these results reveal that Tat directly transactivates Egr-1 expression and suggest that Tat interaction with Egr-1 is probably one of the very upstream molecular events that initiate Tat-induced astrocyte dysfunction and subsequent Tat neurotoxicity.

  13. The Presence of HIV-1 Tat Protein Second Exon Delays Fas Protein-mediated Apoptosis in CD4+ T Lymphocytes

    PubMed Central

    López-Huertas, María Rosa; Mateos, Elena; Sánchez del Cojo, María; Gómez-Esquer, Francisco; Díaz-Gil, Gema; Rodríguez-Mora, Sara; López, Juan Antonio; Calvo, Enrique; López-Campos, Guillermo; Alcamí, José; Coiras, Mayte

    2013-01-01

    HIV-1 replication is efficiently controlled by the regulator protein Tat (101 amino acids) and codified by two exons, although the first exon (1–72 amino acids) is sufficient for this process. Tat can be released to the extracellular medium, acting as a soluble pro-apoptotic factor in neighboring cells. However, HIV-1-infected CD4+ T lymphocytes show a higher resistance to apoptosis. We observed that the intracellular expression of Tat delayed FasL-mediated apoptosis in both peripheral blood lymphocytes and Jurkat cells, as it is an essential pathway to control T cell homeostasis during immune activation. Jurkat-Tat cells showed impairment in the activation of caspase-8, deficient release of mitochondrial cytochrome c, and delayed activation of both caspase-9 and -3. This protection was due to a profound deregulation of proteins that stabilized the mitochondrial membrane integrity, such as heat shock proteins, prohibitin, or nucleophosmin, as well as to the up-regulation of NF-κB-dependent anti-apoptotic proteins, such as BCL2, c-FLIPS, XIAP, and C-IAP2. These effects were observed in Jurkat expressing full-length Tat (Jurkat-Tat101) but not in Jurkat expressing the first exon of Tat (Jurkat-Tat72), proving that the second exon, and particularly the NF-κB-related motif ESKKKVE, was necessary for Tat-mediated protection against FasL apoptosis. Accordingly, the protection exerted by Tat was independent of its function as a regulator of both viral transcription and elongation. Moreover, these data proved that HIV-1 could have developed strategies to delay FasL-mediated apoptosis in infected CD4+ T lymphocytes through the expression of Tat, thus favoring the persistent replication of HIV-1 in infected T cells. PMID:23364796

  14. HIV-1 Tat Primes and Activates Microglial NLRP3 Inflammasome-Mediated Neuroinflammation.

    PubMed

    Chivero, Ernest T; Guo, Ming-Lei; Periyasamy, Palsamy; Liao, Ke; Callen, Shannon E; Buch, Shilpa

    2017-03-29

    Neuroinflammation associated with HIV-1 infection is a problem affecting ∼50% of HIV-infected individuals. NLR family pyrin domain containing 3 (NLRP3) inflammasome has been implicated in HIV-induced microglial activation, but the mechanism(s) remain unclear. Because HIV-1 Transactivator of Transcription (Tat) protein continues to be present despite antiretroviral therapy and activates NF-kB, we hypothesized that Tat could prime the NLRP3 inflammasome. We found a dose- and time-dependent induction of NLRP3 expression in microglia exposed to Tat compared with control. Tat exposure also time-dependently increased the mature caspase-1 and IL-1β levels and enhanced the IL-1β secretion. These in vitro findings were validated in archival brain tissues from Simian Immunodeficiency Virus (SIV)-infected and uninfected rhesus macaques. Further validation of NLRP3 priming in vivo involved administration of lipopolysaccharide (LPS) to HIV transgenic (Tg) rats followed by assessment of IL-1β mRNA expression and inflammasome activation (ASC oligomers and mature IL-1β). Intriguingly, LPS potentiated upregulation of IL-1β mRNA and inflammasome activation in HIV-Tg rats compared with the wild-type controls. Interestingly, we found an inverse relationship in the expression of NLRP3 and its negative regulator, miR-223, suggesting a miR-223-mediated mechanism for Tat-induced NLRP3 priming. Furthermore, blockade of NLRP3 resulted in decreased IL-1β secretion. Collectively, these findings suggest a novel role of Tat in priming and activating the NLRP3 inflammasome. Therefore, NLRP3 can be envisioned as a therapeutic target for ameliorating Tat-mediated neuroinflammation.SIGNIFICANCE STATEMENT Despite successful suppression of viremia with increased longevity in the era of combined antiretroviral therapy, chronic inflammation with underlying neurocognitive impairment continues to afflict almost 50% of infected individuals. Viral, bacterial, and cellular products have all been

  15. PKCθ and HIV-1 Transcriptional Regulator Tat Co-exist at the LTR Promoter in CD4+ T Cells

    PubMed Central

    López-Huertas, María Rosa; Li, Jasmine; Zafar, Anjum; Rodríguez-Mora, Sara; García-Domínguez, Carlota; Mateos, Elena; Alcamí, José; Rao, Sudha; Coiras, Mayte

    2016-01-01

    PKCθ is essential for the activation of CD4+ T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4+ T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T538 residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4+ T cells and could be used as a

  16. FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles.

    PubMed

    Pendergrast, P Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

    2002-03-01

    FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

  17. Inhibition of GABAergic Neurotransmission by HIV-1 Tat and Opioid Treatment in the Striatum Involves μ-Opioid Receptors

    PubMed Central

    Xu, Changqing; Fitting, Sylvia

    2016-01-01

    Due to combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease with high prevalence of mild forms of neurocognitive impairments, also referred to as HIV-associated neurocognitive disorders (HAND). Although opiate drug use can exacerbate HIV-1 Tat-induced neuronal damage, it remains unknown how and to what extent opioids interact with Tat on the GABAergic system. We conducted whole-cell recordings in mouse striatal slices and examined the effects of HIV-1 Tat in the presence and absence of morphine (1 μM) and damgo (1 μM) on GABAergic neurotransmission. Results indicated a decrease in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) by Tat (5–50 nM) in a concentration-dependent manner. The significant Tat-induced decrease in IPSCs was abolished when removing extracellular and/or intracellular calcium. Treatment with morphine or damgo alone significantly decreased the frequency, but not amplitude of IPSCs. Interestingly, morphine but not damgo indicated an additional downregulation of the mean frequency of mIPSCs in combination with Tat. Pretreatment with naloxone (1 μM) and CTAP (1 μM) prevented the Tat-induced decrease in sIPSCs frequency but only naloxone prevented the combined Tat and morphine effect on mIPSCs frequency. Results indicate a Tat- or opioid-induced decrease in GABAergic neurotransmission via μ-opioid receptors with combined Tat and morphine effects involving additional opioid receptor-related mechanisms. Exploring the interactions between Tat and opioids on the GABAergic system may help to guide future research on HAND in the context of opiate drug use. PMID:27877102

  18. The MHC-II transactivator CIITA inhibits Tat function and HIV-1 replication in human myeloid cells.

    PubMed

    Forlani, Greta; Turrini, Filippo; Ghezzi, Silvia; Tedeschi, Alessandra; Poli, Guido; Accolla, Roberto S; Tosi, Giovanna

    2016-04-18

    We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells. U937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants. CIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells. U937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1

  19. Didehydro-Cortistatin A inhibits HIV-1 Tat mediated neuroinflammation and prevents potentiation of cocaine reward in Tat transgenic mice

    PubMed Central

    Mediouni, Sonia; Jablonski, Joseph; Paris, Jason J.; Clementz, Mark A.; Thenin-Houssier, Suzie; McLaughlin, Jay P.; Valente, Susana T.

    2015-01-01

    HIV-1 Tat protein has been shown to have a crucial role in HIV-1-associated neurocognitive disorders (HAND), which includes a group of syndromes ranging from undetectable neurocognitive impairment to dementia. The abuse of psychostimulants, such as cocaine, by HIV infected individuals, may accelerate and intensify neurological damage. On the other hand, exposure to Tat potentiates cocaine-mediated reward mechanisms, which further promotes HAND. Here, we show that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid, crosses the blood-brain barrier, cross-neutralizes Tat activity from several HIV-1 clades and decreases Tat uptake by glial cell lines. In addition, dCA potently inhibits Tat mediated dysregulation of IL-1β, TNF-α and MCP-1, key neuroinflammatory signaling proteins. Importantly, using a mouse model where doxycycline induces Tat expression, we demonstrate that dCA reverses the potentiation of cocaine-mediated reward. Our results suggest that adding a Tat inhibitor, such as dCA, to current antiretroviral therapy may reduce HIV-1-related neuropathogenesis. PMID:25613133

  20. A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

    PubMed Central

    Chen, Yupeng; Zhang, Lirong; Estarás, Conchi; Choi, Seung H.; Moreno, Luis; Karn, Jonathan; Moresco, James J.; Yates, John R.

    2014-01-01

    HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P–RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P–S2P transition at the integrated HIV-1 promoter. PMID:25319827

  1. Detection of human immunodeficiency virus type 1 (HIV-1) Tat protein by aptamer-based biosensors

    NASA Astrophysics Data System (ADS)

    Hashim, Uda; Fatin, M. F.; Ruslinda, A. R.; Gopinath, Subash C. B.; Uda, M. N. A.

    2017-03-01

    A study was conducted to detect the human immunodeficiency virus (HIV-1) Tat protein using interdigitated electrodes. The measurements and images of the IDEs' finger gaps and the images of chitosan-carbon nanotubes deposited on top of the interdigitated electrodes were taken using the Scanning Electron Microscope. The detection of HIV-1 Tat protein was done using split aptamers and aptamer tail. Biosensors were chosen as diagnostic equipment due to their rapid diagnostic capabilities.

  2. MSM ameliorates HIV-1 Tat induced neuronal oxidative stress via rebalance of the glutathione cycle

    PubMed Central

    Kim, Seol-hee; Smith, Adam J; Tan, Jun; Shytle, R Douglas; Giunta, Brian

    2015-01-01

    HIV-1 Tat protein is a key neuropathological element in HIV associated neurogcognitive disorders (HAND); a type of cognitive syndrome thought to be at least partially mediated by increased levels of brain reactive oxygen species (ROS) and nitric oxide (NO). Methylsulfonylmethane (MSM) is a sulfur-containing compound known to reduce oxidative stress. This study was conducted to determine whether administration of MSM attenuates HIV-1 Tat induced oxidative stress in mouse neuronal cells. MSM treatment significantly decreased neuronal cell NO and ROS secretion. Further, MSM significantly reversed HIV-1 Tat mediated reductions in reduced glutathione (GSH) as well as HIV-1 Tat mediated increases in oxidized glutathione (GSSG). In addition, Tat reduced nuclear translocation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), a key nuclear promoter of antioxidant activity, while MSM increased its translocation to the nucleus in the presence of Tat. These results suggest that HIV-1 Tat reduces the resiliency of neuron cells to oxidative stress which can be reversed by MSM. Given the clinical safety of MSM, future preclinical in vivo studies will be required to further confirm these results in effort to validate MSM as a neuroprotectant in patients at risk of, or who are already diagnosed with, HAND. PMID:25893035

  3. Molecular Mechanism of HIV-1 Tat Interacting with Human Dopamine Transporter

    PubMed Central

    Midde, Narasimha M.; Quizon, Pamela M.; Sun, Wei-Lun; Zhu, Jun; Zhan, Chang-Guo

    2015-01-01

    Nearly 70% of HIV-infected individuals suffer from HIV-associated neurocognitive disorders (HAND). HIV-1 transactivator of transcription (Tat) protein is known to synergize with abused drugs and exacerbate the progression of central nervous system (CNS) pathology. Cumulative evidences suggest that the HIV-1 Tat protein exerts the neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. Through computational modeling and molecular dynamics (MD) simulations, we develop a three-dimensional (3D) structural model for HIV-1 Tat binding with hDAT. The model provides novel mechanistic insights concerning how HIV-1 Tat interacts with hDAT and inhibits dopamine uptake in hDAT. In particular, according to the computational modeling, Tat binds most favorably with the outward-open state of hDAT. Residues Y88, K92, and Y470 of hDAT are predicted to be key residues involved in the interaction between hDAT and Tat. The roles of these hDAT residues in the interaction with Tat are validated by experimental tests through site-directed mutagensis and DA uptake assays. The agreement between the computational and experimental data suggests that the computationally predicted hDAT-Tat binding mode and mechanistic insights are reasonable and provide a new starting point to design further pharmacological studies on the molecular mechanism of HIV-associated neurocognitive disorders. PMID:25695767

  4. HIV-1 Tat protein binds to TLR4-MD2 and signals to induce TNF-α and IL-10.

    PubMed

    Ben Haij, Nawal; Leghmari, Kaoutar; Planès, Rémi; Thieblemont, Nathalie; Bahraoui, Elmostafa

    2013-10-28

    HIV-1 infection results in hyper-immune activation and immunological disorders as early as the asymptomatic stage. Here, we hypothesized that during early HIV-1 infection, HIV-1 Tat protein acts on monocytes/macrophages to induce anti-inflammatory and proinflammatory cytokines and participates in immune dysregulation. In this work we showed that Tat protein: i) by its N-terminal domain induces production of both IL-10 and TNF-α in a TLR4-MD2 dependent manner, ii) interacts specifically with TLR4-MD2 and MD2 with high affinity but not with CD14, iii) induces in vivo TNF-α and IL-10 in a TLR4 dependent manner. Collectively, our data showed for the first time that, HIV-1 Tat interacts physically with high affinity with TLR4-MD2 to promote proinflammatory cytokines (TNF-α) and the immunosuppressive cytokine IL-10 both involved in immune dysregulation during early HIV-1 infection and AIDS progression.

  5. HIV-1 Tat and Viral Latency: What We Can Learn from Naturally Occurring Sequence Variations.

    PubMed

    Kamori, Doreen; Ueno, Takamasa

    2017-01-01

    Despite the effective use of antiretroviral therapy, the remainder of a latently HIV-1-infected reservoir mainly in the resting memory CD4(+) T lymphocyte subset has provided a great setback toward viral eradication. While host transcriptional silencing machinery is thought to play a dominant role in HIV-1 latency, HIV-1 protein such as Tat, may affect both the establishment and the reversal of latency. Indeed, mutational studies have demonstrated that insufficient Tat transactivation activity can result in impaired transcription of viral genes and the establishment of latency in cell culture experiments. Because Tat protein is one of highly variable proteins within HIV-1 proteome, it is conceivable that naturally occurring Tat mutations may differentially modulate Tat functions, thereby influencing the establishment and/or the reversal of viral latency in vivo. In this mini review, we summarize the recent findings of Tat naturally occurring polymorphisms associating with host immune responses and we highlight the implication of Tat sequence variations in relation to HIV latency.

  6. HIV-1 Tat and Viral Latency: What We Can Learn from Naturally Occurring Sequence Variations

    PubMed Central

    Kamori, Doreen; Ueno, Takamasa

    2017-01-01

    Despite the effective use of antiretroviral therapy, the remainder of a latently HIV-1-infected reservoir mainly in the resting memory CD4+ T lymphocyte subset has provided a great setback toward viral eradication. While host transcriptional silencing machinery is thought to play a dominant role in HIV-1 latency, HIV-1 protein such as Tat, may affect both the establishment and the reversal of latency. Indeed, mutational studies have demonstrated that insufficient Tat transactivation activity can result in impaired transcription of viral genes and the establishment of latency in cell culture experiments. Because Tat protein is one of highly variable proteins within HIV-1 proteome, it is conceivable that naturally occurring Tat mutations may differentially modulate Tat functions, thereby influencing the establishment and/or the reversal of viral latency in vivo. In this mini review, we summarize the recent findings of Tat naturally occurring polymorphisms associating with host immune responses and we highlight the implication of Tat sequence variations in relation to HIV latency. PMID:28194140

  7. Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4.

    PubMed

    Gu, Jianyou; Babayeva, Nigar D; Suwa, Yoshiaki; Baranovskiy, Andrey G; Price, David H; Tahirov, Tahir H

    2014-01-01

    Developing anti-viral therapies targeting HIV-1 transcription has been hampered by the limited structural knowledge of the proteins involved. HIV-1 hijacks the cellular machinery that controls RNA polymerase II elongation through an interaction of HIV-1 Tat with the positive transcription elongation factor P-TEFb, which interacts with an AF4 family member (AFF1/2/3/4) in the super elongation complex (SEC). Because inclusion of Tat•P-TEFb into the SEC is critical for HIV transcription, we have determined the crystal structure of the Tat•AFF4•P-TEFb complex containing HIV-1 Tat (residues 1-48), human Cyclin T1 (1-266), human Cdk9 (7-332), and human AFF4 (27-69). Tat binding to AFF4•P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5' of Cyclin T1 and the α-3 10 helix of AFF4. The interaction between Tat and AFF4 provides structural constraints that explain tolerated Tat mutations. Analysis of the Tat-binding surface of AFF4 coupled with modeling of all other AF4 family members suggests that AFF1 and AFF4 would be preferred over AFF2 or AFF3 for interaction with Tat•P-TEFb. The structure establishes that the Tat-TAR recognition motif (TRM) in Cyclin T1 interacts with both Tat and AFF4, leading to the exposure of arginine side chains for binding to TAR RNA. Furthermore, modeling of Tat Lys28 acetylation suggests that the acetyl group would be in a favorable position for H-bond formation with Asn257 of TRM, thereby stabilizing the TRM in Cyclin T1, and provides a structural basis for the modulation of TAR RNA binding by acetylation of Tat Lys28.

  8. Interaction between Tat and Drugs of Abuse during HIV-1 Infection and Central Nervous System Disease

    PubMed Central

    Maubert, Monique E.; Pirrone, Vanessa; Rivera, Nina T.; Wigdahl, Brian; Nonnemacher, Michael R.

    2016-01-01

    In many individuals, drug abuse is intimately linked with HIV-1 infection. In addition to being associated with one-third of all HIV-1 infections in the United States, drug abuse also plays a role in disease progression and severity in HIV-1-infected patients, including adverse effects on the central nervous system (CNS). Specific systems within the brain are known to be damaged in HIV-1-infected individuals and this damage is similar to that observed in drug abuse. Even in the era of anti-retroviral therapy (ART), CNS pathogenesis occurs with HIV-1 infection, with a broad range of cognitive impairment observed, collectively referred to as HIV-1-associated neurocognitive disorders (HAND). A number of HIV-1 proteins (Tat, gp120, Nef, Vpr) have been implicated in the etiology of pathogenesis and disease as a result of the biologic activity of the extracellular form of each of the proteins in a number of tissues, including the CNS, even in ART-suppressed patients. In this review, we have made Tat the center of attention for a number of reasons. First, it has been shown to be synthesized and secreted by HIV-1-infected cells in the CNS, despite the most effective suppression therapies available to date. Second, Tat has been shown to alter the functions of several host factors, disrupting the molecular and biochemical balance of numerous pathways contributing to cellular toxicity, dysfunction, and death. In addition, the advantages and disadvantages of ART suppression with regard to controlling the genesis and progression of neurocognitive impairment are currently under debate in the field and are yet to be fully determined. In this review, we discuss the individual and concerted contributions of HIV-1 Tat, drug abuse, and ART with respect to damage in the CNS, and how these factors contribute to the development of HAND in HIV-1-infected patients. PMID:26793168

  9. Homonuclear 1H NMR and circular dichroism study of the HIV-1 Tat Eli variant

    PubMed Central

    Watkins, Jennifer D; Campbell, Grant R; Halimi, Hubert; Loret, Erwann P

    2008-01-01

    Background The HIV-1 Tat protein is a promising target to develop AIDS therapies, particularly vaccines, due to its extracellular role that protects HIV-1-infected cells from the immune system. Tat exists in two different lengths, 86 or 87 residues and 99 or 101 residues, with the long form being predominant in clinical isolates. We report here a structural study of the 99 residue Tat Eli variant using 2D liquid-state NMR, molecular modeling and circular dichroism. Results Tat Eli was obtained from solid-phase peptide synthesis and the purified protein was proven biologically active in a trans-activation assay. Circular dichroism spectra at different temperatures up to 70°C showed that Tat Eli is not a random coil at 20°C. Homonuclear 1H NMR spectra allowed us to identify 1639 NMR distance constraints out of which 264 were interresidual. Molecular modeling satisfying at least 1474 NMR constraints revealed the same folding for different model structures. The Tat Eli model has a core region composed of a part of the N-terminus including the highly conserved Trp 11. The extra residues in the Tat Eli C-terminus protrude from a groove between the basic region and the cysteine-rich region and are well exposed to the solvent. Conclusion We show that active Tat variants share a similar folding pattern whatever their size, but mutations induce local structural changes. PMID:18808674

  10. A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency.

    PubMed Central

    Emiliani, S; Van Lint, C; Fischle, W; Paras, P; Ott, M; Brady, J; Verdin, E

    1996-01-01

    Study of the mechanism of HIV-1 postintegration latency in the ACH2 cell line demonstrates that these cells failed to increase HIV-1 production following treatment with exogenous Tat. Reasoning that the defect in ACH2 cells involves the Tat response, we analyzed the sequence of tat cDNA and Tat responsive element (TAR) from the virus integrated in ACH2. Tat cDNA sequence is closely related to that of HIV LAI, and the encoded protein is fully functional in terms of long terminal repeat (LTR) transactivation. Cloning of a region corresponding to the 5'-LTR from ACH2, however, identified a point mutation (C37 -> T) in TAR. This mutation impaired Tat responsiveness of the LTR in transient transfection assays, and the measured defect was complemented in cells that had been treated with tetradecanoyl phorbol acetate or tumor necrosis factor type alpha (TNF-alpha). A compensatory mutation in TAR (G28 -> A), designed to reestablish base pairing in the TAR hairpin, restored wild-type Tat responsiveness. When the (C37 -> T) mutation was introduced in an infectious clone of HIV-1, no viral production was measured in the absence of TNF-alpha, whereas full complementation was observed when the infection was conducted in the presence of TNF-alpha or when a compensatory mutation (G28 -> A) was introduced into TAR. These experiments identify a novel mutation associated with HIV-1 latency and suggest that alterations in the Tat-TAR axis can be a crucial determinant of the latent phenotype in infected individuals. Images Fig. 3 Fig. 5 PMID:8692823

  11. A mutant Tat protein inhibits infection of human cells by strains from diverse HIV-1 subtypes.

    PubMed

    Rustanti, Lina; Jin, Hongping; Lor, Mary; Lin, Min Hsuan; Rawle, Daniel J; Harrich, David

    2017-03-14

    Nullbasic is a mutant HIV-1 Tat protein that inhibits HIV-1 replication via three independent mechanisms that disrupts 1) reverse transcription of the viral RNA genome into a DNA copy, 2) HIV-1 Rev protein function required for viral mRNA transport from the nucleus to the cytoplasm and 3) HIV-1 mRNA transcription by RNA Polymerase II. The Nullbasic protein is derived from the subtype B strain HIV-1BH10 and has only been tested against other HIV-1 subtype B strains. However, subtype B strains only account for ~10% of HIV-1 infections globally and HIV-1 Tat sequences vary between subtypes especially for subtype C, which is responsible for ~50% HIV-1 infection worldwide. These differences could influence the ability of Tat to interact with RNA and cellular proteins and thus could affect the antiviral activity of Nullbasic. Therefore, Nullbasic was tested against representative HIV-1 strains from subtypes C, D and A/D recombinant to determine if it can inhibit their replication. Nullbasic was delivered to human cells using a self-inactivating (SIN) γ-retroviral system. We evaluated Nullbasic-mCherry (NB-mCh) fusion protein activity against the HIV-1 strains in TZM-bl cell lines for inhibition of transactivation and virus replication. We also examined antiviral activity of Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein against the same strains in primary CD4(+) T cells. The Nullbasic expression was monitored by western blot and flow cytometry. The effects of Nullbasic on primary CD4(+) T cells cytotoxicity, proliferation and apoptosis were also examined. The results show that Nullbasic inhibits Tat-mediated transactivation and virus replication of all the HIV-1 strains tested in TZM-bl cells. Importantly, Nullbasic inhibits replication of the HIV-1 strains in primary CD4(+) T cells without affecting cell proliferation, cytotoxicity or level of apoptotic cells. A SIN-based γ-retroviral vector used to express Nullbasic fusion proteins improved protein expression

  12. A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription.

    PubMed Central

    Bohjanen, P R; Colvin, R A; Puttaraju, M; Been, M D; Garcia-Blanco, M A

    1996-01-01

    Linear TAR RNA has previously been used as a decoy to inhibit HIV-1 transcription in vitro and HIV-1 replication in vivo. A 48 nucleotide circular RNA containing the stem, bulge and loop of the HIV-1 TAR element was synthesized using the self-splicing activity of a group I permuted intron-exon and was tested for its ability to function as a TAR decoy in vitro. This small circular TAR molecule was exceptionally stable in HeLa nuclear extracts, whereas a similar linear TAR molecule was rapidly degraded. The TAR circle bound specifically to Tfr38, a peptide containing the TAR-binding region of Tat. The ability of Tat to trans-activate transcription from the HIV-1 promoter in vitro was efficiently inhibited by circular TAR RNA but not by TAR circles that contained either bulge or loop mutations. TAR circles did not inhibit transactivation exclusively by binding to Tat since this inhibition was not reversed by adding excess Tat to the transcription reaction. Together, these data suggest that TAR circles act as decoys that inhibit transactivation by binding to Tat and at least one cellular factor. These data also demonstrate the utility of small circular RNA molecules as tools for biochemical studies. PMID:8871552

  13. HIV-1 Tat Promotes Lysosomal Exocytosis in Astrocytes and Contributes to Astrocyte-mediated Tat Neurotoxicity.

    PubMed

    Fan, Yan; He, Johnny J

    2016-10-21

    Tat interaction with astrocytes has been shown to be important for Tat neurotoxicity and HIV/neuroAIDS. We have recently shown that Tat expression leads to increased glial fibrillary acidic protein (GFAP) expression and aggregation and activation of unfolded protein response/endoplasmic reticulum (ER) stress in astrocytes and causes neurotoxicity. However, the exact molecular mechanism of astrocyte-mediated Tat neurotoxicity is not defined. In this study, we showed that neurotoxic factors other than Tat protein itself were present in the supernatant of Tat-expressing astrocytes. Two-dimensional gel electrophoresis and mass spectrometry revealed significantly elevated lysosomal hydrolytic enzymes and plasma membrane-associated proteins in the supernatant of Tat-expressing astrocytes. We confirmed that Tat expression and infection of pseudotyped HIV.GFP led to increased lysosomal exocytosis from mouse astrocytes and human astrocytes. We found that Tat-induced lysosomal exocytosis was tightly coupled to astrocyte-mediated Tat neurotoxicity. In addition, we demonstrated that Tat-induced lysosomal exocytosis was astrocyte-specific and required GFAP expression and was mediated by ER stress. Taken together, these results show for the first time that Tat promotes lysosomal exocytosis in astrocytes and causes neurotoxicity through GFAP activation and ER stress induction in astrocytes and suggest a common cascade through which aberrant astrocytosis/GFAP up-regulation potentiates neurotoxicity and contributes to neurodegenerative diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. HIV-1 Tat Inhibits Autotaxin Lysophospholipase D Activity and Modulates Oligodendrocyte Differentiation

    PubMed Central

    Wheeler, Natalie A.; Fuss, Babette; Knapp, Pamela E.

    2016-01-01

    White matter injury has been frequently reported in HIV+ patients. Previous studies showed that HIV-1 Tat (transactivator of transcription), a viral protein that is produced and secreted by HIV-infected cells, is toxic to young, immature oligodendrocytes (OLGs). Adding Tat to the culture medium reduced the viability of immature OLGs, and the surviving OLGs exhibited reduced process networks. OLGs produce and secrete autotaxin (ATX), an ecto-enzyme containing a lysophospholipase D (lysoPLD) activity that converts lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a lipid signaling molecule that stimulates OLG differentiation. We hypothesized that Tat affects OLG development by interfering with the ATX-LPA signaling pathway. Our data show that Tat treatment leads to changes in the expression of OLG differentiation genes and the area of OLG process networks, both of which can be rescued by LPA. Tat-treated OLGs showed no change in LPA receptor expression but significantly decreased extracellular ATX levels and lysoPLD activity. In Tat transgenic mice, expression of Tat in vivo leads to decreased OLG ATX secretion. Furthermore, co-immunoprecipitation experiments revealed a potential physical interaction between Tat and ATX. Together, these data strongly suggest two functional implications of Tat blocking ATX’s lysoPLD activity. On one hand, it attenuates OLG differentiation, and on the other hand it interferes with the protective effects of LPA on OLG process morphology. PMID:27659560

  15. PJA2 ubiquitinates the HIV-1 Tat protein with atypical chain linkages to activate viral transcription

    PubMed Central

    Faust, Tyler B.; Li, Yang; Jang, Gwendolyn M.; Johnson, Jeffrey R.; Yang, Shumin; Weiss, Amit; Krogan, Nevan J.; Frankel, Alan D.

    2017-01-01

    Transcription complexes that assemble at the HIV-1 promoter efficiently initiate transcription but generate paused RNA polymerase II downstream from the start site. The virally encoded Tat protein hijacks positive transcription elongation factor b (P-TEFb) to phosphorylate and activate this paused polymerase. In addition, Tat undergoes a series of reversible post-translational modifications that regulate distinct steps of the transcription cycle. To identify additional functionally important Tat cofactors, we performed RNAi knockdowns of sixteen previously identified Tat interactors and found that a novel E3 ligase, PJA2, ubiquitinates Tat in a non-degradative manner and specifically regulates the step of HIV transcription elongation. Interestingly, several different lysine residues in Tat can function as ubiquitin acceptor sites, and variable combinations of these lysines support both full transcriptional activity and viral replication. Further, the polyubiquitin chain conjugated to Tat by PJA2 can itself be assembled through variable ubiquitin lysine linkages. Importantly, proper ubiquitin chain assembly by PJA2 requires that Tat first binds its P-TEFb cofactor. These results highlight that both the Tat substrate and ubiquitin modification have plastic site usage, and this plasticity is likely another way in which the virus exploits the host molecular machinery to expand its limited genetic repertoire. PMID:28345603

  16. HIV-1 Tat modulates T-bet expression and induces Th1 type of immune response

    SciTech Connect

    Kulkarni, Asavari; Ravi, Dyavar S.; Singh, Kamini; Rampalli, Shravanti; Parekh, Vrajesh; Mitra, Debashis; Chattopadhyay, Samit . E-mail: samit@nccs.res.in

    2005-04-08

    The HIV-1 transactivator Tat performs various viral and cellular functions. Primarily, it induces processive transcription from the HIV-1 LTR promoter. However, Tat secreted from infected cells is known to activate uninfected lymphocytes through receptors. To further delineate the specific target genes, extracellular Tat was expressed on the cell membrane of stimulator cells and co-cultured with human PBMCs. Along with induced CD4{sup +} T cell proliferation and IFN-{gamma} secretion, there was strong upregulation of T-bet expression which is majorly implicated in generating T{sub H}1 type of immune response. To further delineate the effect of extracellular Tat on HIV replication, both p24 analysis and in vivo GFP expression were performed. There was a significant inhibition of HIV-1 replication in human CEM-GFP cell line and hPBMCs. Thus, for the first time we report that apart from its transactivation activity, extracellular Tat acts as a costimulatory molecule that affects viral replication by modulating host immune response through induction of T-bet expression and IFN-{gamma} secretion.

  17. Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice

    PubMed Central

    Fiume, Giuseppe; Scialdone, Annarita; Albano, Francesco; Rossi, Annalisa; Maria Tuccillo, Franca; Rea, Domenica; Palmieri, Camillo; Caiazzo, Elisabetta; Cicala, Carla; Bellevicine, Claudio; Falcone, Cristina; Vecchio, Eleonora; Pisano, Antonio; Ceglia, Simona; Mimmi, Selena; Iaccino, Enrico; Laurentiis, Annamaria de; Pontoriero, Marilena; Agosti, Valter; Troncone, Giancarlo; Mignogna, Chiara; Palma, Giuseppe; Arra, Claudio; Mallardo, Massimo; Maria Buonaguro, Franco; Scala, Giuseppe; Quinto, Ileana

    2015-01-01

    Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4+ and CD8+ T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation. PMID:26343909

  18. A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

    PubMed

    Lin, Min-Hsuan; Sivakumaran, Haran; Jones, Alun; Li, Dongsheng; Harper, Callista; Wei, Ting; Jin, Hongping; Rustanti, Lina; Meunier, Frederic A; Spann, Kirsten; Harrich, David

    2014-12-14

    Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased

  19. The cross-talk of HIV-1 Tat and methamphetamine in HIV-associated neurocognitive disorders

    PubMed Central

    Mediouni, Sonia; Garibaldi Marcondes, Maria Cecilia; Miller, Courtney; McLaughlin, Jay P.; Valente, Susana T.

    2015-01-01

    Antiretroviral therapy has dramatically improved the lives of human immunodeficiency virus 1 (HIV-1) infected individuals. Nonetheless, HIV-associated neurocognitive disorders (HAND), which range from undetectable neurocognitive impairments to severe dementia, still affect approximately 50% of the infected population, hampering their quality of life. The persistence of HAND is promoted by several factors, including longer life expectancies, the residual levels of virus in the central nervous system (CNS) and the continued presence of HIV-1 regulatory proteins such as the transactivator of transcription (Tat) in the brain. Tat is a secreted viral protein that crosses the blood–brain barrier into the CNS, where it has the ability to directly act on neurons and non-neuronal cells alike. These actions result in the release of soluble factors involved in inflammation, oxidative stress and excitotoxicity, ultimately resulting in neuronal damage. The percentage of methamphetamine (MA) abusers is high among the HIV-1-positive population compared to the general population. On the other hand, MA abuse is correlated with increased viral replication, enhanced Tat-mediated neurotoxicity and neurocognitive impairments. Although several strategies have been investigated to reduce HAND and MA use, no clinically approved treatment is currently available. Here, we review the latest findings of the effects of Tat and MA in HAND and discuss a few promising potential therapeutic developments. PMID:26557111

  20. Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

    PubMed

    Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

    2010-10-01

    HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

  1. Crystal structure of HIV-1 Tat complexed with human P-TEFb

    SciTech Connect

    Tahirov, Tahir H.; Babayeva, Nigar D.; Varzavand, Katayoun; Cooper, Jeffrey J.; Sedore, Stanley C.; Price, David H.

    2010-08-23

    Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat-P-TEFb complex containing HIV-1 Tat, human Cdk9 (also known as CDK9), and human cyclin T1 (also known as CCNT1). Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat-P-TEFb complex and block HIV replication.

  2. Bcl-2 upregulation by HIV-1 Tat during infection of primary human macrophages in culture.

    PubMed

    Zhang, Mingjie; Li, Xingxiang; Pang, Xiaowu; Ding, Lina; Wood, Owen; Clouse, Kathleen A; Hewlett, Indira; Dayton, Andrew I

    2002-01-01

    The ability of cells of the human monocyte/macrophage lineage to host HIV-1 replication while resisting cell death is believed to significantly contribute to their ability to serve as a reservoir for viral replication in the host. Although macrophages are generally resistant to apoptosis, interruption of anti-apoptotic pathways can render them susceptible to apoptosis. Here we report that HIV-1(BAL )infection of primary human monocyte-derived macrophages (MDM) upregulates the mRNA and protein levels of the anti-apoptic gene, Bcl-2. Furthermore, this upregulation can be quantitatively mimicked by treating MDM with soluble HIV-1 Tat-86 protein. These results suggest that in infecting cells of the monocyte/macrophage lineage, HIV-1 may be benefiting from additional protection against apoptosis caused by specific upregulation of cellular anti-apoptotic genes.

  3. Involvement of p300 in constitutive and HIV-1 Tat-activated expression of glial fibrillary acidic protein in astrocytes

    PubMed Central

    Zou, Wei; Wang, Zhenyuan; Liu, Ying; Fan, Yan; Zhou, Betty Y.; Yang, X. Frank; He, Johnny J.

    2010-01-01

    HIV-1 Tat protein is an important pathogenic factor in HIV-1-associated neurological diseases. One hallmark of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by elevated GFAP expression in astrocytes. We have shown that Tat activates GFAP expression in astrocytes (Zhou, et al., Mol. Cell. Neurosci. 27:296, 2004) and that GFAP is an important regulator of Tat neurotoxicity (Zou, et. al., Am. J. Pathol. 171:1293, 2007). However, the underlying mechanisms for Tat-mediated GFAP up-regulation are not understood. In the current study, we reported concurrent up-regulation of adenovirus E1a-associated 300 kDa protein p300 and GFAP in Tat-expressing human astroytoma cells and primary astrocytes. We showed that p300 was indeed induced by Tat expression and HIV-1 infection and that the induction occurred at the transcriptional level through the cis-acting elements of early growth response 1 (Egr-1) within its promoter. Using siRNA, we further showed that p300 regulated both constitutive and Tat-mediated GFAP expression. Moreover, we showed that ectopic expression of p300 potentiated Tat transactivation activity and increased proliferation of HIV-1-infected astrocytes, but had little effect on HIV-1 replication in these cells. Taken together, these results demonstrate for the first time that Tat is a positive regulator of p300 expression, which in turn regulates GFAP expression, and suggest that the Tat-Egr-1-p300-GFAP axis likely contributes to Tat neurotoxicity and predisposes astrocytes to be an HIV-1 sanctuary in the CNS. PMID:20578042

  4. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    PubMed Central

    Jin, Hongping; Li, Dongsheng; Sivakumaran, Haran; Lor, Mary; Rustanti, Lina; Cloonan, Nicole; Wani, Shivangi

    2016-01-01

    ABSTRACT Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells. PMID:27381288

  5. Progesterone protects normative anxiety-like responding among ovariectomized female mice that conditionally express the HIV-1 regulatory protein, Tat, in the CNS

    PubMed Central

    Paris, Jason J.; Fenwick, Jason; McLaughlin, Jay P.

    2014-01-01

    Increased anxiety is co-morbid with human immunodeficiency virus (HIV) infection. Actions of the neurotoxic HIV-1 regulatory protein, Tat, may contribute to affective dysfunction. We hypothesized that Tat expression would increase anxiety-like behavior of female GT-tg bigenic mice that express HIV-1 Tat protein in the brain in a doxycycline-dependent manner. Furthermore, given reports that HIV-induced anxiety may occur at lower rates among women, and that the neurotoxic effects of Tat are ameliorated by sex steroids in vitro, we hypothesized that 17β-estradiol and/or progesterone would ameliorate Tat-induced anxiety-like effects. Among naturally-cycling proestrous and diestrous mice, Tat-induction via 7 days of doxycycline treatment significantly increased anxiety-like responding in an open field, elevated plus maze and a marble-burying task, compared to treatment with saline. Proestrous mice demonstrated less anxiety-like behavior than diestrous mice in the open field and elevated plus maze, but these effects did not significantly interact with Tat-induction. Among ovariectomized mice, doxycycline-induced Tat protein significantly increased anxiety-like behavior in an elevated plus maze and a marble burying task compared to saline-treated mice, but not an open field (where anxiety-like responding was already maximal). Co-administration of progesterone (4 mg/kg), but not 17β-estradiol (0.09 mg/kg), with doxycycline significantly ameliorated anxiety-like responding in the elevated plus maze and marble burying tasks. When administered together, 17β-estradiol partially antagonized the protective effects of progesterone on Tat-induced anxiety-like behavior. These findings support evidence of steroid-protection over HIV-1 proteins, and extend them by demonstrating the protective capacity of progesterone on Tat-induced anxiety-like behavior of ovariectomized female mice. PMID:24726788

  6. Tight junction regulation by morphine and HIV-1 tat modulates blood-brain barrier permeability.

    PubMed

    Mahajan, Supriya D; Aalinkeel, Ravikumar; Sykes, Donald E; Reynolds, Jessica L; Bindukumar, B; Fernandez, Stanley F; Chawda, Ramnik; Shanahan, Thomas C; Schwartz, Stanley A

    2008-09-01

    Human immunodeficiency virus (HIV)-1 patients who abuse opiates are at a greater risk of developing neurological complications of AIDS. Alterations in blood-brain barrier (BBB) integrity are associated with cytoskeletal disorganization and disruption of tight junction (TJ) integrity. We hypothesize that opiates in combination with HIV-1 viral proteins can modulate TJ expression in primary brain microvascular endothelial cells (BMVEC), thereby compromising BBB integrity and exacerbating HIV-1 neuropathogenesis. We investigated the effect of morphine and/or tat on the expression of TJ proteins ZO-1, JAM-2, Occludin and P-glycoprotein and the functional effects of TJ modulation in BMVEC. Morphine and/or tat, via the activation of pro-inflammatory cytokines, intracellular Ca(2+) release, and activation of myosin light chain kinase, modulated TJ expression resulting in decreased transendothelial electric resistance and enhanced transendothelial migration across the BBB. These studies may lead to the development of novel anti-HIV-1 therapeutics that target specific TJ proteins, thus preventing TJ disruption in opiate using HIV-1 patients.

  7. HIV-1 Proteins, Tat and gp120, Target the Developing Dopamine System

    PubMed Central

    Fitting, Sylvia; Booze, Rosemarie M.; Mactutus, Charles F.

