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 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 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 Localization of HIV-1 RNA in mammalian nuclei.

1996 ◽  
Vol 135 (1) ◽  
pp. 9-18 ◽  
Author(s):  
G Zhang ◽  
M L Zapp ◽  
G Yan ◽  
M R Green
Keyword(s):  
Nuclear Export ◽  
Splice Junction ◽  
Stable Population ◽  
Beta Globin ◽  
Nuclear Retention ◽  
Viral Rnas ◽  
Immunodeficiency Virus ◽  
Nascent Transcripts ◽  
Hiv 1

The Rev protein of human immunodeficiency virus type 1 (HIV-1) facilitates the nuclear export of unspliced and partially spliced viral RNAs. In the absence of Rev, these intron-containing HIV-1 RNAs are retained in the nucleus. The basis for nuclear retention is unclear and is an important aspect of Rev regulation. Here we use in situ hybridization and digital imaging microscopy to examine the intranuclear distributions of intron-containing HIV RNAs and to determine their spatial relationships to intranuclear structures. HeLa cells were transfected with an HIV-1 expression vector, and viral transcripts were localized using oligonucleotide probes specific for the unspliced or spliced forms of a particular viral RNA. In the absence of Rev, the unspliced viral RNAs were predominantly nuclear and had two distinct distributions. First, a population of viral transcripts was distributed as approximately 10-20 intranuclear punctate signals. Actinomycin D chase experiments indicate that these signals represent nascent transcripts. A second, stable population of viral transcripts was dispersed throughout the nucleoplasm excluding nucleoli. Rev promoted the export of this stable population of viral RNAs to the cytoplasm in a time-dependent fashion. Significantly, the distributions of neither the nascent nor the stable populations of viral RNAs coincided with intranuclear speckles in which splicing factors are enriched. Using splice-junction-specific probes, splicing of human beta-globin pre-mRNA occurred cotranscriptionally, whereas splicing of HIV-1 pre-mRNA did not. Taken together, our results indicate that the nucleolus and intranuclear speckles are not involved in Rev regulation, and provide further evidence that efficient splicing signals are critical for cotranscriptional splicing.

scholarly journals C19ORF66 is an Interferon-Stimulated Gene (ISG) which Inhibits Human Immunodeficiency Virus-1

2016 ◽  
Author(s):  
Weidong Xiong ◽  
Deisy Contreras ◽  
Joseph Ignatius Irudayam ◽  
Ayub Ali ◽  
Otto O. Yang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Export ◽  
Splice Variants ◽  
Nuclear Export Signal ◽  
Full Length ◽  
Type I ◽  
Immune Factor ◽  
Immunodeficiency Virus ◽  
P24 Protein ◽  
Hiv 1

ABSTRACTInnate immunity is the first line of defense against invading microbes1. The type I interferon (IFN) pathway plays a key role in controlling Human Immunodeficiency Virus type 1 (HIV-1) replication2,3. We identified an IFN-α stimulated gene C19ORF66 that we term Suppressor of Viral Activity (SVA). Full length SVA-1 protein inhibits HIV-1 by blocking virion production. SVA splice variants truncated at the C-terminus and/or disrupted at the nuclear export signal (NES) lose antiviral activity and localize to nucleus, while full length SVA-1 co-localizes with HIV-1 p24 protein in the cytoplasmic compartment of infected cells. SVA-1 is structurally and functionally conserved across species, including mouse and chimpanzee. We provide the first description of the effector function of the gene SVA/C190RF66 as an innate immune factor with anti-HIV-1 activity.

scholarly journals Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

2012 ◽  
Vol 86 (16) ◽  
pp. 8402-8411 ◽  
Author(s):  
Iris Kemler ◽  
Dyana Saenz ◽  
Eric Poeschla
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Nuclear Export ◽  
Leptomycin B ◽  
Genomic Rnas ◽  
Intracellular Location ◽  
Export Pathway ◽  
Immunodeficiency Virus ◽  
Nuclear Shuttling ◽  
Rna Interaction ◽  
Hiv 1

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.

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 Direct Participation of Sam68, the 68-Kilodalton Src-Associated Protein in Mitosis, in the CRM1-Mediated Rev Nuclear Export Pathway

2002 ◽  
Vol 76 (16) ◽  
pp. 8374-8382 ◽  
Author(s):  
Jinliang Li ◽  
Ying Liu ◽  
Byung Oh Kim ◽  
Johnny J. He
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Nuclear Export ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Human Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Nuclear Retention ◽  
Export Pathway ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication requires efficient nuclear export of incompletely spliced and unspliced HIV-1 mRNA transcripts, which is achieved by Rev expression at an early stage of the viral life cycle. We have recently shown that expression of Sam68, the 68-kDa Src-associated protein in mitosis, is able to alleviate Rev function block in astrocytes by promoting Rev nuclear export. In the present study, we utilized an antisense RNA expression strategy to down-modulate constitutive Sam68 expression and examined its effect on Rev function, HIV-1 gene expression, and viral replication. These results showed that down-modulation of constitutive Sam68 expression markedly inhibited HIV-1 production in 293T cells and viral replication in T lymphocytes such as Jurkat and CEM cells, as well as human peripheral blood mononuclear cells (PBMCs). Rev-dependent in trans complementation and reporter gene assays further demonstrated that inhibition of HIV-1 gene expression by Sam68 down-modulation was due to impeded Rev activity. Moreover, digital fluorescence microscopic imaging revealed that down-modulation of Sam68 expression caused exclusive nuclear retention and colocalization of both Rev and CRM1. Taken together, these data suggest that adequate Sam68 expression is required for Rev function and, thereby, for HIV-1 gene expression and viral replication, and they support the notion that Sam68 is directly involved in the CRM1-mediated Rev nuclear export pathway.

scholarly journals A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export.

1996 ◽  
Vol 133 (1) ◽  
pp. 5-14 ◽  
Author(s):  
N Visa ◽  
E Izaurralde ◽  
J Ferreira ◽  
B Daneholt ◽  
I W Mattaj
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Transcription Initiation ◽  
Nuclear Pore ◽  
Balbiani Ring ◽  
Cytoplasmic Transport ◽  
Cap Binding Complex ◽  
Rna Export ◽  
Ring Particle

In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.

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