scholarly journals Binding stoichiometry and structural model of the HIV-1 Rev/Importin beta complex

2021 ◽  
Author(s):  
Didier Spittler ◽  
Rose-Laure Indorato ◽  
Elisabetta Boeri Erba ◽  
Elise Delaforge ◽  
Luca Signor ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Structural Model ◽  
Host Protein ◽  
Rna Transcripts ◽  
Importin Beta ◽  
X Ray Scattering ◽  
Binding Epitope ◽  
Hiv 1 ◽  
Binding Behaviour ◽  
Arginine Rich Motif

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin beta (ImpB), but how Rev associates with ImpB is poorly understood. Here we report biochemical, biophysical and structural studies of the ImpB/Rev complex. Binding and mutagenesis data reveal that ImpB associates with two Rev monomers through independent binding sites and that the N-terminal half of Rev's Arginine-Rich Motif (ARM) is a primary ImpB binding epitope. Small-angle X-ray scattering (SAXS), crosslinking mass spectrometry and compensatory mutagenesis data suggest a structural model in which one Rev monomer binds to the C-terminal half of ImpB with Rev helix alpha-2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by ImpB and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular ImpB cargos.

scholarly journals Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4+ T Cells

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Chuan Li ◽  
Hai-Bo Wang ◽  
Wen-Dong Kuang ◽  
Xiao-Xin Ren ◽  
Shu-Ting Song ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Nuclear Export ◽  
Therapeutic Strategy ◽  
Host Protein ◽  
Viral Reactivation ◽  
Viral Transcription ◽  
Dependent Manner ◽  
Ltr Promoter ◽  
Hiv 1

ABSTRACT HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo. Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy. IMPORTANCE HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.

scholarly journals Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function

2015 ◽  
Vol 89 (7) ◽  
pp. 3557-3567 ◽  
Author(s):  
Sona Budhiraja ◽  
Hongbing Liu ◽  
Jacob Couturier ◽  
Anna Malovannaya ◽  
Jun Qin ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Noncoding Rna ◽  
Nuclear Protein ◽  
Protein Complexes ◽  
Critical Role ◽  
Cellular Protein ◽  
Host Protein ◽  
Reporter Plasmid ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACTBy recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 depletion also reduced p24 expression during viral infection, indicating that RBM14 is limiting for Rev function. RBM14 has previously been shown to localize to nuclear paraspeckles, a structure implicated in retaining unspliced HIV-1 transcripts for either Rev-mediated nuclear export or degradation. We found that depletion of NEAT1 RNA, a long noncoding RNA required for paraspeckle integrity, abolished the ability of overexpressed RBM14 to enhance Rev function, indicating the dependence of RBM14 function on paraspeckle integrity. Our study extends the known host cell interactome of Rev and XPO1 and further substantiates a critical role for paraspeckles in the mechanism of action of Rev. Our study also validates the nuclear complexome as a database from which viral cofactors can be mined.IMPORTANCEThis study mined a database of nuclear protein complexes to identify a cellular protein named RBM14 that is associated with XPO1 (CRM1), a nuclear protein that binds to the HIV-1 Rev protein and mediates nuclear export of incompletely spliced viral RNAs. Functional assays demonstrated that RBM14, a protein found in paraspeckle structures in the nucleus, is involved in HIV-1 Rev function. This study validates the nuclear complexome database as a reference that can be mined to identify viral cofactors.

scholarly journals HIV-1 Dynamics in the Host Cell: A Review of Viral- and Host- Protein Interactions and Potential Therapeutic Targets for HIV-1 Infection

2016 ◽  
Vol 22 (1) ◽  
pp. 10
Author(s):  
Masha Sorin ◽  
Ganjam V. Kalpana
Keyword(s):  
Viral Replication ◽  
Host Cell ◽  
Nuclear Export ◽  
Host Factors ◽  
Host Protein ◽  
Restriction Factors ◽  
Human Immune System ◽  
Antiviral Responses ◽  
Retroviral Replication ◽  
Hiv 1

HIV-1, the causative agent of AIDS, is a sophisticated retrovirus that has both evolved to invade the complex human immune system and adapted to utilize the host machinery for its own propagation. A dynamic interaction between the virus and host systems can be observed at every step of the HIV-1 lifecycle. Host factors are involved not only in mounting antiviral responses, but are also hijacked by the virus to enhance viral replication. Host factors are necessary for viral replication during entry, reverse transcription, nuclear import, integration, transcription, nuclear export, translation, assembly, and budding. Recently, a new class of host factors, called “host restriction factors,” has been identified that prevent retroviral replication in a specific host cell environment and constitute an important part of intracellular innate immunity against the virus. These restriction factors act as barriers to retroviral replication at various stages within the infected cell. Nevertheless, the HIV-1 virus has learned to subvert these antiviral responses and successfully propagate within the permissive host environment. This review article describes the identification and mechanism of action of several pro- and anti-HIV-1 host factors. It is likely that we are only beginning to get a glimpse of an ongoing complex battle between HIV-1 and the host, the understanding of which should provide valuable information for the development of novel therapeutic strategies against HIV-1. 

scholarly journals HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

2019 ◽  
Author(s):  
Jordan T. Becker ◽  
Edward L. Evans ◽  
Bayleigh E. Benner ◽  
Stephanie L. Pritzl ◽  
Laura E. Smith ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Host Protein ◽  
Capsid Proteins ◽  
Particle Assembly ◽  
Nonsense Mediated Decay ◽  
Genome Interactions ◽  
Hiv 1