    2015-01-01

    In 2014, 3.2 million children (< 15 years of age) were estimated to be living with HIV and AIDS worldwide, with the 240,000 newly infected children in the past year, i.e., another child infected approximately every two minutes [1]. The primary mode of HIV infection is through mother-to-child transmission (MTCT), occurring either in utero, intrapartum, or during breastfeeding. The effects of HIV-1 on the central nervous system (CNS) are putatively accepted to be mediated, in part, via viral proteins, such as Tat and gp120. The current review focuses on the targets of HIV-1 proteins during the development of the dopamine (DA) system, which appears to be specifically susceptible in HIV-1-infected children. Collectively, the data suggest that the DA system is a clinically relevant target in chronic HIV-1 infection, is one of the major targets in pediatric HIV-1 CNS infection, and may be specifically susceptible during development. The present review discusses the development of the DA system, follows the possible targets of the HIV-1 proteins during the development of the DA system, and suggests potential therapeutic approaches. By coupling our growing understanding of the development of the CNS with the pronounced age-related differences in disease progression, new light may be shed on the neurological and neurocognitive deficits that follow HIV-1 infection. PMID:25613135

  8. HIV-1 Tat protein induces the production of IDO in human monocyte derived-dendritic cells through a direct mechanism: effect on T cells proliferation.

    PubMed

    Planès, Rémi; Bahraoui, Elmostafa

    2013-01-01

    During HIV-1 infection, an increase of indoleamine 2,3 dioxygenase (IDO) expression, and dendritic cells (DC) dysfunction were often associated with AIDS disease progression. In this work, we investigated the effect of HIV-1 Tat protein on the expression of IDO, in MoDCs. We show that Tat induces IDO protein expression and activity in a dose dependent manner by acting at the cell membrane. Using Tat-mutants, we show that the N-Terminal domain, Tat 1-45, but not the central region, Tat 30-72, is sufficient to induce the expression of active IDO. Tat protein is also able to induce several cytokines in MoDCs, including IFN-γ, a strong inducer of IDO. In order to understand whether IDO is induced directly by Tat protein or indirectly following IFN-γ production, complementary experiments were performed and showed that: i) at the kinetic level, Tat induced IDO expression before the production of IFN-γ ii) treatment of MoDCs with Tat-conditioned medium was unable to stimulate IDO expression, iii) coculture of MoDCs in a transwell cell system did not allow IDO expression in MoDCs not previously treated by Tat, iv) direct contact between Tat-treated and untreated MoDCs was not sufficient to induce IDO expression in a Tat-independent manner, and v) treatment of MoDCs in the presence of IFN-γ pathway inhibitors, Jak I and Ly294002, inhibited IFN-γ-induced IDO but had no effect on Tat-induced IDO. At the functional level, our data showed that treatment of MoDCs with Tat led to the inhibition of their capacity to stimulate T cell proliferation. This impairement was totally abolished when the stimulation was performed in the presence of 1MT, an inhibitor of IDO activity, arguing for the implication of the kynurenine pathway. By inducing IDO, Tat protein may be considered, as a viral pathogenic factor, in the dysregulation of the DC functions during HIV-1 infection.

  9. Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters.

    PubMed

    Ziegler, André; Seelig, Joachim

    2004-01-01

    The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal

  10. The Tat Inhibitor Didehydro-Cortistatin A Prevents HIV-1 Reactivation from Latency

    PubMed Central

    Mousseau, Guillaume; Kessing, Cari F.; Fromentin, Rémi; Trautmann, Lydie; Chomont, Nicolas

    2015-01-01

    ABSTRACT Antiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4+ T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4+ T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs. PMID:26152583

  11. The perlecan heparan sulfate proteoglycan mediates cellular uptake of HIV-1 Tat through a pathway responsible for biological activity

    SciTech Connect

    Argyris, Elias G.; Kulkosky, Joseph; Meyer, Marie E.; Xu Yan; Mukhtar, Muhammad; Pomerantz, Roger J. . E-mail: roger.j.pomerantz@jefferson.edu; Williams, Kevin Jon . E-mail: K_Williams@mail.jci.tju.edu

    2004-12-20

    Cell surface heparan sulfate proteoglycans (HSPGs) mediate internalization of HIV-1 Tat. Herein, we report that human WiDr cells, which express perlecan but no other HSPGs, can internalize {sup 125}I-labeled Tat with minimal lysosomal degradation. Pre-treatment of cells with heparitinase almost completely abolished {sup 125}I-Tat surface binding, while the use of an HIV-1 long terminal repeat (LTR) promoter-reporter construct demonstrated that transactivation was potently blocked by pretreatment of cells with heparitinase, indicating an essential role for perlecan in the biologic effects of Tat. We conclude that the perlecan mediates Tat uptake and is required for HIV-1 LTR-directed transactivation in this human cell type.

  12. Enduring cortical alterations after a single in-vivo treatment of HIV-1 Tat

    PubMed Central

    Wayman, Wesley N.; Dodiya, Hemraj B.; Persons, Amanda L.; Kashanchi, Fatah; Kordower, Jeffrey H.; Hu, Xiu-Ti; Napier, T. Celeste

    2013-01-01

    HIV-1 proteins, including the transactivator of transcription (Tat), are believed to be involved in HIV-associated neurocognitive disorders by disrupting Ca2+ homeostasis, which leads to progressive dysregulation, damage, or death of neurons in the brain. We have found previously that bath-applied Tat abnormally increased Ca2+ influx through overactivated, voltage-sensitive L-type Ca2+ channels in pyramidal neurons within the rat medial prefrontal cortex (mPFC). However, it is unknown whether the Tat-induced Ca2+ dysregulation was mediated by increased activity and/or the number of the L-channels. This study tested the hypothesis that transient/early exposure to Tat in vivo promoted enduring L-channel dysregulation in the mPFC without neuron loss. Accordingly, rats were administered a single intracerebroventricular injection of recombinant Tat (80 µg/20 µl; diluted by cerebrospinal fluids to pathophysiological concentrations) or vehicle. Rats were killed 14 days after injection for immunohistochemical assessments of the mPFC, motor cortex, caudate–putamen, and nucleus accumbens. Stereological estimates for positively stained cells indicated a significant increase in the number of cells expressing the pore-forming Cav1.2-α1c subunit of L-channels in the mPFC compared with other regions in Tat-treated or vehicle-treated rat brains. Optical density measurements showed a Tat-induced increase in glial fibrillary acidic protein expression, indicating astrogliosis in the cortical regions. There was no significant loss of neurons in any brain region investigated. These findings indicate that transient Tat exposure in vivo induced enduring L-channel dysregulation and astrogliosis in the mPFC without neuron loss. Such maladaptations may contribute toward dysregulated Ca2+ homeostasis and neuropathology in the PFC in the early stages of HIV infection. PMID:22828409

  13. Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup.

    PubMed

    Ruiz, Arthur P; Prasad, Vinayaka R

    2016-01-01

    HIV-1 Tat protein is secreted from infected cells and is endocytosed by uninfected bystander cells. Subsequently, Tat is translocated to the nucleus and binds to promoters of host cell genes, increasing the production of inflammatory host cytokines and chemokines. This inflammatory activation of uninfected cells by HIV-1 Tat protein contributes to the overall inflammatory burden in the central nervous system (CNS) that leads to the development of HIV-associated neurocognitive disorders (HAND). Here we describe methods to evaluate the uptake and transcriptional impact of HIV-1 Tat on uninfected cells by using a trans-cellular transactivation system. Cell lines transiently transfected with Tat expression constructs secrete Tat into the culture medium. Trans-cellular uptake and transactivation caused by secreted Tat can be measured by co-culturing LTR-responsive reporter cells with Tat-transfected cells. Such Tat-producer cells can also be co-cultured with immune cell lines, such as monocytic THP-1 cells or lymphocytic Jurkat T-cells, to evaluate transcriptional changes elicited by Tat taken up by the uninfected cells.

  14. Reservoir cells no longer detectable after a heterologous SHIV challenge with the synthetic HIV-1 Tat Oyi vaccine

    PubMed Central

    Watkins, Jennifer D; Lancelot, Sophie; Campbell, Grant R; Esquieu, Didier; Mareuil, Jean de; Opi, Sandrine; Annappa, Sylvie; Salles, Jean-Pierre; Loret, Erwann P

    2006-01-01

    Background Extra-cellular roles of Tat might be the main cause of maintenance of HIV-1 infected CD4 T cells or reservoir cells. We developed a synthetic vaccine based on a Tat variant of 101 residues called Tat Oyi, which was identified in HIV infected patients in Africa who did not progress to AIDS. We compared, using rabbits, different adjuvants authorized for human use to test on ELISA the recognition of Tat variants from the five main HIV-1 subtypes. A formulation was tested on macaques followed by a SHIV challenge with a European strain. Results Tat Oyi with Montanide or Calcium Phosphate gave rabbit sera able to recognize all Tat variants. Five on seven Tat Oyi vaccinated macaques showed a better control of viremia compared to control macaques and an increase of CD8 T cells was observed only on Tat Oyi vaccinated macaques. Reservoir cells were not detectable at 56 days post-challenge in all Tat Oyi vaccinated macaques but not in the controls. Conclusion The Tat Oyi vaccine should be efficient worldwide. No toxicity was observed on rabbits and macaques. We show in vivo that antibodies against Tat could restore the cellular immunity and make it possible the elimination of reservoir cells. PMID:16441880

  15. Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein

    PubMed Central

    Siddappa, Nagadenahalli Byrareddy; Venkatramanan, Mohanram; Venkatesh, Prasanna; Janki, Mohanbabu Vijayamma; Jayasuryan, Narayana; Desai, Anita; Ravi, Vasanthapuram; Ranga, Udaykumar

    2006-01-01

    Background Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat. Results Tat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian

  16. Exposure to HIV-1 Tat in brain impairs sensorimotor gating and activates microglia in limbic and extralimbic brain regions of male mice.

    PubMed

    Paris, Jason J; Singh, Harminder D; Carey, Amanda N; McLaughlin, Jay P

    2015-09-15

    Human immunodeficiency virus (HIV) infection is associated with mood disorders and behavioral disinhibition. Impairments in sensorimotor gating and associated neurocognitive disorders are reported, but the HIV-proteins and mechanisms involved are not known. The regulatory HIV-1 protein, Tat, is neurotoxic and its expression in animal models increases anxiety-like behavior concurrent with neuroinflammation and structural changes in limbic and extra-limbic brain regions. We hypothesized that conditional expression of HIV-1 Tat1-86 in the GT-tg bigenic mouse model would impair sensorimotor gating and increase microglial reactivity in limbic and extralimbic brain regions. Conditional Tat induction via doxycycline (Dox) treatment (0-125 mg/kg, i.p., for 1-14 days) significantly potentiated the acoustic startle reflex (ASR) of GT-tg mice and impaired prepulse inhibition (PPI) of this response in a dose-dependent manner when Dox (100mg/kg) was administered for brief (1 day) or prolonged (daily for 7 days) intervals. A greater proportion of active/reactive Iba1-labeled microglia was seen in the anterior cingulate cortex (ACC), dentate gyrus, and nucleus accumbens core when Tat protein was induced under either brief or prolonged expression conditions. Other subregions of the medial prefrontal cortex, amygdala, hippocampal formation, ventral tegmental area, and ventral pallidum also displayed Tat-induced microglial activation, but only the activation observed in the ACC recapitulated the pattern of ASR and PPI behaviors. Tat exposure also increased frontal cortex GFAP. Pretreatment with indomethacin attenuated the behavioral effects of brief (but not prolonged) Tat-exposure. Overall, exposure to HIV-1 Tat protein induced sensorimotor deficits associated with acute and persistent neuroinflammation in limbic/extralimbic brain regions. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Exposure to HIV-1 Tat in brain impairs sensorimotor gating and activates microglia in limbic and extralimbic brain regions of male mice

    PubMed Central

    Paris, Jason J.; Singh, Harminder D.; Carey, Amanda N.; McLaughlin, Jay P.

    2015-01-01

    Human immunodeficiency virus (HIV) infection is associated with mood disorders and behavioral disinhibition. Impairments in sensorimotor gating and associated neurocognitive disorders are reported, but the HIV-proteins and mechanisms involved are not known. The regulatory HIV-1 protein, Tat, is neurotoxic and its expression in animal models increases anxiety-like behavior concurrent with neuroinflammation and structural changes in limbic and extra-limbic brain regions. We hypothesized that conditional expression of HIV-1 Tat1–86 in the GT-tg bigenic mouse model would impair sensorimotor gating and increase microglial reactivity in limbic and extralimbic brain regions. Conditional Tat induction via doxycycline (Dox) treatment (0–125 mg/kg, i.p., for 1–14 days) significantly potentiated the acoustic startle reflex (ASR) of GT-tg mice and impaired prepulse inhibition (PPI) of this response in a dose-dependent manner when Dox (100 mg/kg) was administered for brief (1 day) or prolonged (daily for 7 days) intervals. A greater proportion of active/reactive Iba1-labeled microglia was seen in the anterior cingulate cortex (ACC), dentate gyrus, and nucleus accumbens core when Tat protein was induced under either brief or prolonged expression conditions. Other subregions of the medial prefrontal cortex, amygdala, hippocampal formation, ventral tegmental area, and ventral pallidum also displayed Tat-induced microglial activation, but only the activation observed in the ACC recapitulated the pattern of ASR and PPI behaviors. Tat exposure also increased frontal cortex GFAP. Pretreatment with indomethacin attenuated the behavioral effects of brief (but not prolonged) Tat-exposure. Overall, exposure to HIV-1 Tat protein induced sensorimotor deficits associated with acute and persistent neuroinflammation in limbic/extralimbic brain regions. PMID:26005128

  18. Design and SAR of new substituted purines bearing aryl groups at N9 position as HIV-1 Tat-TAR interaction inhibitors.

    PubMed

    Pang, Ruifang; Zhang, Chunlei; Yuan, Dekai; Yang, Ming

    2008-09-01

    Twenty-four purine derivatives bearing aryl groups at N9 position were designed and synthesized as HIV-1 Tat-TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Ten of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells at a concentration of 30 microM, indicating effective inhibitory activities of blocking the Tat-TAR interaction. The aryl groups at N9 position affected the binding affinities between compounds and TAR RNA, showing some specificities of aryl groups to TAR RNA.

  19. Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells.

    PubMed

    Green, Victoria A; Arbuthnot, Patrick; Weinberg, Marc S

    2012-11-15

    Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target. shRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels. This study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics

  20. HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes.

    PubMed

    Planès, Rémi; Ben Haij, Nawal; Leghmari, Kaoutar; Serrero, Manutea; BenMohamed, Lbachir; Bahraoui, Elmostafa

    2016-07-01

    In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) βII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10. In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement

  1. The 5′ UTR of HIV-1 full-length mRNA and the Tat viral protein modulate the programmed −1 ribosomal frameshift that generates HIV-1 enzymes

    PubMed Central

    Charbonneau, Johanie; Gendron, Karine; Ferbeyre, Gerardo; Brakier-Gingras, Léa

    2012-01-01

    Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed −1 ribosomal frameshift. Here, using dual-luciferase reporters, we investigated whether the highly structured 5′ untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency. We showed that, when the 5′ UTR of HIV-1 mRNA occupies the 5′ end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold in Jurkat T-cells, compared with a control dual-luciferase reporter with a short unstructured 5′ UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5′ end of the messenger. HIV-1 mRNA 5′ UTR also contains an internal ribosome entry site (IRES), but we showed that, when the cap-dependent initiation mode is available, the IRES is not used or is weakly used. However, when the ribosomes have to use the IRES to translate the dual-luciferase reporter, the frameshift efficiency is comparable to that of the control dual-luciferase reporter. The decrease in cap-dependent initiation and the accompanying increase in frameshift efficiency caused by the 5′ UTR of HIV-1 mRNA is antagonized, in a dose-dependent way, by the Tat viral protein. Tat also stimulates the IRES-dependent initiation and decreases the corresponding frameshift efficiency. A model is presented that accounts for the variations in frameshift efficiency depending on the 5′ UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication. PMID:22286970

  2. HIV-1 Tat exacerbates lipopolysaccharide-induced cytokine release via TLR4 signaling in the enteric nervous system

    PubMed Central

    Guedia, Joy; Brun, Paola; Bhave, Sukhada; Fitting, Sylvia; Kang, Minho; Dewey, William L.; Hauser, Kurt F.; Akbarali, Hamid I.

    2016-01-01

    The loss of gut epithelium integrity leads to translocation of microbes and microbial products resulting in immune activation and drives systemic inflammation in acquired immunodeficiency syndrome (AIDS) patients. Although viral loads in HIV patients are significantly reduced in the post-cART era, inflammation and immune activation persist and can lead to morbidity. Here, we determined the interactive effects of the viral protein HIV-1 Tat and lipopolysaccharide (LPS) on enteric neurons and glia. Bacterial translocation was significantly enhanced in Tat-expressing (Tat+) mice. Exposure to HIV-1 Tat in combination with LPS enhanced the expression and release of the pro-inflammatory cytokines IL-6, IL-1β and TNF-α in the ilea of Tat+ mice and by enteric glia. This coincided with enhanced NF-κB activation in enteric glia that was abrogated in glia from TLR4 knockout mice and by knockdown (siRNA) of MyD88 siRNA in wild type glia. The synergistic effects of Tat and LPS resulted in a reduced rate of colonic propulsion in Tat+ mice treated with LPS. These results show that HIV-1 Tat interacts with the TLR4 receptor to enhance the pro-inflammatory effects of LPS leading to gastrointestinal dysmotility and enhanced immune activation. PMID:27491828

  3. Application of novel solid lipid nanoparticle (SLN)-gene vector formulations based on a dimeric HIV-1 TAT-peptide in vitro and in vivo.

    PubMed

    Rudolph, Carsten; Schillinger, Ulrike; Ortiz, Aurora; Tabatt, Kerstin; Plank, Christian; Müller, Rainer H; Rosenecker, Joseph

    2004-09-01

    To optimize gene delivery of SLN-based gene vectors by incorporation of a dimeric HIV-1 TAT peptide (TAT2) into SLN gene vectors. Plasmid DNA was complexed with two SLN preparations either with or without pre-compaction of DNA by TAT2, poly-L-arginine, or the mutant TAT2-M1. DNA complexed with polyethylenimine (PEI) served as a standard. Gene expression was analyzed upon transfection of bronchial epithelial cells in vitro and after intratracheal instillation or aerosol application to the lungs of mice in vivo. Stability of DNA was analyzed by agarose gel electrophoresis. Incorporation of TAT2 into SLN gene vectors induced an up to 100-fold sequence-dependent increase of gene expression as compared with the mutant TAT2-M1 and was 4- to 8-times higher as compared with PEI in vitro. In vivo application of TAT2-SLN gene vectors via jet nebulization increased SLN-based gene expression but was accompanied with DNA degradation. DNA degradation was not observed when an innovative device operating on the principle of a perforated vibrating membrane was used. Incorporation of TAT2 into SLN gene vectors is suitable to optimize gene transfer in vitro. The use of a mild nebulization technology avoids DNA degradation and offers the opportunity for further studies in large animal models.

  4. Extracts from Acacia catechu suppress HIV-1 replication by inhibiting the activities of the viral protease and Tat

    PubMed Central

    2013-01-01

    Background Acacia catechu (Mimosa family) stem bark extracts have been used traditionally as a dietary supplement as well as a folk medicine given its reported anti-inflammatory, immunomodulatory, hepatoprotective, antioxidant, anti-microbial and anti-tumor activities. The present study was undertaken to evaluate the anti-HIV-1 activity of the extracts from stem bark of A. catechu. Methods The aqueous and 50% ethanolic extracts of A. catechu stem bark were prepared and 50% ethanolic extract was further fractioned by successively partitioning with petroleum ether, chloroform and n-butanol. All the extracts and fractions were evaluated for cytotoxicity and anti-HIV-1 activity using different in vitro assays. The active n-butanol fraction was evaluated for its inhibition against HIV-1 reverse transcriptase, integrase, protease, pro-viral genome integration and viral Tat protein mediated transactivation. The effect of n-butanol fraction on the induction of pro-inflammatory cytokines secretion in Vk2/E6E7 cells and transepithelial resistance in Caco-2 and HEC-1A cells was investigated. Results The aqueous and 50% ethanolic extracts of A. catechu showed IC50 values of 1.8 ± 0.18 μg/ml and 3.6 ± 0.31 μg/ml, respectively in cell-free virus based assay using TZM-bl cells and HIV-1NL4.3 (X-4 tropic). In the above assay, n-butanol fraction exhibited anti-HIV-1 activity with an IC50 of 1.7 ± 0.12 μg/ml. The n-butanol fraction showed a dose-dependent inhibition against HIV-1NL4.3 infection of the peripheral blood lymphocytes and against HIV-1BaL(R-5-tropic) as well as two different primary viral isolates of HIV-1 infection of TZM-bl cells. The n-butanol fraction demonstrates a potent inhibitory activity against the viral protease (IC50 = 12.9 μg/ml), but not reverse transcriptase or integrase. Further, in Alu-PCR no effect on viral integration was observed. The n-butanol fraction interfered with the Tat-mediated Long Terminal Repeat transactivation in

  5. HIV-1 Tat Protein Induces Production of Proinflammatory Cytokines by Human Dendritic Cells and Monocytes/Macrophages through Engagement of TLR4-MD2-CD14 Complex and Activation of NF-κB Pathway

    PubMed Central

    Leghmari, Kaoutar; Serrero, Manutea; Delobel, Pierre; Izopet, Jacques; BenMohamed, Lbachir; Bahraoui, Elmostafa

    2015-01-01

    We recently reported that the human immunodeficiency virus type-1 (HIV-1) Tat protein induced the expression of programmed death ligand-1 (PD-L1) on dendritic cells (DCs) through a TLR4 pathway. However, the underlying mechanisms by which HIV-1 Tat protein induces the abnormal hyper-activation of the immune system seen in HIV-1 infected patients remain to be fully elucidated. In the present study, we report that HIV-1 Tat protein induced the production of significant amounts of the pro-inflammatory IL-6 and IL-8 cytokines by DCs and monocytes from both healthy and HIV-1 infected patients. Such production was abrogated in the presence of anti-TLR4 blocking antibodies or soluble recombinant TLR4-MD2 as a decoy receptor, suggesting TLR4 was recruited by Tat protein. Tat-induced murine IL-6 and CXCL1/KC a functional homologue of human IL-8 was abolished in peritoneal macrophages derived from TLR4 KO but not from Wt mice, confirming the involvement of the TLR4 pathway. Furthermore, the recruitment of TLR4-MD2-CD14 complex by Tat protein was demonstrated by the activation of TLR4 downstream pathways including NF-κB and SOCS-1 and by down-modulation of cell surface TLR4 by endocytosis in dynamin and lipid-raft-dependent manners. Collectively, these findings demonstrate, for the first time, that HIV-1 Tat interacts with TLR4-MD2-CD14 complex and activates the NF-κB pathway, leading to overproduction of IL-6 and IL-8 pro-inflammatory cytokines by myeloid cells from both healthy and HIV-1 infected patients. This study reveals a novel mechanism by which HIV-1, via its early expressed Tat protein, hijacks the TLR4 pathway, hence establishing abnormal hyper-activation of the immune system. PMID:26090662

  6. Immunogenicity of HIV-1 IIIB and SHIV 89.6P Tat and Tat toxoids in rhesus macaques: induction of humoral and cellular immune responses.

    PubMed

    Richardson, Max W; Mirchandani, Jyotika; Silvera, Peter; Régulier, Emmanuel G; Capini, Christelle; Bojczuk, Paul M; Hu, Jason; Gracely, Edward J; Boyer, Jean D; Khalili, Kamel; Zagury, Jean-François; Lewis, Mark G; Rappaport, Jay

    2002-09-01

    This study compared immune responses in rhesus macaques immunized with unmodified HIV-1 IIIB Tat, SHIV89.6P Tat, and carboxymethylated IIIB and 89.6P Tat toxoids. Immunization with either IIIB or 89.6P preparation induced high titer and broadly crossreactive serum anti-Tat IgG that recognized HIV-1 subtype-E and SIVmac251 Tat. However, the response was delayed, and titers were lower in 89.6P vaccination groups. Serum anti-Tat IgG recognized peptides corresponding to the amino-terminus, basic domain, and carboxy-terminal region. Cellular proliferative responses to Tat toxoids corresponding to the immunogen were evident in vitro in both IIIB and 89.6P groups. Crossreactive proliferative responses were observed in IIIB groups in response to stimulation with 89.6P or SIVmac251 Tat toxoids, but were much less prevalent in 89.6P groups. The truncated 86 amino acid IIIB Tat appears to be more immunogenic than the 102 amino acid 89.6P Tat with respect to both humoral and cellular immune responses, and may be a better vaccine component. Despite induction of robust humoral and cellular immune responses (including both CD4+ and CD8+ T-cell responses) to Tat, all animals were infected upon intravenous challenge with 30 MID(50) of SHIV89.6P and outcome of vaccine groups was not different from controls. Sequencing both Tat exons from serum viral RNA revealed no evidence of escape mutants. These results suggest that with intravenous SHIV89.6P challenge in rhesus macaques, precipitous CD4+ T-cell decline overwhelms potentially protective immune responses. Alternatively, Tat specific CD8+ T-cell responses may not appropriately recognize infected cells in vivo in this model. In view of evidence demonstrating Tat specific CTLs in the SIV model and in humans infected with HIV-1, results in this pathogenic SHIV model may not apparently predict the efficacy of this approach in human studies. The potency and cross-reactivity of these immune responses confirm Tat toxoid as an excellent

  7. Genetic attributes of blood-derived subtype-C HIV-1 tat and env in India and neurocognitive function.

    PubMed

    Tilghman, Myres W; Bhattacharya, Jayanta; Deshpande, Suprit; Ghate, Manisha; Espitia, Stephen; Grant, Igor; Marcotte, Thomas D; Smith, Davey; Mehendale, Sanjay

    2014-01-01

    Genetic elements in HIV-1 subtype B tat and env are associated with neurotoxicity yet less is known about other subtypes. HIV-1 subtype C tat and env sequences were analyzed to determine viral genetic elements associated with neurocognitive impairment in a large Indian cohort. Population-based sequences of HIV-1 tat (exon 1) and env (C2-V3 coding region) were generated from blood plasma of HIV-infected patients in Pune, India. Participants were classified as cognitively normal or impaired based on neuropsychological assessment. Tests for signature residues, positive and negative selection, entropy, and ambiguous bases were performed using tools available through Los Alamos National Laboratory (http://www.hiv.lanl.gov) and Datamonkey (http://www.datamonkey.org). HIV-1 subtype C tat and env sequences were analyzed for 155 and 160 participants, of which 34-36% were impaired. Two signature residues were unique to impaired participants in exon 1 of tat at codons 29 (arginine) and 68 (proline). Positive selection was noted at codon 29 among normal participants and at codon 68 in both groups. The signature at codon 29 was also a signature for low CD4+ (<200 cells/mm(3)) counts but remained associated with impairment after exclusion of those with low CD4+ counts. No unique genetic signatures were noted in env. In conclusion, two signature residues were identified in exon 1 of HIV-1 subtype C tat that were associated with neurocognitive impairment in India and not completely accounted for by HIV disease progression. These signatures support a linkage between diversifying selection in HIV-1 subtype C tat and neurocognitive impairment. © 2013 Wiley Periodicals, Inc.

  8. Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals

    PubMed Central

    de la Fuente, Cynthia; Santiago, Francisco; Deng, Longwen; Eadie, Carolyne; Zilberman, Irene; Kehn, Kylene; Maddukuri, Anil; Baylor, Shanese; Wu, Kaili; Lee, Chee Gun; Pumfery, Anne; Kashanchi, Fatah

    2002-01-01

    Background Expression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator. Results Utilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins. Conclusions We have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals. PMID:12069692

  9. MH2 domain of Smad3 reduces HIV-1 Tat-induction of cytokine secretion.

    PubMed

    Eldeen, Mazen B; Deshmane, Satish L; Simbiri, Kenneth; Khalili, Kamel; Amini, Shohreh; Sawaya, Bassel E

    2006-07-01

    HIV-1 infection of the central nervous system (CNS) is associated with dysregulation of several important cytokines and chemokines, which are involved in inflammatory process. Earlier studies ascribed a critical role for Tat, a potent viral transcription activator, in this process by enhancing the expression of several immunomodulators including TGFbeta and MCP-1. Investigation of signaling pathways which are controlled by these cytokines led to identification of MH2 domain of Smad3, the downstream activator of TGFbeta pathway, as a modulator of MCP-1 promoter activity. The level of MCP-1 is increased in AIDS patients with neurologic problems, through recruitment of inflammatory cells, which can contribute to neuropathogenesis of AIDS. Therefore, we attempted to investigate the effect of MH2 on expression of MCP-1 and other immunolmodulators in CNS cells. By employing an adenovirus expression vector, we demonstrated that MH2 can decrease the levels of Tat-induced activation of MCP-1 and several other cytokines and chemokines in astrocytic cells. In addition, we showed that MH2 significantly reduced the activity of cytokines produced by cultures of adenovirus-MH2 transduced cells as measured by the transmigration of human PBMC cells. Thus, MH2 domain of Smad3 is a potential agent that may be developed as an inhibitor for the cytokine-mediated inflammatory responses in the brain and may have the potential to prevent transmigration of HIV-1-infected monocytes across the blood brain barrier in AIDS patients.

  10. Imaging HIV-1 Tat Trafficking and Interactions by Engineered Green-Fluorescent-Protein Tagging

    NASA Astrophysics Data System (ADS)

    Beltram, Fabio

    2002-03-01

    The direct monitoring of protein function in live cells under physiologically relevant conditions is one of the most powerful and innovative methodologies for proteomics. Efficient florescent probes fully compatible with human-cell expression are the fundamental tools for these studies and their optimization opens the way to resolution at the single-protein level. Biological events involving protein pairs are also directly accessible thanks to tuning of protein-tag spectral properties and production of complementary pairs. Such pairs are characterized by overlapping absorption (for the acceptor tag) and emission (for the donor tag) spectra. By tagging the proteins of interest with acceptor and donor molecules, protein interaction can be directly visualized by FRET, fluorescent resonant energy transfer. In this talk we shall present the design by molecular dynamics calculations and the application of optimized green fluorescent proteins to the study of the human immunodeficiency virus HIV-1 proteomics. In particular trafficking and cellular interactions of HIV-1 transactivator protein Tat in live human cells will be presented. Tat localization and complex internalization pathways of exogenous molecules will be presented thanks to the peculiar optical properties of mutated GFPs. Cellular protein partners and subcellular interaction sites will be identified and directly visualized. The relevance of such results and of advanced spectroscopic and imaging techniques for a new level of understanding of biological processes and its significance for advancement in molecular biology will be underlined. A. Marcello et al., J. Biol. Chem. 276, 39220 (2001). R. Cinelli et al., Appl. Phys. Lett. 79, 3353 (2001).

  11. Transcription through the HIV-1 nucleosomes: Effects of the PBAF complex in Tat activated transcription

    PubMed Central

    Easley, Rebecca; Carpio, Lawrence; Dannenberg, Luke; Choi, Soyun; Alani, Dowser; Van Duyne, Rachel; Guendel, Irene; Klase, Zachary; Agbottah, Emmanuel; Kehn-Hall, Kylene; Kashanchi, Fatah

    2010-01-01

    The SWI/SNF complex remodels nucleosomes, allowing RNA Polymerase II access to the HIV-1 proviral DNA. It has not been determined which SWI/SNF complex (BAF or PBAF) remodels nucleosomes at the transcription start site. These complexes differ in only three subunits and determining which subunit(s) is required could explain the regulation of Tat activated transcription. We show that PBAF is required for chromatin remodeling at the nuc-1 start site and transcriptional elongation. We find that Baf200 is required to ensure activation at the LTR level and for viral production. Interestingly, the BAF complex was observed on the LTR whereas PBAF was present on both LTR and Env regions. We found that Tat activated transcription facilitates removal of histones H2A and H2B at the LTR, and that the FACT complex may be responsible for their removal. Finally, the BAF complex may play an important role in regulating splicing of the HIV-1 genome. PMID:20599239

  12. Molecular and Genetic Characterization of HIV-1 Tat Exon-1 Gene from Cameroon Shows Conserved Tat HLA-Binding Epitopes: Functional Implications

    PubMed Central

    Teto, Georges; Fonsah, Julius Y.; Tagny, Claude T.; Mbanya, Dora; Nchindap, Emilienne; Kenmogne, Leopoldine; Fokam, Joseph; Njamnshi, Dora M.; Kouanfack, Charles; Njamnshi, Alfred K.; Kanmogne, Georgette D.

    2016-01-01

    HIV-1 Tat plays a critical role in viral transactivation. Subtype-B Tat has potential use as a therapeutic vaccine. However, viral genetic diversity and population genetics would significantly impact the efficacy of such a vaccine. Over 70% of the 37-million HIV-infected individuals are in sub-Saharan Africa (SSA) and harbor non-subtype-B HIV-1. Using specimens from 100 HIV-infected Cameroonians, we analyzed the sequences of HIV-1 Tat exon-1, its functional domains, post-translational modifications (PTMs), and human leukocyte antigens (HLA)-binding epitopes. Molecular phylogeny revealed a high genetic diversity with nine subtypes, CRF22_01A1/CRF01_AE, and negative selection in all subtypes. Amino acid mutations in Tat functional domains included N24K (44%), N29K (58%), and N40K (30%) in CRF02_AG, and N24K in all G subtypes. Motifs and phosphorylation analyses showed conserved amidation, N-myristoylation, casein kinase-2 (CK2), serine and threonine phosphorylation sites. Analysis of HLA allelic frequencies showed that epitopes for HLAs A*0205, B*5301, Cw*0401, Cw*0602, and Cw*0702 were conserved in 58%–100% of samples, with B*5301 epitopes having binding affinity scores > 100 in all subtypes. This is the first report of N-myristoylation, amidation, and CK2 sites in Tat; these PTMs and mutations could affect Tat function. HLA epitopes identified could be useful for designing Tat-based vaccines for highly diverse HIV-1 populations, as in SSA. PMID:27438849

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

    PubMed Central

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

    2017-01-01

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

  14. HIV-1 Tat depresses DNA-PK{sub CS} expression and DNA repair, and sensitizes cells to ionizing radiation

    SciTech Connect

    Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun . E-mail: zhoupk@nic.bmi.ac.cn

    2006-07-01

    Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and {gamma}-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and {gamma}H2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy {gamma}-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G{sub 2}/M arrest as compared with parental cells. In addition, the G{sub 2}/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased

  15. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

    SciTech Connect

    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu

    2014-10-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.

  16. HIV-1 Tat Regulates Occludin and Aβ Transfer Receptor Expression in Brain Endothelial Cells via Rho/ROCK Signaling Pathway

    PubMed Central

    Chen, Yanlan; Jiang, Wenlin; Wu, Xianghong; Ye, Biao; Zhou, Xiaoting

    2016-01-01

    HIV-1 transactivator protein (Tat) has been shown to play an important role in HIV-associated neurocognitive disorders. The aim of the present study was to evaluate the relationship between occludin and amyloid-beta (Aβ) transfer receptors in human cerebral microvascular endothelial cells (hCMEC/D3) in the context of HIV-1-related pathology. The protein expressions of occludin, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in hCMEC/D3 cells were examined using western blotting and immunofluorescent staining. The mRNA levels of occludin, RAGE, and LRP1 were measured using quantitative real-time polymerase chain reaction. HIV-1 Tat at 1 µg/mL and the Rho inhibitor hydroxyfasudil (HF) at 30 µmol/L, with 24 h exposure, had no significant effect on hCMEC/D3 cell viability. Treatment with HIV-1 Tat protein decreased mRNA and protein levels of occludin and LRP1 and upregulated the expression of RAGE; however, these effects were attenuated by HF. These data suggest that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-mediated changes in occludin, RAGE, and LRP1 in hCMEC/D3 cells. HF may have a beneficial influence by protecting the integrity of the blood-brain barrier and the expression of Aβ transfer receptors. PMID:27563375

  17. HIV-1 Tat Recruits HDM2 E3 Ligase To Target IRF-1 for Ubiquitination and Proteasomal Degradation

    PubMed Central

    Remoli, Anna Lisa; Marsili, Giulia; Perrotti, Edvige; Acchioni, Chiara; Sgarbanti, Marco; Borsetti, Alessandra; Hiscott, John

    2016-01-01

    ABSTRACT In addition to its ability to regulate HIV-1 promoter activation, the viral transactivator Tat also functions as a determinant of pathogenesis and disease progression by directly and indirectly modulating the host anti-HIV response, largely through the capacity of Tat to interact with and modulate the activities of multiple host proteins. We previously demonstrated that Tat modulated both viral and host transcriptional machinery by interacting with the cellular transcription factor interferon regulatory factor 1 (IRF-1). In the present study, we investigated the mechanistic basis and functional significance of Tat−IRF-1 interaction and demonstrate that Tat dramatically decreased IRF-1 protein stability. To accomplish this, Tat exploited the cellular HDM2 (human double minute 2 protein) ubiquitin ligase to accelerate IRF-1 proteasome-mediated degradation, resulting in a quenching of IRF-1 transcriptional activity during HIV-1 infection. These data identify IRF-1 as a new target of Tat-induced modulation of the cellular protein machinery and reveal a new strategy developed by HIV-1 to evade host immune responses. PMID:27795392

  18. Mathematical model of the Tat-Rev regulation of HIV-1 replication in an activated cell predicts the existence of oscillatory dynamics in the synthesis of viral components

    PubMed Central

    2014-01-01

    analyzed alternative hypotheses for the re-cycling of the Rev proteins both in the cytoplasm and the nuclear pore complex. Conclusions The quantitative mathematical model of the Tat-Rev regulation of HIV-1 replication predicts the existence of oscillatory dynamics which depends on the efficacy of the Tat and TAR interaction as well as on the Rev-mediated transport processes. The biological relevance of the oscillatory regimes for the HIV-1 life cycle is discussed. PMID:25564443

  19. Development of an Attenuated Tat Protein as a Highly-effective Agent to Specifically Activate HIV-1 Latency

    PubMed Central

    Geng, Guannan; Liu, Bingfeng; Chen, Cancan; Wu, Kang; Liu, Jun; Zhang, Yijun; Pan, Ting; Li, Jun; Yin, Yue; Zhang, Junsong; Huang, Feng; Yu, Fei; Chen, Jingliang; Ma, Xiancai; Zhou, Jie; Kuang, Ersheng; Liu, Chao; Cai, Weiping; Zhang, Hui

    2016-01-01

    Although combined antiretroviral therapy (cART) successfully decreases plasma viremia to undetectable levels, the complete eradication of human immunodeficiency virus type 1 (HIV-1) remains impractical because of the existence of a viral reservoir, mainly in resting memory CD4+ T cells. Various cytokines, protein kinase C activators, and histone deacetylase inhibitors (HDACi) have been used as latency-reversing agents (LRAs), but their unacceptable side effects or low efficiencies limit their clinical use. Here, by a mutation accumulation strategy, we generated an attenuated HIV-1 Tat protein named Tat-R5M4, which has significantly reduced cytotoxicity and immunogenicity, yet retaining potent transactivation and membrane-penetration activity. Combined with HDACi, Tat-R5M4 activates highly genetically diverse and replication-competent viruses from resting CD4+ T lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Thus, Tat-R5M4 has promising potential as a safe, efficient, and specific LRA in HIV-1 treatment. PMID:27434587

  20. HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

    PubMed Central

    Norman, John P.; Perry, Seth W.; Reynolds, Holly M.; Kiebala, Michelle; De Mesy Bentley, Karen L.; Trejo, Margarita; Volsky, David J.; Maggirwar, Sanjay B.; Dewhurst, Stephen; Masliah, Eliezer; Gelbard, Harris A.

    2008-01-01

    Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS. PMID:19009018

  1. β-chemokine production by neural and glial progenitor cells is enhanced by HIV-1 Tat: Effects on microglial migration

    PubMed Central

    Hahn, Yun Kyung; Vo, Phu; Fitting, Sylvia; Block, Michelle L.; Hauser, Kurt F.; Knapp, Pamela E.

    2010-01-01

    HIV-1 neuropathology results from collective effects of viral proteins and inflammatory mediators on several cell types. Significant damage is mediated indirectly through inflammatory conditions promulgated by glial cells, including microglia that are productively infected by HIV-1, and astroglia. Neural and glial progenitors exist in both developing and adult brains. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins Tat or gp120. In the initial screen, ten analytes were basally released by murine striatal progenitors. The beta-chemokines CCL5/RANTES, CCL3/MIP-1α, and CCL4/MIP-1β were increased by 12 h exposure to HIV-1 Tat. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti-CCR5 antibody pretreatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. gp120 did not affect chemokine/cytokine release, although both CCR5 and CXCR4, which serve as gp120 co-receptors, were detected in progenitors. We postulate that chemokine production by progenitors may be a normal, adaptive process that encourages immune inspection of newly generated cells. Pathogens such as HIV might usurp this function to create a maladaptive state, especially during development or regeneration, when progenitors are numerous. PMID:20403075

  2. HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase.

    PubMed

    Kadri, Ferdous; Pacifici, Marco; Wilk, Anna; Parker-Struckhoff, Amanda; Del Valle, Luis; Hauser, Kurt F; Knapp, Pamela E; Parsons, Christopher; Jeansonne, Duane; Lassak, Adam; Peruzzi, Francesca

    2015-12-25

    The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of TAU exon 10 results in tauopathies characterized by alterations in the proportion of TAU isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the TAU molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes TAU 3R up-regulation through increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with TAU RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of TAU exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.

