From the Ribosome to the membrane subcellular trafficking of the Rous sacrcoma virus Gag polyprotein /

by Scheifele, Lisa Z.

iii Retroviruses such as human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV) are the etiological agents of immunodeficiency diseases and cancer. We have been studying the subcellular targeting of the Gag protein of Rous sarcoma virus (RSV), one of the first simple retroviruses identified. The Gag protein directs assembly at the plasma membrane and incorporates the viral genome through association with a cis-acting packaging element in the viral RNA. Although functional domains within Gag that mediate the assembly process have been well defined, the activity of these motifs has not been well correlated with the trafficking of Gag throughout the cell during assembly. We have identified distinct subcellular targeting motifs within the Rous sarcoma virus Gag protein that may influence the retroviral assembly pathway. Within the Gag membrane-binding domain, we have identified an alpha helix that is crucial for plasma membrane targeting of Gag. When this helix is deleted, the Gag protein accumulates at intracellular membranes. Yet membrane affinity is not diminished, indicating that membrane binding and plasma membrane targeting of the RSV Gag polyprotein are genetically separable. We have also identified both a nuclear targeting signal within the N-terminal MA domain of Gag and a nuclear export signal (NES) within the p10 domain. Expression of dominant-negative nuclear pore proteins redistributes the Gag protein from a cytoplasmic and plasma membrane localization to an almost exclusively nuclear localization, confirming that Gag both enters and exits the nucleus. We have further identified the soluble receptor that mediates the cytoplasmic localization of Gag; treatment of virus-expressing cells with leptomycin B, a specific inhibitor of the CRM-1 export pathway results in sequestration of the Gag protein within the nucleus. Single amino-acid substitutions within a leucine-rich iv cluster in the p10 domain of Gag result in accumulation of the protein within the nucleus, confirming the localization of the Gag NES within the p10 region. Interestingly, we find that the NES sequence is highly conserved among avian retroviruses; when naturally occurring variations within this sequence are recreated in the RSV Gag protein, NES function is retained, while artificial substitutions predicted to retain NES function do not. We have sought to identify additional cellular proteins that influence the import and export of Gag from the nucleus by employing the powerful genetic system developed in the yeast Saccharomyces cerevisiae. Expression of Gag linked to a tandem GFP reporter reveals a localization to the cytoplasm of yeast cells with an accumulation in the nucleus not seen at steady-state in avian cells. Not only is the dwell time between the nucleus and cytoplasm altered, but the nuclear export pathway is perturbed as well; yeast cells do not show an enhanced accumulation of Gag proteins within the nucleus following leptomycin B treatment, suggesting that the Crm1 pathway may not be the predominant export pathway utilized by Gag in yeast cells. To further investigate the role of Gag nuclear trafficking in retroviral replication, we have studied the replication of viruses containing mutations in the Gag NES. These mutant Gag proteins are able to direct the budding of virions which contain the normal complement of viral polyproteins and the proper amount of viral genomic RNA. However, the virions that are produced show profound morphological defects, including amorphous and heterogeneously shaped particles, and viral cores that are elongated or granular in appearance. Despite these substantial morphological defects, the particles are able to enter new cells and undergo reverse transcription, producing 2-LTR circles, hallmarks of entry of the synthesized DNA genome into the nucleus. Although the particles are able to complete v reverse transcription, they are non-infectious. Nuclear transport of Gag may therefore crucial not only for the efficient production of viral particles at the plasma membrane, but also for the establishment of a productive infection.