The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway. Retroviruses must gain access to the nucleus to replicate. After receptor binding, entry, and reverse transcription, the integration-competent nucleoprotein complex (called the preintegration complex or PIC) enters the nucleus. For oncoretroviruses like Rous sarcoma virus (RSV) that primarily infect dividing cells, the PIC awaits breakdown of the nuclear envelope during mitosis for nuclear entry. Lentiviruses including HIV-1 infect nondividing cells, and PICs are transported through intact nuclear envelopes. HIV-1 nuclear entry is complex, and redundant signals have been identified in the viral matrix (MA), integrase, and Vpr proteins (reviewed in ref. 1). The recent report that RSV can replicate at low levels in quiescent cells does raise the possibility that a viral protein might mediate active nuclear targeting of the RSV PIC (2).After nuclear entry of the PIC and proviral integration, viral RNA is transcribed, and unspliced genome-length viral mRNAs must exit the nucleus for translation into viral structural proteins and encapsidation into virions. The nuclear export of introncontaining mRNAs is normally inhibited by cellular mechanisms, so retroviruses must circumvent this obstacle. Lentiviruses encode trans-acting factors such as the HIV-1 Rev protein to mediate nuclear export of intron-containing viral RNAs (3). Oncoretroviruses including RSV lack Rev-like transport factors and instead have cis-acting constitutive transport elements to facilitate the export of unspliced viral RNA (4).The regulation ...

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.