Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA

Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (⌬GP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer (ovine pulmonary adenocarcinoma) in sheep. For all retroviruses, full-length viral RNAs are transcribed from integrated viral DNA in the nucleus; unspliced viral RNA then is exported to the cytoplasm where it is translated into viral Gag and Gag-Pol polyproteins or packaged as genomes into new virions. At the same time, full-length viral RNA is also spliced in the nucleus to give mRNA(s) for the synthesis of envelope and (for some retroviruses) other proteins. From this perspective, nuclear full-length viral RNA is an unspliced env mRNA precursor. For most cellular mRNAs, splicing of nuclear mRNA precursors is required for export to the cytoplasm. This results from binding of nuclear RNP splicing complexes to the intron-exon junctions during the splicing process, leading to deposition of cellular factors onto the mRNA that facilitate export (32). Unspliced or incompletely spliced RNAs are typically retained in the nucleus.In order to export unspliced viral RNAs from the nucleus, retroviruses use one of two strategies to overcome the cellular barrier to export of unspliced RNAs (10). For complex retroviruses such as human immunodeficiency virus types 1 and 2 (HIV-1 and -2, respectively) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively), export of fulllength RNAs is facilitated by virally encoded trans-acting factors (Rev and Rex, respectively). Rev is encoded by a doubly spliced HIV-1 mRNA that is transported to the cytoplasm by the same mechanism used for spliced cellular mRNAs (10). Once Rev protein enters the nucleus (by way of a nuclear localization signal [NLS] [8,22]), it mediates the export of full-length HIV-1 RNAs by binding to the RNA at a cis-acting Rev-responsive element (RRE) located in the env gene (19,41,54,61). Rev bound to the R...

Section: Vol 83 2009mentioning

confidence: 99%

“…This binding of Tap, in conjunction with the cofactor p15 (NXT1) (18), facilitates export of unspliced MPMV RNAs from the nucleus. Another cis-acting element with export activity is the direct repeat regions (DRs) of Rous sarcoma virus and Rous-associated virus type 2 (35,47).…”

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confidence: 99%

Jaagsiekte Sheep Retrovirus Encodes a Regulatory Factor, Rej, Required for Synthesis of Gag Protein

Hofacre

Nitta

Fan

2009

“…In support of this hypothesis, RSV Gag mutants that do not traffic through the nucleus fail to properly package their RNA genome and are noninfectious (2,4,15,16). However, another report suggests that RSV Gag does not play a role in the export of unspliced genomic RNA (9).…”

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confidence: 99%

CRM1-Dependent Trafficking of Retroviral Gag Proteins Revisited

Baluyot

Grosse

Lyddon

et al. 2012

We analyzed the nuclear trafficking ability of Gag proteins from six retroviral genera. Contrary to a previous report, human immunodeficiency virus type 1 (HIV-1) Gag showed no propensity to cycle through the nucleus. The only Gag protein that displayed CRM1-dependent nuclear cycling was that of Rous sarcoma virus (RSV). Surprisingly, this cycling could be eliminated without compromising infectivity by replacing the RSV Gag N-terminal matrix (MA) domain with HIV MA.

“…Similarly, alpharetroviruses, such as avian sarcoma/leukemia viruses, have one or two copies of CTEs, which facilitate nuclear export of unspliced viral transcripts (17,18). Overexpression of a dominant negative NXF1 mutant can modestly impair CTE-mediated gene expression (19). However, their CTEs lack any evident homology to the MPMV CTE (17).…”

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confidence: 99%

Murine Leukemia Virus Uses NXF1 for Nuclear Export of Spliced and Unspliced Viral Transcripts

Sakuma

Davila

Malcolm

et al. 2014

Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. IMPORTANCEMurine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is involved in the nuclear export of both spliced and unspliced viral RNAs, and, finally, (iii) depletion of NXF1 results in nuclear retention or degradation of viral RNAs. Our study provides a novel insight into MLV nuclear export.