  3. HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase*

    PubMed Central

    Kadri, Ferdous; Pacifici, Marco; Wilk, Anna; Parker-Struckhoff, Amanda; Del Valle, Luis; Hauser, Kurt F.; Knapp, Pamela E.; Parsons, Christopher; Jeansonne, Duane; Lassak, Adam; Peruzzi, Francesca

    2015-01-01

    The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of TAU exon 10 results in tauopathies characterized by alterations in the proportion of TAU isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the TAU molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes TAU 3R up-regulation through increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with TAU RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of TAU exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection. PMID:26534959

  4. Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake

    PubMed Central

    Yuan, Yaxia; Quizon, Pamela M.; Sun, Wei-Lun; Yao, Jianzhuang; Zhu, Jun; Zhan, Chang-Guo

    2016-01-01

    HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND. PMID:27250920

  5. Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake.

    PubMed

    Yuan, Yaxia; Quizon, Pamela M; Sun, Wei-Lun; Yao, Jianzhuang; Zhu, Jun; Zhan, Chang-Guo

    2016-06-02

    HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND.

  6. HIV-1 Tat protein vaccination in mice infected with Mycobacterium tuberculosis is safe, immunogenic and reduces bacterial lung pathology.

    PubMed

    Cafaro, Aurelio; Piccaro, Giovanni; Altavilla, Giuseppe; Gigantino, Vincenzo; Matarese, Giuseppe; Olivieri, Erika; Ferrantelli, Flavia; Ensoli, Barbara; Palma, Carla

    2016-08-22

    The therapeutic HIV-1 Tat protein vaccine is in advanced clinical development. Tuberculosis, the main AIDS co-infection, is highly endemic in areas where AIDS prevention through vaccination is needed. However, safety and immunogenicity of Tat vaccination in the course of Mycobacterium tuberculosis (Mtb) infection is still unknown and it prevents the possibility to administer the vaccine to Mtb-infected individuals. We addressed the interplay and effects of Tat vaccination on Mtb infection in immunocompetent mice. C57BL/6 mice were vaccinated or not with Bacillus Calmette-Guerin (BCG), the current tuberculosis vaccine, and after 5 weeks were infected with Mtb by intravenous route. The Tat protein was injected intradermally at 1, 2 and 4 weeks after Mtb challenge. Eight weeks after Mtb infection, all mice were sacrificed, and both the degree of pathology and immune responses to Mtb and Tat were evaluated. As additional control, some mice were either vaccinated or not with BCG, were not challenged with Mtb, but received the Tat protein. Statistical significances were evaluated by one-way or two-way ANOVA and Tukey's multiple comparisons post-test. In the lungs of Mtb-infected mice, Tat-vaccine did not favour Mtb replication and indeed reduced both area of cellular infiltration and protein levels of Interferon-γ, Chemokine (C-C motif) ligand-4 and Interleukin-1β, pathological events triggered by Mtb-infection. Moreover, the protection against Mtb infection conferred by BCG remained good after Tat protein treatment. In spleen cells of Mtb-infected mice, Tat vaccination enhanced Mtb-specific Interferon-γ and Interleukin-17 responses, which may have a protective role. Of note, Mtb infection reduced, but did not suppress, the development of anti-Tat antibodies, required for Tat vaccine efficacy and the titer of anti-Tat IgG was potentiated by BCG vaccination in Mtb-free mice. In general, Tat treatment was well tolerated in both Mtb-infected and Mtb-free mice. Tat

  7. Sialic acid associated with αvβ3 integrin mediates HIV-1 Tat protein interaction and endothelial cell proangiogenic activation.

    PubMed

    Chiodelli, Paola; Urbinati, Chiara; Mitola, Stefania; Tanghetti, Elena; Rusnati, Marco

    2012-06-08

    Sialic acid (NeuAc) is a major anion on endothelial cells (ECs) that regulates different biological processes including angiogenesis. NeuAc is present in the oligosaccharidic portion of integrins, receptors that interact with extracellular matrix components and growth factors regulating cell adhesion, migration, and proliferation. Tat is a cationic polypeptide that, once released by HIV-1(+) cells, accumulates in the extracellular matrix, promoting EC adhesion and proangiogenic activation by engaging α(v)β(3). By using two complementary approaches (NeuAc removal by neuraminidase or its masking by NeuAc-binding lectin from Maackia amurensis, MAA), we investigated the presence of NeuAc on endothelial α(v)β(3) and its role in Tat interaction, EC adhesion, and proangiogenic activation. α(v)β(3) immunoprecipitation with biotinylated MAA or Western blot analysis of neuraminidase-treated ECs demonstrated that NeuAc is associated with both the α(v) and the β(3) subunits. Surface plasmon resonance analysis demonstrated that the masking of α(v)β(3)-associated NeuAc by MAA prevents Tat/α(v)β(3) interaction. MAA and neuraminidase prevent α(v)β(3)-dependent EC adhesion to Tat, the consequent FAK and ERK1/2 phosphorylation, and EC proliferation, migration, and regeneration in a wound-healing assay. Finally, MAA inhibits Tat-induced neovascularization in the ex vivo human artery ring sprouting assay. The inhibitions are specific because the NeuAc-unrelated lectin from Ulex europaeus is ineffective on Tat. Also, MAA and neuraminidase affect only weakly integrin-dependent EC adhesion and proangiogenic activation by fibronectin. In conclusion, NeuAc is associated with endothelial α(v)β(3) and mediates Tat-dependent EC adhesion and proangiogenic activation. These data point to the possibility to target integrin glycosylation for the treatment of angiogenesis/AIDS-associated pathologies.

  8. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes.

    PubMed

    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu

    2014-10-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes.

  9. Isolation and characterization of bifunctional Escherichia coli TatA mutant proteins that allow efficient tat-dependent protein translocation in the absence of TatB.

    PubMed

    Blaudeck, Natascha; Kreutzenbeck, Peter; Müller, Matthias; Sprenger, Georg A; Freudl, Roland

    2005-02-04

    In Escherichia coli, the Tat system promotes the membrane translocation of a subset of exported proteins across the cytoplasmic membrane. Four genes (tatA, tatB, tatC, and tatE) have been identified that encode the components of the E. coli Tat translocation apparatus. Whereas TatA and TatE can functionally substitute for each other, the TatB and the TatC proteins have been shown to perform distinct functions. In contrast to Tat systems of the ABC(E) type found in E. coli and many other bacteria, some microorganisms possess a TatAC-type translocase that consists of TatA and TatC only, suggesting that, in these systems, TatB is not required or that one of the remaining components (TatA or TatC) additionally takes over the TatB function. We have addressed the molecular basis for the difference in subunit composition between TatABC(E) and TatAC-type systems by using a genetic approach. A plasmid-encoded E. coli minimal Tat translocase consisting solely of TatA and TatC was shown to mediate a low level translocation of a sensitive Tat-dependent reporter protein. Suppressor mutations in the minimal Tat translocase were isolated that compensate for the absence of TatB and that showed substantial increases in translocation activities. All of the mutations mapped to the extreme amino-terminal domain of TatA. No mutations affecting TatC were identified. These results suggest that in TatAC-type systems, the TatA protein represents a bifunctional component fulfilling both the TatA and TatB functions. Furthermore, our results indicate that the structure of the amino-terminal domain of TatA is decisive for whether or not TatB is required.

  10. A Growth Factor Attenuates HIV-1 Tat and Morphine Induced Damage to Human Neurons: Implication in HIV/AIDS-Drug Abuse Cases

    PubMed Central

    Malik, Shaily; Khalique, Hena; Buch, Shilpa; Seth, Pankaj

    2011-01-01

    The neuropathological abnormalities of human immunodeficiency virus (HIV)-1 patients abusing illicit drugs suggest extensive interactions between the two agents, thereby leading to increased rate of progression to neurodegeneration. The role of HIV-1 transactivating protein, Tat has been elucidated in mediating neuronal damage via apoptosis, a hallmark of HIV-associated dementia (HAD), however the underlying mechanisms involved in enhanced neurodegeneration by illicit drugs remain elusive. In this study, we demonstrated that morphine enhances HIV-Tat induced toxicity in human neurons and neuroblastoma cells. Enhanced toxicity by Tat and morphine was accompanied by increased numbers of TUNEL positive apoptotic neurons, elevated caspase-3 levels and decreased ratio of anti- and pro-apoptotic proteins, Bcl2/Bax. Tat and morphine together elicited high levels of reactive oxygen species that were NADPH dependent. Significant alterations in mitochondrial membrane homeostasis were also observed with co-exposure of these agents. Extensive studies of mitogen activated protein kinase (MAPK) signaling pathways revealed the involvement of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways in enhanced toxicity of Tat and morphine. In addition to this, we found that pre-treatment of cells with platelet derived growth factor (PDGF-BB) protected neurons from HIV-Tat and morphine induced damage. PDGF-BB alleviated ROS production, maintained mitochondrial membrane potential, decreased caspase-3 activation and hence protected the cells from undergoing apoptosis. PDGF-BB mediated protection against Tat and morphine involved the phosphatidylinositol–3 kinase (PI3K) pathway, as specific inhibitor of PI3K abrogated the protection conferred by PDGF-BB. This study demonstrates the mechanism of enhanced toxicity in human neurons subjected to co-exposure of HIV protein Tat and morphine, thus implying its importance in HIV positive drug abusers

  11. Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport

    PubMed Central

    Quizon, Pamela M.; Sun, Wei-Lun; Yuan, Yaxia; Midde, Narasimha M.; Zhan, Chang-Guo; Zhu, Jun

    2016-01-01

    Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission. PMID:27966610

  12. Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport.

    PubMed

    Quizon, Pamela M; Sun, Wei-Lun; Yuan, Yaxia; Midde, Narasimha M; Zhan, Chang-Guo; Zhu, Jun

    2016-12-14

    Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.

  13. CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING

    PubMed Central

    Zhong, Yu; Smart, Eric J.; Weksler, Babette; Couraud, Pierre-Olivier; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    The blood-brain barrier (BBB) is the critical structure for preventing HIV trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1 infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMEC). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMEC. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions upon Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients. PMID:18667611

  14. The effects of CpG-ODNs and Chitosan adjuvants on the elicitation of immune responses induced by the HIV-1-Tat-based candidate vaccines in mice.

    PubMed

    Alipour, Samira; Mahdavi, Atiyeh; Abdoli, Asghar

    2017-03-01

    HIV1-Tat-based vaccines could elicit broad, durable and neutralizing immune responses and are considered as potential AIDS vaccines. The present study aims to formulate CpG-ODNs adjuvant and Chitosan with Tat protein to enhance the immunogenicity of HIV-1-Tat-based candidate vaccines and to investigate their efficacies in mice. To this end, we added CpG-ODNs, Chitosan and Alum as adjuvants to the Tat-based candidate vaccine formulations. Then, we compared frequency and magnitude of both humoral and cellular immune responses from mice immunized with the adjuvant-formulated Tat candidate vaccines against those obtained from mice immunized with recombinant Tat protein alone. Mice were subcutaneously immunized three times at 2-week intervals with the candidate vaccines. Measurements of anti-Tat immune responses showed that all vaccinated groups had a good immunity compared to the control groups and developed high levels of both humoral and cellular responses. However, immunized mice with CpG-ODNs, and Chitosan-adjuvanted Tat vaccines elicited stronger T-cell responses (both humoral and cellular immunity) compared to the others. These data suggest that co-administration of recombinant Tat protein with CpG-ODNs and Chitosan may serve as a potential formulation for enhancing of the Tat vaccine-induced immunity and might have effects on shaping Th polarization induced by HIV1-Tat protein vaccines. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. beta-Chemokine production by neural and glial progenitor cells is enhanced by HIV-1 Tat: effects on microglial migration.

    PubMed

    Hahn, Yun Kyung; Vo, Phu; Fitting, Sylvia; Block, Michelle L; Hauser, Kurt F; Knapp, Pamela E

    2010-07-01

    Human immunodeficiency virus (HIV)-1 neuropathology results from collective effects of viral proteins and inflammatory mediators on several cell types. Significant damage is mediated indirectly through inflammatory conditions promulgated by glial cells, including microglia that are productively infected by HIV-1, and astroglia. Neural and glial progenitors exist in both developing and adult brains. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins transactivator of transcription (Tat) or glycoprotein 120 (gp120). In the initial screen, ten analytes were basally released by murine striatal progenitors. The beta-chemokines CCL5/regulated upon activation, normal T cell expressed and secreted, CCL3/macrophage inflammatory protein-1alpha, and CCL4/macrophage inflammatory protein-1beta were increased by 12-h exposure to HIV-1 Tat. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti-CCR5 antibody pre-treatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. gp120 did not affect chemokine/cytokine release, although both CCR5 and CXCR4, which serve as gp120 co-receptors, were detected in progenitors. We postulate that chemokine production by progenitors may be a normal, adaptive process that encourages immune inspection of newly generated cells. Pathogens such as HIV might usurp this function to create a maladaptive state, especially during development or regeneration, when progenitors are numerous.

  16. Neoflavonoids as Inhibitors of HIV-1 Replication by Targeting the Tat and NF-κB Pathways.

    PubMed

    Olmedo, Dionisio A; López-Pérez, José Luis; Del Olmo, Esther; Bedoya, Luis M; Sancho, Rocío; Alcamí, José; Muñoz, Eduardo; Feliciano, Arturo San; Gupta, Mahabir P

    2017-02-19

    Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter gene. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Seven 4-phenylchromen-2-one derivatives showed HIV transcriptional inhibitory activity but only the phenylchrome-2-one 10 inhibited NF-κB and displayed anti-Tat activity simultaneously. Compounds 10, 14, and 25, inhibited HIV replication in both targets at concentrations <25 μM. The assays of these synthetic 4-phenylchromen-2-ones may aid in the investigation of some aspects of the anti-HIV activity of such compounds and could serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential anti-HIV therapeutic drug.

  17. HIV-1 evolution in patients undergoing immunotherapy with Tat, Rev, and Nef expressing dendritic cells followed by treatment interruption.

    PubMed

    de Goede, Anna L; van Deutekom, Hanneke W M; Vrancken, Bram; Schutten, Martin; Allard, Sabine D; van Baalen, Carel A; Osterhaus, Albert D M E; Thielemans, Kris; Aerts, Joeri L; Keşmir, Can; Lemey, Philippe; Gruters, Rob A

    2013-11-13

    This study aimed to evaluate HIV sequence evolution in whole genes and in CD8 T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates. HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI. HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8 T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution. Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8 T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes. Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8 T-cell epitope level.

  18. Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication.

    PubMed

    Bhargavan, Biju; Woollard, Shawna M; Kanmogne, Georgette D

    2016-02-01

    TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetic modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests that TLR3 can act as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Toll-Like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication

    PubMed Central

    Bhargavan, Biju; Woollard, Shawna M.; Kanmogne, Georgette D.

    2016-01-01

    TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetics modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests TLR3 can acts as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects. PMID:26569339

  20. Modulation of Th1/Th2 immune responses to HIV-1 Tat by new pro-GSH molecules.

    PubMed

    Fraternale, Alessandra; Paoletti, Maria Filomena; Dominici, Sabrina; Buondelmonte, Costantina; Caputo, Antonella; Castaldello, Arianna; Tripiciano, Antonella; Cafaro, Aurelio; Palamara, Anna Teresa; Sgarbanti, Rossella; Garaci, Enrico; Ensoli, Barbara; Magnani, Mauro

    2011-09-16

    We have previously demonstrated that in Ova-immunized mice the increase in intra-macrophage thiol pool induced by pro-GSH molecules modulates the Th1/Th2 balance in favour of a Th1-type immune response. We show now that the same molecules can support a Th1-type over Th2-type immunity against Tat, which is an early HIV-1 regulatory protein and a Th1 polarizing immunomodulator that is increasingly considered in new anti-HIV vaccination strategies. Our results indicate that Tat-immunized mice pre-treated with the C4 (n-butanoyl) derivative of reduced glutathione (GSH-C4) or a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA) (I-152), have decreased levels of anti-Tat IgG1 as well as increased levels of anti-Tat IgG2a and IgG2b isotypes suggesting a Th1-type response. Moreover, Th1-(IFN-γ and IL-2) Ag-specific cellular responses were detected by ELISPOT assay in splenocytes of the same animals as well as an increase of IL-12 levels in the plasma. These findings suggest that the Th1 immune response to HIV-1 Tat could be further polarized by these molecules. These results together with those previously reported suggest that pro-GSH molecules could be used to modulate the immune response towards different antigens and may be further exploited for inducing specific Th1 immune responses against other HIV antigens as well as other intracellular pathogens in new Tat-based vaccination protocols.

  1. Random mutagenesis of the human immunodeficiency virus type-1 trans-activator of transcription (HIV-1 Tat).

    PubMed Central

    Siderovski, D P; Matsuyama, T; Frigerio, E; Chui, S; Min, X; Erfle, H; Sumner-Smith, M; Barnett, R W; Mak, T W

    1992-01-01

    A new method is described for the direct construction of randomly mutagenized genes by applying the polymerase chain reaction (PCR) to an oligonucleotide synthesized using doped nucleotide reservoirs. We have demonstrated the utility of this method by generating a library of mutant HIV-1 tat genes. Several arbitrarily selected, inactive tat clones were sequenced to evaluate the extent of the mutagenesis. Moreover, fourteen recombinants encoding varying levels of transcriptional trans-activator activity were isolated by transient transfection of sub-library pools into a HeLa cell line bearing an HIV-LTR-chloramphenicol acetyltransferase (CAT) reporter gene. Sequence data revealed a spectrum of alterations including nucleotide substitutions, insertions, and deletions, suggesting that mutations arose from both the doped DNA synthesis and the subsequent PCR 'rescue' of full-length product. Sequence comparison between inactive and active Tat clones revealed a selection pressure against amino-acid substitutions within the N-terminal domains of Tat, indicating the importance of this region to trans-activation competence. In addition, single and double missense mutations within the basic-rich, TAR RNA-binding domain were seen to be tolerated within active Tat clones. Images PMID:1437550

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

    SciTech Connect

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

    2009-07-21

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

  3. Effects of Conditional Central Expression of HIV-1 Tat Protein to Potentiate Cocaine-Mediated Psychostimulation and Reward Among Male Mice

    PubMed Central

    Paris, Jason J; Carey, Amanda N; Shay, Christopher F; Gomes, Stacey M; He, Johnny J; McLaughlin, Jay P

    2014-01-01

    As a major neuropathogenic factor associated with human immunodeficiency virus (HIV) infection, HIV-1 Tat protein is known to synergize with psychostimulant drugs of abuse to cause neurotoxicity and exacerbate the progression of central nervous system pathology. However, the functional consequences of the interaction between HIV-1 Tat and abused drugs on behavior are little known. We tested the hypothesis that HIV-1 Tat expression in brain would modulate the psychostimulant effects of cocaine. Using the GT-tg bigenic mouse model, where brain-selective Tat expression is induced by activation of a doxycycline (Dox) promotor, we tested the effects of Tat on cocaine (10 mg/kg, s.c.) induced locomotion and conditioned place preference (CPP). Compared with uninduced littermates or C57BL/6J controls, cocaine-induced hyperlocomotion was sustained for a significantly longer duration among Tat-induced mice. Moreover, although all groups displayed similar saline-CPP, Tat-induced GT-tg mice demonstrated a three-fold increase in cocaine-CPP over the response of either uninduced littermates or Dox-treated C57BL/6J control mice. Induction of Tat also increased the magnitude of a previously established cocaine-CPP after an additional cycle of cocaine place-conditioning. Despite Tat-induced potentiation, extinction of place preference occurred within 21 days, commensurate with cocaine-extinction among saline-treated littermates and C57BL/6J controls. Re-exposure to cocaine produced reinstatement of an equivalent place preference in Tat-induced GT-tg or C57BL/6J mice; however, induction of Tat protein after the extinction of CPP also produced reinstatement without additional exposure to cocaine. Together, these data suggest that central HIV-1 Tat expression can potentiate the psychostimulant behavioral effects of cocaine in mice. PMID:23945478

  4. HIV-1 Tat Induces Unfolded Protein Response and Endoplasmic Reticulum Stress in Astrocytes and Causes Neurotoxicity through Glial Fibrillary Acidic Protein (GFAP) Activation and Aggregation.

    PubMed

    Fan, Yan; He, Johnny J

    2016-10-21

    HIV-1 Tat is a major culprit for HIV/neuroAIDS. One of the consistent hallmarks of HIV/neuroAIDS is reactive astrocytes or astrocytosis, characterized by increased cytoplasmic accumulation of the intermediate filament glial fibrillary acidic protein (GFAP). We have shown that that Tat induces GFAP expression in astrocytes and that GFAP activation is indispensable for astrocyte-mediated Tat neurotoxicity. However, the underlying molecular mechanisms are not known. In this study, we showed that Tat expression or GFAP expression led to formation of GFAP aggregates and induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in astrocytes. In addition, we demonstrated that GFAP up-regulation and aggregation in astrocytes were necessary but also sufficient for UPR/ER stress induction in Tat-expressing astrocytes and for astrocyte-mediated Tat neurotoxicity. Importantly, we demonstrated that inhibition of Tat- or GFAP-induced UPR/ER stress by the chemical chaperone 4-phenylbutyrate significantly alleviated astrocyte-mediated Tat neurotoxicity in vitro and in the brain of Tat-expressing mice. Taken together, these results show that HIV-1 Tat expression leads to UPR/ER stress in astrocytes, which in turn contributes to astrocyte-mediated Tat neurotoxicity, and raise the possibility of developing HIV/neuroAIDS therapeutics targeted at UPR/ER stress.

  5. Morphine Causes Rapid Increases in Glial Activation and Neuronal Injury in the Striatum of Inducible HIV-1 Tat Transgenic Mice

    PubMed Central

    Bruce-Keller, Annadora J.; Turchan-Cholewo, Jadwiga; Smart, Eric J.; Geurin, Theresa; Chauhan, Ashok; Reid, Rollie; Xu, Ruqiang; Nath, Avindra; Knapp, Pamela E.; Hauser, Kurt F.

    2009-01-01

    HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2 day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3-NT co-detection in Mac1-positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase-3, and this was even more significantly elevated by co-administration of morphine. In spite of elevations in caspase-3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co-administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate-abusing AIDS patients. PMID:18551626

  6. Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV-1 Tat transgenic mice.

    PubMed

    Bruce-Keller, Annadora J; Turchan-Cholewo, Jadwiga; Smart, Eric J; Geurin, Theresa; Chauhan, Ashok; Reid, Rollie; Xu, Ruqiang; Nath, Avindra; Knapp, Pamela E; Hauser, Kurt F

    2008-10-01

    HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2-day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3-NT co-detection in Mac1-positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase-3, and this was even more significantly elevated by co-administration of morphine. In spite of elevations in caspase-3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co-administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate-abusing AIDS patients.

  7. Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA.

    PubMed Central

    Unwalla, H; Banerjea, A C

    2001-01-01

    Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10-23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3' end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3' end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3' end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed. PMID:11415445

  8. Low dose HIV-1 Tat improves the defective nuclear factor (NF)-kappaB activity of dendritic cells from persons with spinal cord injury.

    PubMed

    Hsieh, Szu-Min; Wang, Yen-Ho; Chang, Shan-Chwen; Huang, Tien-Shang

    2009-01-01

    Deficits of immune function may be involved in the infections associated with spinal cord injury (SCI). Previous report showed that the impaired maturation potential of dendritic cells (DCs) contributes to immune defect in persons with SCI, especially in those with tetraplegia. To evaluate the roles of cell signaling in the impaired maturation potential of DCs, we assessed the phenotypic and functional maturation potential of DCs in 20 subjects with trauma-induced stable SCI and their neurologically intact healthy control in the presence of DC stimulators, including HIV-1 Tat protein (Tat). Our results showed the tetraplegic subjects had an impaired maturation potential of DCs. The impairment could be attributed to insufficient nuclear factor (NF)-kappaB activity. The maturation potentials and NF-kappaB activity of DCs in response to stimulators could be improved by pretreatment with Tat, although Tat did not increase DC maturation. The improvement by Tat pretreatment was inhibited by a specific NF-kappaB blocker. We concluded that HIV-1 Tat could improve the maturation potentials of DCs from tetraplegic subjects, through Tat-induced enhancement of NF-kappaB activity. These data suggest a potential therapeutic role of HIV-1 Tat in improving immune function in tetraplegic persons.

  9. The Tat-dependent protein translocation pathway.

    PubMed

    Hou, Bo; Brüser, Thomas

    2011-12-01

    The twin-arginine translocation (Tat) pathway is found in bacteria, archaea, and plant chloroplasts, where it is dedicated to the transmembrane transport of fully folded proteins. These proteins contain N-terminal signal peptides with a specific Tat-system binding motif that is recognized by the transport machinery. In contrast to other protein transport systems, the Tat system consists of multiple copies of only two or three usually small (∼8-30 kDa) membrane proteins that oligomerize to two large complexes that transiently interact during translocation. Only one of these complexes includes a polytopic membrane protein, TatC. The other complex consists of TatA. Tat systems of plants, proteobacteria, and several other phyla contain a third component, TatB. TatB is evolutionarily and structurally related to TatA and usually forms tight complexes with TatC. Minimal two-component Tat systems lacking TatB are found in many bacterial and archaeal phyla. They consist of a 'bifunctional' TatA that also covers TatB functionalities, and a TatC. Recent insights into the structure and interactions of the Tat proteins have various important implications.

  10. Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis.

    PubMed

    Ziegler, André; Blatter, Xiaochun Li; Seelig, Anna; Seelig, Joachim

    2003-08-05

    Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K

  11. Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex

    PubMed Central

    Schulze-Gahmen, Ursula; Echeverria, Ignacia; Stjepanovic, Goran; Bai, Yun; Lu, Huasong; Schneidman-Duhovny, Dina; Doudna, Jennifer A; Zhou, Qiang; Sali, Andrej; Hurley, James H

    2016-01-01

    HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM. DOI: http://dx.doi.org/10.7554/eLife.15910.001 PMID:27731797

  12. Programmed neuronal cell death induced by HIV-1 tat and methamphetamine.

    PubMed

    Qi, Li; Gang, Lu; Hang, Kwong Wing; Ling, Choi Heung; Xiaofeng, Zeng; Zhen, Li; Wai, Yew David; Sang, Poon Wai

    2011-12-01

    Apoptosis and autophagy are the two major types of programmed cell death (PCD) in neurons. Homeostatic autophagy often precedes apoptosis, and when apoptosis is blocked, the failure to keep homeostasis will lead to necrosis instead. It has been reported that human immunodeficiency virus (HIV) infected methamphetamine (Meth) abusers represent greater neuropathological abnormalities than Meth abusers or HIV-positive non-Meth users. Recent publications suggest that Tat and Meth when administered together result in greater neuronal damage than when administered separately. However, the cellular events of the combined Tat-Meth effect have not yet been fully characterized. Therefore, we investigated the effects of Tat and/or Meth on apoptosis and autophagy to elucidate whether PCD was involved in Tat and/or Meth-induced neuronal damage. Annexin-V-FITC/PI staining assay was used to detect cellular apoptosis using a neuroblastoma cell line SH-SY5Y. Cellular ultrastructural changes were observed under transmission electron microscopy (TEM). Flow-cytometric data showed apoptosis following Meth treatment, and more extensive apoptosis with Tat + Meth treatment. The most important finding was that the autophagosome and/or multilamellar bodies (MLBs) were most pronounced with Tat + Meth treatment, were less so with Meth treatment, and infrequent with Tat treatment. This suggests the involvement of autophagy and apoptosis in Tat with Meth-elicited cell damage. However, the relation between apoptosis and autophagy remains unknown in this experiment. Further research is needed to analyze the relation among related molecules. A thorough understanding of this multifaceted relationship will be critical for the assessment of therapeutic modalities for patients with HIV with drug abuse. Copyright © 2011 Wiley Periodicals, Inc.

  13. Efficacy of Tat-Conjugated Ritonavir-Loaded Nanoparticles in Reducing HIV-1 Replication in Monocyte-Derived Macrophages and Cytocompatibility with Macrophages and Human Neurons

    PubMed Central

    Borgmann, Kathleen; Rao, Kavitha S.; Labhasetwar, Vinod

    2011-01-01

    Abstract Human immunodeficiency virus (HIV)-1 targets mononuclear phagocytes (MP), which disseminate infection to organs such as brain, spleen and lymph. Thus MP, which include microglia, tissue macrophages and infiltrating monocyte-derived macrophages (MDM), are important target of anti-HIV-1 drug therapy. Most of the currently used antiretroviral drugs are effective in reducing viral loadin the periphery but cannot effectively eradicate infection from tissue reservoirs such as brain MP. HIV-1 infection of the central nervous system can lead to a wide variety of HIV-1-associated neurocognitive disorders. In this study, we demonstrate that ritonavir-loaded nanoparticles (RNPs) are effective in inhibiting HIV-1 infection of MDM. Reduced infection is observed in multiple read-out systems including reduction of cytopathic effects, HIV-1 p24 protein secretion and production of progeny virions. Furthermore, the RNPs retained antiretroviral efficacy after being removed from MDM cultures. As HIV-1-infected cells in the brain are likely to survive for a long period of time, both acute and chronic infection paradigms were evaluated. Tat-peptide-conjugated RNPs (Tat-RNP) were effective in both short-term and long-term HIV-1-infected MDM. Importantly, we confirm that delivery of RNPs, both with and without Tat-peptide conjugation, is toxic neither to MDM nor to neural cells, which may be bystander targets of the nanoformulations. In conclusion, Tat-NPs could be a safe and effective way of delivering anti-HIV-1 drugs for controlling viral replication occurring within brain MP. PMID:21175357

  14. Relaxed specificity of the Bacillus subtilis TatAdCd translocase in Tat-dependent protein secretion.

    PubMed

    Eijlander, Robyn T; Jongbloed, Jan D H; Kuipers, Oscar P

    2009-01-01

    Protein translocation via the twin arginine translocation (TAT) pathway is characterized by the translocation of prefolded proteins across the hydrophobic lipid bilayer of the membrane. In Bacillus subtilis, two different Tat translocases are involved in this process, and both display different substrate specificities: PhoD is secreted via TatAdCd, whereas YwbN is secreted via TatAyCy. It was previously assumed that both TatAy and TatCy are essential for the translocation of the YwbN precursor. Through complementation studies, we now show that TatAy can be functionally replaced by TatAd when the latter is offered to the cells in excess amounts. Moreover, under conditions of overproduction, TatAdCd, in contrast to TatAyCy, shows an increased tolerance toward the acceptance of various Tat-dependent proteins.

  15. Relaxed Specificity of the Bacillus subtilis TatAdCd Translocase in Tat-Dependent Protein Secretion▿

    PubMed Central

    Eijlander, Robyn T.; Jongbloed, Jan D. H.; Kuipers, Oscar P.

    2009-01-01

    Protein translocation via the twin arginine translocation (TAT) pathway is characterized by the translocation of prefolded proteins across the hydrophobic lipid bilayer of the membrane. In Bacillus subtilis, two different Tat translocases are involved in this process, and both display different substrate specificities: PhoD is secreted via TatAdCd, whereas YwbN is secreted via TatAyCy. It was previously assumed that both TatAy and TatCy are essential for the translocation of the YwbN precursor. Through complementation studies, we now show that TatAy can be functionally replaced by TatAd when the latter is offered to the cells in excess amounts. Moreover, under conditions of overproduction, TatAdCd, in contrast to TatAyCy, shows an increased tolerance toward the acceptance of various Tat-dependent proteins. PMID:18978042

  16. Cortical consequences of HIV-1 Tat exposure in rats are enhanced by chronic cocaine

    PubMed Central

    Wayman, Wesley N.; Chen, Lihua; Persons, Amanda L.; Napier, T. Celeste

    2015-01-01

    The life span of individuals that are sero-positive for human immunodeficiency virus (HIV) has greatly improved; however, complications involving the central nervous system (CNS) remain a concern. While HIV does not directly infect neurons, the proteins produced by the virus, including HIV transactivator of transcription (Tat), are released from infected glia; these proteins can be neurotoxic. This neurotoxicity is thought to mediate the pathology underlying HIV-associated neurological impairments. Cocaine abuse is common among HIV infected individuals, and this abuse augments HIV-associated neurological deficits. The brain regions and pathophysiological mechanisms that are dysregulated by both chronic cocaine and Tat are the focus of the current review. PMID:25760043

  17. HIV-1 Tat protein increases the permeability of brain endothelial cells by both inhibiting occludin expression and cleaving occludin via matrix metalloproteinase-9.

    PubMed

    Xu, Ruifen; Feng, Xuyang; Xie, Xin; Zhang, Jin; Wu, Daocheng; Xu, Lixian

    2012-02-03

    Brain homeostasis is maintained by the blood-brain barrier (BBB), which prevents the entrance of circulating molecules and immune cells into the central nervous system. The BBB is formed by specialized brain endothelial cells that are connected by tight junctions (TJ). Previous studies have proven that the Tat protein of human immunodeficiency virus type 1 (HIV-1) alters TJ protein expression. However, the mechanisms by which the alterations occur have not been characterized in detail. In this study, primary human brain microvascular endothelial cells (HBMEC) were exposed to recombinant HIV-1 Tat protein, and the effects on occludin were observed. Tat treatment decreased occludin mRNA and protein levels. This effect was partially abrogated by addition of the RhoA inhibitor C3 exoenzyme and the p160-Rho-associated coiled kinase (ROCK) inhibitor Y-27632. Meanwhile, Tat also induced MMP-9 expression. RNA interference targeting MMP-9 reduced both the paracellular permeability of Tat-treated HBMEC and the concentration of soluble occludin in supernatants from the cells. Taken together, these results show that the HIV-1 Tat protein disrupts BBB integrity, at least in part by decreasing the production of occludin.

  18. Structure-based design of ligands for protein basic domains: application to the HIV-1 Tat protein.

    PubMed

    Filikov, A V; James, T L

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calormetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  19. Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

    NASA Astrophysics Data System (ADS)

    Filikov, Anton V.; James, Thomas L.

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  20. 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.

  1. Chronic low-level expression of HIV-1 Tat promotes a neurodegenerative phenotype with aging.

    PubMed

    Dickens, Alex M; Yoo, Seung Wan; Chin, Alfred C; Xu, Jiadi; Johnson, Tory P; Trout, Amanda L; Hauser, Kurt F; Haughey, Norman J

    2017-08-10

    The widespread use of combinational antiretroviral therapies (cART) in developed countries has changed the course of Human Immunodeficiency Virus (HIV) infection from an almost universally fatal disease to a chronic infection for the majority of individuals. Although cART has reduced the severity of neurological damage in HIV-infected individuals, the likelihood of cognitive impairment increases with age, and duration of infection. As cART does not suppress the expression of HIV non-structural proteins, it has been proposed that a constitutive production of HIV regulatory proteins in infected brain cells may contribute to neurological damage. However, this assumption has never been experimentally tested. Here we take advantage of the leaky tetracycline promoter system in the Tat-transgenic mouse to show that a chronic very low-level expression of Tat is associated with astrocyte activation, inflammatory cytokine expression, ceramide accumulation, reductions in brain volume, synaptic, and axonal damage that occurs over a time frame of 1 year. These data suggest that a chronic low-level production of Tat may contribute to progressive neurological damage in virally suppressed HIV-infected individuals.

  2. Interactive role of human immunodeficiency virus type 1 (HIV-1) clade-specific Tat protein and cocaine in blood-brain barrier dysfunction: implications for HIV-1-associated neurocognitive disorder.

    PubMed

    Gandhi, Nimisha; Saiyed, Zainulabedin M; Napuri, Jessica; Samikkannu, Thangavel; Reddy, Pichili V B; Agudelo, Marisela; Khatavkar, Pradnya; Saxena, Shailendra K; Nair, Madhavan P N

    2010-07-01

    In recent years, increasing interest has emerged to assess the human immunodeficiency virus type 1 (HIV-1) clade C viral pathogenesis due to its anticipated spread in the United States and other western countries. Previous studies suggest that clade C is less neuropathogenic than clade B; however, the underlying mechanism is poorly understood. Additionally, the interactive role of drugs of abuse such as cocaine on clade C-associated neuropathogenesis has not been reported. In the current study, we hypothesize that HIV-1 clade-specific Tat proteins exert differential effects on blood-brain barrier (BBB) integrity and cocaine further differentially aggravates the BBB dysfunction. We evaluated the effect of Tat B and Tat C and/or cocaine on the BBB integrity using an in vitro model constructed with primary human brain microvascular endothelial cells (HBMECs) and astrocytes. The BBB membrane integrity was measured by transendothelial electrical resistance (TEER) and paracellular permeability was measured by fluorescein isothiocyanate (FITC)-dextran transport assay and monocytes transmigration across the BBB. Results indicate that Tat B disrupts BBB integrity to a greater extent compared to Tat C and cocaine further differentially exacerbates the BBB dysfunction. This BBB dysfunction was associated with altered expression of tight junction proteins zona occuldens (ZO-1) and junctional adhesion molecule (JAM)-2. Thus, these results for the first time delineate the differential role of Tat B and Tat C and/or cocaine in BBB dysfunction, which may be correlated with the clade-specific differences observed in HIV-1-associated neurological disorders.

  3. Chronic Central Nervous System Expression of HIV-1 Tat Leads to Accelerated Rarefaction of Neocortical Capillaries and Loss of Red Blood Cell Velocity Heterogeneity

    PubMed Central

    Silva, Jharon N.; Polesskaya, Oksana; Wei, Helen S.; Rasheed, Izad-Yar D.; Chamberlain, Jeffrey M.; Nishimura, Christopher; Feng, Changyong; Dewhurst, Stephen

    2014-01-01

    Objectives HIV-1 infection of the central nervous system is associated with impairment of cerebral blood flow and neurocognitive function, and accelerated signs of aging. Since normal aging is associated with rarefaction of the cerebral vasculature, we set out to examine chronic viral effects on the cerebral vasculature. Methods Doxycycline-inducible HIV-1 Tat transgenic (Tat-tg) and wild-type (WT) control mice were used. Animals were treated with doxycycline for 3 weeks or 5–7 months. Cerebral vessel density and capillary segment length were determined from quantitative image analyses of sectioned cortical tissue. In addition, movement of red blood cells in individual capillaries was imaged in vivo using multiphoton microscopy, to determine red blood cell velocity and flux. Results Mean RBCV was not different between Tat-tg mice and age matched WT controls. However, cortical capillaries from Tat-tg mice showed a significant loss of RBCV heterogeneity and increased RBCF that was attributed to a marked decrease in total cortical capillary length (35–40%) compared to WT mice. Conclusions Cerebrovascular rarefaction is accelerated in HIV-1 Tat transgenic mice, and this is associated with alterations in red cell blood velocity. These changes may have relevance to the pathogenesis of HIV-associated neurocognitive disorders in an aging HIV positive population. PMID:24813724

  4. The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation

    PubMed Central

    Doetsch, Martina; Fürtig, Boris; Gstrein, Thomas; Stampfl, Sabine; Schroeder, Renée

    2011-01-01

    The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44–61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing—both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44–61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal. PMID:21297117

  5. Selective Vulnerability of Striatal D2 versus D1 Dopamine Receptor-Expressing Medium Spiny Neurons in HIV-1 Tat Transgenic Male Mice.

    PubMed

    Schier, Christina J; Marks, William D; Paris, Jason J; Barbour, Aaron J; McLane, Virginia D; Maragos, William F; McQuiston, A Rory; Knapp, Pamela E; Hauser, Kurt F

    2017-06-07

    Despite marked regional differences in HIV susceptibility within the CNS, there has been surprisingly little exploration into the differential vulnerability among neuron types and the circuits they underlie. The dorsal striatum is especially susceptible, harboring high viral loads and displaying marked neuropathology, with motor impairment a frequent manifestation of chronic infection. However, little is known about the response of individual striatal neuron types to HIV or how this disrupts function. Therefore, we investigated the morphological and electrophysiological effects of HIV-1 trans-activator of transcription (Tat) in dopamine subtype 1 (D1) and dopamine subtype 2 (D2) receptor-expressing striatal medium spiny neurons (MSNs) by breeding transgenic Tat-expressing mice to Drd1a-tdTomato- or Drd2-eGFP-reporter mice. An additional goal was to examine neuronal vulnerability early during the degenerative process to gain insight into key events underlying the neuropathogenesis. In D2 MSNs, exposure to HIV-1 Tat reduced dendritic spine density significantly, increased dendritic damage (characterized by swellings/varicosities), and dysregulated neuronal excitability (decreased firing at 200-300 pA and increased firing rates at 450 pA), whereas insignificant morphologic and electrophysiological consequences were observed in Tat-exposed D1 MSNs. These changes were concomitant with an increased anxiety-like behavioral profile (lower latencies to enter a dark chamber in a light-dark transition task, a greater frequency of light-dark transitions, and reduced rearing time in an open field), whereas locomotor behavior was unaffected by 2 weeks of Tat induction. Our findings suggest that D2 MSNs and a specific subset of neural circuits within the dorsal striatum are preferentially vulnerable to HIV-1.SIGNIFICANCE STATEMENT Despite combination antiretroviral therapy (cART), neurocognitive disorders afflict 30-50% of HIV-infected individuals and synaptodendritic injury

  6. 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

  7. Differential effects of Tat proteins derived from HIV-1 subtypes B and recombinant CRF02_AG on human brain microvascular endothelial cells: implications for blood-brain barrier dysfunction.

