scholarly journals Dynamic ensemble of HIV-1 RRE stem IIB reveals non-native conformations that disrupt the Rev-binding site

2019 ◽  
Vol 47 (13) ◽  
pp. 7105-7117 ◽  
Author(s):  
Chia-Chieh Chu ◽  
Raphael Plangger ◽  
Christoph Kreutz ◽  
Hashim M Al-Hashimi
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Dynamic Equilibrium ◽  
Nmr Relaxation ◽  
Conformational Flexibility ◽  
Relaxation Dispersion ◽  
Conformational States ◽  
Rev Response Element ◽  
Local Secondary Structure ◽  
Hiv 1

AbstractThe HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of ∼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.

scholarly journals Dynamic ensemble of HIV-1 RRE stem IIB reveals non-native conformations that disrupt the Rev binding site

2018 ◽  
Author(s):  
Chia-Chieh Chu ◽  
Raphael Plangger ◽  
Christoph Kreutz ◽  
Hashim M. Al-Hashimi
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Dynamic Equilibrium ◽  
Nmr Relaxation ◽  
Conformational Flexibility ◽  
Relaxation Dispersion ◽  
Conformational States ◽  
Rev Response Element ◽  
Local Secondary Structure ◽  
Hiv 1

ABSTRACTThe HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native ‘excited state’ (ES) conformations that have a combined population of ~20%. The ESs disrupt the Rev binding site by changing local secondary structure and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.

scholarly journals Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Ina P. O'Carroll ◽  
Yashna Thappeta ◽  
Lixin Fan ◽  
Edric A. Ramirez-Valdez ◽  
Sean Smith ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Double Helix ◽  
Distal Portion ◽  
Viral Rna ◽  
Functional Assay ◽  
Response Element ◽  
Rev Response Element ◽  
Domain I ◽  
Hiv 1

ABSTRACT The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an “A” shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other. IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is “A” shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.

scholarly journals Characterization of the Multiple Conformational States of Free Monomeric and Trimeric Human Immunodeficiency Virus Envelope Glycoproteins after Fixation by Cross-Linker

2006 ◽  
Vol 80 (14) ◽  
pp. 6725-6737 ◽  
Author(s):  
Wen Yuan ◽  
Jessica Bazick ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Conformational Flexibility ◽  
Envelope Glycoproteins ◽  
Conformational States ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Trimeric Envelope

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) gp120 exterior and gp41 transmembrane envelope glycoproteins assemble into trimers on the virus surface that represent potential targets for antibodies. Potent neutralizing antibodies bind the monomeric gp120 glycoprotein with small changes in entropy, whereas unusually large decreases in entropy accompany gp120 binding by soluble CD4 and less potent neutralizing antibodies. The high degree of conformational flexibility in the free gp120 molecule implied by these observations has been suggested to contribute to masking the trimer from antibodies that recognize the gp120 receptor-binding regions. Here we use cross-linking and recognition by antibodies to investigate the conformational states of gp120 monomers and soluble and cell surface forms of the trimeric HIV-1 envelope glycoproteins. The fraction of monomeric and trimeric envelope glycoproteins able to be recognized after fixation was inversely related to the entropic changes associated with ligand binding. In addition, fixation apparently limited the access of antibodies to the V3 loop and gp41-interactive surface of gp120 only in the context of trimeric envelope glycoproteins. The results support a model in which the unliganded monomeric and trimeric HIV-1 envelope glycoproteins sample several different conformations. Depletion of particular fixed conformations by antibodies allowed characterization of the relationships among the conformational states. Potent neutralizing antibodies recognize the greatest number of conformations and therefore can bind the virion envelope glycoproteins more rapidly and completely than weakly neutralizing antibodies. Thus, the conformational flexibility of the HIV-1 envelope glycoproteins creates thermodynamic and kinetic barriers to neutralization by antibodies directed against the receptor-binding regions of gp120.

scholarly journals Recruitment of the Crm1 Nuclear Export Factor Is Sufficient To Induce Cytoplasmic Expression of Incompletely Spliced Human Immunodeficiency Virus mRNAs

