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 ...

The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E؊). To distinguish between cis and trans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E؊.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E؊, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG؊ was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that the src sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG؊, Myr1E؊, and Myr1E؊.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.All retroviruses incorporate two identical copies of their RNA genome into each virion. The genomic RNA molecules are linked near their 5Ј ends by noncovalent interactions to form a stable structure possessing ordered secondary and tertiary structure. Although there are multiple contact points throughout the two parallel RNA molecules, the most stable linkage is called the dimer linkage structure. The dimer linkage structure can be visualized by electron microscopy and appears to be a region about 50 nucleotides (nt) in length near the 5Ј end of the genome (centered around nucleotide 511 in Rous sarcoma virus [RSV]) (1,20,23). Dimerization is required for infectivity, although precisely how it contributes to the replication cycle remains poorly understood. Dimerization is believed to facilitate recombination during reverse transcription by enabling close approximation of the viral RNA molecules, leading to increased genetic diversity and improved viral fitness (15,16,26). The dimeric RNA structure has also been implicated in inhibiting the translation of unspliced viral RNA so that genomic RNA is available for packaging; however, there is little experimental evidence in support of this idea (26).Because the RNA sequences that are important for dimerization overlap those required for RNA incorporation into virus particles, dimerization and packaging were postu...

The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a "classical " nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.The retroviral Gag polyprotein is sufficient to direct virus assembly, as demonstrated by its ability to form virus-like particles in the absence of all other viral gene products (reviewed in reference 39). Previously, it was thought that Gag proteins of type C retroviruses were synthesized on cytosolic ribosomes and then targeted directly to the plasma membrane, where budding occurs. However, we discovered that the Rous sarcoma virus (RSV) Gag polyprotein traffics through the nucleus (32). Nuclear import is mediated by a nuclear localization signal (NLS) within the N-terminal matrix (MA) domain. A CRM1-dependent nuclear export signal (NES) within the p10 domain of Gag mediates nuclear egress, after which Gag travels to the plasma membrane for particle release. Treatment of cells with leptomycin B (LMB), a specific inhibitor of CRM1, results in the accumulation of Gag proteins in the nucleus (32).Nuclear import of macromolecules, including all proteins greater than 50 kDa, occurs by signal-mediated active transport across the nuclear membrane through the nuclear pore complex. The majority of proteins bearing specific NLS sequences are imported through an interaction with soluble transport factors belonging to the importin superfamily (reviewed in reference 9). The "classical" NLSs in...