    PubMed

    Woollard, Shawna M; Bhargavan, Biju; Yu, Fang; Kanmogne, Georgette D

    2014-06-01

    HIV-1 genetic differences influence viral replication and progression to AIDS. HIV-1 circulating recombinant form (CRF)02_AG is the predominant viral subtype infecting humans in West and Central Africa, but its effects on HIV neuropathogenesis are not known. In the present study, we investigated the effects of Tat proteins from HIV-1 subtype B (Tat.B) and HIV-1 CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major component of the blood-brain barrier (BBB). Using Affymetrix GeneChip Human Gene 1.0.ST arrays, we showed that Tat.AG had minimal effects while Tat.B induced transcriptional upregulation of 90 genes in HBMEC, including proinflammatory chemokines, complement components C3, C7, and complement factor B, matrix metalloproteinases (MMP)-3, MMP-10, and MMP-12. These results were confirmed by real-time PCR. Compared with Tat.AG, Tat.B significantly increased MMP-3, MMP-10, and MMP-12 activities in HBMEC, and the MMPs tissue inhibitor of metalloproteinase-2 blocked Tat-induced increase in MMPs activity. Western blot analyses also showed that Tat increased the expression of C3 and its cleaved fragment C3b in HBMEC. These data suggest that genetic differences between HIV-1 subtypes B and CRF02_AG influence the effects of Tat proteins from these two clades on HBMEC, including molecular and cellular functions, and canonical pathways, which would affect BBB dysfunction and viral neuropathogenesis.

  8. Chronic central nervous system expression of HIV-1 Tat leads to accelerated rarefaction of neocortical capillaries and loss of red blood cell velocity heterogeneity.

    PubMed

    Silva, Jharon N; Polesskaya, Oksana; Wei, Helen S; Rasheed, Izad-Yar D; Chamberlain, Jeffrey M; Nishimura, Christopher; Feng, Changyong; Dewhurst, Stephen

    2014-10-01

    HIV-1 infection of the CNS is associated with impairment of CBF and neurocognitive function, and accelerated signs of aging. As normal aging is associated with rarefaction of the cerebral vasculature, we set out to examine chronic viral effects on the cerebral vasculature. DOX-inducible HIV-1 Tat-tg and WT control mice were used. Animals were treated with DOX for three weeks or five to seven months. Cerebral vessel density and capillary segment length were determined from quantitative image analyses of sectioned cortical tissue. In addition, movement of red blood cells in individual capillaries was imaged in vivo using multiphoton microscopy, to determine RBCV and flux. Mean RBCV was not different between Tat-tg mice and age-matched WT controls. However, cortical capillaries from Tat-tg mice showed a significant loss of RBCV heterogeneity and increased RBCF that was attributed to a marked decrease in total cortical capillary length (35-40%) compared to WT mice. Cerebrovascular rarefaction is accelerated in HIV-1 Tat-transgenic mice, and this is associated with alterations in red cell blood velocity. These changes may have relevance to the pathogenesis of HIV-associated neurocognitive disorders in an aging HIV-positive population. © 2014 John Wiley & Sons Ltd.

  9. Specific HIV-1 TAR RNA loop sequence and functional groups are required for human cyclin T1-Tat-TAR ternary complex formation.

    PubMed

    Richter, Sara; Cao, Hong; Rana, Tariq M

    2002-05-21

    Replication of human immunodeficiency virus requires Tat protein which activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex (P-TEFb). Tat binds directly through its transactivation domain to the CycT1 subunit of the P-TEFb and induces loop sequence specific binding of the P-TEFb onto nascent HIV-1 TAR RNA. By using a gel electrophoresis method and a comprehensive set of TAR loop mutants, we have identified the sequence and structural determinants for high-affinity CycT1-Tat-TAR ternary complex formation. Our results show that CycT1 and Tat binding to TAR RNA is highly cooperative, and a capacity of 85%, a Hill coefficient of 2.7, and a dissociation constant (K(D)) of 2.45 nM were observed. These results indicate that there are three binding sites on TAR RNA. CycT1 does not bind TAR RNA in the absence of Tat, and Tat binding to TAR, while detectable, is very inefficient in the absence of CycT1. It is conceivable that the CycT1-Tat heterodimer directly binds to TAR RNA in the U-rich RNA bulge region and this binding facilitates the interactions of the CycT1-Tat heterodimer at the other two sites in the RNA loop region. On the basis of our results, we suggest a model where CycT1 interacts with Tat protein and positions the protein complex to make contacts with the G34 region of the loop sequence; G34 is critical for CycT1-Tat binding and forms a C30.G34 base pair. Two functional groups, O6 and N7, at nucleotide positions 32 and 34 in the TAR loop are essential for CycT1-Tat interactions with TAR RNA. The identity of two nucleotides, U31 and G33, is not critical, but they contribute to the stabilization of the RNA-protein complex. The presence of a single-nucleotide bulge of A35 or C35 is essential for distortion of the backbone RNA structure as well as the accessibility of functional groups in the major groove of the double

  10. Soy Isoflavones Genistein and Daidzein Exert Anti-Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat1–86 Exposure

    PubMed Central

    Adams, Sheila M.; Aksenova, Marina V.; Aksenov, Michael Y.; Mactutus, Charles F.; Booze, Rosemarie M.

    2012-01-01

    Background HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity. Methodology/Principal Findings We demonstrate in primary cortical cell cultures that 17β-estradiol or isoflavones (genistein or daidzein) attenuate Tat1–86-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERα or ERβ specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERβ selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling. Conclusions/Significance Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons. PMID:22629415

  11. Soy isoflavones genistein and daidzein exert anti-apoptotic actions via a selective ER-mediated mechanism in neurons following HIV-1 Tat(1-86) exposure.

    PubMed

    Adams, Sheila M; Aksenova, Marina V; Aksenov, Michael Y; Mactutus, Charles F; Booze, Rosemarie M

    2012-01-01

    HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity. We demonstrate in primary cortical cell cultures that 17β-estradiol or isoflavones (genistein or daidzein) attenuate Tat(1-86)-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERα or ERβ specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERβ selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling. Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons.

  12. Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.

    PubMed

    Rose, Patrick; Fröbel, Julia; Graumann, Peter L; Müller, Matthias

    2013-01-01

    The twin-arginine translocation (Tat) pathway guides fully folded proteins across membranes of bacteria, archaea and plant chloroplasts. In Escherichia coli, Tat-specific transport is executed in a still largely unknown manner by three functionally diverse membrane proteins, termed TatA, TatB, and TatC. In order to follow the intracellular distribution of the TatABC proteins in live E. coli cells, we have individually expressed fluorophore-tagged versions of each Tat protein in addition to a set of chromosomally encoded TatABC proteins. In this way, a Tat translocase could form from the native TatABC proteins and be visualized via the association of a fluorescent Tat variant. A functionally active TatA-green fluorescent protein fusion was found to re-locate from a uniform distribution in the membrane into a few clusters preferentially located at the cell poles. Clustering was absolutely dependent on the co-expression of functional Tat substrates, the proton-motive force, and the cognate TatBC subunits. Likewise, polar cluster formation of a functional TatB-mCherry fusion required TatA and TatC and that of a functional TatC-mCherry fusion a functional Tat substrate. Furthermore we directly demonstrate the co-localization of TatA and TatB in the same fluorescent clusters. Our collective results are consistent with distinct Tat translocation sites dynamically forming in vivo in response to newly synthesized Tat substrates.

  13. HIV-1 Tat and opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines.

    PubMed

    El-Hage, Nazira; Wu, Guanghan; Wang, Juan; Ambati, Jayakrishna; Knapp, Pamela E; Reed, Janelle L; Bruce-Keller, Annadora J; Hauser, Kurt F

    2006-01-15

    Opiates exacerbate human immunodeficiency virus type 1 (HIV-1) Tat(1-72)-induced release of key proinflammatory cytokines by astrocytes, which may accelerate HIV neuropathogenesis in opiate abusers. The release of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), in particular, is potentiated by opiate-HIV Tat interactions in vitro. Although MCP-1 draws monocytes/macrophages to sites of CNS infection, and activated monocytes/microglia release factors that can damage bystander neurons, the role of MCP-1 in neuro-acquired immunodeficiency syndrome (neuroAIDS) progression in opiate abusers, or nonabusers, is uncertain. Using a chemotaxis assay, N9 microglial cell migration was found to be significantly greater in conditioned medium from mouse striatal astrocytes exposed to morphine and/or Tat(1-72) than in vehicle-, mu-opioid receptor (MOR) antagonist-, or inactive, mutant Tat(delta31-61)-treated controls. Conditioned medium from astrocytes treated with morphine and Tat caused the greatest increase in motility. The response was attenuated using conditioned medium immunoneutralized with MCP-1 antibodies, or medium from MCP-1(-/-) astrocytes. In the presence of morphine (time-release, subcutaneous implant), intrastriatal Tat increased the proportion of neural cells that were astroglia and F4/80+ macrophages at 7 days post-injection. This was not seen after treatment with Tat alone, or with morphine plus inactive Tat(delta31-61) or naltrexone. Glia displayed increased MOR and MCP-1 immunoreactivity after morphine and/or Tat exposure. The findings indicate that MCP-1 underlies most of the response of microglia, suggesting that one way in which opiates exacerbate neuroAIDS is by increasing astroglial-derived proinflammatory chemokines at focal sites of CNS infection and promoting macrophage entry and local microglial activation. Importantly, increased glial expression of MOR can trigger an opiate-driven amplification/positive feedback of MCP-1 production and

  14. Penetration of HIV-1 Tat47–57 into PC/PE Bilayers Assessed by MD Simulation and X-ray Scattering

    PubMed Central

    Neale, Chris; Huang, Kun; García, Angel E.; Tristram-Nagle, Stephanie

    2015-01-01

    The interactions of the basic, cell-penetrating region (Y47GRKKRRQRRR57) of the HIV-1 Tat protein with dioleoylphosphatidylcholine (DOPC) bilayers were previously assessed by comparing experimental X-ray diffuse scattering with atomistic molecular dynamics simulations. Here, we extend this investigation by evaluating the influence of phosphatidylethanolamine (PE) lipids. Using experimental bilayer form factors derivedfrom X-ray diffuse scattering data as a guide, our simulations indicate that Tat peptides localize close to the carbonyl-glycerol group in the headgroup region of bilayers composed of either DOPC or DOPC:DOPE (1:1) lipid. Our results also suggest that Tat peptides may more frequently insert into the hydrophobic core of bilayers composed of PC:PE (1:1) lipids than into bilayers composed entirely of PC lipids. PE lipids may facilitate peptide translocation across a lipid bilayer by stabilizing intermediate states in which hydrated peptides span the bilayer. PMID:26402709

  15. On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation.

    PubMed

    Verhoef, K; Bauer, M; Meyerhans, A; Berkhout, B

    1998-11-20

    Tat is an essential protein of human immunodeficiency virus type 1 (HIV-1) and activates transcription from the viral long terminal repeat (LTR) promoter. The tat gene is composed of two coding exons of which the first, corresponding to the N-terminal 72 amino acid residues, has been reported to be sufficient for its transcription function. We introduced a stop codon at the end of the first Tat-coding exon in an expression vector that produces a truncated 71-amino acid Tat protein. This Q72stop mutant displays reduced transcriptional activity of approximately 54% in transient LTR-CAT transfection assays. To test the contribution of the second Tat-coding exon to virus replication, the Q72stop mutation was also introduced in the infectious pLAI molecular clone. The effect on virus replication was analyzed in primary cells and in a transformed T cell line. The fitness of the mutant virus was calculated to be approximately 75% compared with the wild-type control. Thus, a small contribution of the C-terminal Tat domain to viral fitness was measured. It has been proposed that the second Tat-coding exon is involved in transcriptional downregulation of the MHC class I gene of the infected host cell. Cell surface expression of the MHC protein was analyzed in T cells infected with the wild-type LAI virus and the replication-competent Q72stop mutant. MHC expression was transiently reduced on infection with either virus, indicating that the second Tat-coding exon is not involved in this downregulation.

  16. A stromal pool of TatA promotes Tat-dependent protein transport across the thylakoid membrane.

    PubMed

    Frielingsdorf, Stefan; Jakob, Mario; Klösgen, Ralf Bernd

    2008-12-05

    In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transport-incompetent due to extraction with solutions of chaotropic salts.

  17. The 73 kDa subunit of the CPSF complex binds to the HIV-1 LTR promoter and functions as a negative regulatory factor that is inhibited by the HIV-1 Tat protein.

    PubMed

    de la Vega, Laureano; Sánchez-Duffhues, Gonzalo; Fresno, Manuel; Schmitz, M Lienhard; Muñoz, Eduardo; Calzado, Marco A

    2007-09-14

    Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs. The cleavage and polyadenylation specificity factor (CPSF) is critical for this process and its 73 kDa subunit (CPSF-73) mediates cleavage coupled to polyadenylation and histone pre-mRNA processing. Using CPSF-73 over-expression and siRNA-mediated knockdown experiments, this study identifies CPSF-73 as an important regulatory protein that represses the basal transcriptional activity of the HIV-1 LTR promoter. Similar results were found with over-expression of the CPSF-73 homologue RC-68, but not with CPSF 100 kDa subunit (CPSF-100) and RC-74. Chromatin immunoprecipitation assays revealed the physical interaction of CPSF-73 with the HIV-1 LTR promoter. Further experiments revealed indirect CPSF-73 binding to the region between -275 to -110 within the 5' upstream region. Functional assays revealed the importance for the 5' upstream region (-454 to -110) of the LTR for CPSF-73-mediated transcription repression. We also show that HIV-1 Tat protein interacts with CPSF-73 and counteracts its repressive activity on the HIV-1 LTR promoter. Our results clearly show a novel function for CPSF-73 and add another candidate protein for explaining the molecular mechanisms underlying HIV-1 latency.

  18. Interactive HIV-1 Tat and Morphine-Induced Synaptodendritic Injury Is Triggered through Focal Disruptions in Na+ Influx, Mitochondrial Instability, and Ca2+ Overload

    PubMed Central

    Knapp, Pamela E.; Zou, Shiping; Marks, William D.; Bowers, M. Scott; Akbarali, Hamid I.; Hauser, Kurt F.

    2014-01-01

    Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na+]i) and calcium ([Ca2+]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca2+]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the μ-opioid receptor antagonist CTAP and were not observed in neurons cultured from μ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca2+]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na+]i, mitochondrial instability, excessive Ca2+ influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via μ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca2+]i and by further disrupting [Ca2+]i homeostasis. We hypothesize that the spatiotemporal relationship of μ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS. PMID:25232120

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

    PubMed

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

    2012-07-12

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

  20. A Peptide Nucleic Acid-Aminosugar Conjugate Targeting Transactivation Response Element of HIV-1 RNA Genome Shows a High Bioavailability in Human Cells and Strongly Inhibits Tat-mediated Transactivation of HIV-1 Transcription

    PubMed Central

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

    2012-01-01

    The 6-aminoglucosamine ring of the aminoglycoside antibiotic neomycin B (ring II) was conjugated to a 16 mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. For this purpose we prepared the aminoglucosamine monomer 15 and attached it to the protected PNA prior to its cleavage from the solid support. We found that the resulting PNA-aminoglucosamine conjugate is stable under acidic condition, efficiently taken up by the human cells and fairly distributed in both cytosol and nucleus without endosomal entrapment since co-treatment with endosome-disrupting agent had no effect on its cellular distribution. The conjugate displayed very high target specificity in vitro and strongly inhibited Tat mediated transactivation of HIV-1 LTR transcription in cell culture system. The unique properties of this new class of PNA conjugate suggest it to be a potential candidate for therapeutic application. PMID:22698070

  1. An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

    PubMed Central

    Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C.

    2014-01-01

    Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and

  2. HIV-1 Tat affects the programming and functionality of human CD8⁺ T cells by modulating the expression of T-box transcription factors.

    PubMed

    Sforza, Fabio; Nicoli, Francesco; Gallerani, Eleonora; Finessi, Valentina; Reali, Eva; Cafaro, Aurelio; Caputo, Antonella; Ensoli, Barbara; Gavioli, Riccardo

    2014-07-31

    HIV infection is characterized by several immune dysfunctions of both CD8⁺ and CD4⁺ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8⁺ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4⁺ T cells; however, its effects on CD8⁺ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8⁺ T-cell functionality and programming. CD8⁺ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8⁺ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8⁺ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Tat deeply alters the programming and functionality of CD8⁺ T lymphocytes.

  3. An attenuated herpes simplex virus type 1 (HSV1) encoding the HIV-1 Tat protein protects mice from a deadly mucosal HSV1 challenge.

    PubMed

    Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C

    2014-01-01

    Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and

  4. HIV-1 protein Tat potentiation of methamphetamine-induced decreases in evoked overflow of dopamine in the striatum of the rat.

    PubMed

    Cass, Wayne A; Harned, Michael E; Peters, Laura E; Nath, Avindra; Maragos, William F

    2003-09-12

    HIV-1 infection of the brain can lead to the development of clinical syndromes reminiscent of Parkinson's disease, suggesting that HIV infection may damage nigrostriatal dopamine (DA) neurons. Although the responsible mechanisms have not been well defined, neurotoxic viral proteins, such as Tat, released from infected cells may be involved. Drug abuse is a major risk factor for contracting HIV infection. Methamphetamine (METH), a psychostimulant with high abuse potential, may also be toxic to brain DA neurons. Thus, the combination of METH abuse and HIV infection may lead to substantial alterations in DA neuron functioning. The present experiments examined how Tat, alone and with METH, affects DA release in the striatum. Male rats were given an intrastriatal injection of Tat (25 micro g) or vehicle 24 h before treatment with saline or neurotoxic doses of METH. Seven days later microdialysis studies were carried out to measure potassium- and amphetamine-evoked overflow of DA from the striatum. The Tat treatment alone led to no change in potassium-evoked overflow of DA, a 20% decrease in amphetamine-evoked overflow of DA, and a 16% decrease in striatal DA content. The METH alone led to a 37-42% decrease in striatal DA overflow and content. The combined treatment with Tat and METH led to significantly greater 70-78% decreases in striatal DA overflow and content. These results indicate that Tat enhances METH-induced reductions in striatal DA release and content, possibly in a synergistic manner, and suggest that METH abusers infected with HIV may be at increased risk for basal ganglia dysfunction.

  5. Substrate-Dependent Assembly of the Tat Translocase as Observed in Live Escherichia coli Cells

    PubMed Central

    Rose, Patrick; Fröbel, Julia; Graumann, Peter L.; Müller, Matthias

    2013-01-01

    The twin-arginine translocation (Tat) pathway guides fully folded proteins across membranes of bacteria, archaea and plant chloroplasts. In Escherichia coli, Tat-specific transport is executed in a still largely unknown manner by three functionally diverse membrane proteins, termed TatA, TatB, and TatC. In order to follow the intracellular distribution of the TatABC proteins in live E. coli cells, we have individually expressed fluorophore-tagged versions of each Tat protein in addition to a set of chromosomally encoded TatABC proteins. In this way, a Tat translocase could form from the native TatABC proteins and be visualized via the association of a fluorescent Tat variant. A functionally active TatA-green fluorescent protein fusion was found to re-locate from a uniform distribution in the membrane into a few clusters preferentially located at the cell poles. Clustering was absolutely dependent on the co-expression of functional Tat substrates, the proton-motive force, and the cognate TatBC subunits. Likewise, polar cluster formation of a functional TatB-mCherry fusion required TatA and TatC and that of a functional TatC-mCherry fusion a functional Tat substrate. Furthermore we directly demonstrate the co-localization of TatA and TatB in the same fluorescent clusters. Our collective results are consistent with distinct Tat translocation sites dynamically forming in vivo in response to newly synthesized Tat substrates. PMID:23936332

  6. Methamphetamine potentiates HIV-1 Tat protein-mediated activation of redox-sensitive pathways in discrete regions of the brain.

    PubMed

    Flora, Govinder; Lee, Yong Woo; Nath, Avindra; Hennig, Bernhard; Maragos, William; Toborek, Michal

    2003-01-01

    Tat is a major regulatory protein encoded by human immunodeficiency viral genome, which has been implicated in the pathogenesis of HIV infection, including neurologic complications associated with this disease. In addition, drug abuse has been identified as a major risk factor of HIV infection. We hypothesize that abusive drugs, such as methamphetamine (METH), can directly influence specific molecular processes that can further contribute to toxic effects of Tat. To elucidate the molecular signaling pathways of Tat- and/or METH-induced toxicity, we investigated the effects of a single injection of Tat (25 microg/microl into the right hippocampus) and/or METH (10 mg/kg, intraperitoneally) on the generation of cellular oxidative stress, DNA-binding activity of specific redox-responsive transcription factors, and expression of inflammatory genes. Administration of Tat or METH resulted in stimulation of cellular oxidative stress and activation of redox-regulated transcription factors in the cortical, striatal, and hippocampal regions of the mouse brain. In addition, DNA-binding activities of NF-kappaB, AP-1, and CREB in the frontal cortex and hippocampus were more pronounced in mice injected with Tat plus METH compared to the effects of Tat or METH alone. Intercellular adhesion molecule-1 gene expression also was upregulated in a synergistic manner in cortical, striatal, and hippocampal regions in mice which received injections of Tat combined with METH compared to the effects of these agents alone. Moreover, synergistic effects of Tat plus METH on the tumor necrosis factor-alpha and interleukin-1beta mRNA levels were observed in the striatal region. These results indicate that Tat and METH can cross-amplify their cellular effects, leading to alterations of redox-regulated inflammatory pathways in the brain. Such synergistic proinflammatory stimulation may have significant implications in HIV-infected patients who abuse drugs.

  7. Viral diversity and diversification of major non-structural genes vif, vpr, vpu, tat exon 1 and rev exon 1 during primary HIV-1 subtype C infection.

    PubMed

    Rossenkhan, Raabya; Novitsky, Vladimir; Sebunya, Theresa K; Musonda, Rosemary; Gashe, Berhanu A; Essex, M

    2012-01-01

    To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; -0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.

  8. Allosteric modulatory effects of SRI-20041 and SRI-30827 on cocaine and HIV-1 Tat protein binding to human dopamine transporter.

    PubMed

    Sun, Wei-Lun; Quizon, Pamela M; Yuan, Yaxia; Zhang, Wei; Ananthan, Subramaniam; Zhan, Chang-Guo; Zhu, Jun

    2017-06-16

    Dopamine transporter (DAT) is the target of cocaine and HIV-1 transactivator of transcription (Tat) protein. Identifying allosteric modulatory molecules with potential attenuation of cocaine and Tat binding to DAT are of great scientific and clinical interest. We demonstrated that tyrosine 470 and 88 act as functional recognition residues in human DAT (hDAT) for Tat-induced inhibition of DA transport and transporter conformational transitions. Here we investigated the allosteric modulatory effects of two allosteric ligands, SRI-20041 and SRI-30827 on cocaine binding on wild type (WT) hDAT, Y470 H and Y88 F mutants. Effect of SRI-30827 on Tat-induced inhibition of [(3)H]WIN35,428 binding was also determined. Compared to a competitive DAT inhibitor indatraline, both SRI-compounds displayed a similar decrease (30%) in IC50 for inhibition of [(3)H]DA uptake by cocaine in WT hDAT. The addition of SRI-20041 or SRI-30827 following cocaine slowed the dissociation rate of [(3)H]WIN35,428 binding in WT hDAT relative to cocaine alone. Moreover, Y470H and Y88F hDAT potentiate the inhibitory effect of cocaine on DA uptake and attenuate the effects of SRI-compounds on cocaine-mediated dissociation rate. SRI-30827 attenuated Tat-induced inhibition of [(3)H]WIN35,428 binding. These observations demonstrate that tyrosine 470 and 88 are critical for allosteric modulatory effects of SRI-compounds on the interaction of cocaine with hDAT.

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

    NASA Astrophysics Data System (ADS)

    Tassew, Nardos Gobena

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

  10. Twin arginine translocation (Tat)-dependent export in the apparent absence of TatABC or TatA complexes using modified Escherichia coli TatA subunits that substitute for TatB.

    PubMed

    Barrett, Claire M L; Freudl, Roland; Robinson, Colin

    2007-12-14

    The twin arginine translocation pathway exports folded proteins across the cytoplasmic membrane of many bacteria. In Escherichia coli and other Gram-negative bacteria, TatA, TatB, and TatC are all essential for efficient translocation, and current models suggest that separate TatABC and TatA complexes coalesce at the point of translocation. However, other microbes appear only to possess tatA and tatC genes. In Escherichia coli, virtually no translocation is observed when only TatA and TatC are present, but several mutations at the extreme N terminus of TatA were shown to support translocation. Here we show that these apparently bifunctional mutant TatA variants can function as typical TatA components because translocation is observed when they are co-expressed with TatBC, and they assemble into large, heterogeneous complexes that resemble wild type TatA complexes. However, cells expressing TatC plus the mutant TatA variants do not contain complexes that resemble the expected 370-kDa TatABC complex, clearly indicating that the mutant TatA forms cannot assemble efficiently, or stably, into this complex. The simultaneous expression of wild type TatA furthermore blocks translocation activity, suggesting that the mutant TatA forms preferentially bind to other TatA molecules rather than TatC. Surprisingly, we observe translocation in the absence of detectable free TatA, when translational fusions of the mutant TatAs with TatC are expressed. Transport can thus proceed in the simultaneous absence of TatABC and TatA complexes at detectable levels, and we conclude that the active translocon may be formed from dynamic twin arginine translocation complexes, one or more of which may await characterization.

  11. Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones

    PubMed Central

    Pascale, Lise; Azoulay, Stéphane; Di Giorgio, Audrey; Zenacker, Laura; Gaysinski, Marc; Clayette, Pascal; Patino, Nadia

    2013-01-01

    RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous ‘polyamide amino acids’ (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy–entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets. PMID:23605042

  12. HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas.

    PubMed

    Luzzi, Anna; Morettini, Federica; Gazaneo, Sara; Mundo, Lucia; Onnis, Anna; Mannucci, Susanna; Rogena, Emily A; Bellan, Cristiana; Leoncini, Lorenzo; De Falco, Giulia

    2014-01-01

    A close association between HIV infection and the development of cancer exists. Although the advent of highly active antiretroviral therapy has changed the epidemiology of AIDS-associated malignancies, a better understanding on how HIV can induce malignant transformation will help the development of novel therapeutic agents. HIV has been reported to induce the expression of DNMT1 in vitro, but still no information is available about the mechanisms regulating DNMT expression in HIV-related B-cell lymphomas. In this paper, we investigated the expression of DNMT family members (DNMT1, DNMT3a/b) in primary cases of aggressive B-cell lymphomas of HIV-positive subjects. Our results confirmed the activation of DNMT1 by HIV in vivo, and reported for the first time a marked up-regulation of DNMT3a and DNMT3b in HIV-positive aggressive B-cell lymphomas. DNMT up-regulation in HIV-positive tumors correlated with down-regulation of specific microRNAs, as the miR29 family, the miR148-152 cluster, known to regulate their expression. Literature reports the activation of DNMTs by the human polyomavirus BKV large T-antigen and adenovirus E1a, through the pRb/E2F pathway. We have previously demonstrated that the HIV Tat protein is able to bind to the pocket proteins and to inactivate their oncosuppressive properties, resulting in uncontrolled cell proliferation. Therefore, we focused on the role of Tat, due to its capability to be released from infected cells and to dysregulate uninfected ones, using an in vitro model in which Tat was ectopically expressed in B-cells. Our findings demonstrated that the ectopic expression of Tat was per se sufficient to determine DNMT up-regulation, based on microRNA down-regulation, and that this results in aberrant hypermethylation of target genes and microRNAs. These results point at a direct role for Tat in participating in uninfected B-cell lymphomagenesis, through dysregulation of the epigenetical control of gene expression.

  13. Density-dependent decay in HIV-1 dynamics.

    PubMed

    Holte, Sarah E; Melvin, Ann J; Mullins, James I; Tobin, Nicole H; Frenkel, Lisa M

    2006-03-01

    The decay of HIV-1-infected cell populations after treatment with antiretroviral therapy has been measured using simple exponential decay models. These models are unlikely to be realistic over periods longer than a few months, however, because the population dynamics of HIV are complex. We considered an alternate model developed by Perelson and colleagues that extends the standard model for biphasic viral load decline and allows for nonlinear log decay of infected cell populations. Using data from 6 children on highly active antiretroviral therapy (HAART) and a single parameter in the new model, the assumption of log linear decay of infected cell populations is tested. Our analysis indicates that the short-lived and long-lived infected cell populations do not decay according to a simple exponential model. Furthermore, the resulting estimates of time to eradication of infected cell compartments are dramatically longer than those previously reported (eg, decades vs. years for long-lived infected cell populations and years vs. weeks for short-lived infected cell populations). Furthermore, estimates of the second-phase decay rates are significantly different than 0 for most children when obtained using the Perelson biphasic decay model. In contrast, this rate is not significantly different than 0 when the density-dependent decay model is used for parameter estimation and inference. Thus, the density-dependent decay model but not the simple exponential decay model is consistent with recent data showing that even under consistent HAART-mediated suppression of viral replication, decay rates of infected cell reservoirs decay little over several years. This suggests that conclusions about long-term viral dynamics of HIV infection based on simple exponential decay models should be carefully re-evaluated.

  14. HIV-1-Specific T Cell-Dependent Natural Killer (NK) Cell Activation: Major Contribution by NK Cells to Interferon-γ Production in Response to HIV-1 Antigens

    PubMed Central

    Loo, Christopher P.; Long, Brian R.; Hecht, Frederick M.; Nixon, Douglas F.

    2009-01-01

    Abstract Natural killer (NK) cells can directly recognize virus-infected cells. Here, we demonstrate that NK cells also produce interferon (IFN)-γ in an HIV-1-specific, T cell-dependent manner. After stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected individuals with HIV-1-derived peptides, up to half of the IFN-γ-producing PBMCs are NK cells. These results indicate that T cell-dependent NK cell IFN-γ production can be important for immune control of HIV-1, and have implications for the interpretation of data from vaccine trials using ELISPOT and ELISA. PMID:19500013

  15. Quantitative proteomic analysis of HIV-1 Tat-induced dysregulation in SH-SY5Y neuroblastoma cells.

    PubMed

    Ganief, Tariq; Gqamana, Putuma; Garnett, Shaun; Hoare, Jackie; Stein, Dan J; Joska, John; Soares, Nelson; Blackburn, Jonathan M

    2017-03-01

    Despite affecting up to 70% of HIV-positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV-associated neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC-based quantitative proteomic analysis on HIV-Tat treated SH-SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS-PAGE and analyzed by nLC-MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation-based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV-Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation-synapse loss.

  16. Differential Codon Usage Pattern of HIV-1 tat Gene in Those with Slower and Faster Rates of Disease Progression.

    PubMed

    Vidyavijayan, K K; Anangi, Brahmaiah; Banu, Brindha; Thiruvengadam, Kannan; Hassan, Sameer; Hanna, Luke Elizabeth

    2017-09-01

    Codon usage has been identified as one of the most important factors that influence gene expression. The frequencies with which the different codons are used vary significantly between different organisms and also between the genes within the same organism. HIV has a remarkable nucleotide composition with an above average percentage of "A" nucleotides resulting in a codon usage pattern different from that of the human host. In this study, we compared the codon usage pattern of HIV-1 genes among different groups of HIV disease progressors to understand the influence of differential codon usage pattern on the pathogenic manifestation in the host.

  17. M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner

    PubMed Central

    Wang, Qin; Zhang, Xiaolin; Han, Yuling; Wang, Xinlu; Gao, Guangxia

    2016-01-01

    M2BP (also called 90K) is an interferon-stimulated gene product that is upregulated in HIV-1 infection. A recent study revealed that M2BP reduces the infectivity of HIV-1 by inhibiting the processing of the viral envelope protein. Here we report that in addition to reducing viral infectivity, M2BP inhibits HIV-1 virion production. We provide evidence showing that M2BP inhibits HIV-1 Gag trafficking to the plasma membrane in a vimentin-dependent manner. When vimentin filaments were collapsed by treating cells with acrylamide or by overexpression of a dominant-negative mutant of vimentin, M2BP inhibition of HIV-1 virion production was significantly relieved. We further show that M2BP interacts with both HIV-1 Gag and vimentin and thereby mediates their interactions. We propose that M2BP traps HIV-1 Gag to vimentin filaments to inhibit the transportation of HIV-1 Gag to the plasma membrane. These findings uncover a novel mechanism by which a host antiviral factor inhibits HIV-1 virion production. PMID:27604950

  18. Syphilis Infection Differentially Regulates the Phenotype and Function of γδ T Cells in HIV-1-Infected Patients Depends on the HIV-1 Disease Stage

    PubMed Central

    Li, Zhen; Lu, Xiaofan; Hu, Zhiliang; Luo, Zhenwu; Jiang, Wei; Wu, Hao; Gao, Yanqing; Yan, Junling; Zhang, Qiuyue; Song, Aixin; Huang, Xiaojie; Mou, Danlei; Su, Bin; Zhang, Tong

    2017-01-01

    A rapidly escalating outbreak of syphilis infection has been affected men who have sex with men, particularly those with HIV-1 infection. γδ T cells are unconventional immune cells with two main subsets, Vδ1 T cells and Vδ2 T cells, which possess a combination of innate and adaptive immune features allowing them against HIV-1. However, whether syphilis infection affects the phenotype and function of γδ T cells in HIV-1-infected patients remains unclear, especially in acute HIV-1 infection (AHI). In this study, we enrolled 57 HIV-1-infected patients (24 with HIV-1 infection only and 33 coinfected with syphilis) from an acute HIV-1-infected cohort in Beijing (PRIMO). A comprehensive analysis of γδ T-cell phenotype and function was performed by flow cytometry. We found syphilis coinfection could reverse the imbalance of Vδ1/Vδ2 ratio in AHI. Syphilis infection results in decreased γδ T-cell activation in AHI, but increased γδ T-cell activation in chronic HIV-1 infection (CHI). Moreover, patients with CHI had larger numbers of IL-17-producing γδ T cells than those with AHI, regardless of syphilis status. Thus, syphilis affected the γδ T-cell immune response differently in patients depending on the stages of HIV-1 disease. In addition, the percentage of IL-17-producing γδ T cells was positively correlated with the percentage of neutrophils. These results suggest that the γδ T-cell/IL-17/neutrophil axis is involved in HIV-1 pathogenesis and disease progression. Taken together, our observations provide new insight into the roles of γδ T cells in immunopathogenesis of syphilis and HIV-1 coinfection, particularly during AHI, and our findings may be helpful for the prevention of syphilis and other sexually transmitted infections and highlight the great significance on the remedy of patients coinfected with HIV-1. PMID:28871259

  19. Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells.

    PubMed

    Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B; Park, Jongwoo; Courter, Joel R; Melillo, Bruno N; Sodroski, Joseph G; Kaufmann, Daniel E; Finzi, Andrés; Parsons, Matthew S; Kent, Stephen J

    2015-12-09

    Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4(+) T cells from HIV-1(+) subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1(+) serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed "shock and kill," aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune responses within HIV-1

  20. Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

    PubMed Central

    Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B.; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno N.; Sodroski, Joseph G.; Kaufmann, Daniel E.; Parsons, Matthew S.

    2015-01-01

    ABSTRACT Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+ T cells from HIV-1+ subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+ serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. IMPORTANCE An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune

  1. TAT-pathway-dependent lipoproteins as a niche-based adaptation in prokaryotes.

    PubMed

    Shruthi, Hamsanathan; Babu, Mohan Madan; Sankaran, Krishnan

    2010-04-01

    Bacterial lipoproteins, characterized by the N-terminal N-acyl S-diacylglyceryl Cysteine, are key membrane proteins in bacterial homeostasis. It is generally thought that during the modification lipoprotein precursors are translocated via the Sec-machinery in an unfolded state. The recent discovery of twin-arginine translocation (TAT) machinery, meant for exporting folded-proteins, and the presence of TAT-type signal sequences in co-factor-containing (hence already folded) lipoproteins, prompted us to investigate its role and significance in lipoprotein biosynthesis. We systematically analyzed 696 prokaryotic genomes using an algorithm based on DOLOP and TatP rules to predict TAT-pathway-dependent lipoprotein substrates. Occurrence of the deduced TAT-pathway-dependent lipoprotein substrates in relation to genome size, presence or absence of TAT machinery, and extent of its usage for lipoprotein export and habitat types revealed that unlike the host-obligates, the free-living prokaryotes in complex hostile environments (e.g., soil) depend more on TAT-exported lipoproteins. Functional classification of the predicted TAT-dependent lipoproteins revealed enrichment in hydrolases and oxido-reductases, which are fast-folding and co-factor-containing proteins. The role of the TAT pathway in the export of folded-lipoproteins and in niche-specific adaptation for survival has important implications not only in lipoprotein biosynthesis, but also for protein and metabolic engineering applications.

  2. Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway.

    PubMed

    Sancho, Rocío; Márquez, Nieves; Gómez-Gonzalo, Marta; Calzado, Marco A; Bettoni, Giorgio; Coiras, Maria Teresa; Alcamí, José; López-Cabrera, Manuel; Appendino, Giovanni; Muñoz, Eduardo

    2004-09-03

    Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.

  3. HIV-1-infected blood mononuclear cells form an integrin- and agrin-dependent viral synapse to induce efficient HIV-1 transcytosis across epithelial cell monolayer.

    PubMed

    Alfsen, Annette; Yu, Huifeng; Magérus-Chatinet, Aude; Schmitt, Alain; Bomsel, Morgane

    2005-09-01

    The heparan sulfate proteoglycan agrin and adhesion molecules are key players in the formation of neuronal and immune synapses that evolved for efficient communication at the sites of cell-cell contact. Transcytosis of infectious virus across epithelial cells upon contact between HIV-1-infected cells and the mucosal pole of the epithelial cells is one mechanism for HIV-1 entry at mucosal sites. In contrast, transcytosis of cell-free HIV-1 is not efficient. A synapse between HIV-1-infected cells and the mucosal epithelial surface that resembles neuronal and immune synapses is visualized by electron microscopy. We have termed this the "viral synapse." Similarities of the viral synapse also extend to the functional level. HIV-1-infected cell-induced transcytosis depends on RGD-dependent integrins and efficient cell-free virus transcytosis is inducible upon RGD-dependent integrin cross-linking. Agrin appears differentially expressed at the apical epithelial surface and acts as an HIV-1 attachment receptor. Envelope glycoprotein subunit gp41 binds specifically to agrin, reinforcing the interaction of gp41 to its epithelial receptor galactosyl ceramide.

  4. HIV-1-infected Blood Mononuclear Cells Form an Integrin- and Agrin-dependent Viral Synapse to Induce Efficient HIV-1 Transcytosis across Epithelial Cell Monolayer

    PubMed Central

    Alfsen, Annette; Yu, Huifeng; Magérus-Chatinet, Aude; Schmitt, Alain; Bomsel, Morgane

    2005-01-01

    The heparan sulfate proteoglycan agrin and adhesion molecules are key players in the formation of neuronal and immune synapses that evolved for efficient communication at the sites of cell-cell contact. Transcytosis of infectious virus across epithelial cells upon contact between HIV-1-infected cells and the mucosal pole of the epithelial cells is one mechanism for HIV-1 entry at mucosal sites. In contrast, transcytosis of cell-free HIV-1 is not efficient. A synapse between HIV-1-infected cells and the mucosal epithelial surface that resembles neuronal and immune synapses is visualized by electron microscopy. We have termed this the “viral synapse.” Similarities of the viral synapse also extend to the functional level. HIV-1-infected cell-induced transcytosis depends on RGD-dependent integrins and efficient cell-free virus transcytosis is inducible upon RGD-dependent integrin cross-linking. Agrin appears differentially expressed at the apical epithelial surface and acts as an HIV-1 attachment receptor. Envelope glycoprotein subunit gp41 binds specifically to agrin, reinforcing the interaction of gp41 to its epithelial receptor galactosyl ceramide. PMID:15975901

  5. Ligand-gated purinergic receptors regulate HIV-1 Tat and morphine related neurotoxicity in primary mouse striatal neuron-glia co-cultures.

