Intermolecular interactions between retroviral Gag proteins in the nucleus.
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.
Kenney
Scott P
SP
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
Lochmann
Timothy L
TL
Schmid
Cullen L
CL
Parent
Leslie J
LJ
D002467:Cell Nucleus / Q000737:chemistry / Q000821:virology*; D031541:Fluorescence Resonance Energy Transfer; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D052736:Rous sarcoma virus / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods
D015683:Gene Products, gag