ABSTRACTHIV-1 RNA genomes interact with diverse RNA binding proteins (RBPs) in the cytoplasm including antiviral factor APOBEC3G (A3G) that, in the absence of viral Vif proteins, is packaged into virions. Where and when HIV-1-A3G interactions are initiated for packaging inside the cell is unknown, and the relative contributions of genome vs. Gag capsid proteins to this process remains controversial. Here we visualized A3G responses to HIV-1 infection over an entire replication cycle using long-term (up to 72 h) live single cell imaging. We show that Vif-deficient HIV-1 dramatically shifts A3G and a second RNA surveillance factor, MOV10, from the cytoplasm to virus particle assembly sites with little to no net discernible effects on general markers of cytoplasmic processing bodies (DCP1A), stress granules (TIA-1), or a marker of the nonsense-mediated decay machinery (UPF1). Using a new live cell RNA-protein interaction assay based on RNA tethering (the in-cell RNA-protein interaction protocol, or IC-IP), we provide evidence that A3G- and MOV10- genome interactions are selective, strong, occur in presence or absence of Gag, and are initiated in the cytosol soon if not immediately after genome nuclear export. Finally, although Gag is sufficient to package A3G into virions even in the absence of genomes, single virion imaging indicates that selective A3G-genome interactions promote much more consistent per virion delivery of A3G to assembly sites. Collectively, these studies suggest a paradigm for early, strong, and persistent cytosolic detection of select HIV-1 RNA signatures by A3G, MOV10 and other host RBPs that are enriched in virions.IMPORTANCEHost-pathogen interactions determine the success of viral replication. While extensive work has identified many interactions between HIV-1 and cellular factors, our understanding of where these interactions in cells occur during the course of infection is incomplete. Here, we show that multiple RNA-binding proteins (including the antiviral restriction factor, APOBEC3G, and MOV10 helicase) bind HIV-1 RNA genomes in the cytoplasm and co-traffic with them into progeny virions. Furthermore, we show that these interactions with HIV-1 RNA occur in the absence of Gag and are sufficiently strong to recruit these to otherwise non-native subcellular locales. Together, these data begin to illuminate the intracellular trafficking pathways shared by host RNA binding proteins and the viral RNAs they preferentially bind.

scholarly journals A proposal for a new HIV-1 DLS structural model

2012 ◽  
Vol 40 (11) ◽  
pp. 5012-5022 ◽  
Author(s):  
Jun-ichi Sakuragi ◽  
Hirotaka Ode ◽  
Sayuri Sakuragi ◽  
Tatsuo Shioda ◽  
Hironori Sato
Keyword(s):  
Structural Model ◽  
Hiv 1

scholarly journals Structural characterization of the N-terminal part of the MERS-CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering

2016 ◽  
Vol 72 (2) ◽  
pp. 192-202 ◽  
Author(s):  
Nicolas Papageorgiou ◽  
Julie Lichière ◽  
Amal Baklouti ◽  
François Ferron ◽  
Marion Sévajol ◽  
...  
Keyword(s):  
Structural Model ◽  
Structural Features ◽  
Terminal Region ◽  
X Ray Diffraction ◽  
Terminal Part ◽  
Multifunctional Protein ◽  
X Ray ◽  
N Protein ◽  
Intrinsically Disordered ◽  
X Ray Scattering

The N protein of coronaviruses is a multifunctional protein that is organized into several domains. The N-terminal part is composed of an intrinsically disordered region (IDR) followed by a structured domain called the N-terminal domain (NTD). In this study, the structure determination of the N-terminal region of the MERS-CoV N proteinviaX-ray diffraction measurements is reported at a resolution of 2.4 Å. Since the first 30 amino acids were not resolved by X-ray diffraction, the structural study was completed by a SAXS experiment to propose a structural model including the IDR. This model presents the N-terminal region of the MERS-CoV as a monomer that displays structural features in common with other coronavirus NTDs.

scholarly journals The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

2005 ◽  
Vol 79 (21) ◽  
pp. 13463-13472 ◽  
Author(s):  
Danso Ako-Adjei ◽  
Marc C. Johnson ◽  
Volker M. Vogt
Keyword(s):  
Nuclear Export ◽  
Leukemia Virus ◽  
Particle Diameter ◽  
Particle Morphology ◽  
Structural Protein ◽  
Wild Type ◽  
Virus Like Particles ◽  
Gag Proteins ◽  
Hiv 1

ABSTRACT The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

scholarly journals Defective HIV-1 proviruses produce viral proteins

2020 ◽  
Vol 117 (7) ◽  
pp. 3704-3710 ◽  
Author(s):  
Hiromi Imamichi ◽  
Mindy Smith ◽  
Joseph W. Adelsberger ◽  
Taisuke Izumi ◽  
Francesca Scrimieri ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Viral Proteins ◽  
Open Reading Frames ◽  
Peripheral Blood Mononuclear ◽  
Hiv Rna ◽  
Rna Transcripts ◽  
Extracellular Virus ◽  
Hiv 1

HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as “defective” by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant “graveyard” of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4+ T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4+ T cells and T cell–cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.

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