Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

Butterfield-Gerson, Kristin L.; Scheifele, Lisa Z.; Ryan, Eileen P.; Hopper, Anita K.; Parent, Leslie J.

doi:10.1128/jvi.80.4.1798-1806.2006

Cited by 57 publications

(91 citation statements)

References 57 publications

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“…Parent and colleagues have shown in a series of studies that Rous sarcoma virus (RSV) Gag shuttles (6,17,20,(45)(46)(47), and experiments with RSV Gag mutants that do not undergo nuclear cycling provided evidence that this is needed for efficient gRNA encapsidation (17,45). RSV Gag nuclear import is mediated by NLSs in MA and NC (45).…”

Section: Discussionmentioning

confidence: 99%

“…(K to Q) Subcellular localization of FIV MA-CFP, CA-CFP, and NCp2-CFP. Transfected HeLa cells were treated or not with LMB for 3 h. The nuclear/total MFI (Q) was determined as described above for panel J for 158 cells (6,17,25,23,15,15,21,16,11, and 9 cells for columns from left to right, respectively). Images are representative of at least three independent experiments.…”

Section: Discussionmentioning

confidence: 99%

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Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

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“…Our earlier studies identified the nuclear import receptors for the isolated MA and NC domains in yeast (13). To test whether the mammalian homologues of these karyopherins were associated with MA and NC in avian cells, the natural host for RSV, MA-GFP and YFP-NC (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…S1). Genetic analysis using Saccharomyces cerevisiae revealed that nuclear import of NC depends on the importin-α/β (imp-α/β) pathway, whereas Kap120p and Mtr10p [transportin-SR and importin-11 (imp-11), respectively, in higher eukaryotes] are import receptors for MA (13). Toxicity of the Gag protein in yeast prevented determination of whether it interacts with the same karyopherins as the isolated MA and NC domains.…”

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confidence: 99%

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Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

Gudleski¹,

Flanagan²,

Ryan

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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105

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Retroviral Gag polyproteins coopt host factors to traffic from cytosolic ribosomes to the plasma membrane, where virions are released. Before membrane transport, the multidomain Gag protein of Rous sarcoma virus (RSV) undergoes importin-mediated nuclear import and CRM1-dependent nuclear export, an intrinsic step in the assembly pathway. Transient nuclear trafficking of Gag is required for efficient viral RNA (vRNA) encapsidation, suggesting that Gag: vRNA binding might occur in the nucleus. Here, we show that Gag is imported into the nucleus through direct interactions of the Gag NC domain with importin-α (imp-α) and the MA domain with importin-11 (imp-11). The vRNA packaging signal, known as ψ, inhibited imp-α binding to Gag, indicating that the NC domain does not bind to imp-α and vRNA simultaneously. Unexpectedly, vRNA binding also prevented the association of imp-11 with both the MA domain alone and with Gag, suggesting that the MA domain may bind to the vRNA genome. In contrast, direct binding of Gag to the nuclear export factor CRM1, via the CRM1-RanGTP heterodimer, was stimulated by ψRNA. These findings suggest a model whereby the genomic vRNA serves as a switch to regulate the ordered association of host import/export factors that mediate Gag nucleocytoplasmic trafficking for virion assembly. The Gag:vRNA interaction appears to serve multiple critical roles in assembly: specific selection of the vRNA genome for packaging, stimulating the formation of Gag dimers, and triggering export of viral ribonucleoprotein complexes from the nucleus.protein-RNA binding | retrovirus assembly | importin-α/β | importin-11 | CRM1

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Rous Sarcoma Virus: Contributions of a Chicken Virus to Tumor Biology, Human Cancer Therapeutics, and Retrovirology

Parent

2011

Cancer Associated Viruses

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Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

Cited by 57 publications

References 57 publications

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

Rous Sarcoma Virus: Contributions of a Chicken Virus to Tumor Biology, Human Cancer Therapeutics, and Retrovirology

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