    PubMed

    Sorrell, Mary E; Hauser, Kurt F

    2014-03-01

    Emerging evidence suggests that opioid drugs, such as morphine and heroin, can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that opioid drugs acting via μ opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. Key aspects of microglial function are regulated by the P2X family of ATP activated ligand-gated ion channels. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, which prompted us to investigate whether the cation-permeable P2X receptors contribute to the neurotoxic effects of HIV and morphine. To address this question, neuron survival, as well as other endpoints including changes in dendritic length, extracellular ATP levels, and intracellular calcium levels, were assayed in primary neuron-glia co-cultures from mouse striatum. Treatment with TNP-ATP, a non-selective P2X antagonist, prevented the neurotoxic effects of exposure to morphine and/or HIV Tat, or ATP alone, suggesting P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. Although P2X7, and perhaps P2X1, receptor activation decreases neuron survival, neither P2X1, P2X3, nor P2X7 selective receptor antagonists prevented Tat and/or morphine-induced neurotoxicity. These and other experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and provide circumstantial evidence implicating P2X4 receptors in particular. Our findings reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompanies neuroAIDS and opiate abuse.

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

    SciTech Connect

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

    2016-01-15

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

  7. Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²⁺ overload.

    PubMed

    Fitting, Sylvia; Knapp, Pamela E; Zou, Shiping; Marks, William D; Bowers, M Scott; Akbarali, Hamid I; Hauser, Kurt F

    2014-09-17

    Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the μ-opioid receptor antagonist CTAP and were not observed in neurons cultured from μ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via μ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of μ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

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

    PubMed Central

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

    2016-01-01

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

  9. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    SciTech Connect

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

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

    PubMed Central

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

    2016-01-01

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

  11. Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins*

    PubMed Central

    Cooper, Itzik; Sasson, Keren; Teichberg, Vivian I.; Schnaider-Beeri, Michal; Fridkin, Mati; Shechter, Yoram

    2012-01-01

    Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway. PMID:23150670

  12. Peptide derived from HIV-1 TAT protein destabilizes a monolayer of endothelial cells in an in vitro model of the blood-brain barrier and allows permeation of high molecular weight proteins.

    PubMed

    Cooper, Itzik; Sasson, Keren; Teichberg, Vivian I; Schnaider-Beeri, Michal; Fridkin, Mati; Shechter, Yoram

    2012-12-28

    Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3-0.6 μmol/ml of this C-TAT peptide, for a period of 1-2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.

  13. A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association

    PubMed Central

    Huang, Huachao; Liu, Shuai; Jean, Maxime; Simpson, Sydney; Huang, He; Merkley, Mark; Hayashi, Tsuyoshi; Kong, Weili; Rodríguez-Sánchez, Irene; Zhang, Xiaofeng; Yosief, Hailemichael O.; Miao, Hongyu; Que, Jianwen; Kobie, James J.; Bradner, James; Santoso, Netty G.; Zhang, Wei; Zhu, Jian

    2017-01-01

    While combinatory antiretroviral therapy (cART) can effectively reduce HIV-1 viremia, it cannot eliminate HIV-1 infection. In the presence of cART, viral reservoirs remain latent, impeding the cure of HIV-1/AIDS. Recently, latency-reversing agents (LRAs) have been developed with the intent of purging latent HIV-1, providing an intriguing strategy for the eradication of the residual viral reservoirs. Our earlier studies show that the first-generation, methyl-triazolo bromodomain, and extra-terminal domain inhibitor (BETi), JQ1, facilitates the reversal of HIV-1 latency. BETis have emerged as a new class of compounds that are promising for this HIV-1 “shock and kill” eradication approach. However, when used as a single drug, JQ1 only modestly reverses HIV-1 latency, which complicates studying the underlining mechanisms. Meanwhile, it has been widely discussed that the induction of latent proviruses is stochastic (Ho et al., 2013). Thus, new BETis are currently under active development with focus on improving potency, ease of synthesis and structural diversity. Using fluorous-tagged multicomponent reactions, we developed a novel second-generation, 3,5-dimethylisoxazole BETi based on an imidazo[1,2-a] pyrazine scaffold, UMB-32. Furthermore, we screened 37 UMB-32 derivatives and identified that one, UMB-136, reactivates HIV-1 in multiple cell models of HIV-1 latency with better efficiency than either JQ1 or UMB-32. UMB-136 enhances HIV-1 transcription and increases viral production through the release of P-TEFb. Importantly, UMB-136 enhances the latency-reversing effects of PKC agonists (prostratin, bryostatin-1) in CD8-depleted PBMCs containing latent viral reservoirs. Our results illustrate that structurally improved BETis, such as UMB-136, may be useful as promising LRAs for HIV-1 eradication. PMID:28638377

  14. CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

    SciTech Connect

    Pritschet, Kathrin; Donhauser, Norbert; Schuster, Philipp; Ries, Moritz; Haupt, Sabrina; Kittan, Nicolai A.; Korn, Klaus; Poehlmann, Stefan; Holland, Gudrun; Bannert, Norbert; Bogner, Elke; Schmidt, Barbara

    2012-02-20

    Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

  15. Trimethylguanosine capping selectively promotes expression of Rev-dependent HIV-1 RNAs.

    PubMed

    Yedavalli, Venkat S R K; Jeang, Kuan-Teh

    2010-08-17

    5'-mRNA capping is an early modification that affects pre-mRNA synthesis/splicing, RNA cytoplasmic transport, and mRNA translation and turnover. In eukaryotes, a 7-methylguanosine (m7G) cap is added to newly transcribed RNA polymerase II (RNAP II) transcripts. A subset of RNAP II-transcribed cellular RNAs, including small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and telomerase RNA, is further hypermethylated at the exocyclic N2 of the guanosine to create a trimethylguanosine (TMG)-capped RNA. Some of these TMG-capped RNAs are transported within the nucleus and from the nucleus to the cytoplasm by the CRM-1 (required for chromosome region maintenance) protein. CRM-1 is also used to export Rev/RRE-dependent unspliced/ partially spliced HIV-1 RNAs. Here we report that like snRNAs and snoRNAs, some Rev/RRE-dependent HIV-1 RNAs are TMG-capped. The methyltransferase responsible for TMG modification of HIV-1 RNAs is the human PIMT (peroxisome proliferator-activated receptor-interacting protein with methyltransferase) protein. TMG capping of unspliced/partially spliced HIV-1 RNAs represents a new regulatory mechanism for selective expression.

  16. Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

    SciTech Connect

    Endsley, Mark A.; Somasunderam, Anoma D.; Li, Guangyu; Oezguen, Numan; Thiviyanathan, Varatharasa; Murray, James L.; Rubin, Donald H.; Hodge, Thomas W.; and others

    2014-04-15

    Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.

  17. 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.

  18. Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells

    NASA Astrophysics Data System (ADS)

    Yu, Xinwei; Feizpour, Amin; Ramirez, Nora-Guadalupe P.; Wu, Linxi; Akiyama, Hisashi; Xu, Fangda; Gummuluru, Suryaram; Reinhard, Björn M.

    2014-06-01

    Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

  19. The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking

    PubMed Central

    Dumas, Audrey; Lê-Bury, Gabrielle; Marie-Anaïs, Florence; Herit, Floriane; Mazzolini, Julie; Guilbert, Thomas; Bourdoncle, Pierre; Russell, David G.; Benichou, Serge; Zahraoui, Ahmed

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1–infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150Glued, and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150Glued, hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1–infected macrophages, leading to strong alterations in phagolysosome biogenesis. PMID:26504171

  20. Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein

    PubMed Central

    Karpowicz, Przemysław; Osmulski, Paweł A.; Witkowska, Julia; Sikorska, Emilia; Giżyńska, Małgorzata; Belczyk-Ciesielska, Agnieszka; Gaczynska, Maria E.; Jankowska, Elżbieta

    2015-01-01

    The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme’s inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4–5 and 8–9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8–9 position was necessary to significantly improve the inhibitory potency. PMID:26575189

  1. The Productive Entry Pathway of HIV-1 in Macrophages Is Dependent on Endocytosis through Lipid Rafts Containing CD4

    PubMed Central

    van Wilgenburg, Bonnie; Moore, Michael D.; James, William S.; Cowley, Sally A.

    2014-01-01

    Macrophages constitute an important reservoir of HIV-1 infection, yet HIV-1 entry into these cells is poorly understood due to the difficulty in genetically manipulating primary macrophages. We developed an effective genetic approach to manipulate the sub-cellular distribution of CD4 in macrophages, and investigated how this affects the HIV-1 entry pathway. Pluripotent Stem Cells (PSC) were transduced with lentiviral vectors designed to manipulate CD4 location and were then differentiated into genetically modified macrophages. HIV-1 infection of these cells was assessed by performing assays that measure critical steps of the HIV-1 lifecycle (fusion, reverse transcription, and expression from HIV-1 integrants). Expression of LCK (which tethers CD4 to the surface of T cells, but is not normally expressed in macrophages) in PSC-macrophages effectively tethered CD4 at the cell surface, reducing its normal endocytic recycling route, and increasing surface CD4 expression 3-fold. This led to a significant increase in HIV-1 fusion and reverse transcription, but productive HIV-1 infection efficiency (as determined by reporter expression from DNA integrants) was unaffected. This implies that surface-tethering of CD4 sequesters HIV-1 into a pathway that is unproductive in macrophages. Secondly, to investigate the importance of lipid rafts (as detergent resistant membranes - DRM) in HIV-1 infection, we generated genetically modified PSC-macrophages that express CD4 mutants known to be excluded from DRM. These macrophages were significantly less able to support HIV-1 fusion, reverse-transcription and integration than engineered controls. Overall, these results support a model in which productive infection by HIV-1 in macrophages occurs via a CD4-raft-dependent endocytic uptake pathway. PMID:24465876

  2. IFITM1 targets HIV-1 latently infected cells for antibody-dependent cytolysis

    PubMed Central

    Raposo, Rui André Saraiva; de Mulder Rougvie, Miguel; Brailey, Phillip M.; Cabido, Vinicius D.; Zdinak, Paul M.; Thomas, Allison S.; Beckerle, Greta A.; Jones, Richard B.; Nixon, Douglas F.

    2017-01-01

    HIV-1 persistence in latent reservoirs during antiretroviral therapy (ART) is the main obstacle to virus eradication. To date, there is no marker that adequately identifies latently infected CD4+ T cells in vivo. Using a well-established ex vivo model, we generated latently infected CD4+ T cells and identified interferon-induced transmembrane protein 1 (IFITM1), a transmembrane antiviral factor, as being overexpressed in latently infected cells. By targeting IFITM1, we showed the efficient and specific killing of a latently infected cell line and CD4+ T cells from ART-suppressed patients through antibody-dependent cytolysis. We hypothesize that IFITM1 could mark natural reservoirs, identifying an immune target for killing of latently infected cells. These novel insights could be explored to develop clinical therapeutic approaches to effectively eradicate HIV-1. PMID:28097226

  3. Thylakoid targeting of Tat passenger proteins shows no delta pH dependence in vivo.

    PubMed

    Finazzi, Giovanni; Chasen, Claudia; Wollman, Francis-André; de Vitry, Catherine

    2003-02-17

    The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane delta pH. In this paper, we assess the delta pH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the delta pH in vivo. Using experimental conditions in which the trans-thylakoid delta pH was almost zero, we found no evidence for a delta pH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a delta pH under physiological conditions, but becomes delta pH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors.

  4. Thylakoid targeting of Tat passenger proteins shows no ΔpH dependence in vivo

    PubMed Central

    Finazzi, Giovanni; Chasen, Claudia; Wollman, Francis-André; de Vitry, Catherine

    2003-01-01

    The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane ΔpH. In this paper, we assess the ΔpH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the ΔpH in vivo. Using experimental conditions in which the trans-thylakoid ΔpH was almost zero, we found no evidence for a ΔpH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a ΔpH under physiological conditions, but becomes ΔpH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors. PMID:12574117

  5. D186/D190 is an allele-dependent determinant of HIV-1 Nef function.

    PubMed

    Imle, Andrea; Stolp, Bettina; Böhmer, Verena; Geyer, Matthias; Raz, Erez; Fackler, Oliver T

    2016-11-01

    The HIV-1 pathogenesis factor Nef interacts with numerous ligands to affect cellular vesicular transport, signal transduction and cytoskeletal dynamics. While most Nef functions depend on multivalent protein interaction motifs, disrupting actin dynamics requires a motif that specifically recruits the host kinase PAK2. An adjacent aspartate was recently predicted to mediate Nef-β-catenin interactions. We report here that β-catenin can be co-immunoprecipitated with Nef.GFP from Jurkat T cell lysates. This association is conserved among lentiviral Nef proteins but does not involve classical Nef protein interaction motifs, including the critical aspartate. While aspartate-to-alanine mutations impaired cell surface receptor downregulation and interference with actin dynamics and cell motility by HIV-1 NA7 Nef, analogous mutations did not affect HIV-1 SF2 Nef function. These allelic differences were determined by a proximal lysine/arginine polymorphism. These results emphasize differences between Nef alleles regarding the functional role of individual residues and underscore the need for allele-specific structure-function analyses. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. 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

  7. 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.

  8. Productive Entry of HIV-1 during Cell-to-Cell Transmission via Dynamin-Dependent Endocytosis

    PubMed Central

    Sloan, Richard D.; Kuhl, Björn D.; Mesplède, Thibault; Münch, Jan; Donahue, Daniel A.

    2013-01-01

    HIV-1 can be transmitted as cell-free virus or via cell-to-cell contacts. Cell-to-cell transmission between CD4+ T cells is the more efficient mode of transmission and is predominant in lymphoid tissue, where the majority of virus resides. Yet the cellular mechanisms underlying productive cell-to-cell transmission in uninfected target cells are unclear. Although it has been demonstrated that target cells can take up virus via endocytosis, definitive links between this process and productive infection remain undefined, and this route of transmission has been proposed to be nonproductive. Here, we report that productive cell-to-cell transmission can occur via endocytosis in a dynamin-dependent manner and is sensitive to clathrin-associated antagonists. These data were obtained in a number of CD4+ T-cell lines and in primary CD4+ T cells, using both CXCR4- and CCR5-tropic virus. However, we also found that HIV-1 demonstrated flexibility in its use of such endocytic pathways as certain allogeneic transmissions were seen to occur in a dynamin-dependent manner but were insensitive to clathrin-associated antagonists. Also, depleting cells of the clathrin accessory protein AP180 led to a viral uptake defect associated with enhanced infection. Collectively, these data demonstrate that endosomal uptake of HIV-1 during cell-to-cell transmission leads to productive infection, but they are also indicative of a flexible model of viral entry during cell-to-cell transmission, in which the virus can alter its entry route according to the pressures that it encounters. PMID:23678185

  9. Productive entry of HIV-1 during cell-to-cell transmission via dynamin-dependent endocytosis.

    PubMed

    Sloan, Richard D; Kuhl, Björn D; Mesplède, Thibault; Münch, Jan; Donahue, Daniel A; Wainberg, Mark A

    2013-07-01

    HIV-1 can be transmitted as cell-free virus or via cell-to-cell contacts. Cell-to-cell transmission between CD4(+) T cells is the more efficient mode of transmission and is predominant in lymphoid tissue, where the majority of virus resides. Yet the cellular mechanisms underlying productive cell-to-cell transmission in uninfected target cells are unclear. Although it has been demonstrated that target cells can take up virus via endocytosis, definitive links between this process and productive infection remain undefined, and this route of transmission has been proposed to be nonproductive. Here, we report that productive cell-to-cell transmission can occur via endocytosis in a dynamin-dependent manner and is sensitive to clathrin-associated antagonists. These data were obtained in a number of CD4(+) T-cell lines and in primary CD4(+) T cells, using both CXCR4- and CCR5-tropic virus. However, we also found that HIV-1 demonstrated flexibility in its use of such endocytic pathways as certain allogeneic transmissions were seen to occur in a dynamin-dependent manner but were insensitive to clathrin-associated antagonists. Also, depleting cells of the clathrin accessory protein AP180 led to a viral uptake defect associated with enhanced infection. Collectively, these data demonstrate that endosomal uptake of HIV-1 during cell-to-cell transmission leads to productive infection, but they are also indicative of a flexible model of viral entry during cell-to-cell transmission, in which the virus can alter its entry route according to the pressures that it encounters.

  10. Therapeutic Immunization with HIV-1 Tat Reduces Immune Activation and Loss of Regulatory T-Cells and Improves Immune Function in Subjects on HAART

    PubMed Central

    Ensoli, Barbara; Bellino, Stefania; Tripiciano, Antonella; Longo, Olimpia; Francavilla, Vittorio; Marcotullio, Simone; Cafaro, Aurelio; Picconi, Orietta; Paniccia, Giovanni; Scoglio, Arianna; Arancio, Angela; Ariola, Cristina; Ruiz Alvarez, Maria J.; Campagna, Massimo; Scaramuzzi, Donato; Iori, Cristina; Esposito, Roberto; Mussini, Cristina; Ghinelli, Florio; Sighinolfi, Laura; Palamara, Guido; Latini, Alessandra; Angarano, Gioacchino; Ladisa, Nicoletta; Soscia, Fabrizio; Mercurio, Vito S.; Lazzarin, Adriano; Tambussi, Giuseppe; Visintini, Raffaele; Mazzotta, Francesco; Di Pietro, Massimo; Galli, Massimo; Rusconi, Stefano; Carosi, Giampiero; Torti, Carlo; Di Perri, Giovanni; Bonora, Stefano; Ensoli, Fabrizio; Garaci, Enrico

    2010-01-01

    Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4+ T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4+ and CD8+ cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4+ T cells and B cells with reduction of CD8+ T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4+ and CD8+ T cells were accompanied by increases of CD4+ and CD8+ T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the

  11. HIV-1 stimulates nuclear entry of amyloid beta via dynamin dependent EEA1 and TGF-β/Smad signaling

    SciTech Connect

    András, Ibolya E. Toborek, Michal

    2014-04-15

    Clinical evidence indicates increased amyloid deposition in HIV-1-infected brains, which contributes to neurocognitive dysfunction in infected patients. Here we show that HIV-1 exposure stimulates amyloid beta (Aβ) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood–brain barrier (BBB). Treatment with HIV-1 and/or Aβ resulted in concurrent increase in early endosomal antigen-1 (EEA1), Smad, and phosphorylated Smad (pSmad) in nuclear fraction of HBMEC. A series of inhibition and silencing studies indicated that Smad and EEA1 closely interact by influencing their own nuclear entry; the effect that was attenuated by dynasore, a blocker of GTP-ase activity of dynamin. Importantly, inhibition of dynamin, EEA1, or TGF-β/Smad effectively attenuated HIV-1-induced Aβ accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aβ involves the dynamin-dependent EEA1 and TGF-β/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level. - Highlights: • HIV-1 induces nuclear accumulation of amyloid beta (Aβ) in brain endothelial cells. • EEA-1 and TGF-Β/Smad act in concert to regulate nuclear entry of Aβ. • Dynamin appropriates the EEA-1 and TGF-Β/Smad signaling. • Dynamin serves as a master regulator of HIV-1-induced nuclear accumulation of Aβ.

  12. Cutting edge: An antibody recognizing ancestral endogenous virus glycoproteins mediates antibody-dependent cellular cytotoxicity on HIV-1-infected cells.

    PubMed

    Michaud, Henri-Alexandre; SenGupta, Devi; de Mulder, Miguel; Deeks, Steven G; Martin, Jeffrey N; Kobie, James J; Sacha, Jonah B; Nixon, Douglas F

    2014-08-15

    The failure of antiviral vaccines is often associated with rapid viral escape from specific immune responses. In the past, conserved epitope or algorithmic epitope selections, such as mosaic vaccines, have been designed to diversify immunity and to circumvent potential viral escape. An alternative approach is to identify conserved stable non-HIV-1 self-epitopes present exclusively in HIV-1-infected cells. We showed previously that human endogenous retroviral (HERV) mRNA transcripts and protein are found in cells of HIV-1-infected patients and that HERV-K (HML-2)-specific T cells can eliminate HIV-1-infected cells in vitro. In this article, we demonstrate that a human anti-HERV-K (HML-2) transmembrane protein Ab binds specifically to HIV-1-infected cells and eliminates them through an Ab-dependent cellular cytotoxicity mechanism in vitro. Thus, Abs directed against epitopes other than HIV-1 proteins may have a role in eliminating HIV-1-infected cells and could be targeted in novel vaccine approaches or immunotherapeutic modalities.

  13. Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1.

    PubMed Central

    SenGupta, D N; Berkhout, B; Gatignol, A; Zhou, A M; Silverman, R H

    1990-01-01

    Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids. HIV-1 leader RNA was found to inhibit translation through two mechanisms. A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA. By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2'-5')oligoadenylate synthetase, were suppressed. In contrast, purified HIV-1 Tat protein produced in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly(A)+ RNA. Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat. The enhancement of translation by Tat was largely due to relief of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1. Images PMID:2120701

  14. HIV-1 infection induces strong production of IP-10 through TLR7/9-dependent pathways

    PubMed Central

    SIMMONS, Rachel P.; SCULLY, Eileen P.; GRODEN, Erin E.; BENEDICT, Kelly F.; CHANG, J. Judy; LANE, Kim; LIFSON, Jeff; ROSENBERG, Eric; LAUFFENBURGER, Douglas A.; ALTFELD, Marcus

    2014-01-01

    Objective To study the cytokine/chemokine profiles in response to HIV-1 viremia, and elucidate the pathways leading to HIV-1-induced inflammation. Design/Methods Plasma levels of 19 cytokines in individuals with early HIV-1 infection and individuals undergoing treatment interruptions were evaluated via multiplex assay. To investigate the cellular sources of relevant cytokines, sorted cells from HIV-1 infected individuals were assessed for mRNA expression. Relevant signaling pathways were assessed by comparing cytokine production patterns of PBMCs stimulated with intact HIV-1 or specific TLR stimulants with and without a TLR7/9 antagonist. Results IP-10 plasma concentration was most significantly associated with HIV-1 viral load and was the most significant contributor in a multivariate model. IP-10 mRNA was highly expressed in monocytes and mDCs and these cells were the dominant producers after in vitro stimulation with TLR7/8 ligands (CL097 and ssRNAGag1166), AT-2 HIV-1, and HIV-1NL43 virus. Partial least square discriminant analysis of culture supernatants revealed distinct cytokine/chemokine secretion profiles associated with intact viruses compared to TLR7/8 ligands alone, with IP-10 production linked to the former. A TLR7/9 antagonist blocked IP-10 production following whole virus stimulation, suggesting the involvement of TLR7/9 in the recognition of HIV-1 by these cells. Conclusions Monocytes and mDCs produce significant amounts of IP-10 in response to HIV-1 viremia and after in vitro stimulation with HIV-1. Stimulation with HIV-1-derived TLR7/8-ligands versus HIV-1 resulted in distinct cytokine/chemokine profiles, indicating additional pathways other than TLR7/8 that lead to the activation of innate immune cells by HIV-1. PMID:24096630

  15. Copper inhibits the HIV-1 protease by both oxygen-dependent and oxygen-independent mechanisms

    SciTech Connect

    Karlstroem, A.R.; Levine, R.L. )

    1991-03-11

    The protease encoded by HIV-1 is essential for the processing of the viral polyproteins encoded by the gag and pol genes into mature viral proteins. Mutation or deletion of the protease gene blocks replication of the virus, making the protease an attractive target for antiviral therapy. The authors found that the HIV-1 protease is inhibited by micromolar concentrations of Cu{sup 2+}. Protease was 50% inhibited by exposure to 5 {mu}M copper for 5 min while exposure to 25 {mu}M caused complete inhibition. This inhibition was not oxygen-dependent and was not reversed by treatment with EDTA, presumably due to the slow off-rate of copper from the protease. Consistent with this interpretation, enzyme activity was recovered after denaturation and refolding of the copper exposed protease. Titration of the inactivated enzyme with Ellman's reagent demonstrated a loss of one of the two sulfhydryl groups present in the molecule, suggesting that copper inhibition was mediated through binding to a cysteine. This was confirmed in studies with a chemically synthesize, mutant protease in which the two cysteine residues were replaced by {alpha}-amino butyrate: The mutant protease was not inhibited by copper. However, both the wild-type and mutant protease were inactivated when exposed to copper, oxygen, and dithiothreitol. This inactivation required oxygen. Thus, the protease can also be inactivated by metal catalyzed oxidation (MCO), a presumably irreversible covalent modification.

  16. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Veillette, Maxime; Désormeaux, Anik; Medjahed, Halima; Gharsallah, Nour-Elhouda; Coutu, Mathieu; Baalwa, Joshua; Guan, Yongjun; Lewis, George; Ferrari, Guido; Hahn, Beatrice H; Haynes, Barton F; Robinson, James E; Kaufmann, Daniel E; Bonsignori, Mattia; Sodroski, Joseph; Finzi, Andrés

    2014-03-01

    Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins. HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.

  17. Functional analysis of TatA and TatB in Streptomyces lividans.

    PubMed

    De Keersmaeker, Sophie; Van Mellaert, Lieve; Lammertyn, Elke; Vrancken, Kristof; Anné, Jozef; Geukens, Nick

    2005-09-30

    Recently, genes encoding TatA, TatB, and TatC homologues were identified in Streptomyces lividans and the functionality of the twin-arginine translocation (Tat) pathway was demonstrated. Previously, we have shown that TatC is indispensable for Tat-dependent secretion in S. lividans. In the present work, we demonstrate that as TatB, S. lividans TatA is important but not essential for efficient secretion of xylanase C and tyrosinase. The results presented here indicate that in the presence of TatC, still partially functional translocation systems composed of TatAC or TatBC can be formed, suggesting that TatA and TatB have at least partially overlapping activities. However, the dissimilar effect caused by a tatA deletion or a tatB deletion on Tat-dependent secretion together with the fact that TatA cannot fully functionally substitute TatB and vice versa indicates that in S. lividans TatA and TatB are not functionally equivalent. Interestingly, soluble GST-tagged TatA and TatB were able to specifically bind Tat-dependent preproteins. The ability to bind Tat-dependent preproteins together with their cytoplasmic localization in S. lividans strongly suggests that both TatA and TatB, independently or associated, serve to recruit Tat-dependent preproteins to the translocase.

  18. Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease.

    PubMed

    Kipp, D Randal; Silva, Rafael G; Schramm, Vern L

    2011-12-07

    Protein motions that occur on the microsecond to millisecond time scale have been linked to enzymatic rates observed for catalytic turnovers, but not to transition-state barrier crossing. It has been hypothesized that enzyme motions on the femtosecond time scale of bond vibrations play a role in transition state formation. Here, we perturb femtosecond motion by substituting all nonexchangeable carbon, nitrogen, and hydrogen atoms with (13)C, (15)N, and (2)H and observe the catalytic effects in HIV-1 protease. According to the Born-Oppenheimer approximation, isotopic substitution alters vibrational frequency with unchanged electrostatic properties. With the use of a fluorescent peptide to report on multiple steps in the reaction, we observe significantly reduced rates in the heavy enzyme relative to the light enzyme. A possible interpretation of our results is that there exists a dynamic link between mass-dependent bond vibrations of the enzyme and events in the reaction coordinate. © 2011 American Chemical Society

  19. Expression of HIV-Tat protein is associated with learning and memory deficits in the mouse

    PubMed Central

    Carey, Amanda N.; Sypek, Elizabeth I.; Singh, Harminder D.; Kaufman, Marc J.; McLaughlin, Jay P.

    2012-01-01

    HIV-Tat protein has been implicated in the pathogenesis of HIV-1 neurological complications (i.e., neuroAIDS), but direct demonstrations of the effects of Tat on behavior are limited. GT-tg mice with a doxycycline (Dox)-inducible and brain-selective tat gene coding for Tat protein were used to test the hypothesis that the activity of Tat in brain is sufficient to impair learning and memory processes. Western blot analysis of GT-tg mouse brains demonstrated an increase in Tat antibody labeling that seemed to be dependent on the dose and duration of Dox pretreatment. Dox-treated GT-tg mice tested in the Barnes maze demonstrated longer latencies to find an escape hole and displayed deficits in probe trial performance, versus uninduced GT-tg littermates, suggesting Tat-induced impairments of spatial learning and memory. Reversal learning was also impaired in Tat-induced mice. Tat-induced mice additionally demonstrated long-lasting (up to one month) deficiencies in novel object recognition learning and memory performance. Furthermore, novel object recognition impairment was dependent on the dose and duration of Dox exposure, suggesting that Tat exposure progressively mediated deficits. These experiments provide evidence that Tat protein expression is sufficient to mediate cognitive abnormalities seen in HIV-infected individuals. Moreover, the genetically engineered GT-tg mouse may be useful for improving our understanding of the neurological underpinnings of neuroAIDS-related behaviors. PMID:22197678

  20. Provirus activation plus CD59 blockage triggers antibody-dependent complement-mediated lysis of latently HIV-1-infected cells.

    PubMed

    Lan, Jie; Yang, Kai; Byrd, Daniel; Hu, Ningjie; Amet, Tohti; Shepherd, Nicole; Desai, Mona; Gao, Jimin; Gupta, Samir; Sun, Yongtao; Yu, Qigui

    2014-10-01

    Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1. Copyright © 2014 by The American Association of Immunologists, Inc.

  1. HIV-1 Inhibits Autophagy in Bystander Macrophage/Monocytic Cells through Src-Akt and STAT3

    PubMed Central

    Van Grol, Jennifer; Subauste, Cecilia; Andrade, Rosa M.; Fujinaga, Koh; Nelson, Julie; Subauste, Carlos S.

    2010-01-01

    Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, β-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1+ patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection. PMID:20661303

  2. PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate Response to HIV-1.

    PubMed

    Yoh, Sunnie M; Schneider, Monika; Seifried, Janna; Soonthornvacharin, Stephen; Akleh, Rana E; Olivieri, Kevin C; De Jesus, Paul D; Ruan, Chunhai; de Castro, Elisa; Ruiz, Pedro A; Germanaud, David; des Portes, Vincent; García-Sastre, Adolfo; König, Renate; Chanda, Sumit K

    2015-06-04

    Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. PQBP1 is a Proximal Sensor of the cGAS-dependent Innate Response to HIV-1

    PubMed Central

    Yoh, Sunnie M.; Schneider, Monika; Akleh, Rana E.; Olivieri, Kevin C.; De Jesus, Paul D.; Ruan, Chunhai; de Castro, Elisa; Ruiz, Pedro A.; Germanaud, David; des Portes, Vincent; García-Sastre, Adolfo; König, Renate; Chanda, Sumit K.

    2015-01-01

    Summary Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning Syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection. PMID:26046437

  4. Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG.

    PubMed

    Kramski, Marit; Lichtfuss, Gregor F; Navis, Marjon; Isitman, Gamze; Wren, Leia; Rawlin, Grant; Center, Rob J; Jaworowski, Anthony; Kent, Stephen J; Purcell, Damian F J

    2012-10-01

    Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Enhanced nuclear factor-kappa B activation induced by tumour necrosis factor-alpha in stably tat-transfected cells is associated with the presence of cell-surface-bound Tat protein.

    PubMed

    Ramazzotti, E; Vignoli, M; Re, M C; Furlini, G; La Placa, M

    1996-05-01

    An enhanced nuclear factor (NF)-kappa B activation in response to tumour necrosis factor (TNF)-alpha has been observed in stably tat-transfected cells. Recent experimental evidence suggests that Tat may autocrinously influence both cellular physiology and HIV-1 long terminal repeat-directed gene expression in Tat-producing cells. Therefore, the possible association of a Tat autocrinous loop with the enhanced NF-kappa B-binding activity induced by TNF-alpha in Tat-producing cells was studied by anti-Tat antibody blocking experiments. Permanently tat-transfected Jurkat cells, maintained either in the presence or absence of anti-Tat antibody, were studied for the presence of TNF-alpha-induced NF-kappa B-binding activity (quantified by electrophoretic mobility shift assays) and the presence of cell-surface-bound Tat (determined by flow cytometry and confocal microscopy of anti-Tat immunofluorescence-stained cell preparations. The enhanced production of TNF-alpha-induced NF-kappa B binding activity exhibited by tat-transfected Jurkat cells was completely abolished in cell cultures maintained in the presence of anti-Tat antibody, thus indicating that the increased TNF-alpha-induced NF-kappa B binding activity observed in Jurkat-tat cells was dependent on the presence of Tat protein in an antibody-accessible location. In accordance with these findings, immunofluorescence-stained preparations of unfixed tat-transfected Jurkat cells showed the presence of cell-surface-bound Tat protein which was completely absent in cells incubated in the presence of anti-Tat antibodies. This study demonstrates that the enhanced NF-kappa B activation exhibited by stably tat-transfected cells in response to TNF-alpha, is associated with cell surface interaction of extracellularly released Tat protein. These data add further evidence to the possible relevance of a Tat autocrinous loop in the physiology of Tat-producing cells and suggest that in HIV-1-infected cells Tat is likely to behave as

  6. A Novel Class of HIV-1 Antiviral Agents Targeting HIV via a SUMOylation-Dependent Mechanism

    PubMed Central

    Madu, Ikenna G.; Li, Shirley; Li, Baozong; Li, Haitang; Chang, Tammy; Li, Yi-Jia; Vega, Ramir; Rossi, John; Yee, Jiing-Kuan; Zaia, John; Chen, Yuan

    2015-01-01

    We have recently identified a chemotype of small ubiquitin-like modifier (SUMO)-specific protease (SENP) inhibitors. Prior to the discovery of their SENP inhibitory activity, these compounds were found to inhibit HIV replication, but with an unknown mechanism. In this study, we investigated the mechanism of how these compounds inhibit HIV-1. We found that they do not affect HIV-1 viral production, but significantly inhibited the infectivity of the virus. Interestingly, virions produced from cells treated with these compounds could gain entry and carry out reverse transcription, but could not efficiently integrate into the host genome. This phenotype is different from the virus produced from cells treated with the class of anti-HIV-1 agents that inhibit HIV protease. Upon removal of the SUMO modification sites in the HIV-1 integrase, the compound no longer alters viral infectivity, indicating that the effect is related to SUMOylation of the HIV integrase. This study identifies a novel mechanism for inhibiting HIV-1 integration and a new class of small molecules that inhibits HIV-1 via such mechanism that may contribute a new strategy for cure of HIV-1 by inhibiting the production of infectious virions upon activation from latency. PMID:26643614

  7. HIV-1 stimulates nuclear entry of amyloid beta via dynamin dependent EEA1 and TGF-β/Smad signaling.

    PubMed

    András, Ibolya E; Toborek, Michal

    2014-04-15

    Clinical evidence indicates increased amyloid deposition in HIV-1-infected brains, which contributes to neurocognitive dysfunction in infected patients. Here we show that HIV-1 exposure stimulates amyloid beta (Aβ) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood-brain barrier (BBB). Treatment with HIV-1 and/or Aβ resulted in concurrent increase in early endosomal antigen-1 (EEA1), Smad, and phosphorylated Smad (pSmad) in nuclear fraction of HBMEC. A series of inhibition and silencing studies indicated that Smad and EEA1 closely interact by influencing their own nuclear entry; the effect that was attenuated by dynasore, a blocker of GTP-ase activity of dynamin. Importantly, inhibition of dynamin, EEA1, or TGF-β/Smad effectively attenuated HIV-1-induced Aβ accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aβ involves the dynamin-dependent EEA1 and TGF-β/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level.

  8. CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription.

    PubMed Central

    Suñé, C; Hayashi, T; Liu, Y; Lane, W S; Young, R A; Garcia-Blanco, M A

    1997-01-01

    Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150. PMID:9315662

  9. 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.

  10. 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

  11. HIC1 controls cellular- and HIV-1- gene transcription via interactions with CTIP2 and HMGA1

    PubMed Central

    Le Douce, Valentin; Forouzanfar, Faezeh; Eilebrecht, Sebastian; Van Driessche, Benoit; Ait-Ammar, Amina; Verdikt, Roxane; Kurashige, Yoshihito; Marban, Céline; Gautier, Virginie; Candolfi, Ermanno; Benecke, Arndt G.; Van Lint, Carine; Rohr, Olivier; Schwartz, Christian

    2016-01-01

    Among many cellular transcriptional regulators, Bcl11b/CTIP2 and HGMA1 have been described to control the establishment and the persistence of HIV-1 latency in microglial cells, the main viral reservoir in the brain. In this present work, we identify and characterize a transcription factor i.e. HIC1, which physically interacts with both Bcl11b/CTIP2 and HMGA1 to co-regulate specific subsets of cellular genes and the viral HIV-1 gene. Our results suggest that HIC1 represses Tat dependent HIV-1 transcription. Interestingly, this repression of Tat function is linked to HIC1 K314 acetylation status and to SIRT1 deacetylase activity. Finally, we show that HIC1 interacts and cooperates with HGMA1 to regulate Tat dependent HIV-1 transcription. Our results also suggest that HIC1 repression of Tat function happens in a TAR dependent manner and that this TAR element may serve as HIC1 reservoir at the viral promoter to facilitate HIC1/TAT interaction. PMID:27725726

  12. Extensive complement-dependent enhancement of HIV-1 by autologous non-neutralising antibodies at early stages of infection

    PubMed Central

    2011-01-01

    Background Non-neutralising antibodies to the envelope glycoprotein are elicited during acute HIV-1 infection and are abundant throughout the course of disease progression. Although these antibodies appear to have negligible effects on HIV-1 infection when assayed in standard neutralisation assays, they have the potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Here we report that non-neutralising antibodies produced early in response to HIV-1 infection can enhance viral infectivity. Results We investigated this complement-mediated antibody-dependent enhancement (C'-ADE) of early HIV infection by carrying out longitudinal studies with primary viruses and autologous sera derived sequentially from recently infected individuals, using a T cell line naturally expressing the complement receptor 2 (CR2; CD21). The C'-ADE was consistently observed and in some cases achieved infection-enhancing levels of greater than 350-fold, converting a low-level infection to a highly destructive one. C'-ADE activity declined as a neutralising response to the early virus emerged, but later virus isolates that had escaped the neutralising response demonstrated an increased capacity for enhanced infection by autologous antibodies. Moreover, sera with autologous enhancing activity were capable of C'ADE of heterologous viral isolates, suggesting the targeting of conserved epitopes on the envelope glycoprotein. Ectopic expression of CR2 on cell lines expressing HIV-1 receptors was sufficient to render them sensitive to C'ADE. Conclusions Taken together, these results suggest that non-neutralising antibodies to the HIV-1 envelope that arise during acute infection are not 'passive', but in concert with complement and complement receptors may have consequences for HIV-1 dissemination and pathogenesis. PMID:21401915

  13. Epigenetic alterations in the brain associated with HIV-1 infection and methamphetamine dependence.

    PubMed

    Desplats, Paula; Dumaop, Wilmar; Cronin, Peter; Gianella, Sara; Woods, Steven; Letendre, Scott; Smith, David; Masliah, Eliezer; Grant, Igor

    2014-01-01

    HIV involvement of the CNS continues to be a significant problem despite successful use of combination antiretroviral therapy (cART). Drugs of abuse can act in concert with HIV proteins to damage glia and neurons, worsening the neurotoxicity caused by HIV alone. Methamphetamine (METH) is a highly addictive psychostimulant drug, abuse of which has reached epidemic proportions and is associated with high-risk sexual behavior, increased HIV transmission, and development of drug resistance. HIV infection and METH dependence can have synergistic pathological effects, with preferential involvement of frontostriatal circuits. At the molecular level, epigenetic alterations have been reported for both HIV-1 infection and drug abuse, but the neuropathological pathways triggered by their combined effects are less known. We investigated epigenetic changes in the brain associated with HIV and METH. We analyzed postmortem frontal cortex tissue from 27 HIV seropositive individuals, 13 of which had a history of METH dependence, in comparison to 14 cases who never used METH. We detected changes in the expression of DNMT1, at mRNA and protein levels, that resulted in the increase of global DNA methylation. Genome-wide profiling of DNA methylation in a subset of cases, showed differential methylation on genes related to neurodegeneration; dopamine metabolism and transport; and oxidative phosphorylation. We provide evidence for the synergy of HIV and METH dependence on the patterns of DNA methylation on the host brain, which results in a distinctive landscape for the comorbid condition. Importantly, we identified new epigenetic targets that might aid in understanding the aggravated neurodegenerative, cognitive, motor and behavioral symptoms observed in persons living with HIV and addictions.

