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

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.

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Nuclear RNA Export and Packaging Functions of HIV-1 Rev Revisited

Journal of Virology ◽

10.1128/jvi.02264-09 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6598-6604 ◽

Cited By ~ 57

Author(s):

Maik Blissenbach ◽

Bastian Grewe ◽

Bianca Hoffmann ◽

Sabine Brandt ◽

Klaus Überla

Keyword(s):

Nuclear Export ◽

General Notion ◽

Main Function ◽

Hiv Replication ◽

Major Function ◽

Nuclear Rna ◽

Rna Export ◽

Nuclear Rna Export ◽

Hiv 1 ◽

Rev Protein

ABSTRACT Although the viral Rev protein is necessary for HIV replication, its main function in the viral replication cycle has been controversial. Reinvestigating the effect of Rev on the HIV-1 RNA distribution in various cell lines and primary cells revealed that Rev enhanced cytoplasmic levels of the unspliced HIV-1 RNA, mostly 3- to 12-fold, while encapsidation of the RNA and viral infectivity could be stimulated >1,000-fold. Although this clearly questions the general notion that the nuclear export of viral RNAs is the major function of Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to enhanced accessibility of the cytoplasmic HIV-1 RNA for translation and encapsidation. This might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects.

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Inhibition of Human Immunodeficiency Virus Rev and Human T-Cell Leukemia Virus Rex Function, but Not Mason-Pfizer Monkey Virus Constitutive Transport Element Activity, by a Mutant Human Nucleoporin Targeted to Crm1

Journal of Virology ◽

10.1128/jvi.72.11.8627-8635.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8627-8635 ◽

Cited By ~ 102

Author(s):

Hal P. Bogerd ◽

Asier Echarri ◽

Ted M. Ross ◽

Bryan R. Cullen

Keyword(s):

Nuclear Export ◽

Leukemia Virus ◽

Constitutive Transport Element ◽

Human T Cell ◽

Immunodeficiency Virus ◽

Nuclear Rna ◽

T Cell Leukemia Virus ◽

Rna Export ◽

Nuclear Rna Export ◽

Hiv 1

ABSTRACT The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran · GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed ΔCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, ΔCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, ΔCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.

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The Human Tap Nuclear RNA Export Factor Contains a Novel Transportin-dependent Nuclear Localization Signal That Lacks Nuclear Export Signal Function

Journal of Biological Chemistry ◽

10.1074/jbc.274.45.32167 ◽

1999 ◽

Vol 274 (45) ◽

pp. 32167-32171 ◽

Cited By ~ 45

Author(s):

Ray Truant ◽

Yibin Kang ◽

Bryan R. Cullen ◽

◽

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Signal Function ◽

Nuclear Rna ◽

Rna Export ◽

Localization Signal ◽

Nuclear Rna Export ◽

Export Signal

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HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment

Nucleic Acids Research ◽

10.1093/nar/gku304 ◽

2014 ◽

Vol 42 (10) ◽

pp. 6645-6658 ◽

Cited By ~ 19

Author(s):

Ichiro Taniguchi ◽

Naoto Mabuchi ◽

Mutsuhito Ohno

Keyword(s):

Viral Rna ◽

Export Pathway ◽

Rna Export ◽

Hiv 1 ◽

Rev Protein

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

10.1101/559096 ◽

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.

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Human Endogenous Retrovirus Expression Is Inversely Associated with Chronic Immune Activation in HIV-1 Infection

PLoS ONE ◽

10.1371/journal.pone.0041021 ◽

2012 ◽

Vol 7 (8) ◽

pp. e41021 ◽

Cited By ~ 19

Author(s):

Christopher E. Ormsby ◽

Devi SenGupta ◽

Ravi Tandon ◽

Steven G. Deeks ◽

Jeffrey N. Martin ◽

...

Keyword(s):

Immune Activation ◽

Endogenous Retrovirus ◽

Human Endogenous Retrovirus ◽

Chronic Immune Activation ◽

Hiv 1

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Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA

Chemistry & Biology ◽

10.1016/s1074-5521(97)90257-x ◽

1997 ◽

Vol 4 (2) ◽

pp. 139-147 ◽

Cited By ~ 474

Author(s):

Barbara Wolff ◽

Jean-Jacques Sanglier ◽

Ying Wang

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nuclear Export ◽

Leptomycin B ◽

Immunodeficiency Virus ◽

Cytoplasmic Translocation ◽

Hiv 1 ◽

Rev Protein

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Membrane Binding and Subcellular Localization of Retroviral Gag Proteins Are Differentially Regulated by MA Interactions with Phosphatidylinositol-(4,5)-Bisphosphate and RNA

mBio ◽

10.1128/mbio.02202-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 25

Author(s):

Jingga Inlora ◽

David R. Collins ◽

Marc E. Trubin ◽

Ji Yeon J. Chung ◽

Akira Ono

Keyword(s):

Virus Assembly ◽

Membrane Binding ◽

Endogenous Retrovirus ◽

Single Amino Acid ◽

Human Endogenous Retrovirus ◽

Structural Protein ◽

Acidic Phospholipid ◽

Specific Localization ◽

Hiv 1

ABSTRACTThe matrix (MA) domain of HIV-1 mediates proper Gag localization and membrane binding via interaction with a plasma-membrane (PM)-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine, a prevalent cellular acidic phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking nonspecific interactions with cellular membranes that do not contain PI(4,5)P2. To examine whether PI(4,5)P2dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the significance of their interrelationships, we compared a panel of Gag-leucine zipper constructs (GagLZ) containing MA of different retroviruses. We found thatin vitromembrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus (RSV) MA is both PI(4,5)P2dependent and susceptible to RNA-mediated inhibition. The PM-specific localization and virus-like particle (VLP) release of these GagLZ proteins are severely impaired by overexpression of a PI(4,5)P2-depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV). In contrast, membrane binding of GagLZ constructs that contain human T-lymphotropic virus type 1 (HTLV-1) MA, murine leukemia virus (MLV) MA, and human endogenous retrovirus K (HERV-K) MA is PI(4,5)P2independent and not blocked by RNA. The PM localization and VLP release of these GagLZ chimeras were much less sensitive to 5ptaseIV expression. Notably, single amino acid substitutions that confer a large basic patch rendered HTLV-1 MA susceptible to the RNA-mediated block, suggesting that RNA readily blocks MA containing a large basic patch, such as HIV-1 and RSV MA. Further analyses of these MA mutants suggest a possibility that HIV-1 and RSV MA acquired PI(4,5)P2dependence to alleviate the membrane binding block imposed by RNA.IMPORTANCEMA basic residues in the HIV-1 structural protein Gag interact with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and RNA. RNA inhibits HIV-1 MA binding to non-PI(4,5)P2acidic lipids. This inhibition may promote PM specificity of Gag membrane binding, an early essential step in virus assembly. However, whether and how relationships between these interactions have developed among retroviruses are poorly understood. In this study, by comparing diverse retroviral MA domains, we elucidated a strong correlation among PI(4,5)P2dependence, susceptibility to RNA-mediated inhibition, and cellular behaviors of Gag. Mutagenesis analyses suggest that a large basic patch on MA is sufficient to confer susceptibility to RNA-mediated inhibition but not for PI(4,5)P2-dependent membrane binding. Our findings highlight RNA’s role as a general blocker of large basic patches and suggest a possibility that some retroviruses, including HIV-1, have evolved to bind PI(4,5)P2, while others have adopted smaller basic patches on their MA domains, to overcome the RNA-mediated restriction of membrane binding.

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