scholarly journals ANP32A and ANP32B are key factors in the Rev dependent CRM1 pathway for nuclear export of HIV-1 unspliced mRNA

2019 ◽  
Author(s):  
Yujie Wang ◽  
Haili Zhang ◽  
Lei Na ◽  
Cheng Du ◽  
Zhenyu Zhang ◽  
...  
Keyword(s):  
Viral Replication ◽  
Nuclear Export ◽  
Posttranscriptional Regulation ◽  
Late Phase ◽  
Viral Rna ◽  
Viral Mrna ◽  
Key Factors ◽  
Gag Protein ◽  
Rna Export ◽  
Hiv 1

AbstractThe nuclear export receptor CRM1 is an important regulator involved in the shuttling of various cellular and viral RNAs between the nucleus and the cytoplasm. HIV-1 Rev interacts with CRM1 in the late phase of HIV-1 replication to promote nuclear export of unspliced and single spliced HIV-1 transcripts. However, the knowledge of cellular factors that are involved in the CRM1-dependent viral RNA nuclear export remains inadequate. Here, we identified that ANP32A and ANP32B mediate the export of unspliced or partially spliced viral mRNA via interacting with Rev and CRM1. We found that double, but not single, knockout of ANP32A and ANP32B, significantly decreased the expression of gag protein. Reconstitution of either ANP32A or ANP32B restored the viral production equally. Disruption of both ANP32A and ANP32B expression led to a dramatic accumulation of unspliced viral mRNA in the nucleus. We further identified that ANP32A and ANP32B interact with both Rev and CRM1 to promote RNA transport and that this function is Rev/RRE-dependent, but not CTE-dependent. Together our data suggests that ANP32A and ANP32B are required for HIV-1 unspliced RNA export in the Rev-CRM1 pathway.Author summaryPosttranscriptional regulation of HIV-1 genome is very important for viral protein expression and viral replication. HIV-1 Rev protein bind to RRE structure of viral RNA and interacts with the mammalian nuclear export factor Chromosomal Maintenance 1 (CRM1) in the late phase of HIV-1 replication to promote nuclear export of unspliced and single spliced HIV-1 transcripts. The REV/RRE-CRM1 pathway has been investigated for years and many host factors have been reported to be involved, but the complicated complex and procedure remain largely unknown. Here the authors report that two host proteins, ANP32A and ANP32B, are novel key factors that support export of unspliced and partial spliced viral RNA from the nucleus to the cytosol. ANP32A/B can interact with both Rev and CRM1, and this interaction is necessary for Rev/RRE-CRM1 dependent viral RNA export. These results suggest that ANP32A and ANP32B are important in viral replication and could be potential targets for novel antiviral strategy.

scholarly journals Mutational Definition of Functional Domains within the Rev Homolog Encoded by Human Endogenous Retrovirus K

2000 ◽  
Vol 74 (20) ◽  
pp. 9353-9361 ◽  
Author(s):  
Hal P. Bogerd ◽  
Heather L. Wiegand ◽  
Jin Yang ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Export ◽  
Biological Activities ◽  
Endogenous Retrovirus ◽  
Viral Rna ◽  
Human Endogenous Retrovirus ◽  
Nuclear Rna ◽  
Rna Export ◽  
Nuclear Rna Export ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACT Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the ∼25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

scholarly journals HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1281
Author(s):  
Kevin M. Tuffy ◽  
Rebecca J. Kaddis Maldonado ◽  
Jordan Chang ◽  
Paul Rosenfeld ◽  
Alan Cochrane ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Integration Site ◽  
Leukemia Virus ◽  
Foamy Virus ◽  
Regulatory Protein ◽  
Viral Rna ◽  
Gag Protein ◽  
Ribonucleoprotein Complexes ◽  
Immunodeficiency Virus ◽  
Hiv 1

The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.

scholarly journals Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rebecca J. Kaddis Maldonado ◽  
Breanna Rice ◽  
Eunice C. Chen ◽  
Kevin M. Tuffy ◽  
Estelle F. Chiari ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Rous Sarcoma Virus ◽  
Nuclear Export ◽  
Live Cell ◽  
Viral Rna ◽  
Wild Type ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome. IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

scholarly journals Selective and Nonselective Packaging of Cellular RNAs in Retrovirus Particles

2007 ◽  
Vol 81 (12) ◽  
pp. 6623-6631 ◽  
Author(s):  
Samuel J. Rulli ◽  
Catherine S. Hibbert ◽  
Jane Mirro ◽  
Thoru Pederson ◽  
Shyam Biswal ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Signal Recognition Particle ◽  
Viral Rna ◽  
Genomic Rna ◽  
Gag Protein ◽  
Mrna Species ◽  
Reverse Transcription Pcr ◽  
Cellular Mrnas ◽  
A Minor ◽  
Hiv 1