  14. Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

    PubMed Central

    Desplats, Paula; Dumaop, Wilmar; Cronin, Peter; Gianella, Sara; Woods, Steven; Letendre, Scott; Smith, David; Masliah, Eliezer; Grant, Igor

    2014-01-01

    HIV involvement of the CNS continues to be a significant problem despite successful use of combination antiretroviral therapy (cART). Drugs of abuse can act in concert with HIV proteins to damage glia and neurons, worsening the neurotoxicity caused by HIV alone. Methamphetamine (METH) is a highly addictive psychostimulant drug, abuse of which has reached epidemic proportions and is associated with high-risk sexual behavior, increased HIV transmission, and development of drug resistance. HIV infection and METH dependence can have synergistic pathological effects, with preferential involvement of frontostriatal circuits. At the molecular level, epigenetic alterations have been reported for both HIV-1 infection and drug abuse, but the neuropathological pathways triggered by their combined effects are less known. We investigated epigenetic changes in the brain associated with HIV and METH. We analyzed postmortem frontal cortex tissue from 27 HIV seropositive individuals, 13 of which had a history of METH dependence, in comparison to 14 cases who never used METH. We detected changes in the expression of DNMT1, at mRNA and protein levels, that resulted in the increase of global DNA methylation. Genome-wide profiling of DNA methylation in a subset of cases, showed differential methylation on genes related to neurodegeneration; dopamine metabolism and transport; and oxidative phosphorylation. We provide evidence for the synergy of HIV and METH dependence on the patterns of DNA methylation on the host brain, which results in a distinctive landscape for the comorbid condition. Importantly, we identified new epigenetic targets that might aid in understanding the aggravated neurodegenerative, cognitive, motor and behavioral symptoms observed in persons living with HIV and addictions. PMID:25054922

  15. DYRK1A Controls HIV-1 Replication at a Transcriptional Level in an NFAT Dependent Manner

    PubMed Central

    Booiman, Thijs; Loukachov, Vladimir V.; van Dort, Karel A.; van ’t Wout, Angélique B.; Kootstra, Neeltje A.

    2015-01-01

    Background Transcription of the HIV-1 provirus is regulated by both viral and host proteins and is very important in the context of viral latency. In latently infected cells, viral gene expression is inhibited as a result of the sequestration of host transcription factors and epigenetic modifications. Results In our present study we analyzed the effect of host factor dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) on HIV-1 replication. We show that DYRK1A controls HIV-1 replication by regulating provirus transcription. Downregulation or inhibition of DYRK1A increased LTR-driven transcription and viral replication in cell lines and primary PBMC. Furthermore, inhibition of DYRK1A resulted in reactivation of latent HIV-1 provirus to a similar extent as two commonly used broad-spectrum HDAC inhibitors. We observed that DYRK1A regulates HIV-1 transcription via the Nuclear Factor of Activated T-cells (NFAT) by promoting its translocation from the nucleus to the cytoplasm. Therefore, inhibition of DYRK1A results in increased nuclear levels of NFAT and increased NFAT binding to the viral LTR and thus increasing viral transcription. Conclusions Our data indicate that host factor DYRK1A plays a role in the regulation of viral transcription and latency. Therefore, DYRK1A might be an attractive candidate for therapeutic strategies targeting the viral reservoir. PMID:26641855

  16. Functional advantage of educated KIR2DL1(+) natural killer cells for anti-HIV-1 antibody-dependent activation.

    PubMed

    Gooneratne, S L; Center, R J; Kent, S J; Parsons, M S

    2016-04-01

    Evidence from the RV144 HIV-1 vaccine trial implicates anti-HIV-1 antibody-dependent cellular cytotoxicity (ADCC) in vaccine-conferred protection from infection. Among effector cells that mediate ADCC are natural killer (NK) cells. The ability of NK cells to be activated in an antibody-dependent manner is reliant upon several factors. In general, NK cell-mediated antibody-dependent activation is most robust in terminally differentiated CD57(+) NK cells, as well as NK cells educated through ontological interactions between inhibitory killer immunoglobulin-like receptors (KIR) and their major histocompatibility complex class I [MHC-I or human leucocyte antigen (HLA-I)] ligands. With regard to anti-HIV-1 antibody-dependent NK cell activation, previous research has demonstrated that the epidemiologically relevant KIR3DL1/HLA-Bw4 receptor/ligand combination confers enhanced activation potential. In the present study we assessed the ability of the KIR2DL1/HLA-C2 receptor/ligand combination to confer enhanced activation upon direct stimulation with HLA-I-devoid target cells or antibody-dependent stimulation with HIV-1 gp140-pulsed CEM.NKr-CCR5 target cells in the presence of an anti-HIV-1 antibody source. Among donors carrying the HLA-C2 ligand for KIR2DL1, higher interferon (IFN)-γ production was observed within KIR2DL1(+) NK cells than in KIR2DL1(-) NK cells upon both direct and antibody-dependent stimulation. No differences in KIR2DL1(+) and KIR2DL1(-) NK cell activation were observed in HLA-C1 homozygous donors. Additionally, higher activation in KIR2DL1(+) than KIR2DL1(-) NK cells from HLA-C2 carrying donors was observed within less differentiated CD57(-) NK cells, demonstrating that the observed differences were due to education and not an overabundance of KIR2DL1(+) NK cells within differentiated CD57(+) NK cells. These observations are relevant for understanding the regulation of anti-HIV-1 antibody-dependent NK cell responses.

  17. Translation of Pur-α is targeted by cellular miRNAs to modulate the differentiation-dependent susceptibility of monocytes to HIV-1 infection.

    PubMed

    Shen, Chan-Juan; Jia, Yan-Hui; Tian, Ren-Rong; Ding, Ming; Zhang, Chiyu; Wang, Jian-Hua

    2012-11-01

    The postentry restriction of HIV-1 replication in monocytes can be relieved when they differentiate to dendritic cells (DCs) or macrophages. Multiple mechanisms have been proposed to interpret the differentiation-dependent susceptibility of monocytes to HIV-1 infection, and the absence of host-cell-encoded essential factors for HIV-1 completing the life cycle may provide an explanation. We have analyzed the gene expression profile in monocytes by mRNA microarray and compared it with that of differentiated DCs. We demonstrated that purine-rich element binding protein α (Pur-α), a host-cell-encoded ubiquitous, sequence-specific DNA- and RNA-binding protein, showed inadequate expression in monocytes, and the translation of Pur-α mRNA was repressed by cell-expressed microRNA (miRNA). These Pur-α-targeted miRNAs modulated the differentiation-dependent susceptibility of monocytes/DCs to HIV-1 infection, because rescue of Pur-α expression by transfection of miRNA inhibitors relieved the restriction of HIV-1 infection in monocytes, and ectopic input of miRNA mimics significantly reduced HIV-1 infection of monocyte-derived DCs (MDDCs). Collectively, our data emphasized that inadequate host factors contribute to HIV-1 restriction in monocytes, and cellular miRNAs modulate differentiation-dependent susceptibility of host cells to HIV-1 infection. Elaboration of HIV-1 restriction in host cells facilitates our understanding of viral pathogenesis and the search for a new antiviral strategy.

  18. Environmental Salinity Determines the Specificity and Need for Tat-Dependent Secretion of the YwbN Protein in Bacillus subtilis

    PubMed Central

    van der Ploeg, René; Mäder, Ulrike; Homuth, Georg; Schaffer, Marc; Denham, Emma L.; Monteferrante, Carmine G.; Miethke, Marcus; Marahiel, Mohamed A.; Harwood, Colin R.; Winter, Theresa; Hecker, Michael; Antelmann, Haike; van Dijl, Jan Maarten

    2011-01-01

    Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described “minimal Tat translocase” consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate. PMID:21479178

  19. Activation of tat-defective human immunodeficiency virus by ultraviolet light

    SciTech Connect

    Sadaie, M.R.; Tschachler, E.; Valerie, K.; Rosenberg, M.; Felber, B.K.; Pavlakis, G.N.; Klotman, M.E.; Wong-Staal, F. )

    1990-05-01

    Ultraviolet light (UV) is known to cause activation of gene expression from the human immunodeficiency virus type 1 (HIV-1) promoter. To address the question of whether tat-defective HIV-1 provirus could be rescued by UV irradiation we examined its effect on HeLa cells containing integrated proviruses with tat mutations. Exposure of these cells to an optimal dose of UV resulted in the production of infectious viruses. The degree of UV activation and reversion to infectious virus appeared to depend on the nature of the original tat mutation. Two of the mutants required cocultivation with tat-expressing cells to fully generate replication competent viruses, while a third mutant required only cocultivation with H9 cells. Sequencing of cDNA from cells infected with this last mutant demonstrated that the parental mutant sequence was retained and that genotypic revertants to the wild-type as well as new mutant sequences were generated. These results suggest that tat-defective HIV-1 provirus can be activated by UV and can subsequently revert to wild-type virus. This study raises the possibility that UV exposure of immune cells in the skin plays a role in the activation of defective HIV-1 in vivo.

  20. Glycosylation patterns of HIV-1 gp120 depend on the type of expressing cells and affect antibody recognition.

    PubMed

    Raska, Milan; Takahashi, Kazuo; Czernekova, Lydie; Zachova, Katerina; Hall, Stacy; Moldoveanu, Zina; Elliott, Matt C; Wilson, Landon; Brown, Rhubell; Jancova, Dagmar; Barnes, Stephen; Vrbkova, Jana; Tomana, Milan; Smith, Phillip D; Mestecky, Jiri; Renfrow, Matthew B; Novak, Jan

    2010-07-02

    Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.

  1. Host Proteins Ku and HMGA1 As Participants of HIV-1 Transcription

    PubMed Central

    Shadrina, O. A.; Knyazhanskaya, E. S.; Korolev, S.P.; Gottikh, M. B.

    2016-01-01

    Human immunodeficiency virus type 1 is known to use the transcriptional machinery of the host cell for viral gene transcription, and the only viral protein that partakes in this process is Tat, the viral trans-activator of transcription. During acute infection, the binding of Tat to the hairpin at the beginning of the transcribed viral RNA recruits the PTEFb complex, which in turn hyperphosphorylates RNA-polymerase II and stimulates transcription elongation. Along with acute infection, HIV-1 can also lead to latent infection that is characterized by a low level of viral transcription. During the maintenance and reversal of latency, there are no detectable amounts of Tat protein in the cell and the mechanism of transcription activation in the absence of Tat protein remains unclear. The latency maintenance is also a problematic question. It seems evident that cellular proteins with a yet unknown nature or role regulate both transcriptional repression in the latent phase and its activation during transition into the lytic phase. The present review discusses the role of cellular proteins Ku and HMGA1 in the initiation of transcription elongation of the HIV-1 provirus. The review presents data regarding Ku-mediated HIV-1 transcription and its dependence on the promoter structure and the shape of viral DNA. We also describe the differential influence of the HMGA1 protein on the induced and basal transcription of HIV-1. Finally, we offer possible mechanisms for Ku and HMGA1 proteins in the proviral transcription regulation. PMID:27099783

  2. A Family History of Substance Dependence Obscures the Group Differences in Brain Function Associated with HIV-1 and ART

    PubMed Central

    Bauer, L.O.

    2012-01-01

    BACKGROUND Recently, the NIH called for additional research on the topic of viral and host factors contributing to impaired cognitive and neural function in HIV/AIDS patients and their response to antiretroviral treatment. This investigation responds to that call by examining a host factor, a family history of substance dependence, often overlooked in cognitive and neuroimaging studies of HIV/AIDS. METHODS We categorized 146 HIV-1 seropositive patients receiving antiretroviral treatment (ART) and 92 seronegative volunteers by the presence or absence of alcohol, cocaine, or heroin dependence affecting a biological parent. Seropositive patients were further categorized by the estimated ability of their individual ART regimens to penetrate the CNS. The indicator of brain function was a 3–7 Hz oscillatory electroencephalographic response (theta ERO) evoked by target stimuli presented during a simple selective attention task. RESULTS The analysis revealed that the presence of a family history of substance dependence obscured the reduction in frontal theta ERO power accompanying the presence of HIV-1 as well as the improvement in frontal theta ERO power accompanying treatment with ART agents estimated to have greater (n=41) versus lesser (n=105) CNS penetrance. Secondary analyses employing sLORETA source localization techniques revealed that the source of the theta ERO response was similarly reduced by the presence of either HIV-1 or a family history of substance dependence. CONCLUSIONS We conclude that a family history of substance dependence complicates and obscures the subtle neurophysiological changes which typically accompany HIV/AIDS and ART. Studies of new therapeutic agents for HIV-1-associated cognitive and neurophysiological impairments must consider this complication and exclude or control it. PMID:22749564

  3. A family history of substance dependence obscures the group differences in brain function associated with HIV-1 and ART.

    PubMed

    Bauer, L O

    2013-01-01

    Recently, the NIH called for additional research on the topic of viral and host factors contributing to impaired cognitive and neural function in HIV/AIDS patients and their response to antiretroviral treatment. This investigation responds to that call by examining a host factor, a family history of substance dependence, often overlooked in cognitive and neuroimaging studies of HIV/AIDS. We categorized 146 HIV-1 seropositive patients receiving antiretroviral treatment (ART) and 92 seronegative volunteers by the presence or absence of alcohol, cocaine, or heroin dependence affecting a biological parent. Seropositive patients were further categorized by the estimated ability of their individual ART regimens to penetrate the CNS. The indicator of brain function was a 3-7Hz oscillatory electroencephalographic response (theta ERO) evoked by target stimuli presented during a simple selective attention task. The analysis revealed that the presence of a family history of substance dependence obscured the reduction in frontal theta ERO power accompanying the presence of HIV-1 as well as the improvement in frontal theta ERO power accompanying treatment with ART agents estimated to have greater (n=41) versus lesser (n=105) CNS penetrance. Secondary analyses employing sLORETA source localization techniques revealed that the source of the theta ERO response was similarly reduced by the presence of either HIV-1 or a family history of substance dependence. We conclude that a family history of substance dependence complicates and obscures the subtle neurophysiological changes which typically accompany HIV/AIDS and ART. Studies of new therapeutic agents for HIV-1-associated cognitive and neurophysiological impairments must consider this complication and exclude or control it. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  4. RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates.

    PubMed

    Kerr, S G; Anderson, K S

    1997-11-18

    The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.

  5. Molecular Architecture of the Cleavage-Dependent Mannose Patch on a Soluble HIV-1 Envelope Glycoprotein Trimer

    PubMed Central

    Behrens, Anna-Janina; Harvey, David J.; Milne, Emilia; Cupo, Albert; Kumar, Abhinav; Zitzmann, Nicole; Struwe, Weston B.; Moore, John P.

    2016-01-01

    ABSTRACT The formation of a correctly folded and natively glycosylated HIV-1 viral spike is dependent on protease cleavage of the gp160 precursor protein in the Golgi apparatus. Cleavage induces a compact structure which not only renders the spike capable of fusion but also limits further maturation of its extensive glycosylation. The redirection of the glycosylation pathway to preserve underprocessed oligomannose-type glycans is an important feature in immunogen design, as glycans contribute to or influence the epitopes of numerous broadly neutralizing antibodies. Here we present a quantitative site-specific analysis of a recombinant, trimeric mimic of the native HIV-1 viral spike (BG505 SOSIP.664) compared to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. We present a detailed molecular map of a trimer-associated glycan remodeling that forms a localized subdomain of the native mannose patch. The formation of native trimers is a critical design feature in shaping the glycan epitopes presented on recombinant vaccine candidates. IMPORTANCE The envelope spike of human immunodeficiency virus type 1 (HIV-1) is a target for antibody-based neutralization. For some patients infected with HIV-1, highly potent antibodies have been isolated that can neutralize a wide range of circulating viruses. It is a goal of HIV-1 vaccine research to elicit these antibodies by immunization with recombinant mimics of the viral spike. These antibodies have evolved to recognize the dense array of glycans that coat the surface of the viral molecule. We show how the structure of these glycans is shaped by steric constraints imposed upon them by the native folding of the viral spike. This information is important in guiding the development of vaccine candidates. PMID:27807235

  6. Expansion of monocytic myeloid-derived suppressor cells dampens T cell function in HIV-1-seropositive individuals.

    PubMed

    Qin, Aiping; Cai, Weiping; Pan, Ting; Wu, Kang; Yang, Qiong; Wang, Nina; Liu, Yufeng; Yan, Dehong; Hu, Fengyu; Guo, Pengle; Chen, Xiaoping; Chen, Ling; Zhang, Hui; Tang, Xiaoping; Zhou, Jie

    2013-02-01

    T lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1 infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLA-DR(-/low) CD11b(+) CD33(+/high) CD14(+) CD15(-) cells) in the peripheral blood of HIV-1-seropositive subjects (n = 61) compared with healthy controls (n = 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1(+) subjects correlated with prognostic HIV-1 disease markers, including the HIV-1 load (r = 0.5957; P < 0.0001), CD4(+) T cell loss (r = -0.5312; P < 0.0001), and activated T cells (r = 0.4421; P = 0.0004). Functional studies showed that M-MDSCs from HIV-1(+) subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manners; this effect was dependent on the induction of arginase 1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generation in vitro, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating that a positive-feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechanism of T cell dysfunction in HIV-1-infected individuals and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.

  7. 1E7-03, a low MW compound targeting host protein phosphatase-1, inhibits HIV-1 transcription

    PubMed Central

    Ammosova, Tatyana; Platonov, Maxim; Ivanov, Andrei; Kont, Yasemin Saygideğer; Kumari, Namita; Kehn-Hall, Kylene; Jerebtsova, Marina; Kulkarni, Amol A; Üren, Aykut; Kovalskyy, Dmytro; Nekhai, Sergei

    2014-01-01

    Background and Purpose HIV-1 transcription is activated by the Tat protein which recruits the cyclin-dependent kinase CDK9/cyclin T1 to TAR RNA. Tat binds to protein phosphatase-1 (PP1) through the Q35VCF38 sequence and translocates PP1 to the nucleus. PP1 dephosphorylates CDK9 and activates HIV-1 transcription. We have synthesized a low MW compound 1H4, that targets PP1 and prevents HIV-1 Tat interaction with PP1 and inhibits HIV-1 gene transcription. Here, we report our further work with the 1H4-derived compounds and analysis of their mechanism of action. Experimental Approach Using the 1H4-PP1 complex as a model, we iteratively designed and synthesized follow-up libraries that were analysed for the inhibition of HIV-1 transcription and toxicity. We also confirmed the mechanism of action of the PP1-targeting molecules by determining the affinity of binding of these molecules to PP1, by analysing their effects on PP1 activity, disruption of PP1 binding to Tat and shuttling of PP1 to the nucleus. Key Results We identified a tetrahydroquinoline derivative, compound 7, which disrupted the interaction of Tat with PP1. We further optimized compound 7 and obtained compound 7c, renamed 1E7-03, which inhibited HIV-1 with low IC50 (fivefold lower than the previously reported compound, 1H4), showed no cytotoxicity and displayed a plasma half-life greater than 8 h in mice. 1E7-03 bound to PP1 in vitro and prevented shuttling of PP1 into the nucleus. Conclusions and Implications Our study shows that low MW compounds that functionally mimic the PP1-binding RVxF peptide can inhibit HIV-1 transcription by deregulating PP1. PMID:25073485

  8. Expression of an RNA glycosidase inhibits HIV-1 transactivation of transcription.

    PubMed

    Kutky, Meherzad; Hudak, Katalin A

    2017-10-05

    HIV-1 (human immunodeficiency virus) transcription is primarily controlled by the virally encoded Tat (transactivator of transcription) protein and its interaction with the viral TAR (transcription response element) RNA element. Specifically, binding of a Tat-containing complex to TAR recruits cellular factors that promote elongation of the host RNA polymerase engaging the viral DNA template. Disruption of this interaction halts viral RNA transcription. In the present study, we investigated the effect of pokeweed antiviral protein (PAP), an RNA glycosidase (EC#: 3.2.2.22) synthesized by the pokeweed plant (Phytolacca americana), on transcription of HIV-1 mRNA. We show that co-expression of PAP with a proviral clone in culture cells resulted in a Tat-dependent decrease in viral mRNA levels. PAP reduced HIV-1 transcriptional activity by inhibiting Tat protein synthesis. The effects of PAP expression on host factors AP-1 (activator protein 1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) and specificity protein 1, which modulate HIV-1 transcription by binding to the viral LTR (5'-long terminal repeat), were also investigated. Only AP-1 showed a modest JNK pathway-dependent increase in activity in the presence of PAP; however, this activation was not sufficient to significantly enhance transcription from a partial viral LTR containing AP-1 binding sites. Therefore, the primary effect of PAP on HIV-1 transcription is to reduce viral RNA synthesis by decreasing the abundance of Tat. These findings provide a mechanistic explanation for the observed decrease in viral RNAs in cells expressing PAP and contribute to our understanding of the antiviral effects of this plant protein. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  9. Decay dynamics of HIV-1 depend on the inhibited stages of the viral life cycle.

    PubMed

    Sedaghat, Ahmad R; Dinoso, Jason B; Shen, Lin; Wilke, Claus O; Siliciano, Robert F

    2008-03-25

    The time to suppression of HIV-1 viremia to below the limit of detection of standard clinical assays is an important prognostic indicator for patients on highly active antiretroviral therapy (HAART). Recent clinical trials of the integrase inhibitor raltegravir have demonstrated more rapid viral decay than previously seen with reverse transcriptase (RT) or protease inhibitor-based regimens. Because of the therapeutic importance of drugs that target different steps in the virus life cycle, it is imperative to consider whether viral dynamics are affected by the stage of the viral life cycle at which an antiretroviral drug acts. We use a mathematical model to investigate the effects of various drug classes on the dynamics of HIV-1 decay and show that the stage at which a drug acts affects the dynamics of viral decay. We find that the drug class acting latest in the viral life cycle dictates the dynamics of HIV-1 decay. In general, we find that the later in the life cycle an inhibitor acts, the more rapid the decay in viremia, and we illustrate this by comparing the effect of RT and integrase inhibitors on viral dynamics. We conclude that the rapid decay observed in patients on integrase-inhibitor-containing regimens is not necessarily an indication of greater drug efficacy but rather an expected consequence of the fact that this drug acts later in the life cycle. We propose that clinically observed viral decay rates for HAART regimens should be evaluated in the context of the drug classes that are represented.

  10. Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

    PubMed Central

    Reh, Lucia; Magnus, Carsten; Schanz, Merle; Weber, Jacqueline; Uhr, Therese; Rusert, Peter; Trkola, Alexandra

    2015-01-01

    An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the

  11. Conformation of trimeric envelope glycoproteins: the CD4-dependent membrane fusion mechanism of HIV-1.

    PubMed

    Yingliang, Wu; Hong, Yi; Zhijian, Cao; Wenxin, Li

    2007-08-01

    The HIV-1 envelope glycoproteins are assembled by the trimeric gp120s and gp41s proteins. The gp120 binds sequentially to CD4 and coreceptor for initiating virus entry. Because of noncovalent interaction and heavy glycosylation for envelope glycoproteins, it is highly difficult to determine entire envelope glycoproteins structure now. Such question extremely limits our good understanding of HIV-1 membrane fusion mechanism. Here, a novel and reasonable assembly model of trimeric gp120s and gp41s was proposed based on the conformational dynamics of trimeric gp120-gp41 complex and gp41, respectively. As for gp41, the heptad repeat sequences in the gp41 C-terminal is of enormous flexibility. On the contrary, the heptad repeat sequences in the gp41 N-terminal likely present stable three-helical bundle due to strong nonpolar interaction, and they were predicted to associate three alpha1 helixes from the non-neutralizing face of the gp120 inner domain, which is quite similar to gp41 fusion core structure. Such interaction likely leads to the formation of noncovalent gp120-gp41 complex. In the proposed assembly of trimeric gp120-gp41 complex, three gp120s present not only perfectly complementary and symmetrical distribution around the gp41, but also different flexibility degree in the different structural domains. Thus, the new model can well explain numerous experimental phenomena, present plenty of structural information, elucidate effectively HIV-1 membrane fusion mechanism, and direct to further develop vaccine and novel fusion inhibitors.

  12. Tat acetylation modulates assembly of a viral-host RNA–protein transcription complex

    PubMed Central

    D'Orso, Iván; Frankel, Alan D.

    2009-01-01

    HIV-1 Tat enhances viral transcription elongation by forming a ribonucleoprotein complex with transactivating responsive (TAR) RNA and P-TEFb, an elongation factor composed of cyclin T1 (CycT1) and Cdk9 that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have shown that Lys-28 in the activation domain (AD) of Tat is essential for HIV-1 transcription and replication and is acetylated by p300/CBP-associated factor (PCAF), but the mechanistic basis of the Lys-28 requirement is unknown. Here, we show that Lys-28 acetylation modulates the affinity and stability of HIV-1 Tat–CycT1–TAR complexes by enhancing an interaction with the CycT1 Tat–TAR recognition motif. High-affinity assembly correlates strongly with stimulation of transcription elongation in vitro and Tat activation in vivo. In marked contrast, bovine lentiviral Tat proteins have evolved a high-affinity TAR interaction that does not require PCAF-mediated acetylation of the Tat AD or CycT1 for RNA binding, whereas HIV-2 Tat has evolved an intermediate mechanism that uses a duplicated TAR element and CycT1 to enhance RNA affinity and consequently transcription activation. The coevolution of Tat acetylation, CycT1 dependence, and TAR binding affinity is seen in viral replication assays using Tat proteins that rely on CycT1 for TAR binding but are acetylation deficient, where compensatory mutations rapidly accrue in TAR to generate high-affinity, CycT1-independent complexes reminiscent of the bovine viruses. Thus, lysine acetylation can be used to modulate and evolve the strength of a viral-host RNA–protein complex, thereby tuning the levels of transcription elongation. PMID:19223581

  13. 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.

  14. Soluble Envelope Glycoprotein Trimers from a CD4-Independent HIV-1 Elicit Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Guinea Pigs

    PubMed Central

    Murray, Marta K.; Teran, Victor A.; Chapleau, Jean-Philippe; Wang, Baomin; Kim, Su Hyon; LaBranche, Celia C.; Richard, Jonathan; Montefiori, David C.

    2015-01-01

    CD4-independent HIV-1 variants can infect coreceptor-expressing cells lacking CD4. The envelope (Env) glycoproteins on these HIV-1 variants expose a coreceptor binding site that overlaps some CD4-induced (CD4i) epitopes. Reports have demonstrated that CD4i antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). Here we investigated the immunogenicity of soluble Env trimers (sgp140) from a CD4-independent HIV-1 in guinea pigs and found that the sgp140 elicited ADCC-mediating antibodies. Therefore, these sgp140 might be useful in vaccine regimens aimed at eliciting ADCC responses. PMID:26246571

  15. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  16. HIV-1 Vpu Antagonism of Tetherin Inhibits Antibody-Dependent Cellular Cytotoxic Responses by Natural Killer Cells

    PubMed Central

    Alvarez, Raymond A.; Hamlin, Rebecca E.; Monroe, Anthony; Moldt, Brian; Hotta, Mathew T.; Rodriguez Caprio, Gabriela; Fierer, Daniel S.; Simon, Viviana

    2014-01-01

    ABSTRACT The type I interferon-inducible factor tetherin retains virus particles on the surfaces of cells infected with vpu-deficient human immunodeficiency virus type 1 (HIV-1). While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpu-deficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating FcγRIIIa signaling and enhanced NK cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1-infected primary CD4+ T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive-patient-derived antibodies. The magnitudes of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surfaces of infected primary CD4 T cells. The affinity of antibody binding to FcγRIIIa was also found to be critical in mediating efficient Fc activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibody-mediated clearance of virally infected cells. IMPORTANCE The ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports the hypothesis that a major function of Vpu is to decrease the recognition of infected cells by anti-HIV antibodies at the cell surface, thereby reducing recognition by antibody-dependent clearance by natural killer cells. PMID:24623433

  17. B and T lymphocyte attenuator down-regulation by HIV-1 depends on type I interferon and contributes to T-cell hyperactivation.

    PubMed

    Zhang, Zheng; Xu, Xiangsheng; Lu, Jiyun; Zhang, Shuye; Gu, Lanlan; Fu, Junliang; Jin, Lei; Li, Haiying; Zhao, Min; Zhang, Jiyuan; Wu, Hao; Su, Lishan; Fu, Yang-Xin; Wang, Fu-Sheng

    2011-06-01

    Nonspecific T-cell hyperactivation is the main driving force for human immunodeficiency virus (HIV)-1 disease progression, but the reasons why the excess immune response is not properly shut off are poorly defined. Eighty-five HIV-1-infected individuals were enrolled to characterize B and T lymphocyte attenuator (BTLA) expression and function. Infection and blockade assays were used to dissect the factors that influenced BTLA signaling in vitro. BTLA expression on overall CD4(+) and CD8(+) T cells was progressively decreased in HIV-1 infection, which was directly correlated with disease progression and CD4(+) T-cell differentiation and activation. BTLA(+)CD4(+) T cells from HIV-1-infected patients also displayed an altered immune status, which was indicated by reduced expression of naive markers but increased activation and exhaustion markers. Cross-linking of BTLA can substantially decrease CD4(+) T-cell activation in vitro. This responsiveness of CD4(+) T cells to BTLA-mediated inhibitory signaling was further found to be impaired in HIV-1-infected patients. Furthermore, HIV-1 NL4-3 down-regulated BTLA expression on CD4(+) T cells dependent on plasmacytoid dendritic cell (pDC)-derived interferon (IFN)-α. Blockade of IFN-α or depletion of pDCs prevents HIV-1-induced BTLA down-regulation. HIV-1 infection potentially impairs BTLA-mediated signaling dependent on pDC-derived IFN-α, which may contribute to broad T-cell hyperactivation induced by chronic HIV-1 infection.

  18. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    SciTech Connect

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-12-15

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  19. A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies

    PubMed Central

    Ding, Shilei; Veillette, Maxime; Coutu, Mathieu; Prévost, Jérémie; Scharf, Louise; Bjorkman, Pamela J.; Ferrari, Guido; Robinson, James E.; Stürzel, Christina; Hahn, Beatrice H.; Sauter, Daniel; Kirchhoff, Frank; Lewis, George K.; Pazgier, Marzena

    2015-01-01

    Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals. PMID:26637462

  20. DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication.

    PubMed

    Edgcomb, Stephen P; Carmel, Andrew B; Naji, Souad; Ambrus-Aikelin, Geza; Reyes, Jason R; Saphire, Andrew C S; Gerace, Larry; Williamson, James R

    2012-01-06

    The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. HIV-1 selectively targets gut-homing CCR6+CD4+ T cells via mTOR-dependent mechanisms

    PubMed Central

    Planas, Delphine; Zhang, Yuwei; Monteiro, Patricia; Goulet, Jean-Philippe; Gosselin, Annie; Hope, Thomas J.; Routy, Jean-Pierre

    2017-01-01

    Gut-associated lymphoid tissues are enriched in CCR6+ Th17-polarized CD4+ T cells that contribute to HIV-1 persistence during antiretroviral therapy (ART). This raises the need for Th17-targeted immunotherapies. In an effort to identify mechanisms governing HIV-1 permissiveness/persistence in gut-homing Th17 cells, we analyzed the transcriptome of CCR6+ versus CCR6– T cells exposed to the gut-homing inducer retinoic acid (RA) and performed functional validations in colon biopsies of HIV-infected individuals receiving ART (HIV+ART). Although both CCR6+ and CCR6– T cells acquired gut-homing markers upon RA exposure, the modulation of unique sets of genes coincided with preferential HIV-1 replication in RA-treated CCR6+ T cells. This molecular signature included the upregulation of HIV-dependency factors acting at entry/postentry levels, such as the CCR5 and PI3K/Akt/mTORC1 signaling pathways. Of note, mTOR expression/phosphorylation was distinctively induced by RA in CCR6+ T cells. Consistently, mTOR inhibitors counteracted the effect of RA on HIV replication in vitro and viral reactivation in CD4+ T cells from HIV+ART individuals via postentry mechanisms independent of CCR5. Finally, CCR6+ versus CCR6– T cells infiltrating the colons of HIV+ART individuals expressed unique molecular signatures, including higher levels of CCR5, integrin β7, and mTOR phosphorylation. Together, our results identify mTOR as a druggable key regulator of HIV permissiveness in gut-homing CCR6+ T cells. PMID:28768913

  2. HIV-1 and its gp120 inhibits the influenza A(H1N1)pdm09 life cycle in an IFITM3-dependent fashion.

    PubMed

    Mesquita, Milene; Fintelman-Rodrigues, Natalia; Sacramento, Carolina Q; Abrantes, Juliana L; Costa, Eduardo; Temerozo, Jairo R; Siqueira, Marilda M; Bou-Habib, Dumith Chequer; Souza, Thiago Moreno L

    2014-01-01

    HIV-1-infected patients co-infected with A(H1N1)pdm09 surprisingly presented benign clinical outcome. The knowledge that HIV-1 changes the host homeostatic equilibrium, which may favor the patient resistance to some co-pathogens, prompted us to investigate whether HIV-1 infection could influence A(H1N1)pdm09 life cycle in vitro. We show here that exposure of A(H1N1)pdm09-infected epithelial cells to HIV-1 viral particles or its gp120 enhanced by 25% the IFITM3 content, resulting in a decrease in influenza replication. This event was dependent on toll-like receptor 2 and 4. Moreover, knockdown of IFITM3 prevented HIV-1 ability to inhibit A(H1N1)pdm09 replication. HIV-1 infection also increased IFITM3 levels in human primary macrophages by almost 100%. Consequently, the arrival of influenza ribonucleoproteins (RNPs) to nucleus of macrophages was inhibited, as evaluated by different approaches. Reduction of influenza RNPs entry into the nucleus tolled A(H1N1)pdm09 life cycle in macrophages earlier than usual, limiting influenza's ability to induce TNF-α. As judged by analysis of the influenza hemagglutin (HA) gene from in vitro experiments and from samples of HIV-1/A(H1N1)pdm09 co-infected individuals, the HIV-1-induced reduction of influenza replication resulted in delayed viral evolution. Our results may provide insights on the mechanisms that may have attenuated the clinical course of Influenza in HIV-1/A(H1N1)pdm09 co-infected patients during the recent influenza form 2009/2010.

  3. HIV-1 and Its gp120 Inhibits the Influenza A(H1N1)pdm09 Life Cycle in an IFITM3-Dependent Fashion

    PubMed Central

    Mesquita, Milene; Fintelman-Rodrigues, Natalia; Sacramento, Carolina Q.; Abrantes, Juliana L.; Costa, Eduardo; Temerozo, Jairo R.; Siqueira, Marilda M.; Bou-Habib, Dumith Chequer; Souza, Thiago Moreno L.

    2014-01-01

    HIV-1-infected patients co-infected with A(H1N1)pdm09 surprisingly presented benign clinical outcome. The knowledge that HIV-1 changes the host homeostatic equilibrium, which may favor the patient resistance to some co-pathogens, prompted us to investigate whether HIV-1 infection could influence A(H1N1)pdm09 life cycle in vitro. We show here that exposure of A(H1N1)pdm09-infected epithelial cells to HIV-1 viral particles or its gp120 enhanced by 25% the IFITM3 content, resulting in a decrease in influenza replication. This event was dependent on toll-like receptor 2 and 4. Moreover, knockdown of IFITM3 prevented HIV-1 ability to inhibit A(H1N1)pdm09 replication. HIV-1 infection also increased IFITM3 levels in human primary macrophages by almost 100%. Consequently, the arrival of influenza ribonucleoproteins (RNPs) to nucleus of macrophages was inhibited, as evaluated by different approaches. Reduction of influenza RNPs entry into the nucleus tolled A(H1N1)pdm09 life cycle in macrophages earlier than usual, limiting influenza's ability to induce TNF-α. As judged by analysis of the influenza hemagglutin (HA) gene from in vitro experiments and from samples of HIV-1/A(H1N1)pdm09 co-infected individuals, the HIV-1-induced reduction of influenza replication resulted in delayed viral evolution. Our results may provide insights on the mechanisms that may have attenuated the clinical course of Influenza in HIV-1/A(H1N1)pdm09 co-infected patients during the recent influenza form 2009/2010. PMID:24978204

  4. Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication

    PubMed Central

    Taylor, Harry E.; Simmons, Glenn E.; Mathews, Thomas P.; Khatua, Atanu K.; Popik, Waldemar; Lindsley, Craig W.; D’Aquila, Richard T.; Brown, H. Alex

    2015-01-01

    Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis. PMID:26020637

  5. HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons.

    PubMed

    Kofman, Alexander; Graf, Marcus; Bojak, Alexandra; Deml, Ludwig; Bieler, Kurt; Kharazova, Alexandra; Wolf, Hans; Wagner, Ralf

    2003-01-01

    There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The

  6. Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

    PubMed Central

    Kumari, Namita; Iordanskiy, Sergey; Kovalskyy, Dmytro; Breuer, Denitra; Niu, Xiaomei; Lin, Xionghao; Xu, Min; Gavrilenko, Konstantin; Kashanchi, Fatah; Dhawan, Subhash

    2014-01-01

    HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-κB expression, and HIV-1- and NF-κB-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of IκB-α, with increased accumulation of cytoplasmic NF-κB. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-κB-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection. PMID:25155598

  7. HIV-1 Cis Enhancing Sequence (CES) enhances CTE-dependent Gag expression.

    PubMed

    Suptawiwat, Ornpreya; Lee, Tun-Hou; Auewarakul, Prasert

    2005-11-10

    In order to export intron-containing RNA from nucleus, retroviruses use either viral trans-acting factors or constitutive cellular factors interacting with cis-elements in their intron-containing RNA. We have previously identified a Cis Enhancing Sequence (CES) in HIV-1 env region that could co-operate with Rev and RRE to enhance Gag expression by promoting RNA stabilization and exportation. In this study, we found that CES could function in a Rev-independent manner by co-operating with a Constitutive Transport Element (CTE) of Mason-Pfizer monkey viruses (MPMV). RRE and CTE promote intron-containing RNA exportation through different pathways. The fact that CES could function in both pathways of RNA export suggested that CES might function at a common step either up- or downstream to Rev/RRE or CTE functions. Known hnRNP-A1-binding sites as well as other 3 highly conserved sequences in the CES were found to be required for its activity.

  8. 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

  9. Women with Cervicovaginal Antibody-Dependent Cell-Mediated Cytotoxicity Have Lower Genital HIV-1 RNA Loads

    PubMed Central

    Nag, Pratip; Kim, Jenney; Sapiega, Vytautas; Landay, Alan L.; Bremer, James W.; Mestecky, Jiri; Reichelderfer, Patricia; Kovacs, Andrea; Cohn, Jonathan; Weiser, Barbara; Baum, Linda L.

    2011-01-01

    Antibodies that mediate human immunodeficiency virus (HIV)-specific antibody-dependent cell-mediated cytotoxicity (ADCC) are present in the cervical fluid of many HIV-positive women; however, the role that these antibodies play in host defense against HIV is not known. To understand the contribution of ADCC in cervical secretions as a protective mechanism against HIV, we evaluated ADCC titers in paired serum and cervical-lavage (CVL) samples from >300 HIV-1–positive women who participated in the multicenter Division of AIDS Treatment Research Initiative Study 009. The present study demonstrates that women with CVL ADCC activity had lower genital viral loads than did women with serum ADCC activity only. Women with CVL ADCC activity were likely to have HIV-1 gp120–specific immunoglobulin (Ig) G, but not IgA, in their cervical fluid. This finding suggests that specific IgG in cervical fluid can mediate ADCC activity that inversely correlates with genital viral load. PMID:15529262

  10. Structural Definition of an Antibody-Dependent Cellular Cytotoxicity Response Implicated in Reduced Risk for HIV-1 Infection

    PubMed Central

    Acharya, Priyamvada; Tolbert, William D.; Gohain, Neelakshi; Wu, Xueji; Yu, Lei; Liu, Tongyun; Huang, Wensheng; Huang, Chih-chin; Kwon, Young Do; Louder, Robert K.; Luongo, Timothy S.; McLellan, Jason S.; Pancera, Marie; Yang, Yongping; Zhang, Baoshan; Flinko, Robin; Foulke, James S.; Sajadi, Mohammad M.; Kamin-Lewis, Roberta; Robinson, James E.; Martin, Loïc; Kwong, Peter D.; Guan, Yongjun; DeVico, Anthony L.; Lewis, George K.

    2014-01-01

    ABSTRACT The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic, as infected or vaccinated individuals frequently produce antibodies specific for these determinants. Antibody titers, as measured by enzyme-linked immunosorbent assay (ELISA) against these epitopes, however, do not consistently correlate with protection. Here, we report crystal structures of CD4-stabilized gp120 cores complexed with the Fab fragments of two nonneutralizing, A32-like monoclonal antibodies (MAbs), N5-i5 and 2.2c, that compete for antigen binding and have similar antigen-binding affinities yet exhibit a 75-fold difference in ADCC potency. We find that these MAbs recognize overlapping epitopes formed by mobile layers 1 and 2 of the gp120 inner domain, including the C1 and C2 regions, but bind gp120 at different angles via juxtaposed VH and VL contact surfaces. A comparison of structural and immunological data further showed that antibody orientation on bound antigen and the capacity to form multivalent antigen-antibody complexes on target cells were key determinants of ADCC potency, with the latter process having the greater impact. These studies provide atomic-level definition of A32-like epitopes implicated as targets of protective antibodies in RV144. Moreover, these studies establish that epitope structure and mode of antibody binding can dramatically affect the potency of Fc-mediated effector function against HIV-1. These results provide key insights for understanding, refining, and improving the outcome of HIV vaccine trials, in which relevant immune responses are facilitated by A32-like elicited responses. IMPORTANCE HIV-1 Env is a primary target for antibodies elicited during infection. Although a small number of infected individuals elicit broadly neutralizing antibodies, the bulk of the humoral response

  11. Structural definition of an antibody-dependent cellular cytotoxicity response implicated in reduced risk for HIV-1 infection.

