Lucie Pessel-Vivares

Cellular mRNAs are exported from the nucleus as fully spliced RNAs. Proofreading mechanisms eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Retroviruses need to export partially spliced and unspliced full-length RNAs to the cytoplasm where they serve as templates for protein synthesis and/or as encapsidated RNA in progeny viruses. Genetically complex retroviruses such as HIV-1 use Rev-equivalent proteins to export intron-retaining RNA from the nucleus using the cellular CRM1-driven nuclear export machinery. By contrast, genetically simpler retroviruses such as murine leukemia virus (MLV) recruit the NXF1 RNA export machinery. In this study, we reveal for the first time that MLV hijacks both NXF1 and CRM1-dependent pathways to achieve optimal replication capacity. The CRM1-pathway marks the MLV full-length RNA (FL RNA) for packaging, while NXF1-driven nuclear export is coupled to translation. Thus, the cytoplasmic function of the viral RNA is determined early in the nucleus. Depending of the nature of ribonucleoprotein complex formed on FL RNA cargo in the nucleus, the FL RNA will be addressed to the translation machinery sites or to the virus-assembly sites at the plasma membrane.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced post-entry inhibitor of human immunodeficiency virus type-1 (HIV-1) infection. While the precise mechanism of viral inhibition remains unclear, MX2 is localized to the nuclear envelope, and blocks the nuclear import of viral cDNAs. The amino-terminus of MX2 (N-MX2) is essential for anti-viral function, and mutation of a triple arginine motif at residues 11 to 13 abrogates anti-HIV-1 activity. In this study, we sought to investigate the role of N-MX2 in anti-viral activity by identifying functionally relevant host-encoded interaction partners through yeast-two-hybrid screening. Remarkably, five out of seven primary candidate interactors were nucleoporins or nucleoporin-like proteins, though none of these candidates were identified when screening with a mutant RRR11-13A N-MX2 fragment. Interactions were confirmed by co-immunoprecipitation, and RNA silencing experiments in cell lines and primary CD4⁺ T cells demonstrated that multiple components of the nuclear pore complex and nuclear import machinery can impact MX2 anti-viral activity. In particular, the phenylalanine-glycine (FG) repeat containing cytoplasmic filament nucleoporin NUP214, and transport receptor transportin-1 (TNPO1) were consistently required for full MX2, and interferon-mediated, anti-viral function. Both proteins were shown to interact with the triple arginine motif, and confocal fluorescence microscopy revealed that their simultaneous depletion resulted in diminished MX2 accumulation at the nuclear envelope. We therefore propose a model whereby multiple components of the nuclear import machinery and nuclear pore complex help position MX2 at the nuclear envelope to promote MX2-mediated restriction of HIV-1. The movement of large molecules into the cell nucleus is regulated at specific sites within the nuclear envelope termed nuclear pores. To infect cells productively, human immunodeficiency virus type-1 (HIV-1) must traverse the nuclear envelope to enable integration of the viral DNA into the genomic DNA of host cells. We, and others, have previously identified a cell-encoded protein, human myxovirus resistance 2 (MX2), which is expressed upon initiation of an innate immune response and prevents accumulation of HIV-1 DNA within the nucleus, thus imposing a block to infection. Here, we reveal that components of the nuclear pore complex, and nuclear import machinery, are required for MX2-dependent inhibition of HIV-1 infection. We show that MX2, which is localized at the cytoplasmic face of the nuclear envelope, interacts with multiple protein components of the nuclear pore complex, as well as transport receptor transportin-1, via a functionally required triple arginine motif at its amino-terminus. We speculate that these interactions facilitate MX2-mediated inhibition of HIV-1 nuclear import by situating the protein at the nuclear envelope.