ABSTRACT Assembly of retrovirus particles normally entails the selective encapsidation of viral genomic RNA. However, in the absence of packageable viral RNA, assembly is still efficient, and the released virus-like particles (termed “Ψ−” particles) still contain roughly normal amounts of RNA. We have proposed that cellular mRNAs replace the genome in Ψ− particles. We have now analyzed the mRNA content of Ψ− and Ψ+ murine leukemia virus (MLV) particles using both microarray analysis and real-time reverse transcription-PCR. The majority of mRNA species present in the virus-producing cells were also detected in Ψ− particles. Remarkably, nearly all of them were packaged nonselectively; that is, their representation in the particles was simply proportional to their representation in the cells. However, a small number of low-abundance mRNAs were greatly enriched in the particles. In fact, one mRNA species was enriched to the same degree as Ψ+ genomic RNA. Similar results were obtained with particles formed from the human immunodeficiency virus type 1 (HIV-1) Gag protein, and the same mRNAs were enriched in MLV and HIV-1 particles. The levels of individual cellular mRNAs were ∼5- to 10-fold higher in Ψ− than in Ψ+ MLV particles, in agreement with the idea that they are replacing viral RNA in the former. In contrast, signal recognition particle RNA was present at the same level in Ψ− and Ψ+ particles; a minor fraction of this RNA was weakly associated with genomic RNA in Ψ+ MLV particles.

scholarly journals PACS1 is an HIV-1 cofactor that functions in Rev-mediated nuclear export of viral RNA

Virology ◽  
2020 ◽  
Vol 540 ◽  
pp. 88-96 ◽  
Author(s):  
Hongbing Liu ◽  
Pei-Wen Hu ◽  
Sona Budhiraja ◽  
Anisha Misra ◽  
Jacob Couturier ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Viral Rna ◽  
Hiv 1

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 Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 394 ◽  
Author(s):  
Tomas Kroupa ◽  
Siddhartha A. K. Datta ◽  
Alan Rein
Keyword(s):  
Salt Concentration ◽  
Electrostatic Interactions ◽  
Rna Binding ◽  
High Specificity ◽  
Viral Rna ◽  
Gag Protein ◽  
Gag Polyprotein ◽  
The Individual ◽  
Hiv 1

Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.

scholarly journals The prototype γ-2 herpesvirus nucleocytoplasmic shuttling protein, ORF 57, transports viral RNA through the cellular mRNA export pathway

2005 ◽  
Vol 387 (2) ◽  
pp. 295-308 ◽  
Author(s):  
Ben J. L. WILLIAMS ◽  
James R. BOYNE ◽  
Delyth J. GOODWIN ◽  
Louise ROADEN ◽  
Guillaume M. HAUTBERGUE ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Mrna Export ◽  
Dominant Negative ◽  
Viral Rna ◽  
Exon Junction Complex ◽  
Independent Manner ◽  
Exon Junction ◽  
Export Pathway ◽  
Rna Export

HVS (herpesvirus saimiri) is the prototype γ-2 herpesvirus. This is a subfamily of herpesviruses gaining importance since the identification of the first human γ-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS ORF 57 (open reading frame 57) protein is a multifunctional transregulatory protein homologous with genes identified in all classes of herpesviruses. Recent work has demonstrated that ORF 57 has the ability to bind viral RNA, shuttles between the nucleus and cytoplasm and promotes the nuclear export of viral transcripts. In the present study, we show that ORF 57 shuttles between the nucleus and cytoplasm in a CRM-1 (chromosomal region maintenance 1)-independent manner. ORF 57 interacts with the mRNA export factor REF (RNA export factor) and two other components of the exon junction complex, Y14 and Magoh. The association of ORF 57 with REF stimulates recruitment of the cellular mRNA export factor TAP (Tip-associated protein), and HVS infection triggers the relocalization of REF and TAP from the nuclear speckles to several large clumps within the cell. Using a dominant-negative form of TAP and RNA interference to deplete TAP, we show that it is essential for bulk mRNA export in mammalian cells and is required for ORF 57-mediated viral RNA export. Furthermore, we show that the disruption of TAP reduces viral replication. These results indicate that HVS utilizes ORF 57 to recruit components of the exon junction complex and subsequently TAP to promote viral RNA export through the cellular mRNA export pathway.

The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA

Nature ◽  
1989 ◽  
Vol 338 (6212) ◽  
pp. 254-257 ◽  
Author(s):  
Michael H. Malim ◽  
Joachim Hauber ◽  
Shu-Yun Le ◽  
Jacob V. Maizel ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Export ◽  
Target Sequence ◽  
Viral Mrna ◽  
Hiv 1

scholarly journals The Cellular RNA Export Receptor TAP/NXF1 Is Required for ICP27-Mediated Export of Herpes Simplex Virus 1 RNA, but the TREX Complex Adaptor Protein Aly/REF Appears To Be Dispensable

2009 ◽  
Vol 83 (13) ◽  
pp. 6335-6346 ◽  
Author(s):  
Lisa A. Johnson ◽  
Ling Li ◽  
Rozanne M. Sandri-Goldin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Yellow Fluorescent Protein ◽  
Adaptor Protein ◽  
Viral Rna ◽  
Herpes Simplex Virus 1 ◽  
Rna Export ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)+ RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)+ RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

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