    PubMed

    Acharya, Priyamvada; Tolbert, William D; Gohain, Neelakshi; Wu, Xueji; Yu, Lei; Liu, Tongyun; Huang, Wensheng; Huang, Chih-Chin; Kwon, Young Do; Louder, Robert K; Luongo, Timothy S; McLellan, Jason S; Pancera, Marie; Yang, Yongping; Zhang, Baoshan; Flinko, Robin; Foulke, James S; Sajadi, Mohammad M; Kamin-Lewis, Roberta; Robinson, James E; Martin, Loïc; Kwong, Peter D; Guan, Yongjun; DeVico, Anthony L; Lewis, George K; Pazgier, Marzena

    2014-11-01

    The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic, as infected or vaccinated individuals frequently produce antibodies specific for these determinants. Antibody titers, as measured by enzyme-linked immunosorbent assay (ELISA) against these epitopes, however, do not consistently correlate with protection. Here, we report crystal structures of CD4-stabilized gp120 cores complexed with the Fab fragments of two nonneutralizing, A32-like monoclonal antibodies (MAbs), N5-i5 and 2.2c, that compete for antigen binding and have similar antigen-binding affinities yet exhibit a 75-fold difference in ADCC potency. We find that these MAbs recognize overlapping epitopes formed by mobile layers 1 and 2 of the gp120 inner domain, including the C1 and C2 regions, but bind gp120 at different angles via juxtaposed VH and VL contact surfaces. A comparison of structural and immunological data further showed that antibody orientation on bound antigen and the capacity to form multivalent antigen-antibody complexes on target cells were key determinants of ADCC potency, with the latter process having the greater impact. These studies provide atomic-level definition of A32-like epitopes implicated as targets of protective antibodies in RV144. Moreover, these studies establish that epitope structure and mode of antibody binding can dramatically affect the potency of Fc-mediated effector function against HIV-1. These results provide key insights for understanding, refining, and improving the outcome of HIV vaccine trials, in which relevant immune responses are facilitated by A32-like elicited responses. HIV-1 Env is a primary target for antibodies elicited during infection. Although a small number of infected individuals elicit broadly neutralizing antibodies, the bulk of the humoral response consists of antibodies

  12. HIV-1 envelope-dependent restriction of CXCR4-using viruses in child but not adult untransformed CD4+ T-lymphocyte lines.

    PubMed

    Mariani, Samanta A; Brigida, Immacolata; Kajaste-Rudnitski, Anna; Ghezzi, Silvia; Rocchi, Alessia; Plebani, Anna; Vicenzi, Elisa; Aiuti, Alessandro; Poli, Guido

    2012-03-01

    Phytohemagglutin-stimulated child and adult leukocytes equally supported CCR5-dependent (R5) and CXCR4-dependent (X4) HIV-1 replication. In contrast, when phytohemagglutin-stimulated leukocytes from either healthy or congenitally immunodeficient children were cultured on feeder cells, they well supported R5, but not X4 HIV-1 replication, whereas both viruses equally spread in adult cells maintained in similar conditions. Both child and adult cells showed similar levels of proliferation and surface expression of CD4, CCR5, CXCR4, CD25, CD69, and HLA-DR. Lack of X4 HIV-1 replication in child versus adult cells was not caused by a differential expression of several known HIV-1 restriction factors. Similar levels of HIV DNA synthesis occurred in child cells infected with R5 and X4 viruses up to 48 hours after infection when R5 HIV-1 showed a significantly superior capacity to spread in culture than X4 virus. Cultured child cells well supported single round vescicular stomatitis virus-G pseudotyped virus replication, whereas superinfection of R5-infected cells with X4 HIV-1 (or vice versa) rescued the replication of this latter virus. Thus, child cells exposed to feeder cell culture represent a novel model system in which the superior capacity of R5 versus X4 viruses to spread can be investigated in primary, untransformed CD4(+) cells.

  13. Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail

    PubMed Central

    Muranyi, Walter; Malkusch, Sebastian; Müller, Barbara; Heilemann, Mike; Kräusslich, Hans-Georg

    2013-01-01

    The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse. PMID:23468635

  14. B and T Lymphocyte Attenuator Down-regulation by HIV-1 Depends on Type I Interferon and Contributes to T-Cell Hyperactivation

    PubMed Central

    Zhang, Zheng; Xu, Xiangsheng; Lu, Jiyun; Zhang, Shuye; Gu, Lanlan; Fu, Junliang; Jin, Lei; Li, Haiying; Zhao, Min; Zhang, Jiyuan; Wu, Hao; Su, Lishan; Fu, Yang-Xin

    2011-01-01

    Background. Nonspecific T-cell hyperactivation is the main driving force for human immunodeficiency virus (HIV)–1 disease progression, but the reasons why the excess immune response is not properly shut off are poorly defined. Methods. Eighty-five HIV-1–infected individuals were enrolled to characterize B and T lymphocyte attenuator (BTLA) expression and function. Infection and blockade assays were used to dissect the factors that influenced BTLA signaling in vitro. Results. BTLA expression on overall CD4+ and CD8+ T cells was progressively decreased in HIV-1 infection, which was directly correlated with disease progression and CD4+ T-cell differentiation and activation. BTLA+CD4+ T cells from HIV-1–infected patients also displayed an altered immune status, which was indicated by reduced expression of naive markers but increased activation and exhaustion markers. Cross-linking of BTLA can substantially decrease CD4+ T-cell activation in vitro. This responsiveness of CD4+ T cells to BTLA-mediated inhibitory signaling was further found to be impaired in HIV-1–infected patients. Furthermore, HIV-1 NL4-3 down-regulated BTLA expression on CD4+ T cells dependent on plasmacytoid dendritic cell (pDC)-derived interferon (IFN)-α. Blockade of IFN-α or depletion of pDCs prevents HIV-1-induced BTLA down-regulation. Conclusions. HIV-1 infection potentially impairs BTLA-mediated signaling dependent on pDC-derived IFN-α, which may contribute to broad T-cell hyperactivation induced by chronic HIV-1 infection. PMID:21592997

  15. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    PubMed

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  16. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans

    PubMed Central

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients’ depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host. PMID:26200356

  17. TAR RNA binding properties and relative transactivation activities of human immunodeficiency virus type 1 and 2 Tat proteins.

    PubMed Central

    Rhim, H; Rice, A P

    1993-01-01

    Using gel shift assays, we found that the human immunodeficiency virus type 1 (HIV-1) Tat protein (Tat-1) bound both HIV-1 and HIV-2 TAR RNAs with similar high affinities. In contrast, the HIV-2 Tat protein (Tat-2) bound only TAR-2 RNA with high affinity. We conclude that the weak in vivo activity of Tat-2 on the HIV-1 long terminal repeat that has been observed previously is likely the result of low affinity for TAR-1 RNA. Additionally, TAR-2 RNA was found to contain multiple specific binding sites for Tat proteins. GAL4-Tat fusion proteins were analyzed to compare the relative transactivation activities of Tat-1 and Tat-2 in the absence of requirements for binding to TAR RNAs. The GAL4-Tat-2 protein was found to transactivate synthetic promoters containing GAL4 binding sites at levels severalfold higher than did the GAL4-Tat-1 protein. Images PMID:8419640

  18. Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity

    PubMed Central

    Richard, Jonathan; Veillette, Maxime; Ding, Shilei; Zoubchenok, Daria; Alsahafi, Nirmin; Coutu, Mathieu; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno; Smith, Amos B.; Shaw, George M.; Hahn, Beatrice H.; Sodroski, Joseph; Kaufmann, Daniel E.; Finzi, Andrés

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells. PMID:26870823

  19. Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells

    PubMed Central

    Giacaman, Rodrigo A; Asrani, Anil C; Gebhard, Kristin H; Dietrich, Elizabeth A; Vacharaksa, Anjalee; Ross, Karen F; Herzberg, Mark C

    2008-01-01

    Background Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. Results Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. Conclusion P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1. PMID:18371227

  20. Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat.

    PubMed Central

    Taylor, J P; Pomerantz, R J; Raj, G V; Kashanchi, F; Brady, J N; Amini, S; Khalili, K

    1994-01-01

    The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed. Images PMID:8189531

  1. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs.

    PubMed

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-12-01

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. A Tat ménage à trois--The role of Bacillus subtilis TatAc in twin-arginine protein translocation.

    PubMed

    Goosens, Vivianne J; De-San-Eustaquio-Campillo, Alba; Carballido-López, Rut; van Dijl, Jan Maarten

    2015-10-01

    The twin-arginine translocation system (Tat) is a protein transport system that moves fully folded and cofactor-containing proteins across membranes of bacteria, archaea and thylakoids. The minimal Tat pathway is composed of two subunits, TatA and TatC. In some organisms TatA has been duplicated and evolved to form a third specialized subunit, TatB. The Bacillus subtilis genome encodes two TatC subunits (TatCd and TatCy) and three TatA subunits (TatAd, TatAy and TatAc). These subunits combine to form two parallel minimal pathways, TatAy-TatCy and TatAd-TatCd. The purpose and role of the third TatA component, TatAc, has remained ambiguous. In this study we examined the translocation of two natively expressed TatAy-TatCy-dependent substrates, EfeB and QcrA, in various Tat-deficient genetic backgrounds. More specifically, we examined the ability of different mutated TatAy subunits to complement for the absence of wild-type TatAy. We further detailed a graded growth phenotype associated with the functional translocation of EfeB. We found that in various instances where specific amino acid substitutions were made in TatAy, a definite TatAc-associated growth phenotype occurred in genetic backgrounds lacking TatAc. Altogether, our findings show that TatAy and TatAc interact and that this TatAy-TatAc interaction, although not essential, supports the translocation of the Tat substrate EfeB when TatAy function is compromised. This implies that the third TatA-like protein in B. subtilis could represent an intermediate evolutionary step in TatA-TatB specialization. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

    PubMed Central

    2012-01-01

    Background The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals. PMID:22828074

  4. Transcytosis of HIV-1 through vaginal epithelial cells is dependent on trafficking to the endocytic recycling pathway.

    PubMed

    Kinlock, Ballington L; Wang, Yudi; Turner, Tiffany M; Wang, Chenliang; Liu, Bindong

    2014-01-01

    While it is accepted that viruses can enter epithelial cells by endocytosis, the lack of an established biological mechanism for the trafficking of infectious virions through vaginal epithelial cells and their release from the plasma membrane has contributed to ongoing controversy about whether endocytosis is a mere artifact of some cell culture systems and whether squamous vaginal epithelial cells are even relevant as it pertains to HIV-1 transmission. In this study, we investigated the intracellular trafficking pathway that HIV-1 exploits to transcytose vaginal epithelial cells. The reduction of endosome tubulation by recycling endosome inhibitors blocked transcytosis of HIV-1 in a cell culture and transwell system. In addition, we demonstrate that although heat-inactivated virus was endocytosed as efficiently as native virus, heat-inactivated virus was trafficked exclusively to the lysosomal pathway for degradation following endocytosis. Lysosomal protease-specific inhibitors blocked the degradation of inactivated virions. Immunofluorescence analysis not only demonstrated that HIV-1 was inside the cells but the different colocalization pattern of native vs. heat inactivated virus with transferrin provided conclusive evidence that HIV-1 uses the recycling pathway to get across vaginal epithelial cells. Altogether, our findings demonstrate the precise intracellular trafficking pathway utilized by HIV-1 in epithelial cells, confirms that HIV-1 transcytosis through vaginal epithelial cells is a biological phenomenon and brings to light the differential intracellular trafficking of native vs heat-inactivated HIV-1 which with further exploration could prove to provide valuable insights that could be used in the prevention of transcytosis/transmission of HIV-1 across the mucosal epithelia.

  5. Prothymosin-α Variants Elicit Anti-HIV-1 Response via TLR4 Dependent and Independent Pathways

    PubMed Central

    Gusella, G. Luca; Teixeira, Avelino; Aberg, Judith; Uversky, Vladimir N.; Mosoian, Arevik

    2016-01-01

    Background Prothymosin α (ProTα) (isoform 2: iso2) is a widely distributed, small acidic protein with intracellular and extracellular-associated functions. Recently, we identified two new ProTα variants with potent anti-HIV activity from CD8+ T cells and cervicovaginal lavage. The first is a splice variant of the ProTα gene known as isoB and the second is the product of ProTα pseudogene 7 (p7). Similarly to iso2, the anti-HIV activity of both variants is mediated by type I IFN. Here we tested whether the immunomodulatory activity of isoB and p7 are also TLR4 dependent and determined their kinetic of release in response to HIV-1 infection. Methods Type I, type III, TNF-α and IL-6 mRNA inducing activity was determined in macrophages from wild type and TLR4 knockout mice treated with recombinant ProTα variants. Supernatants from mock and HIV infected cells were analyzed by mass spectrometry in positive and negative modes for the presence of ProTα variants. In silico structural and functional analysis of ProTα variants were performed. Results We show that both isoB and p7 upregulate IFN-β, IFN-λ1, IL-6, TNF-α and RANTES mRNAs in primary human macrophages. The potent stimulation of IFN-β by the recombinant ProTα variants in human macrophages is dependent on the TLR4 pathway, whereas the induction of TNF-α and IL-6 may also occur independently of TLR4, suggesting the interaction of ProTα variants with other signaling molecules/receptors. In silico analyses confirmed that the novel isoB and p7 variants are intrinsically disordered proteins, which lack the NLS and mass spectrometry showed release of ProTα variants within minutes post HIV-1 infection. These features are consistent with the function of ProTα variants as damage associate molecular patterns (DAMPs). Conclusions Our findings indicate that ProTα variants strongly inhibit viral replication mainly, but not exclusively, through TLR4 signaling and that they are released within minutes of viral

  6. Human immunodeficiency virus-1 Tat protein increases the number of inhibitory synapses between hippocampal neurons in culture.

    PubMed

    Hargus, Nicholas J; Thayer, Stanley A

    2013-11-06

    Synaptodendritic damage correlates with cognitive decline in many neurodegenerative diseases, including human immunodeficiency virus-1 (HIV-1)-associated neurocognitive disorders (HAND). Because HIV-1 does not infect neurons, viral-mediated toxicity is indirect, resulting from released neurotoxins such as the HIV-1 protein transactivator of transcription (Tat). We compared the effects of Tat on inhibitory and excitatory synaptic connections between rat hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding proteins gephyrin or PSD95 fused to GFP. Tat (24 h) increased the number of GFP-gephyrin puncta and decreased the number of PSD95-GFP puncta. The effects of Tat on inhibitory and excitatory synapse number were mediated via the low-density lipoprotein receptor-related protein and subsequent Ca(2+) influx through GluN2A-containing NMDA receptors (NMDARs). The effects of Tat on synapse number required cell-autonomous activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Ca(2+) buffering experiments suggested that loss of excitatory synapses required activation of CaMKII in close apposition to the NMDAR, whereas the increase in inhibitory synapses required Ca(2+) diffusion to a more distal site. The increase in inhibitory synapses was prevented by inhibiting the insertion of GABAA receptors into the membrane. Synaptic changes induced by Tat (16 h) were reversed by blocking either GluN2B-containing NMDARs or neuronal nitric oxide synthase, indicating changing roles for pathways activated by NMDAR subtypes during the neurotoxic process. Compensatory changes in the number of inhibitory and excitatory synapses may serve as a novel mechanism to reduce network excitability in the presence of HIV-1 neurotoxins; these changes may inform the development of treatments for HAND.

  7. Contribution of Phe-7 to Tat-Dependent Export of β-Lactamase in Xanthomonas campestris

    PubMed Central

    Lee, Chen-Wei; Tseng, Yi-Hsuan; Deng, Fu-Seng; Lin, Juey-Wen

    2012-01-01

    Strains of Xanthomonas campestris pv. campestris isolated in Taiwan are commonly resistant to ampicillin owing to the constitutive expression of a chromosomally encoded β-lactamase that is secreted into the periplasm. In this study, we found that levels of β-lactamase vary among X. campestris pv. campestris strains, a difference that can be attributed to amino acid substitutions at least at positions 7 and 206, with the former having the major impact. Bioinformatic and PCR analyses indicated that X. campestris pv. campestris possesses tatABC genes and that the signal peptide of X. campestris pv. campestris pre-Bla contains the typical twin-arginine motif (N-R-R-Q-F-L at amino acid residues 3 to 8 in strain X. campestris pv. campestris strain 11), suggesting that Bla is secreted via the Tat pathway. To assess the importance of Phe7 in the efficient export of X. campestris pv. campestris Bla, we prepared mutant constructs containing amino acid substitutions and monitored their expression by measuring enzyme activity and detecting Bla protein by Western blotting. The results indicate that replacement of Phe7 with Leu severely inhibited Bla export whereas replacement with Pro almost abolished it. Although a change to Arg caused moderate inhibition of export, replacement with Tyr had no effect. These results suggest that for efficient export of Bla by X. campestris pv. campestris, the aromatic-aromatic interactions and stability of protein structure around the twin-arginine motif are important, since only proteins that can attain a folded state in the cytoplasm are competent for export via the Tat pathway. PMID:22526303

  8. Contribution of Phe-7 to Tat-dependent export of β-lactamase in Xanthomonas campestris.

    PubMed

    Lee, Chen-Wei; Tseng, Yi-Hsuan; Deng, Fu-Seng; Lin, Juey-Wen; Tseng, Yi-Hsiung; Weng, Shu-Fen

    2012-07-01

    Strains of Xanthomonas campestris pv. campestris isolated in Taiwan are commonly resistant to ampicillin owing to the constitutive expression of a chromosomally encoded β-lactamase that is secreted into the periplasm. In this study, we found that levels of β-lactamase vary among X. campestris pv. campestris strains, a difference that can be attributed to amino acid substitutions at least at positions 7 and 206, with the former having the major impact. Bioinformatic and PCR analyses indicated that X. campestris pv. campestris possesses tatABC genes and that the signal peptide of X. campestris pv. campestris pre-Bla contains the typical twin-arginine motif (N-R-R-Q-F-L at amino acid residues 3 to 8 in strain X. campestris pv. campestris strain 11), suggesting that Bla is secreted via the Tat pathway. To assess the importance of Phe(7) in the efficient export of X. campestris pv. campestris Bla, we prepared mutant constructs containing amino acid substitutions and monitored their expression by measuring enzyme activity and detecting Bla protein by Western blotting. The results indicate that replacement of Phe(7) with Leu severely inhibited Bla export whereas replacement with Pro almost abolished it. Although a change to Arg caused moderate inhibition of export, replacement with Tyr had no effect. These results suggest that for efficient export of Bla by X. campestris pv. campestris, the aromatic-aromatic interactions and stability of protein structure around the twin-arginine motif are important, since only proteins that can attain a folded state in the cytoplasm are competent for export via the Tat pathway.

  9. The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 TAT protein

    PubMed Central

    1994-01-01

    Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL- 6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat- expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA- protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT- induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6- dependent mouse cell line, stably expressing the tat gene

  10. A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and Dengue virus.

    PubMed

    Varga, Norbert; Sutkeviciute, Ieva; Ribeiro-Viana, Renato; Berzi, Angela; Ramdasi, Rasika; Daghetti, Anna; Vettoretti, Gerolamo; Amara, Ali; Clerici, Mario; Rojo, Javier; Fieschi, Franck; Bernardi, Anna

    2014-04-01

    DC-SIGN is a C-type lectin receptor on antigen presenting cells (dendritic cells) which has an important role in some viral infection, notably by HIV and Dengue virus (DV). Multivalent presentation of carbohydrates on dendrimeric scaffolds has been shown to inhibit DC-SIGN binding to HIV envelope glycoprotein gp120, thus blocking viral entry. This approach has interesting potential applications for infection prophylaxis. In an effort to develop high affinity inhibitors of DC-SIGN mediated viral entry, we have synthesized a group of glycodendrimers of different valency that bear different carbohydrates or glycomimetic DC-SIGN ligands and have studied their DC-SIGN binding activity and antiviral properties both in an HIV and a Dengue infection model. Surface Plasmon Resonance (SPR) competition studies have demonstrated that the materials obtained bind efficiently to DC-SIGN with IC50s in the μm range, which depend on the nature of the ligand and on the valency of the scaffold. In particular, a hexavalent presentation of the DC-SIGN selective antagonist 4 displayed high potency, as well as improved accessibility and chemical stability relative to previously reported dendrimers. At low μm concentration the material was shown to block both DC-SIGN mediated uptake of DV by Raji cells and HIV trans-infection of T cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Increased excitability in tat-transgenic mice: role of tat in HIV-related neurological disorders.

    PubMed

    Zucchini, Silvia; Pittaluga, Anna; Brocca-Cofano, Egidio; Summa, Maria; Fabris, Marina; De Michele, Rita; Bonaccorsi, Angela; Busatto, Graziella; Barbanti-Brodano, Giuseppe; Altavilla, Giuseppe; Verlengia, Gianluca; Cifelli, Pierangelo; Corallini, Alfredo; Caputo, Antonella; Simonato, Michele

    2013-07-01

    HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Genetic Selection for Context-Dependent Stochastic Phenotypes: Sp1 and TATA Mutations Increase Phenotypic Noise in HIV-1 Gene Expression

    PubMed Central

    Shah, Priya S.; Arkin, Adam P.; Schaffer, David V.

    2013-01-01

    The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly ‘switch’ between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify ‘Switching’ phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus

  13. Genetic selection for context-dependent stochastic phenotypes: Sp1 and TATA mutations increase phenotypic noise in HIV-1 gene expression.

    PubMed

    Miller-Jensen, Kathryn; Skupsky, Ron; Shah, Priya S; Arkin, Adam P; Schaffer, David V

    2013-01-01

    The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly 'switch' between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify 'Switching' phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a

  14. Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses.

    PubMed

    Prévost, Jérémie; Zoubchenok, Daria; Richard, Jonathan; Veillette, Maxime; Pacheco, Beatriz; Coutu, Mathieu; Brassard, Nathalie; Parsons, Matthew S; Ruxrungtham, Kiat; Bunupuradah, Torsak; Tovanabutra, Sodsai; Hwang, Kwan-Ki; Moody, M Anthony; Haynes, Barton F; Bonsignori, Mattia; Sodroski, Joseph; Kaufmann, Daniel E; Shaw, George M; Chenine, Agnès L; Finzi, Andrés

    2017-04-01

    HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV(+)) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity

  15. P-TEFb Kinase Complex Phosphorylates Histone H1 to Regulate Expression of Cellular and HIV-1 Genes*

    PubMed Central

    O'Brien, Siobhan K.; Cao, Hong; Nathans, Robin; Ali, Akbar; Rana, Tariq M.

    2010-01-01

    Transcription of HIV-1 genes depends on the RNA polymerase II kinase and elongation factor positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We identify histone H1 as a substrate for P-TEFb involved in cellular and HIV-1 transcription. We show that P-TEFb interacts with H1 and that P-TEFb inhibition by RNAi, flavopiridol, or dominant negative CDK9 expression correlates with loss of phosphorylation and mobility of H1 in vivo. Importantly, P-TEFb directs H1 phosphorylation in response to wild-type HIV-1 infection, but not Tat-mutant HIV-1 infection. Our results show that P-TEFb phosphorylates histone H1 at a specific C-terminal phosphorylation site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb also disrupts Tat transactivation in an HIV reporter cell line as well as transcription of the c-fos and hsp70 genes in HeLa cells. We identify histone H1 as a novel P-TEFb substrate, and our results suggest new roles for P-TEFb in both cellular and HIV-1 transcription. PMID:20551309

  16. 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.

  17. Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-ĸB-dependent mechanism.

    PubMed

    Díaz, Laura; Martínez-Bonet, Marta; Sánchez, Javier; Fernández-Pineda, Alejandra; Jiménez, José Luis; Muñoz, Eduardo; Moreno, Santiago; Álvarez, Susana; Muñoz-Fernández, Ma Ángeles

    2015-07-22

    Multiple studies have shown that HIV-1 patients may develop virus reservoirs that impede eradication; these reservoirs include the central nervous system (CNS). Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. To broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as a latent HIV-1 activator. We used primary astrocytes, NHA cells, and astrocytoma cells U-87. Infected cells with HIV-1(NL4.3) were treated with bryostatin alone or in combination with different inhibitors. HIV-1 production was quantified by using ELISA. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of the LTR promoter with the active NF-κB member p65/relA. To confirm the NF-κB role, Western blot and confocal microscopy were performed. Bryostatin reactivates latent viral infection in the NHA and U87 cells via activation of protein kinase C (PKC)-alpha and -delta, because the PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. No alteration in cell proliferation was found. Moreover, bryostatin strongly stimulated LTR transcription by activating the transcription factor NF-κB. Bryostatin could be a beneficial adjunct to the treatment of HIV-1 brain infection.

  18. Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-ĸB-dependent mechanism

    PubMed Central

    Díaz, Laura; Martínez-Bonet, Marta; Sánchez, Javier; Fernández-Pineda, Alejandra; Jiménez, José Luis; Muñoz, Eduardo; Moreno, Santiago; Álvarez, Susana; Muñoz-Fernández, Mª Ángeles

    2015-01-01

    Multiple studies have shown that HIV-1 patients may develop virus reservoirs that impede eradication; these reservoirs include the central nervous system (CNS). Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. To broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as a latent HIV-1 activator. We used primary astrocytes, NHA cells, and astrocytoma cells U-87. Infected cells with HIV-1NL4.3 were treated with bryostatin alone or in combination with different inhibitors. HIV-1 production was quantified by using ELISA. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of the LTR promoter with the active NF-κB member p65/relA. To confirm the NF-κB role, Western blot and confocal microscopy were performed. Bryostatin reactivates latent viral infection in the NHA and U87 cells via activation of protein kinase C (PKC)-alpha and -delta, because the PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. No alteration in cell proliferation was found. Moreover, bryostatin strongly stimulated LTR transcription by activating the transcription factor NF-κB. Bryostatin could be a beneficial adjunct to the treatment of HIV-1 brain infection. PMID:26199173

  19. Celastrol Inhibits Tat-mediated Human Immunodeficiency Virus (HIV) Transcription and Replication

    PubMed Central

    Narayan, Vivek; Kodihalli, Ravindra C.; Chiaro, Chris; Cary, Daniele; Aggarwal, Bharat B.; Henderson, Andrew J.; Prabhu, K. Sandeep

    2011-01-01

    Current drugs used for anti-retroviral therapy against HIV have a narrow spectrum of activity, and more often have associated toxicities and severe side effects in addition to developing resistance. Thus, there is a need to develop new therapeutic strategies against HIV/AIDS to complement the already existing ones. Surprisingly, Tat, an early virus encoded protein required for the efficient transcription of the HIV genome, has not been developed as a target for small molecular therapeutics. We have previously described the ability of an endogenous Michael acceptor electrophile (MAE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to inhibit Tat-dependent transcription, by targeting its cysteine (Cys) rich domain. In an effort to identify other MAEs possessing inhibitory activity against HIV-1 Tat, we tested a collection of plant-derived compounds with electrophilic properties, including curcumin, rosmarinic acid, and gambogic acid, for their ability to inhibit Tat. Celastrol (Cel), a triterpenoid MAE isolated from T. wilfordii, exhibited the highest inhibitory activity against Tat. Using biochemical techniques, we demonstrate that Cel by covalently modifying the cysteine thiols inhibits Tat transactivation function. Using circular dichroism (CD) spectroscopy, we show that alklylation of Tat brought about a change in the secondary structure of Tat, which inhibited the transcription elongation of the HIV proviral genome by effecting mechanisms other than Tat-TAR interaction. Our results demonstrate the underlying mechanism of anti-retroviral activity of the plant-derived MAEs, and suggest that Cel could serve as a lead compound to develop novel anti-viral therapeutics. PMID:21763500

  20. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    PubMed

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  1. Characterization of a Monoclonal Antibody to a Novel Glycan-Dependent Epitope in the V1/V2 Domain of the HIV-1 Envelope Protein, gp120

    PubMed Central

    To, Briana; Morin, Trevor J.; Theolis, Richard; O’Rourke, Sara M.; Yu, Bin; Mesa, Kathryn A.; Berman, Phillip W.

    2014-01-01

    Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of

  2. HIV-1 Triggers WAVE2 Phosphorylation in Primary CD4 T Cells and Macrophages, Mediating Arp2/3-dependent Nuclear Migration*

    PubMed Central

    Spear, Mark; Guo, Jia; Turner, Amy; Yu, Dongyang; Wang, Weifeng; Meltzer, Beatrix; He, Sijia; Hu, Xiaohua; Shang, Hong; Kuhn, Jeffrey; Wu, Yuntao

    2014-01-01

    The human immunodeficiency virus type 1 (HIV-1) initiates receptor signaling and early actin dynamics during viral entry. This process is required for viral infection of primary targets such as resting CD4 T cells. WAVE2 is a component of a multiprotein complex linking receptor signaling to dynamic remodeling of the actin cytoskeleton. WAVE2 directly activates Arp2/3, leading to actin nucleation and filament branching. Although several bacterial and viral pathogens target Arp2/3 for intracellular mobility, it remains unknown whether HIV-1 actively modulates the Arp2/3 complex through virus-mediated receptor signal transduction. Here we report that HIV-1 triggers WAVE2 phosphorylation at serine 351 through gp120 binding to the chemokine coreceptor CXCR4 or CCR5 during entry. This phosphorylation event involves both Gαi-dependent and -independent pathways, and is conserved both in X4 and R5 viral infection of resting CD4 T cells and primary macrophages. We further demonstrate that inhibition of WAVE2-mediated Arp2/3 activity through stable shRNA knockdown of Arp3 dramatically diminished HIV-1 infection of CD4 T cells, preventing viral nuclear migration. Inhibition of Arp2/3 through a specific inhibitor, CK548, also drastically inhibited HIV-1 nuclear migration and infection of CD4 T cells. Our results suggest that Arp2/3 and the upstream regulator, WAVE2, are essential co-factors hijacked by HIV for intracellular migration, and may serve as novel targets to prevent HIV transmission. PMID:24415754

  3. CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    PubMed Central

    2012-01-01

    Background HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. Results The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. Conclusion Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1

  4. Twin-arginine translocation system in Helicobacter pylori: TatC, but not TatB, is essential for viability.

    PubMed

    Benoit, Stéphane L; Maier, Robert J

    2014-01-21

    The twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects. This work reports the first characterization of the twin-arginine translocation (Tat) system in the gastric pathogen Helicobacter pylori. While tatB mutants were

  5. Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

    PubMed Central

    Setz, Christian; Friedrich, Melanie; Schlößer, Stefan; Kölle, Julia; Spranger, Robert; Rauch, Pia; Reif, Tatjana; Karius-Fischer, Julia; Balasubramanyam, Ashok; Henklein, Petra; Fossen, Torgils; Schubert, Ulrich

    2017-01-01

    There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1. PMID:28388673

  6. Purification of TAT-CC-HA protein under native condition, and its transduction analysis and biological effects on BCR-ABL positive cells.

    PubMed

    Huang, Zhenglan; Ji, Maosheng; Peng, Zhi; Huang, Shifeng; Xiao, Qing; Li, Chunli; Zeng, Jianming; Gao, Miao; Feng, Wenli

    2011-06-01

    BCR-ABL oncoprotein is the cause of chronic myeloid leukemia. The homologous oligomerization of BCR-ABL protein mediated by BCR coiled-coil (CC) domain plays an important role in ABL kinase activation. The HIV-1 TAT peptide has been used extensively for the introduction of proteins into cells. We recombinated a TAT-CC-HA protein to interrupt the homologous oligomerization of BCR-ABL. The expression conditions for TAT-CC-HA were optimized. The TAT-CC-HA fusion protein was purified with Ni+-NTA resin. TAT-CC-HA fusion protein was added into the cultures of Ba/F3-p210, 32D-p210, K562, KU812, Ba/F3, 32D, and HL-60 cells. It was found that TAT-CC-HA could transduce into these cells. It was confirmed that TAT-CC-HA fusion protein was internalized by Ba/F3-p210, K562, and Ba/F3 cells and located in the cytoplasm observed by confocal laser scanning fluorescence microscope. The transduction of TAT-CC-HA fusion protein into K562 cells was in a dose-dependent and time-dependent manner. The result of coimmunoprecipitation assay indicated that TAT-CC-HA could interact with BCR-ABL in K562 cells. The effects of TAT-CC-HA fusion protein on cell growth and apoptosis were detected by MTT test and flow cytometry. Our findings suggested that TAT-CC-HA fusion protein could specifically inhibit the growth of BCR-ABL positive cells, and specifically induce apoptosis of BCR-ABL positive cells, while not affect the growth and apoptosis of BCR-ABL negative cells.

  7. Induced degradation of Tat by nucleocapsid (NC) via the proteasome pathway and its effect on HIV transcription.

    PubMed

    Hong, Hye-Won; Lee, Seong-Wook; Myung, Heejoon

    2013-04-23

    Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 Tat protein upregulates transcriptional transactivation. The nucleocapsid protein NC of HIV-1 is a component of virion and plays a key role in genome packaging. Herein, we have demonstrated the interaction between NC and Tat by means of a yeast two-hybrid assay, GST pull-down analysis, co-immunoprecipitation and subcellular colocalization analysis. We observed that the level of Tat was significantly reduced in the presence of NC. But NC did not affect mRNA expression level of Tat. The level of Tat in the presence of NC was increased by treating cells with a proteasome inhibitor, MG132. The ubiquitination state of Tat was not seen to increase in the presence of NC, suggesting the proteasomal degradation was independent of ubiquitination. Lowered level of Tat in the presence of NC led to a decrease in Tat-mediated transcriptional transactivation.

  8. HIV-1 Phenotypic Reverse Transcriptase Inhibitor Drug Resistance Test Interpretation Is Not Dependent on the Subtype of the Virus Backbone

    PubMed Central

    Bronze, Michelle; Steegen, Kim; Wallis, Carole L.; De Wolf, Hans; Papathanasopoulos, Maria A.; Van Houtte, Margriet; Stevens, Wendy S.; de Wit, Tobias Rinke; Stuyver, Lieven J.

    2012-01-01

    To date, the majority of HIV-1 phenotypic resistance testing has been performed with subtype B virus backbones (e.g. HXB2). However, the relevance of using this backbone to determine resistance in non-subtype B HIV-1 viruses still needs to be assessed. From 114 HIV-1 subtype C clinical samples (36 ARV-naïve, 78 ARV-exposed), pol amplicons were produced and analyzed for phenotypic resistance using both a subtype B- and C-backbone in which the pol fragment was deleted. Phenotypic resistance was assessed in resulting recombinant virus stocks (RVS) for a series of antiretroviral drugs (ARV's) and expressed as fold change (FC), yielding 1660 FC comparisons. These Antivirogram® derived FC values were categorized as having resistant or sensitive susceptibility based on biological cut-off values (BCOs). The concordance between resistance calls obtained for the same clinical sample but derived from two different backbones (i.e. B and C) accounted for 86.1% (1429/1660) of the FC comparisons. However, when taking the assay variability into account, 95.8% (1590/1660) of the phenotypic data could be considered as being concordant with respect to their resistance call. No difference in the capacity to detect resistance associated with M184V, K103N and V106M mutations was noted between the two backbones. The following was concluded: (i) A high level of concordance was shown between the two backbone phenotypic resistance profiles; (ii) Assay variability is largely responsible for discordant results (i.e. for FC values close to BCO); (iii) Confidence intervals should be given around the BCO's, when assessing resistance in HIV-1 subtype C; (iv) No systematic resistance under- or overcalling of subtype C amplicons in the B-backbone was observed; (v) Virus backbone subtype sequence variability outside the pol region does not contribute to phenotypic FC values. In conclusion the HXB2 virus backbone remains an acceptable vector for phenotyping HIV-1 subtype C pol amplicons. PMID

  9. Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis

    PubMed Central

    Rivero, Matias; Cohen, Éric A.; Lopez-Lastra, Marcelo; Mouland, Andrew J.

    2013-01-01

    The precursor group-specific antigen (pr55Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55Gag expression, the synthesis of other HIV-1 proteins, including that of pr160Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55Gag for virus assembly and production. PMID:23861855

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-16

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

  12. The Cellular TAR RNA Binding Protein, TRBP, Promotes HIV-1 Replication Primarily by Inhibiting the Activation of Double-Stranded RNA-Dependent Kinase PKR▿

    PubMed Central

    Sanghvi, Viraj R.; Steel, Laura F.

    2011-01-01

    The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5′-cap or by activating the dsRNA-dependent kinase, PKR. Several mechanisms by which TRBP can facilitate translation of viral transcripts have been proposed, including the binding and unwinding of TAR and the suppression of PKR activation. Further, TRBP has been identified as a cofactor of Dicer in the processing of microRNAs (miRNAs), and sequestration of TRBP by TAR in infected cells has been proposed as a viral countermeasure to potential host cell RNA interference-based antiviral activities. Here, we have addressed the relative importance of these various roles for TRBP in HIV-1 replication. Using Jurkat T cells, primary human CD4+ T cells, and additional cultured cell lines, we show that depletion of TRBP has no effect on viral replication when PKR activation is otherwise blocked. Moreover, the presence of TAR-containing mRNAs does not affect the efficacy of cellular miRNA silencing pathways. These results establish that TRBP, when expressed at physiological levels, promotes HIV-1 replication mainly by suppressing the PKR-mediated antiviral response, while its contribution to HIV-1 replication through PKR-independent pathways is minimal. PMID:21937648

  13. A TatABC-type Tat translocase is required for unimpaired aerobic growth of Corynebacterium glutamicum ATCC13032.

    PubMed

    Oertel, Dan; Schmitz, Sabrina; Freudl, Roland

    2015-01-01

    The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.

  14. A TatABC-Type Tat Translocase Is Required for Unimpaired Aerobic Growth of Corynebacterium glutamicum ATCC13032

    PubMed Central

    Oertel, Dan; Schmitz, Sabrina; Freudl, Roland

    2015-01-01

    The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria. PMID:25837592

  15. Modulation of the splicing regulatory function of SRSF10 by a novel compound that impairs HIV-1 replication.

    PubMed

    Shkreta, Lulzim; Blanchette, Marco; Toutant, Johanne; Wilhelm, Emmanuelle; Bell, Brendan; Story, Benjamin A; Balachandran, Ahalya; Cochrane, Alan; Cheung, Peter K; Harrigan, P Richard; Grierson, David S; Chabot, Benoit

    2017-04-20

    We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro. Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2β, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2β, concentrations greater than those needed to inhibit HIV-1 replication were required to elicit significant alterations. Thus, the ability of 1C8 to alter the SRSF10-dependent splicing of HIV-1 transcripts, with minor effects on cellular splicing, supports the view that SRSF10 may be used as a target for the development of new anti-viral agents. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function.

    PubMed

    Wong, Raymond W; Balachandran, Ahalya; Haaland, Matthew; Stoilov, Peter; Cochrane, Alan

    2013-11-01

    Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.

  17. Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

    PubMed Central

    Wu, Yuntao

    2010-01-01

    Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells. PMID:20972394

  18. Metal-dependent inhibition of HIV-1 integrase by 5CITEP inhibitor: A theoretical QM/MM approach

    NASA Astrophysics Data System (ADS)

    do Nascimento, Josenaide P.; Araújo Silva, José Rogério; Lameira, Jerônimo; Alves, Cláudio N.

    2013-09-01

    HIV-1 integrase (IN) is a potential target for developing drugs against AIDS. In this letter, QM/MM approach was used to study the inhibition of IN by 5CITEP inhibitor in presence of divalent cations (Mg2+ or Mn2+). In addition, the main interactions occurring in 5CITEP-IN complex and the influence of divalent cations (Mg2+ or Mn2+) in enzymatic inhibition were investigated using B3LYP/6-31+G(d,p)/MM. The results suggest that the Asp64, Asp116 and four crystal water molecules plays a crucial role in cation (Mg2+ or Mn2+) coordination sphere.

  19. Expression Profile of Human Fc Receptors in Mucosal Tissue: Implications for Antibody-Dependent Cellular Effector Functions Targeting HIV-1 Transmission

    PubMed Central

    Cheeseman, Hannah M.; Carias, Ann M.; Evans, Abbey B.; Olejniczak, Natalia J.; Ziprin, Paul; King, Deborah F. L.; Hope, Thomas J.; Shattock, Robin J.

    2016-01-01

    The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in

  20. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  1. Synthesis of a new class of HIV-1 inhibitors.

    PubMed

    Farese-Di Giorgio, A; Pairot, S; Patino, N; Condom, R; Di Giorgio, C; Aumelas, A; Aubertin, A M; Guedj, R

    1999-02-01

    A new family of molecules potentially inhibitors of the HIV-1 Tat-TAR complex was prepared. These compounds are constituted by dinucleotide analogs (PNA dimer) bound, through a linker, to an arginine residue. In this series, several molecules inhibit viral development in cell culture with a micromolar IC50 and without cellular toxicity until 200 microM concentration.

