Evolution of a Species-Specific Determinant within Human CRM1 that Regulates the Post-transcriptional Phases of HIV-1 Replication

Sherer, Nathan M.; Swanson, Chad M.; Hué, Stéphane; Roberts, Roland G.; Bergeron, Julien R. C.; Malim, Michael H.

doi:10.1371/journal.ppat.1002395

Cited by 34 publications

(65 citation statements)

References 67 publications

(110 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The latter phenomenon is particularly evident in the case of HIV-1 gRNA trafficking and particle assembly in mouse cells, which proceed inefficiently (3, 38, 52). Indeed, variant residues in murine CRM1 itself have been reported to account for a substantial portion of this assembly-side block (50). We therefore considered that FIV Gag nuclear trafficking in human and African green monkey cells might proceed differently than in cells of the natural feline host.…”

Section: Nuclear Import and Crm1-dependent Export Of Fiv Gag-cfpmentioning

confidence: 99%

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

View full text Add to dashboard Cite

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.G ag is both necessary and sufficient for retrovirus particle formation and budding. The protein is translated on free polysomes and targeted to the plasma membrane, where virus particles are assembled (16). A central question is whether Gag and the viral genomic RNA (gRNA) associate at the plasma membrane or earlier during assembly and, if so, whether this occurs in the cytosol; in association with organelles, e.g., cotranslationally; or even in the nucleus, as was described previously for an alpharetrovirus (17). Human immunodeficiency virus type 1 (HIV-1) gRNAs become anchored at the plasma membrane before particle assembly is detectable, but it is not clear whether the Gag-gRNA complexes of this and other lentiviruses first form in the cytoplasm and then transit to the plasma membrane or the gRNA traffics there independently (26,27). Biochemical experiments supported the former scenario, with a monomer or low-order multimers forming on HIV-1 gRNAs and higher-order multimer formation depending on subsequent plasma membrane interactions (33).Gag is encoded by the full-length unspliced viral RNA. To circumvent the cellular checkpoint that prevents the export of intron-containing mRNA, lentiviruses express Rev, which functions as an adaptor between the cellular karyopherin CRM1/exportin-1 and a Rev response element (RRE) located in the 3= region of unspliced viral RNAs (9). However, the main cellular function of the CRM1 nuclear export pathway is the export of cellular proteins containing a...

show abstract

Section: Nuclear Import and Crm1-dependent Export Of Fiv Gag-cfpmentioning

confidence: 99%

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

View full text Add to dashboard Cite

show abstract

“…It is well established that the HIV-1 Rev protein recognizes the RRE localized in intron-containing viral RNAs and recruits the cellular export factor CRM1 to mediate translocation across the pore. Nucleocytoplasmic export of unspliced HIV-1 RNA has been the subject of many studies that addressed the biochemical or structural characteristics of the HIV-1 RNA/Rev/ CRM1 export complex (Yi et al 2002;Yedavalli et al 2004;Daugherty et al 2010;Sherer et al 2011). These studies provide important insights into the mechanisms of nucleocytoplasmic transport, but the spatiotemporal relationship between Rev, CRM1, and unspliced HIV-1 RNA remains unknown.…”

Section: Discussionmentioning

confidence: 99%

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Nawroth¹,

Mueller²,

Basyuk³

et al. 2013

RNA

View full text Add to dashboard Cite

The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post-or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.

show abstract

“…This block, among others, was of long-standing interest both due to the dearth of knowledge regarding cellular contributions to HIV-1's posttranscriptional stages, as well as the persisting need for a genetically tractable small animal model for studying HIV-1 infection. Although Rev is not wholly inactive in murine cells (43), the exogenous provision of human CRM1 (hCRM1) enhanced Rev activity and yielded virus particle production up to 30-fold in mouse or rat cell lines relative to rodent CRM1 (39,41,42). These effects correlated with up to 5-fold increases in the cytoplasmic abundance of Rev/RRE-dependent viral mRNAs, consistent with the rescue of vRNP nuclear export.…”

mentioning

confidence: 99%

“…We and two other groups recently identified the murine ortholog of CRM1 (mCRM1) as the source of a profound, speciesspecific block to HIV-1 virion production evident in cells derived from muroid rodents (e.g., mice, rats, and hamsters) (39)(40)(41)(42). This block, among others, was of long-standing interest both due to the dearth of knowledge regarding cellular contributions to HIV-1's posttranscriptional stages, as well as the persisting need for a genetically tractable small animal model for studying HIV-1 infection.…”

mentioning

confidence: 99%

“…However, the CRM1 binding cleft that engages Rev's NES is invariant between mCRM1 and hCRM1 (42,(44)(45)(46). Instead, mouse-human CRM1 chimeras were used to map the species-specific activity to a transferable, patch-like configuration of amino acids located 19 to 30 Å from the NES binding cleft and located between CRM1 HEAT repeats 9 and 10 (39,41,42). Phylogenetic considerations based on sequence comparisons of CRM1 orthologues from 17 mammalian species indicated that the evolution of the mCRM1 "patch domain" was likely a result of positive selection pressure, suggesting a role for this domain as an ancient cellular interface with one or more pathogenic elements, possibly of viral nature (41).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

Aligeti

Behrens

Pocock

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCECells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression. T he nuclear pore complex (NPC) represents a key barrier to the replication of many viruses that infect eukaryotic cells (1-3). For retroviruses such as the primate lentivirus human immunodeficiency virus type 1 (HIV-1), the late, productive phases of the viral life cycle require efficient nuclear export of unspliced, and thus intron-containing, viral genomic RNAs (gRNAs) that (i) encode the group-specific antigen (Gag) and Gag-Pol polyproteins that form ...

show abstract

Evolution of a Species-Specific Determinant within Human CRM1 that Regulates the Post-transcriptional Phases of HIV-1 Replication

Cited by 34 publications

References 67 publications

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

Contact Info

Product

Resources

About