The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

Abstract I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif.

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Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Nucleic Acids Research ◽

10.1093/nar/gkv897 ◽

2015 ◽

Vol 43 (18) ◽

pp. 8884-8897 ◽

Cited By ~ 63

Author(s):

Elena Tosoni ◽

Ilaria Frasson ◽

Matteo Scalabrin ◽

Rosalba Perrone ◽

Elena Butovskaya ◽

...

Keyword(s):

Transcriptional Activity ◽

Cellular Protein ◽

Spectrometric Analysis ◽

Viral Transcription ◽

Mobility Shift ◽

Binding Preference ◽

G Quadruplex ◽

Mobility Shift Assay ◽

Ltr Promoter ◽

Hiv 1

Abstract Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

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Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4+ T Lymphocytes by Combination of Cytokines

Journal of Virology ◽

10.1128/jvi.75.23.11336-11343.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11336-11343 ◽

Cited By ~ 70

Author(s):

Romi Ghose ◽

Li-Ying Liou ◽

Christine H. Herrmann ◽

Andrew P. Rice

Keyword(s):

T Lymphocytes ◽

Rna Polymerase Ii ◽

Interleukin 2 ◽

Cellular Protein ◽

Peripheral Blood Lymphocytes ◽

Tat Protein ◽

Necrosis Factor Alpha ◽

Cyclin T1 ◽

Protein Levels ◽

Hiv 1

ABSTRACT Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4+ T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4+ T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4+ T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4+ T lymphocytes harboring a latent provirus.

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Phosphoserine acidic cluster motifs in the cytoplasmic domains of transmembrane proteins bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

10.1101/286633 ◽

2018 ◽

Author(s):

Rajendra Singh ◽

Charlotte Stoneham ◽

Christopher Lim ◽

Xiaofei Jia ◽

Javier Guenaga ◽

...

Keyword(s):

Protein Trafficking ◽

Transmembrane Proteins ◽

Adaptor Protein ◽

Cellular Protein ◽

Membrane Lipid ◽

Dependent Manner ◽

Cytoplasmic Domains ◽

Clathrin Adaptor ◽

Hiv 1

AbstractProtein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease Furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of Furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro. The sites of these serines vary among mammals in a manner consistent with host-pathogen conflict, yet the Serinc3-PSAC seems dispensible for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu, Furin, and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

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Functional Differences between Human and Bovine Immunodeficiency Virus Tat Transcription Factors

Journal of Virology ◽

10.1128/jvi.74.10.4666-4671.2000 ◽

2000 ◽

Vol 74 (10) ◽

pp. 4666-4671 ◽

Cited By ~ 15

Author(s):

Hal P. Bogerd ◽

Heather L. Wiegand ◽

Paul D. Bieniasz ◽

Bryan R. Cullen

Keyword(s):

Cooperative Interaction ◽

Bovine Immunodeficiency Virus ◽

Viral Gene ◽

Tat Protein ◽

Cyclin T1 ◽

Immunodeficiency Virus ◽

Cellular Cofactor ◽

Ltr Promoter ◽

Hiv 1

ABSTRACT Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.

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Long noncoding RNA MALAT1 releases epigenetic silencing of HIV-1 replication by displacing the polycomb repressive complex 2 from binding to the LTR promoter

Nucleic Acids Research ◽

10.1093/nar/gkz117 ◽

2019 ◽

Vol 47 (6) ◽

pp. 3013-3027 ◽

Cited By ~ 26

Author(s):

Di Qu ◽

Wei-Wei Sun ◽

Li Li ◽

Li Ma ◽

Li Sun ◽

...

Keyword(s):

Long Noncoding Rna ◽

Noncoding Rna ◽

Epigenetic Silencing ◽

Polycomb Repressive Complex ◽

Polycomb Repressive Complex 2 ◽

Ltr Promoter ◽

Hiv 1

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NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

Retrovirology ◽

10.1186/s12977-014-0067-y ◽

2014 ◽

Vol 11 (1) ◽

Cited By ~ 11

Author(s):

Hye-Young Kim ◽

Byeong-Sun Choi ◽

Sung Soon Kim ◽

Tae-Young Roh ◽

Jihwan Park ◽

...

Keyword(s):

Crucial Role ◽

Viral Transcription ◽

Ltr Promoter ◽

Hiv 1

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SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T Cells

Journal of Virology ◽

10.1128/jvi.00292-18 ◽

2018 ◽

Vol 92 (15) ◽

Cited By ~ 12

Author(s):

Jenna M. Antonucci ◽

Sun Hee Kim ◽

Corine St. Gelais ◽

Serena Bonifati ◽

Tai-Wei Li ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Leukemia Virus ◽

Viral Latency ◽

Viral Reservoir ◽

Latently Infected ◽

Ltr Promoter ◽

Hiv 1

ABSTRACT Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T cells, which are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 long terminal repeat (LTR) activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D R207N [HD/RN]) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not the T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro. These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4+ T cells. IMPORTANCE A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in nondividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T cells.

