Retroviruses recruit viral and cellular RNAs into assembling virions. This dissertation addresses where and how RNAs are first recruited in cells by the retrovirus Moloney murine leukemia virus (MLV). Although differences exist between retroviruses, findings in MLV may further our understanding of RNA recruitment by other retroviruses, such as human immunodeficiency virus type 1 (HIV-1).

MLV recruits high levels of cellular noncoding mouse Y RNAs (mY RNAs), the RNA component of Ro ribonucleoproteins (Ro RNPs). In cells, Ro RNPs likely function in noncoding RNA quality control. The Ro60 protein is the main protein component of Ro RNPs. Ro60 knockout mouse embryonic fibroblasts (MEFs) contain ∼30-fold less mY RNAs than wild type MEFs. Residual mY1 RNAs in Ro60 knockout MEFs were redistributed between the nucleus and cytoplasm, in contrast to the mostly cytoplasmic localization of mY1 RNAs in wild type MEFs. Suprisingly, virions from Ro60 knockout MEFs continued to package high levels of mY RNAs. Together, these data suggest that MLV recruits mY RNAs early in their biogenesis.

Work performed here also addressed where MLV genomic RNAs (gRNAs) are first recruited. Here, specific tetracycline-regulated repression of MLV expression produced a more rapid decline in gRNA packaging than virion production. This is consistent with previous actinomycin D studies, and it suggests that MLV unspliced RNAs bisect into non-equilibrating pools of mRNAs and gRNAs. Additionally, transcription inhibition increased the randomness of gRNA dimer partner associations and recombination rates, with residual biases consistent with ongoing partitioning between mRNAs and genomes. These observations support a model of nuclear MLV gRNA dimerization that functions as an initial step in recruitment.

Work reported here also addressed how MLV gRNAs are first recruited. Mutations to the gRNA packaging signal (Ψ), which stabilize Ψ stem loops and disrupt dimerization and nucleocapsid (NC) binding in vitro, led to ∼100-fold decrease in gRNA packaging in a tissue culture system. This supports an RNA switch mechanism whereby dimerization exposes high affinity NC binding sites that recruit the gRNA into assembling virions.

Overall, findings in this dissertation suggest cellular mY RNAs and viral gRNAs are initially recruited from cell nuclei.