  2. HIV-1 tropism for the central nervous system: Brain-derived envelope glycoproteins with lower CD4 dependence and reduced sensitivity to a fusion inhibitor

    SciTech Connect

    Martin-Garcia, Julio . E-mail: julio.martin-garcia@drexelmed.edu; Cao, Wei; Varela-Rohena, Angel; Plassmeyer, Matthew L.; Gonzalez-Scarano, Francisco

    2006-03-01

    We previously described envelope glycoproteins of an HIV-1 isolate adapted in vitro for growth in microglia that acquired a highly fusogenic phenotype and lower CD4 dependence, as well as resistance to inhibition by anti-CD4 antibodies. Here, we investigated whether similar phenotypic changes are present in vivo. Envelope clones from the brain and spleen of an HIV-1-infected individual with neurological disease were amplified, cloned, and sequenced. Phylogenetic analysis demonstrated clustering of sequences according to the tissue of origin, as expected. Functional clones were then used in cell-to-cell fusion assays to test for CD4 and co-receptor utilization and for sensitivity to various antibodies and inhibitors. Both brain- and spleen-derived envelope clones mediated fusion in cells expressing both CD4 and CCR5 and brain envelopes also used CCR3 as co-receptor. We found that the brain envelopes had a lower CD4 dependence, since they efficiently mediated fusion in the presence of low levels of CD4 on the target cell membrane, and they were significantly more resistant to blocking by anti-CD4 antibodies than the spleen-derived envelopes. In contrast, we observed no difference in sensitivity to the CCR5 antagonist TAK-779. However, brain-derived envelopes were significantly more resistant than those from spleen to the fusion inhibitor T-1249 and concurrently showed slightly greater fusogenicity. Our results suggest an increased affinity for CD4 of brain-derived envelopes that may have originated from in vivo adaptation to replication in microglial cells. Interestingly, we note the presence of envelopes more resistant to a fusion inhibitor in the brain of an untreated, HIV-1-infected individual.

  3. TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.

    PubMed

    Bolhuis, A; Mathers, J E; Thomas, J D; Barrett, C M; Robinson, C

    2001-06-08

    In Escherichia coli, a subset of periplasmic proteins is exported via the twin-arginine translocation (Tat) pathway. In the present study, we have purified the Tat complex from E. coli, and we show that it contains only TatA, TatB, and TatC. Within the purified complex, TatB and TatC are present in a strict 1:1 ratio, suggesting a functional association. This has been confirmed by expression of a translational fusion between TatB and TatC. This Tat(BC) chimera supports efficient Tat-dependent export, indicating that TatB and TatC act as a unit in both structural and functional terms. The purified Tat complex contains varying levels of TatA, suggesting a gradual loss during isolation and a looser association. The molecular mass of the complex is approximately 600 kDa, demonstrating the presence of multiple copies of TatA, B, and C. Co-immunoprecipitation experiments show that TatC is required for the interaction of TatA with TatB, suggesting that TatA may interact with the complex via binding to TatC.

  4. Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

    PubMed Central

    Kim, Dong-Hyun; Kim, Baek

    2011-01-01

    The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

  5. 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

  6. Understanding the molecular mechanism of sequence dependent Tenofovir removal by HIV-1 reverse transcriptase: differences in primer binding site versus polypurine tract

    PubMed Central

    Iyidogan, Pinar; Anderson, Karen S.

    2012-01-01

    Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor (NtRTI) that is often administered as first-line therapy against human immunodeficiency virus type-1 (HIV-1) infection and acts as a chain terminator when incorporated into viral DNA. However, HIV-1 reverse transcriptase (RT) excises TFV in the presence of either ATP or pyrophosphate, which is an important drug resistance mechanism that would interfere with the effective treatment. Previous studies have shown conflicting results on excision efficiencies for TFV-terminated primer-templates derived from either primer binding site (PBS) or polypurine tract (PPT) sequences. To provide mechanistic insight into the variation in TFV removal from both sequences that are vital for the HIV-1 life cycle, we compared the efficiencies of removal reaction in response to sequence dependence via utilizing blocked PBS and PPT primer-templates. We found an enhanced TFV excision with PPT sequence over PBS sequence through ATP-mediated removal and a subsequent incorporation of ATP into the unblocked primers. Furthermore, the rate of pyrophosphorolytic excision of TFV from PPT sequence was 21-fold higher than that for the PBS sequence. However, the addition of efavirenz, nonnucleoside reverse transcriptase inhibitor (NNRTI), to the removal reaction effectively inhibits the TFV excision from both primers by forming a stable complex that would leave TFV inaccessible for excision. These results illuminate the degree of primer-template sequence contribution on TFV removal as well as increase our understanding of the molecular mechanism for the beneficial effects of widely used combinations of antiretroviral regimens in the context of synergistic antiviral activity and drug resistance. PMID:22664235

  7. The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA.

    PubMed Central

    Wu-Baer, F; Lane, W S; Gaynor, R B

    1995-01-01

    Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation. Images PMID:8846792

  8. Cell-dependent gag mutants of HIV-1 are crucially defective at the stage of uncoating/reverse transcription in non-permissive cells.

    PubMed

    Koh, K; Miyaura, M; Yoshida, A; Sakurai, A; Fujita, M; Adachi, A

    2000-10-01

    We have previously shown that some of the human immunodeficiency virus type 1 (HIV-1) gag matrix (MA), capsid (CA), and nucleocapsid (NC) mutants display host-cell-dependent replication potential, and that they are defective at the early phase of the virus replication cycle in non-permissive cells. To determine the defective replication stage of the cell-dependent mutants precisely, the processes of virus entry into cells and virus DNA synthesis were monitored by the highly sensitive enzyme-linked immunosorbent assay and polymerase chain reaction amplification analysis. The results obtained indicated that all the cell-dependent MA, CA and NC mutants are defective at the stage of uncoating/reverse transcription, and that a cellular factor(s) is involved in this process.

  9. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

    SciTech Connect

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.; Kwong, Peter D.; Morris, Lynn; Kirchhoff, F.

    2016-08-31

    ABSTRACT

    All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.

    IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of

  10. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

    PubMed Central

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.

    2016-01-01

    ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site

  11. Anti-Tat and anti-HIV activities of trimers of n-alkylglycines.

    PubMed

    Márquez, Nieves; Sancho, Rocío; Macho, Antonio; Moure, Alejandra; Masip, Isabel; Messeguer, Angel; Muñoz, Eduardo

    2006-02-28

    Transcription of human immunodeficiency virus (HIV-1) is activated by viral Tat protein which regulates HIV-LTR transcription and elongation. In the present report, the evaluation of the anti-Tat activity of a combinatorial library composed of 5120 N-trialkylglycines is reported. The antiviral activity was studied through luciferase-based assays targeting the HIV-1 promoter activation induced by the HIV-1 Tat protein. We identified five peptoids with specific anti-HIV-1 Tat activity; none of these peptoids affected the binding of HIV-1 Tat protein to the viral TAR RNA. Using a recombinant-virus assay in which luciferase activity correlates with the rate of HIV-1 transcription we have detected that one of the five selected peptoids, NC37-37-15C, is a potent inhibitor of HIV-1-LTR transcription in both primary T lymphocytes and transformed cell lines. The inhibitory effect of NC37-37-15C, which is additive with azidothymidine (AZT), correlates with its ability to inhibit CTD phosphorylation and shows a suitable profile for development of novel anti-HIV-1 drugs. Likewise, the structural simplicity of N-alkylglycine oligomers makes these peptidomimetics amenable to structural manipulation, thus facilitating the optimisation of lead molecules for drug-like properties.

  12. Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells*

    PubMed Central

    Koch, Sabrina; Fritsch, Maximilian J.; Buchanan, Grant; Palmer, Tracy

    2012-01-01

    The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport. PMID:22399293

  13. Escherichia coli TatA and TatB proteins have N-out, C-in topology in intact cells.

    PubMed

    Koch, Sabrina; Fritsch, Maximilian J; Buchanan, Grant; Palmer, Tracy

    2012-04-27

    The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.

  14. Twin-arginine translocation-arresting protein regions contact TatA and TatB.

    PubMed

    Taubert, Johannes; Brüser, Thomas

    2014-07-01

    Tat systems translocate folded proteins across biological membranes of prokaryotes and plant plastids. TatBC complexes recognize N-terminal Tat signal peptides that contain a sequence motif with two conserved arginines (RR-motif), and transport takes place after a recruitment of TatA. Unfolded Tat substrate domains lower translocation efficiency and too long linkers lead to translocation arrest. To identify the components that interact with transported proteins during their passage through the translocon, we used a Tat substrate that arrests translocation at a long unfolded linker region, and we chose in vivo site-directed photo cross-linking to specifically detect the interactions of this linker region. For comparison, we included the interactions of the signal peptide and of the folded domain at the C-terminus of this construct. The data show that the linker contacts only two, structurally similar Tat components, namely TatA and TatB. These contacts depend on the recognition of the Tat-specific signal peptide. Only when membrane translocation of the globular domain was allowed--i.e., in the absence of the linker--we observed the same TatAB-contacts also to the globular domain. The data thus suggest that mature protein domains are translocated through a TatAB environment.

  15. A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

    PubMed Central

    Fujinaga, Koh; Irwin, Dan; Taube, Ran; Zhang, Fan; Geyer, Matthias; Peterlin, B. Matija

    2002-01-01

    The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex. PMID:12438619

  16. Chelation Motifs Affecting Metal-dependent Viral Enzymes: N′-acylhydrazone Ligands as Dual Target Inhibitors of HIV-1 Integrase and Reverse Transcriptase Ribonuclease H Domain

    PubMed Central

    Carcelli, Mauro; Rogolino, Dominga; Gatti, Anna; Pala, Nicolino; Corona, Angela; Caredda, Alessia; Tramontano, Enzo; Pannecouque, Christophe; Naesens, Lieve; Esposito, Francesca

    2017-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection, still represent a serious global health emergency. The chronic toxicity derived from the current anti-retroviral therapy limits the prolonged use of several antiretroviral agents, continuously requiring the discovery of new antiviral agents with innovative strategies of action. In particular, the development of single molecules targeting two proteins (dual inhibitors) is one of the current main goals in drug discovery. In this contest, metal-chelating molecules have been extensively explored as potential inhibitors of viral metal-dependent enzymes, resulting in some important classes of antiviral agents. Inhibition of HIV Integrase (IN) is, in this sense, paradigmatic. HIV-1 IN and Reverse Transcriptase-associated Ribonuclease H (RNase H) active sites show structural homologies, with the presence of two Mg(II) cofactors, hence it seems possible to inhibit both enzymes by means of chelating ligands with analogous structural features. Here we present a series of N′-acylhydrazone ligands with groups able to chelate the Mg(II) hard Lewis acid ions in the active sites of both the enzymes, resulting in dual inhibitors with micromolar and even nanomolar activities. The most interesting identified N′-acylhydrazone analog, compound 18, shows dual RNase H-IN inhibition and it is also able to inhibit viral replication in cell-based antiviral assays in the low micromolar range. Computational modeling studies were also conducted to explore the binding attitudes of some model ligands within the active site of both the enzymes. PMID:28373864

  17. 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.

  18. 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…

  19. HIV-1 viral protein r induces ERK and caspase-8 dependent apoptosis in renal tubular epithelial cells

    PubMed Central

    Snyder, Alexandra; Alsauskas, Zygimantas C.; Leventhal, Jeremy S.; Rosenstiel, Paul E.; Gong, Pengfei; Chan, Justin JK; Barley, Kevin; He, John C.; Klotman, Mary E.; Ross, Michael J.; Klotman, Paul E.

    2010-01-01

    Objective HIV-associated nephropathy is the most common cause of end stage renal disease in persons with HIV/AIDS and is characterized by focal glomerulosclerosis and dysregulated renal tubular epithelial cell (RTEC) proliferation and apoptosis. HIV-1 viral protein r (Vpr) has been implicated in HIV-induced RTEC apoptosis but the mechanisms of Vpr-induced RTEC apoptosis are unknown. The aim of this study was therefore to determine the mechanisms of Vpr-induced apoptosis in RTEC. Methods Apoptosis and caspase activation were analyzed in human RTEC cells (HK2) after transduction with Vpr-expressing and control lentiviral vectors. Bax and BID were inhibited with lentiviral shRNA, and ERK activation was blocked with the MEK1,2 inhibitor, U0126. Results Vpr induced apoptosis as indicated by caspase 3/7 activation, PARP-1 cleavage and mitochondrial injury. Vpr activated both caspases-8 and 9. Inhibition of Bax reduced Vpr-induced apoptosis, as reported in other cell types. Additionally, Vpr induced cleavage of BID to tBID and suppression of BID expression prevented Vpr-induced apoptosis. Since sustained ERK activation can activate caspase-8 in some cell types, we studied the role of ERK in Vpr-induced caspase-8 activation. Vpr induced sustained ERK activation in HK2 cells and incubation with U0126 reduced Vpr-induced caspase-8 activation, BID cleavage and apoptosis. We detected phosphorylated ERK in RTEC in HIVAN biopsy specimens by immunohistochemistry. Conclusions These studies delineate a novel pathway of Vpr-induced apoptosis in RTEC, which is mediated by sustained ERK activation, resulting in caspase 8-mediated cleavage of BID to tBID, thereby facilitating Bax-mediated mitochondrial injury and apoptosis. PMID:20404718

  20. 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

  1. 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

  2. High-salinity growth conditions promote Tat-independent secretion of Tat substrates in Bacillus subtilis.

    PubMed

    van der Ploeg, René; Monteferrante, Carmine G; Piersma, Sjouke; Barnett, James P; Kouwen, Thijs R H M; Robinson, Colin; van Dijl, Jan Maarten

    2012-11-01

    The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.

  3. HIV-1 incidence rates and risk factors in agricultural workers and dependents in rural Kenya: 36-month follow-up of the Kericho HIV cohort study.

    PubMed

    Shaffer, Douglas N; Ngetich, Ignatius K; Bautista, Christian T; Sawe, Frederick K; Renzullo, Philip O; Scott, Paul T; Kibaya, Rukia M; Imbuki, Kennedy O; Michael, Nelson L; Birx, Deborah L; Wasunna, Monique K; Robb, Merlin L

    2010-04-01

    Incidence data from prospective cohort studies using rigorous laboratory methods are important in designing and evaluating HIV vaccine and therapeutic clinical trials and health care programs. We report 36-month HIV-1 incidence rates and demographic and psychosocial risks from the Kericho cohort in rural Kenya's southern Rift Valley Province. Thirty-six month, prospective, closed, observational cohort study of adult plantation workers and dependents followed biannually. HIV-1 incidence rates per 100 person-years (py) were calculated, and Cox regression analyses were used to estimate hazards ratios (HR) associated with seroconversion. Two thousand four hundred volunteers (mean age +/- SD = 30.1 +/- 8.5 years; 36.5% women) participated. Twenty-nine new HIV cases were identified in year 1 of follow-up, which increased to cumulative totals of 49 and 63 cases in years 2 and 3, respectively. The corresponding 1-, 2-, and 3-year incidence rates were 1.41 [95% confidence interval (CI) = 0.95-2.02], 1.16 (95% CI = 0.86-1.54), and 1.00 (95% CI = 0.77-1.28) per 100 py. Risk factors associated with HIV seroconversion included the following: of the Luo tribe (HR = 3.31; 95% CI = 1.65-6.63), marriage more than once (HR = 2.83; 95% CI = 1.20-6.69), self-reported male circumcision (HR = 0.32; 95% CI = 0.17-0.60), history of sexually transmitted infection (HR = 2.40; 95% CI = 1.09-5.26), history of substance abuse during sex (HR = 2.44; 95% CI = 1.16-5.13), and history of transactional sex (HR = 3.30; 95% CI = 1.79-6.09). HIV-1 incidence rates were relatively low in adult plantation workers and dependents in rural Kenya. Cohorts including higher risk populations (eg, commercial sex workers) warrant consideration for regional HIV preventive vaccine trials. Even low incidence, well-described cohorts generate valuable epidemiological clinical trial data.

  4. Monoclonal Antibodies to V2, V3, the CD4-binding site and gp41 HIV-1 Mediate Phagocytosis in a Dose-dependent Manner.

    PubMed

    Musich, Thomas; Li, Liuzhe; Liu, Lily; Zolla-Pazner, Susan; Robert-Guroff, Marjorie; Gorny, Miroslaw K

    2017-01-25

    In light of weak or absent neutralizing activity mediated by anti-V2 monoclonal Abs (mAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP) which is an important element of anti-HIV-1 immunity. We tested six anti-V2 mAbs and compared them with 21 mAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from HIV-1 chronically infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664 or BG505 DS-SOSIP.664 complexed with mAbs. The ADCP activity measured by the area under the curve showed significantly higher activity of anti-gp41 mAbs compared to three other groups of mAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 mAbs were dominant over anti-V3 and anti-CD4bs against clade C gp120ZM109. ADCP mediated by V2 and V3 mAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting a closed envelope conformation better exposes the variable loops. Two IgG3 mAbs against V2 and V3 regions displayed dominant ADCP activity over a panel of IgG1 mAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 mAbs were compared to IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers with some higher activity of anti-V2 mAbs over anti-V3 and anti-CD4bs mAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy.

  5. Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein.

    PubMed

    Sargent, F; Stanley, N R; Berks, B C; Palmer, T

    1999-12-17

    In Escherichia coli, transmembrane translocation of proteins can proceed by a number of routes. A subset of periplasmic proteins are exported via the Tat pathway to which proteins are directed by N-terminal "transfer peptides" bearing the consensus (S/T)RRXFLK "twin-arginine" motif. The Tat system involves the integral membrane proteins TatA, TatB, TatC, and TatE. Of these, TatA, TatB, and TatE are homologues of the Hcf106 component of the DeltapH-dependent protein import system of plant thylakoids. Deletion of the tatB gene alone is sufficient to block the export of seven endogenous Tat substrates, including hydrogenase-2. Complementation analysis indicates that while TatA and TatE are functionally interchangeable, the TatB protein is functionally distinct. This conclusion is supported by the observation that Helicobacter pylori tatA will complement an E. coli tatA mutant, but not a tatB mutant. Analysis of Tat component stability in various tat deletion backgrounds shows that TatC is rapidly degraded in the absence of TatB suggesting that TatC complexes, and is stabilized by, TatB.

  6. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond.

    PubMed

    Lee, Soo-Jin; Yoon, Sung-Hwa; Doh, Kyung-Oh

    2011-08-01

    Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

  7. Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton.

    PubMed

    Cameron, Paul U; Saleh, Suha; Sallmann, Georgina; Solomon, Ajantha; Wightman, Fiona; Evans, Vanessa A; Boucher, Genevieve; Haddad, Elias K; Sekaly, Rafick-Pierre; Harman, Andrew N; Anderson, Jenny L; Jones, Kate L; Mak, Johnson; Cunningham, Anthony L; Jaworowski, Anthony; Lewin, Sharon R

    2010-09-28

    Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4(+) T cells. We now show that HIV-1 latency can be established in resting CD4(+) T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4(+) T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4(+) T cells during normal chemokine-directed recirculation of CD4(+) T cells between blood and tissue.

  8. An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

    PubMed

    Bennett, Ryan P; Stewart, Ryan A; Hogan, Priscilla A; Ptak, Roger G; Mankowski, Marie K; Hartman, Tracy L; Buckheit, Robert W; Snyder, Beth A; Salter, Jason D; Morales, Guillermo A; Smith, Harold C

    2016-12-01

    Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Overlapping Regions in HIV-1 Genome Act as Potential Sites for Host–Virus Interaction

    PubMed Central

    Saha, Deeya; Podder, Soumita; Ghosh, Tapash C.

    2016-01-01

    More than a decade, overlapping genes in RNA viruses became a subject of research which has explored various effect of gene overlapping on the evolution and function of viral genomes like genome size compaction. Additionally, overlapping regions (OVRs) are also reported to encode elevated degree of protein intrinsic disorder (PID) in unspliced RNA viruses. With the aim to explore the roles of OVRs in HIV-1 pathogenesis, we have carried out an in-depth analysis on the association of gene overlapping with PID in 35 HIV1- M subtypes. Our study reveals an over representation of PID in OVR of HIV-1 genomes. These disordered residues endure several vital, structural features like short linear motifs (SLiMs) and protein phosphorylation (PP) sites which are previously shown to be involved in massive host–virus interaction. Moreover, SLiMs in OVRs are noticed to be more functionally potential as compared to that of non-overlapping region. Although, density of experimentally verified SLiMs, resided in 9 HIV-1 genes, involved in host–virus interaction do not show any bias toward clustering into OVR, tat and rev two important proteins mediates host–pathogen interaction by their experimentally verified SLiMs, which are mostly localized in OVR. Finally, our analysis suggests that the acquisition of SLiMs in OVR is mutually exclusive of the occurrence of disordered residues, while the enrichment of PPs in OVR is solely dependent on PID and not on overlapping coding frames. Thus, OVRs of HIV-1 genomes could be demarcated as potential molecular recognition sites during host–virus interaction. PMID:27867372

  10. Involvement of cytokines in human immunodeficiency virus-1 protein Tat and methamphetamine interactions in the striatum.

    PubMed

    Theodore, Shaji; Cass, Wayne A; Maragos, William F

    2006-06-01

    Human immunodeficiency virus-1 (HIV-1) infection of the brain causes elevation in pro-inflammatory cytokines and inflammatory changes in the striatum. HIV-1-infected individuals who also abuse drugs including the psychostimulant methamphetamine (MA) develop more severe encephalitis and neuronal damage compared to HIV-1-infected patients who do not abuse drugs. In previous studies, we demonstrated that the HIV-1 protein Tat and MA interacted to cause enhanced loss of dopamine in the rat striatum via the destruction of dopaminergic terminals. Since both Tat and MA activate glia and induce cytokine production, we investigated the role of cytokines in the synergistic neurotoxicity induced by Tat and MA using cytokine arrays. Significant increases in monocyte chemotactic protein (MCP-1), interleukin-1 alpha (IL-1alpha) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were noted 4 h following Tat + MA treatment compared to saline, Tat or MA. MCP-1 and TIMP-1 levels remained elevated 16 h after Tat + MA compared to saline or MA but were not different from the Tat-treated group at this time point. Weak, but significant elevations in cytokine-induced neutrophil chemoattractant-3 (CINC-3), ciliary neurotrophic factor (CNTF) and macrophage inflammatory protein-3 alpha (MIP-3alpha) were also noted with Tat + MA. The interaction of Tat and MA was prevented in mice genetically deficient in MCP-1 with a consequent attenuation of Tat + MA neurotoxicity. Our findings suggest that HIV-1 infection with concurrent drug abuse might profoundly increase chemokine levels in the striatum resulting in enhanced damage to the dopaminergic system.

  11. Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome.

    PubMed

    Witkowska, Julia; Karpowicz, Przemysław; Gaczynska, Maria; Osmulski, Pawel A; Jankowska, Elżbieta

    2014-08-01

    Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R(49) KKRRQRR(56) , supplemented with Q(66) DPI(69) fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  12. Tripartite containing motif 32 modulates proliferation of human neural precursor cells in HIV-1 neurodegeneration

    PubMed Central

    Fatima, M; Kumari, R; Schwamborn, J C; Mahadevan, A; Shankar, S K; Raja, R; Seth, P

    2016-01-01

    In addition to glial cells, HIV-1 infection occurs in multipotent human neural precursor cells (hNPCs) and induces quiescence in NPCs. HIV-1 infection of the brain alters hNPC stemness, leading to perturbed endogenous neurorestoration of the CNS following brain damage by HIV-1, compounding the severity of dementia in adult neuroAIDS cases. In pediatric neuroAIDS cases, HIV-1 infection of neural stem cell can lead to delayed developmental milestones and impaired cognition. Using primary cultures of human fetal brain-derived hNPCs, we gained novel insights into the role of a neural stem cell determinant, tripartite containing motif 32 (TRIM32), in HIV-1 Tat-induced quiescence of NPCs. Acute HIV-1 Tat treatment of hNPCs resulted in proliferation arrest but did not induce differentiation. Cellular localization and levels of TRIM32 are critical regulators of stemness of NPCs. HIV-1 Tat exposure increased nuclear localization and levels of TRIM32 in hNPCs. The in vitro findings were validated by studying TRIM32 localization and levels in frontal cortex of HIV-1-seropositive adult patients collected at post mortem as well as by infection of hNPCs by HIV-1. We observed increased percentage of cells with nuclear localization of TRIM32 in the subventricular zone (SVZ) as compared with age-matched controls. Our quest for probing into the mechanisms revealed that TRIM32 is targeted by miR-155 as downregulation of miR-155 by HIV-1 Tat resulted in upregulation of TRIM32 levels. Furthermore, miR-155 or siRNA against TRIM32 rescued HIV-1 Tat-induced quiescence in NPCs. Our findings suggest a novel molecular cascade involving miR-155 and TRIM32 leading to HIV-1 Tat-induced attenuated proliferation of hNPCs. The study also uncovered an unidentified role for miR-155 in modulating human neural stem cell proliferation, helping in better understanding of hNPCs and diseased brain. PMID:26586575

  13. Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability

    PubMed Central

    Benoit, Stéphane L.; Maier, Robert J.

    2014-01-01

    ABSTRACT The twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects. PMID:24449753

  14. Activation of HIV-1 with Nanoparticle-Packaged Small-Molecule Protein Phosphatase-1-Targeting Compound

    PubMed Central

    Smith, Kahli A.; Lin, Xionghao; Bolshakov, Oleg; Griffin, James; Niu, Xiaomei; Kovalskyy, Dmytro; Ivanov, Andrey; Jerebtsova, Marina; Taylor, Robert E.; Akala, Emmanuel; Nekhai, Sergei

    2015-01-01

    Complete eradication of HIV-1 infection is impeded by the existence of latent HIV-1 reservoirs in which the integrated HIV-1 provirus is transcriptionally inactive. Activation of HIV-1 transcription requires the viral Tat protein and host cell factors, including protein phosphatase-1 (PP1). We previously developed a library of small compounds that targeted PP1 and identified a compound, SMAPP1, which induced HIV-1 transcription. However, this compound has a limited bioavailability in vivo and may not be able to reach HIV-1-infected cells and induce HIV-1 transcription in patients. We packaged SMAPP1 in polymeric polyethylene glycol polymethyl methacrylate nanoparticles and analyzed its release and the effect on HIV-1 transcription in a cell culture. SMAPP1 was efficiently packaged in the nanoparticles and released during a 120-hr period. Treatment of the HIV-1-infected cells with the SMAPP1-loaded nanoparticles induced HIV-1 transcription. Thus, nanoparticles loaded with HIV-1-targeting compounds might be useful for future anti-HIV-1 therapeutics. PMID:26839837

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

    SciTech Connect

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

    2013-11-27

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

  16. Blockage of CD59 Function Restores Activities of Neutralizing and Nonneutralizing Antibodies in Triggering Antibody-Dependent Complement-Mediated Lysis of HIV-1 Virions and Provirus-Activated Latently Infected Cells

    PubMed Central

    Yang, Kai; Lan, Jie; Shepherd, Nicole; Hu, Ningjie; Xing, Yanyan; Byrd, Daniel; Amet, Tohti; Jewell, Corlin; Gupta, Samir; Kounga, Carole

    2015-01-01

    ABSTRACT Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs), including 2G12, 2F5, and 4E10, and non-nAbs, including 2.2C, A32, N5-i5, and N12-i15, to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs, 2.2C and N12-i15, reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the enzyme-linked immunosorbent assay-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells. IMPORTANCE There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and

  17. Blockage of CD59 Function Restores Activities of Neutralizing and Nonneutralizing Antibodies in Triggering Antibody-Dependent Complement-Mediated Lysis of HIV-1 Virions and Provirus-Activated Latently Infected Cells.

    PubMed

    Yang, Kai; Lan, Jie; Shepherd, Nicole; Hu, Ningjie; Xing, Yanyan; Byrd, Daniel; Amet, Tohti; Jewell, Corlin; Gupta, Samir; Kounga, Carole; Gao, Jimin; Yu, Qigui

    2015-09-01

    Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs), including 2G12, 2F5, and 4E10, and non-nAbs, including 2.2C, A32, N5-i5, and N12-i15, to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs, 2.2C and N12-i15, reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the enzyme-linked immunosorbent assay-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells. There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and infected cells

  18. Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis.

    PubMed

    Eijlander, Robyn T; Kolbusz, Magdalena A; Berendsen, Erwin M; Kuipers, Oscar P

    2009-06-01

    Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the B. subtilis TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the B. subtilis Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and Escherichia coli TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for E. coli TatC and implications for substrate binding and translocation are discussed.

  19. Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture.

    PubMed

    Neuveut, C; Jeang, K T

    1996-08-01

    Human immunodeficiency virus type 1 (HIV-1) Tat is essential for virus replication and is a potent trans activator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Extensive structure-function studies of Tat in subgenomic settings with point mutagenesis and transient transfection readouts have been performed. These reporter assays have defined certain amino acid residues as being important for trans activation of reporter plasmids. However, they have not directly addressed functions related to virus replication. Here, we have studied Tat structure-function in the setting of replicating viruses. We characterized mutations that emerged in Tat during HIV-1 infections of T lymphocytes. To ensure that the selection pressure for change was directed toward protein function, we constructed HIV-Is in which the Tat reading frame was freed from constraints exerted by overlapping with the reading frames of vpr, rev, and env. When these recombinant viruses were passaged in T cells, 26 novel nucleotide changes in tat were observed from sequencing of 220 independently isolated clones. Recloning of these changes into a pNL4-3 molecular background allowed for the characterization of residues in Tat important for virus replication. Interestingly, many of the changes that affected replication when they were assayed in transient trans activation of plasmid reporters were found to be relatively neutral. We conclude that the structure-function of Tat in virus replication is incompletely reflected by activity measurements based only on subgenomic transient transfections.

  20. Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture.

    PubMed Central

    Neuveut, C; Jeang, K T

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) Tat is essential for virus replication and is a potent trans activator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Extensive structure-function studies of Tat in subgenomic settings with point mutagenesis and transient transfection readouts have been performed. These reporter assays have defined certain amino acid residues as being important for trans activation of reporter plasmids. However, they have not directly addressed functions related to virus replication. Here, we have studied Tat structure-function in the setting of replicating viruses. We characterized mutations that emerged in Tat during HIV-1 infections of T lymphocytes. To ensure that the selection pressure for change was directed toward protein function, we constructed HIV-Is in which the Tat reading frame was freed from constraints exerted by overlapping with the reading frames of vpr, rev, and env. When these recombinant viruses were passaged in T cells, 26 novel nucleotide changes in tat were observed from sequencing of 220 independently isolated clones. Recloning of these changes into a pNL4-3 molecular background allowed for the characterization of residues in Tat important for virus replication. Interestingly, many of the changes that affected replication when they were assayed in transient trans activation of plasmid reporters were found to be relatively neutral. We conclude that the structure-function of Tat in virus replication is incompletely reflected by activity measurements based only on subgenomic transient transfections. PMID:8764071

  1. HIV-1 Env- and Vpu-Specific Antibody-Dependent Cellular Cytotoxicity Responses Associated with Elite Control of HIV.

    PubMed

    Madhavi, Vijaya; Wines, Bruce D; Amin, Janaki; Emery, Sean; Lopez, Ester; Kelleher, Anthony; Center, Rob J; Hogarth, P Mark; Chung, Amy W; Kent, Stephen J; Stratov, Ivan

    2017-09-15

    Studying HIV-infected individuals who control HIV replication (elite controllers [ECs]) enables exploration of effective anti-HIV immunity. HIV Env-specific and non-Env-specific antibody-dependent cellular cytotoxicity (ADCC) may contribute to protection from progressive HIV infection, but the evidence is limited. We recruited 22 ECs and matched them with 44 viremic subjects. HIV Env- and Vpu-specific ADCC responses in sera were studied using a novel enzyme-linked immunosorbent assay (ELISA)-based dimeric recombinant soluble FcγRIIIa (rsFcγRIIIa)-binding assay, surface plasmon resonance, antibody-dependent natural killer (NK) cell activation assays, and ADCC-mediated killing assays. ECs had higher levels of HIV Env-specific antibodies capable of binding FcγRIIIa, activating NK cells, and mediating granzyme B activity (all P < 0.01) than viremic subjects. ECs also had higher levels of antibodies against a C-terminal 13-mer Vpu peptide capable of mediating FcγRIIIa binding and NK cell activation than viremic subjects (both P < 0.05). Our data associate Env-specific and Vpu epitope-specific ADCC in effective immune responses against HIV among ECs. Our findings have implications for understanding the role of ADCC in HIV control.IMPORTANCE Understanding immune responses associated with elite control of HIV may aid the development of immunotherapeutic and vaccine strategies for controlling HIV infection. Env is a major HIV protein target of functional antibody responses that are heightened in ECs. Interestingly, EC antibodies also target Vpu, an accessory protein crucial to HIV, which degrades CD4 and antagonizes tetherin. Antibodies specific to Vpu are a common feature of the immune response of ECs that may prove to be of functional importance to the design of improved ADCC-based immunotherapy and preventative HIV vaccines. Copyright © 2017 American Society for Microbiology.

  2. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

    PubMed Central

    Molinos-Albert, Luis M.; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A.; Contreras, F.-Xabier; Floor, Martin; Cardona, Pere J.; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-01

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants. PMID:28084464

  3. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif.

    PubMed

    Molinos-Albert, Luis M; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A; Contreras, F-Xabier; Floor, Martin; Cardona, Pere J; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-13

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

  4. 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

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

    PubMed

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

    2012-02-01

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

  6. 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

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

    PubMed

    Li, Zichong; Lu, Huasong; Zhou, Qiang

    2016-02-01

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

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

    PubMed Central

    Li, Zichong; Lu, Huasong

    2016-01-01

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

  9. Indian Long-term Non-Progressors Show Broad ADCC Responses with Preferential Recognition of V3 Region of Envelope and a Region from Tat Protein

    PubMed Central

    Kulkarni, Archana; Kurle, Swarali; Shete, Ashwini; Ghate, Manisha; Godbole, Sheela; Madhavi, Vijaya; Kent, Stephen J.; Paranjape, Ramesh; Thakar, Madhuri

    2017-01-01

    HIV-specific antibody-dependent cell cytotoxicity (ADCC) is likely to be important in governing protection from human immunodeficiency virus (HIV) and slowing disease progression. Little is known about the ADCC responses to HIV-1 subtype C. We characterized ADCC responses in HIV-1 subtype C-infected Indian subjects with slow disease progression and identified the dominant antigenic regions recognized by these antibodies. ADCC responses were measured in plasma from 34 long-term non-progressors (LTNPs), who were asymptomatic and maintained CD4 count above 500 cells/mm3 for the last 7 years in the absence of antiretroviral therapy (ART), and 58 ART naïve progressors with CD4 count <500 cells/mm3 against overlapping HIV-1 peptides using a flow cytometry-based antibody-dependent natural killer (NK) cell activation assay. The assay measured CD107a expression on NK cells as a marker of antibody-dependent NK cell activation and IFN-γ secretion by NK cells upon activation. The ADCC epitopes were mapped using the matrix of overlapping peptides. Indian LTNPs showed higher and broader ADCC responses compared to the progressors. The Env-C and Tat-specific ADCC responses were associated with lower plasma viral load, whereas the Env-C responses were also associated with higher CD4 counts. Five of 10 LTNP responders targeted epitopes in the V3 region (amino acids 288–330) of Env-C. Additionally, three Tat regions were targeted by ADCC antibodies from LTNPs. ADCC responses were associated with slow HIV progression in Indian subtype C-infected cohort. The frequently recognized peptides from the V3 loop of Env and the novel epitopes from Tat by the LTNPs warrants further study to understand the role of ADCC responses to these regions in control and prevention of HIV-1 infection. PMID:28154562

  10. Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton

    PubMed Central

    Cameron, Paul U.; Saleh, Suha; Sallmann, Georgina; Solomon, Ajantha; Wightman, Fiona; Evans, Vanessa A.; Boucher, Genevieve; Haddad, Elias K.; Sekaly, Rafick-Pierre; Harman, Andrew N.; Anderson, Jenny L.; Jones, Kate L.; Mak, Johnson; Cunningham, Anthony L.; Jaworowski, Anthony; Lewin, Sharon R.

    2010-01-01

    Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4+ T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4+ T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4+ T cells during normal chemokine-directed recirculation of CD4+ T cells between blood and tissue. PMID:20837531

  11. HIV-1 Envelope Glycoprotein Resistance to Monoclonal Antibody 2G12 Is Subject-Specific and Context-Dependent in Macaques and Humans

    PubMed Central

    Malherbe, Delphine C.; Sanders, Rogier W.; van Gils, Marit J.; Park, Byung; Gomes, Michelle M.; Schuitemaker, Hanneke; Barnett, Susan; Haigwood, Nancy L.

    2013-01-01

    HIV-1 Envelope (Env) protein is the sole target of neutralizing antibodies (NAbs) that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many common alterations are known to affect sensitivity to NAbs, including residues encoding potential N-linked glycosylation sites (PNGS). Knowledge of Env motifs associated with neutralization resistance is valuable for the design of an effective Env-based vaccine so we characterized Envs isolated longitudinally from a SHIVSF162P4 infected macaque for sensitivity to neutralizing monoclonal antibodies (MAbs) B12, 2G12, 4E10 and 2F5. The early Env, isolated from plasma at day 56 after infection, was the most sensitive and the late Env, from day 670, was the most resistant to MAbs. We identified four PNGS in these Envs that accumulated over time at positions 130, 139, 160 and 397. We determined that removal of these PNGS significantly increased neutralization sensitivity to 2G12, and conversely, we identified mutations by in silico analyses that contributed resistance to 2G12 neutralization. In order to expand our understanding of these PNGS, we analyzed Envs from clade B HIV-infected human subjects and identified additional glycan and amino acid changes that could affect neutralization by 2G12 in a context-dependent manner. Taken together, these in vitro and in silico analyses of clade B Envs revealed that 2G12 resistance is achieved by previously unrecognized PNGS substitutions in a context-dependent manner and by subject-specific pathways. PMID:24040404

  12. Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    PubMed Central

    Chan, Vera S. F.; Chung, Nancy P. Y.; Wang, Shu-Rong; Li, Zhongye; Ma, Jing; Lin, Chia-Wei; Hsieh, Ya-Ju; Chang, Kao-Ping; Kung, Sui-Sum; Wu, Yi-Chia; Chu, Cheng-Wei; Tai, Hsiao-Ting; Gao, George F.; Zheng, Bojian; Yokoyama, Kazunari K.; Austyn, Jonathan M.; Lin, Chen-Lung S.

    2013-01-01

    During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to

  13. A Re-Examination of Global Suppression of RNA Interference by HIV-1

    PubMed Central

    Sanghvi, Viraj R.; Steel, Laura F.

    2011-01-01

    The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing. PMID:21386885

  14. Identification of protein-protein interactions between the TatB and TatC subunits of the twin-arginine translocase system and respiratory enzyme specific chaperones.

    PubMed

    Kuzniatsova, Lalita; Winstone, Tara M L; Turner, Raymond J

    2016-04-01

    The Twin-arginine translocation (Tat) pathway serves for translocation of fully folded proteins across the cytoplasmic membrane in bacterial and chloroplast thylakoid membranes. The Escherichia coli Tat system consists of three core components: TatA, TatB, and TatC. The TatB and TatC subunits form the receptor complex for Tat dependent proteins. The TatB protein is composed of a single transmembrane helix and cytoplasmic domain. The structure of TatC revealed six transmembrane helices. Redox Enzyme Maturation Proteins (REMPs) are system specific chaperones, which play roles in the maturation of Tat dependent respiratory enzymes. Here we applied the in vivo bacterial two-hybrid technique to investigate interaction of REMPs with the TatBC proteins, finding that all but the formate dehydrogenase REMP dock to TatB or TatC. We focused on the NarJ subfamily, where DmsD--the REMP for dimethyl sulfoxide reductase in E. coli--was previously shown to interact with TatB and TatC. We found that these REMPs interact with TatC cytoplasmic loops 1, 2 and 4, with the exception of NarJ, that only interacts with 1 and 4. An in vitro isothermal titration calorimetry study was applied to confirm the evidence of interactions between TatC fragments and DmsD chaperone. Using a peptide overlapping array, it was shown that the different NarJ subfamily REMPs interact with different regions of the TatB cytoplasmic domains. The results demonstrate a role of REMP chaperones in targeting respiratory enzymes to the Tat system. The data suggests that the different REMPs may have different mechanisms for this task. Copyright © 2016 Elsevier B.V. All rights reserved

  15. TatBC-Independent TatA/Tat Substrate Interactions Contribute to Transport Efficiency

    PubMed Central

    Taubert, Johannes; Hou, Bo; Risselada, H. Jelger; Mehner, Denise; Lünsdorf, Heinrich; Grubmüller, Helmut; Brüser, Thomas

    2015-01-01

    The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of Escherichia coli and plant plastids, a third component – TatB – is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells. PMID:25774531

  16. 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.

  17. 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

  18. Determination of the minimal amount of Tat activity required for human immunodeficiency virus type 1 replication.

    PubMed

    Verhoef, K; Koper, M; Berkhout, B</