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The HIV-1 capsid-binding host factor CPSF6 is post-transcriptionally regulated by the cellular microRNA miR-125b

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010534 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5081-5094

Author(s):

Evan Chaudhuri ◽

Sabyasachi Dash ◽

Muthukumar Balasubramaniam ◽

Adrian Padron ◽

Joseph Holland ◽

...

Keyword(s):

Molecular Mechanisms ◽

Mammalian Species ◽

Cellular Protein ◽

Luciferase Reporter ◽

Down Regulation ◽

Host Interaction ◽

Cleavage And Polyadenylation ◽

Specificity Factor ◽

Viral Dna Integration ◽

Hiv 1

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in mRNA processing. Emerging evidence suggests that CPSF6 also plays key roles in HIV-1 infection, specifically during nuclear import and integration targeting. However, the cellular and molecular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post-transcriptional mechanism that regulates CPSF6 via the cellular microRNA miR-125b. An in silico analysis revealed that the 3′UTR of CPSF6 contains a miR-125b–binding site that is conserved across several mammalian species. Because miRNAs repress protein expression, we tested the effects of miR-125b expression on CPSF6 levels in miR-125b knockdown and over-expression experiments, revealing that miR-125b and CPSF6 levels are inversely correlated. To determine whether miR-125b post-transcriptionally regulates CPSF6, we introduced the 3′UTR of CPSF6 mRNA into a luciferase reporter and found that miR-125b negatively regulates CPSF6 3′UTR-driven luciferase activity. Accordingly, mutations in the miR-125b seed sequence abrogated the regulatory effect of the miRNA on the CPSF6 3′UTR. Finally, pulldown experiments demonstrated that miR-125b physically interacts with CPSF6 3′UTR. Interestingly, HIV-1 infection down-regulated miR-125b expression concurrent with up-regulation of CPSF6. Notably, miR-125b down-regulation in infected cells was not due to reduced pri-miRNA or pre-miRNA levels. However, miR-125b down-regulation depended on HIV-1 reverse transcription but not viral DNA integration. These findings establish a post-transcriptional mechanism that controls CPSF6 expression and highlight a novel function of miR-125b during HIV-host interaction.

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SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1 Particles

Journal of Virology ◽

10.1128/jvi.00594-20 ◽

2020 ◽

Vol 94 (20) ◽

Cited By ~ 1

Author(s):

Austin Featherstone ◽

Christopher Aiken

Keyword(s):

Transmembrane Domain ◽

Antiviral Effect ◽

Cellular Protein ◽

Cell Protein ◽

Specific Effects ◽

Glycoprotein Gp120 ◽

Env Protein ◽

Hiv 1 ◽

Soluble Cd4 ◽

Viral Accessory Protein

ABSTRACT SERINC5 is a 10-transmembrane-domain cellular protein that is incorporated into budding HIV-1 particles and reduces HIV-1 infectivity by inhibiting virus-cell fusion. HIV-1 susceptibility to SERINC5 is determined by sequences in the viral Env glycoprotein gp120, and the antiviral effect of SERINC5 is counteracted by the viral accessory protein Nef. While the precise mechanism by which SERINC5 inhibits HIV-1 infectivity is unclear, previous studies have suggested that SERINC5 affects Env conformation. To define the effects of SERINC5 on Env conformation, we quantified the binding of HIV-1 particles to immobilized Env-specific monoclonal antibodies. We observed that SERINC5 reduced the binding of HIV-1 particles bearing a SERINC5-susceptible Env to antibodies that recognize the V3 loop, a soluble CD4 (sCD4)-induced epitope, and an N-linked glycan. In contrast, SERINC5 did not alter the capture of HIV-1 particles bearing the SERINC5-resistant Env protein. Moreover, the effect of SERINC5 on antibody-dependent virus capture was abrogated by Nef expression. Our results indicate that SERINC5 inhibits HIV-1 infectivity by altering the conformation of gp120 on virions and/or physical masking of specific HIV-1 Env epitopes. IMPORTANCE SERINC5 is a host cell protein that inhibits the infectivity of HIV-1 by a novel and poorly understood mechanism. Here, we provide evidence that the SERINC5 protein alters the conformation of the HIV-1 Env proteins and that this action is correlated with SERINC5’s ability to inhibit HIV-1 infectivity. Defining the specific effects of SERINC5 on the HIV-1 glycoprotein conformation may be useful for designing new antiviral strategies targeting Env.

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