Intermolecular Interactions between Retroviral Gag Proteins in the Nucleus

Abstract

The retroviral Gag polyprotein directs virus particle assembly, resulting in the release of virions from the plasma membranes of infected cells. The earliest steps in assembly, those immediately following Gag synthesis, are very poorly understood. For Rous sarcoma virus (RSV), Gag proteins are synthesized in the cytoplasm and then undergo transient nuclear trafficking before returning to the cytoplasm for transport to the plasma membrane. Thus, RSV provides a useful model to study the initial steps in assembly because the early and later stages are spatially separated by the nuclear envelope. We previously described mutants of RSV Gag that are defective in nuclear export, thereby isolating these "trapped" Gag proteins at an early assembly step. Using the nuclear export mutants, we asked whether Gag protein-protein interactions occur within the nucleus. Complementation experiments revealed that the wild-type Gag protein could partially rescue export-defective Gag mutants into virus-like particles (VLPs). Additionally, the export mutants had a trans-dominant negative effect on wild-type Gag, interfering with its release into VLPs. Confocal imaging of wild-type and mutant Gag proteins bearing different fluorescent tags suggested that complementation between Gag proteins occurred in the nucleus. Additional evidence for nuclear Gag-Gag interactions was obtained using fluorescence resonance energy transfer, and we found that the formation of intranuclear Gag complexes was dependent on the NC domain. Bimolecular fluorescence complementation allowed the direct visualization of intranuclear Gag-Gag dimers. Together, these experimental results strongly suggest that RSV Gag proteins are capable of interacting within the nucleus.

Nucleolar Trafficking of the Mouse Mammary Tumor Virus Gag Protein Induced by Interaction with Ribosomal Protein L9

“…For photobleaching, nuclei were selected as the region of interest, and mCherry was photobleached using the 543-nm laser at 100% power until the fluorescence intensity was reduced to 20% of prebleach levels or for 5 min. Förster resonance energy transfer (FRET) efficiency was calculated as previously described (33). FRET analysis was performed using a minimum of 10 different cells on two different days, and the mean, standard error of the mean, and P values were calculated using an unpaired t test with GraphPad Prism 5 (GraphPad Software, Inc.).…”

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

“…Images were equally adjusted for intensity using CorelDraw ×3 (Corel Corporation). Quantification of the nuclear fluorescence intensity of Gag proteins was calculated by dividing the fluorescence intensity of the nucleus by the fluorescence intensity of the entire cell (Leica Microsystems software) (30).…”

“…CRM1 likely associates with a dimer of Gag, based on studies showing that nucleic acid binding promotes Gag dimerization (36,42) and Gag dimers form in the nucleus (30). However, the crystal structure of the p10-CA Gag dimer shows that the NES is buried in hydrophobic interactions with CA, and is therefore unavailable to bind to CRM1 (17).…”

“…Once in the nucleus, Gag is released from the import factors and binds to genomic vRNA, primarily through interaction of the NC domain with ψ. MA might also bind to vRNA, likely at a different site. On vRNA binding, Gag forms a dimer or oligomer (30) that exposes the p10 NES or increases its affinity for CRM1-RanGTP, leading to export of the viral ribonucleoprotein complex through the nuclear pore. Although ψ-RNA selectively promotes Gag:CRM1 binding, considering that only two copies of vRNA are incorporated into each virion, it is possible that other RNAs in the nucleus [e.g., U6 and other small RNAs enriched in retroviral particles (31)(32)(33)(34)] might also bind Gag to stimulate nuclear export.…”

Cell biology of retroviral RNA packaging

“…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.…”