A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as ''scaffolding'' in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.murine leukemia virus ͉ packaging cell lines ͉ retrovirus assembly ͉ RNA packaging ͉ Semliki Forest virus gene expression system

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an ␣-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.

The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.