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

Abstract

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

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

“…The formation of infectious virus, as assessed by the ratio of transduced to transfected cells in a reporter virus plasmid-transfected monolayer, was not blocked, although it was reduced 50 to 70% (2). The binding of RSV Gag to the gRNA in the nucleus exposes an NES in the Gag p10 domain, which in turn promotes CRM1-RanGTP binding, which results in the nuclear export of the complex (20,45,47). The nuclear encapsidation process of this alpharetrovirus so far remains unique among retroviruses (40).…”

“…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).…”

Cell biology of retroviral RNA packaging

“…Thus, the complex can be directed to the plasma membrane to complete particle assembly. 76 Altogether, these data suggest that very few Gag molecules initiate the recognition of the genome, 61,73 at a variety of cellular sites, such as the nucleus (RSV), the nuclear membrane (FIV) or the cytosol (HIV-1) (Fig. 3).…”

“…Based on some in vitro studies it seems that Gag interacts with the importin receptors as a monomer. 76 Once in the nucleus, Gag recruits the gRNA 76 via an interaction between NC and the Psi site of the unspliced gRNA (Fig. 3).…”

“…The formation of this complex seems to promote Gag dimerization or oligomerization. 77 It was proposed that Gag multimerization induces a change of conformation of the complex exposing the nuclear export signal (NES), 76 which resides in a leucine-rich region of 18 residues within the p10 sequence of Gag. The NES interacts with the CRM1 nuclear export receptor which promotes the export of the Gag/RNA cargo from the nucleus to the cytoplasm by the CRM1 export pathway 78,79 (Fig.…”