2002 ◽  
Vol 76 (5) ◽  
pp. 2036-2042 ◽  
Author(s):  
Rui Yi ◽  
Hal P. Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Export ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Binding Domain ◽  
Viral Mrnas ◽  
Rev Response Element ◽  
Cytoplasmic Expression ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Cytoplasmic expression of the incompletely spliced RNA transcripts that encode the late, structural proteins of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev regulatory protein. General agreement exists that Rev acts, at least in part, by recruiting the cellular Crm1 nuclear export factor to HIV-1 transcripts bearing the Rev response element RNA target, and thereby inducing their nuclear egress. However, several groups have argued that Crm1 recruitment may not be sufficient for Rev function. Thus, several additional candidate cofactors for Rev have been proposed, and Rev has also been suggested to also inhibit the nuclear splicing of HIV-1 transcripts and/or to directly enhance their cytoplasmic translation. To examine whether Crm1 recruitment is, instead, sufficient to activate the nuclear export of viral mRNAs, we targeted a leucine-rich Crm1 binding domain, derived from a heterologous protein that normally plays no role in RNA metabolism, to HIV-1 RNAs and showed that this tethered Crm1 binding domain is sufficient to induce the nuclear export and cytoplasmic translation of late HIV-1 mRNA species. More importantly, we show that direct tethering of the Crm1 nuclear export factor to target mRNAs, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins.

scholarly journals Cofactor Requirements for Nuclear Export of Rev Response Element (Rre)–And Constitutive Transport Element (Cte)–Containing Retroviral Rnas

2001 ◽  
Vol 152 (5) ◽  
pp. 895-910 ◽  
Author(s):  
Wilma Hofmann ◽  
Beate Reichart ◽  
Andrea Ewald ◽  
Eleonora Müller ◽  
Iris Schmitt ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Kinase Inhibitor ◽  
Initiation Factor ◽  
Nuclear Pore ◽  
Xenopus Laevis Oocytes ◽  
Binding Studies ◽  
Rev Response Element ◽  
Constitutive Transport Element ◽  
Hiv 1

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)–mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev–NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

scholarly journals HIV-1gag-polmRNA localization regulates the site of virion assembly

2016 ◽  
Author(s):  
Jordan T. Becker ◽  
Nathan M. Sherer
Keyword(s):  
Virus Particle ◽  
Nuclear Export ◽  
Genetic Material ◽  
Regulatory Elements ◽  
Capsid Proteins ◽  
Viral Pathogen ◽  
Genomic Rnas ◽  
Rev Response Element ◽  
The Right ◽  
Hiv 1

ABSTRACTHIV-1 full-length, unspliced genomic RNAs (gRNAs) serve both as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as the genetic material packaged by Gag into virions that assemble at the plasma membrane (PM). Whether localized Gag synthesis contributes to assembly at the PM is unknown. Here we show that artificially tethering gRNAs or surrogategag-pol mRNAs to non-PM membranes or the actin cytoskeleton can markedly affect Gag’s distribution in the cytoplasm, causing aberrant subcellular sites of assembly and severe reductions to virus particle output. Onlygag-polmRNAs competent for translation were capable of altering Gag’s distribution within the cell, and the activity mapped to twocis-acting RNA regulatory elements; the 5’ packaging signal (Psi) bound by Gag and, unexpectedly, the Rev response element (RRE) that regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, our results suggest a model wherein localized translation of gRNAs at the PM helps to compartmentalize Gag-gRNA interactions, thereby promoting efficient genome encapsidation.AUTHOR SUMMARYThe spatial distribution of messenger RNAs (mRNAs) within the cytoplasm can be a crucial determinant of gene expression. Here we provide evidence that a devastating viral pathogen, human immunodeficiency virus type 1 (HIV-1), exploits localized translation to favor the formation of infectious, transmissible virions at the surface of infected cells. Artificially tethering viral mRNAs encoding the Gag and Gag-Pol capsid proteins (gag-polmRNAs) to alternative regions of the cell such as cytoplasmic vesicles or the actin cytoskeletion markedly alters Gag subcellular distribution, perturbs sites of assembly, and reduces virus particle production. These and additional findings suggest a model for HIV-1 assembly wherein localized Gag/Gag-Pol translation coupled to confined interactions between Gag and viral genomes ensures infectious virion production at the right place and the right time. Perturbing HIV-1 mRNA subcellular localization could represent a novel antiviral strategy.

