The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/GagPol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of GagPol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA.
IMPORTANCEViruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell-and thus dictated by the components' availabilities within the cell-or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.