mRNA subcellular localization is a crucial determinant of eukaryotic gene expression. For retroviruses including HIV-1, the unspliced genomic RNA (gRNA) serves both as the mRNA encoding Gag/Gag-Pol capsid proteins and the genetic material packaged by Gag into virions assembling at the plasma membrane. Gag is sufficient to drive the assembly of virus-like particles in the absence of gRNA binding. Thus, what role gRNA cytoplasmic trafficking plays during assembly is unknown. Here we demonstrate that aberrantly tethering HIV-1 gRNAs to membranes or the actin cytoskeleton alters Gag subcellular distribution and reduces virus particle production. This block was restricted to gRNAs competent for Gag synthesis and mapped to the Rev response element; a cis-acting RNA structure that regulates gRNA nuclear export. These results expose an unexpected mechanistic link between HIV-1 gRNA nuclear history, cytoplasmic trafficking, and Gag assembly. Perturbing viral mRNA subcellular distribution could represent a novel antiviral strategy.