scholarly journals Impact of Nuclear Export Pathway on Cytoplasmic HIV-1 RNA Transport Mechanism and Distribution

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Jianbo Chen ◽  
Chijioke Umunnakwe ◽  
David Q. Sun ◽  
Olga A. Nikolaitchik ◽  
Vinay K. Pathak ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Transport Mechanism ◽  
Full Length ◽  
Rna Transport ◽  
Transport Mechanisms ◽  
Rev Response Element ◽  
Export Pathway ◽  
Cytoplasmic Transport ◽  
Hiv 1

ABSTRACT HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions. IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.

scholarly journals A Nuclear Kinesin-Like Protein Interacts with and Stimulates the Activity of the Leucine-Rich Nuclear Export Signal of the Human Immunodeficiency Virus Type 1 Rev Protein

2003 ◽  
Vol 77 (13) ◽  
pp. 7236-7243 ◽  
Author(s):  
L. K. Venkatesh ◽  
T. Gettemeier ◽  
G. Chinnadurai
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Cell Leukemia Virus Type ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACT The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

scholarly journals RNA-directed remodeling of the HIV-1 protein Rev orchestrates assembly of the Rev–Rev response element complex

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Bhargavi Jayaraman ◽  
David C Crosby ◽  
Christina Homer ◽  
Isabel Ribeiro ◽  
David Mavor ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nuclear Export ◽  
Rna Binding ◽  
Rna Recognition ◽  
Protein Interface ◽  
Viral Mrnas ◽  
Response Element ◽  
Rev Response Element ◽  
Functional Complex ◽  
Hiv 1

The HIV-1 protein Rev controls a critical step in viral replication by mediating the nuclear export of unspliced and singly-spliced viral mRNAs. Multiple Rev subunits assemble on the Rev Response Element (RRE), a structured region present in these RNAs, and direct their export through the Crm1 pathway. Rev-RRE assembly occurs via several Rev oligomerization and RNA-binding steps, but how these steps are coordinated to form an export–competent complex is unclear. Here, we report the first crystal structure of a Rev dimer-RRE complex, revealing a dramatic rearrangement of the Rev-dimer upon RRE binding through re-packing of its hydrophobic protein–protein interface. Rev-RNA recognition relies on sequence-specific contacts at the well-characterized IIB site and local RNA architecture at the second site. The structure supports a model in which the RRE utilizes the inherent plasticity of Rev subunit interfaces to guide the formation of a functional complex.

scholarly journals Matrix Mediates the Functional Link between Human Immunodeficiency Virus Type 1 RNA Nuclear Export Elements and the Assembly Competency of Gag in Murine Cells

2009 ◽  
Vol 83 (17) ◽  
pp. 8525-8535 ◽  
Author(s):  
Nathan M. Sherer ◽  
Chad M. Swanson ◽  
Stelios Papaioannou ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Particle Production ◽  
Functional Link ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembles poorly in murine cells, reflecting inefficient targeting of the Gag structural polyprotein to the plasma membrane. Virus particle production can be restored by replacing the cis-acting Rev response element (RRE) in Gag-Pol mRNAs with multiple copies of the CTE (4×CTE), suggesting a mechanistic link between HIV-1 RNA trafficking and productive Gag assembly. In this report, we demonstrate that Gag molecules generated from RRE-dependent transcripts are intrinsically defective for assembly in murine 3T3 cells. When controlled for the intracellular Gag level, modulations of the Gag matrix (MA) domain that enhance Gag membrane association (e.g., deletion of the MA globular head) substantially improve assembly for Gag derived from RRE- but not 4×CTE-dependent transcripts. Gag mutants carrying a leucine zipper replacement of the nucleocapsid (NC) domain remain largely assembly defective when derived from RRE-dependent transcripts, indicating that the defect does not reflect aberrant NC/RNA-driven Gag multimerization. We further demonstrate that single changes in uncharged amino acids implicated in Gag/MA myristoyl switch regulation, most notably replacing the leucine at position 21 with serine, improve assembly for Gag derived from RRE-dependent transcripts. In sum, we provide genetic evidence to suggest that HIV-1 RNA metabolism specifically modulates the activation of MA-dependent membrane targeting.

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