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

Abstract

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.

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

“…Plasmid MLV-Gag-mCherry is similar to MLV-GagPol except for a DNA fragment encoding a short linker fused to the mCherry gene, which was inserted immediately before the stop codon of gag to express a Gag-mCherry fusion protein. Although it does not express the pol gene product, MLV-Gag-mCherry still has the pol gene sequence, which has been shown to be important for efficient MLV Gag expression (26)(27)(28). The MLV-GagPol plasmid contains an E231G mutation at the C-terminal region of CA; a plasmid lacking the mutation generated results similar to those shown in Fig.…”

“…We also generated a structurally similar MLV-Gag-mCherry construct that expresses MLV Gag-mCherry by inserting an mCherry gene at the end of Gag (Fig. 3B); although not translated, the pol gene sequence is maintained in this construct to facilitate Gag expression (26)(27)(28). Equal amount of MLV-GagPol and MLV-Gag-mCherry helper plasmids, neither of which contains the MLV packaging signal, were transfected into 293T cells alone ("no RNA" sample), with the 1-RNA, or with M⌿-RNA construct.…”

Insights into the nuclear export of murine leukemia virus intron-containing RNA

“…The export pathway used by MLV RNA was identified very recently, in 2014. [26][27][28][29][30] Different complementary approaches such as RNA silencing, dominant-negative mutant of Tap and RNA competitor harboring 4 copies of the CTE sequence have been used either in human or murine cells lines, infected or transfected with MLV. All results concur that the Tap pathway is strictly required for MLV expression (Fig.…”

Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns

“…Using bioinformatic tools, we have not been able to find any genes outside of the NXF family that possess CTEs based on sequence homology. However, the genomes of several mammalian and endogenous retroviruses have been shown to contain non-homologous elements that function as CTEs in conjunction with Nxf1/Nxt (Ogert and Beemon 1995;Yang and Cullen 1999;Nappi et al 2001;Legiewicz et al 2010;Sakuma et al 2014). In addition, we have previously used retroviral vector "trap" strategies to identify several host cell CTEs that lack apparent homology with the MPMV/NXF1 CTE, but which still function in conjunction with Nxf1/Nxt1 .…”