Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. Despite decades of research, it is still unclear how these viruses establish a lifelong, asymptomatic infection in their rodent hosts while infection of humans often results in severe disease. Unable to enter a state of bona fide latency, the transcription and replication of the viral genomic RNA is likely highly regulated in time and subcellular space. Moreover, we hypothesize that the viral nucleoprotein (NP), responsible for the encapsidation of the viral RNA and the most highly expressed viral gene product, plays a key role in the regulation of the viral gene expression program. Further, exploring host-virus interactions may elucidate the basic aspects of arenavirus biology and how they cause such severe disease in humans. To explore these questions in greater detail, this dissertation has pursued three main avenues. First, to better understand lymphocytic choriomeningitis mammarenavirus (LCMV) genome replication and transcription at the single-cell level, we established a high-throughput, single-molecule (sm)FISH image acquisition and analysis pipeline and followed viral RNA species from viral entry through the late stages of persistent infection in vitro. This work provided support for a cyclical model of persistence where individual cells are initially transiently infected, clear active infection, and become re-infected from neighboring reservoir cells within the population. Second, we used FISH to visualize viral genomic RNA to describe the subcellular sites where LCMV RNAs localize during infection. We observed that, viral RNA concentrates in large subcellular structures located near the cellular microtubule organizing center and colocalizes with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane bound organelle as a site for the pre-assembly of viral components including genomic RNA and viral glycoprotein prior to their transport to the plasma membrane where new particles will bud. Last, we used mass spectrometry to identify human proteins that interact with the NPs of LCMV and Junín mammareanavirus (JUNV) strain Candid #1. We provided a detailed map of the host machinery engaged by arenavirus NPs, and in particular, showed that NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. We demonstrated that JUNV antagonizes the antiviral activity of PKR completely, effectively abrogating the antiviral activity of this surveillance pathway. In sum, the work composing this dissertation has given us fresh insight into how arenaviruses establish and maintain persistence; the nature of the subcellular site where viral genomic RNA is transcribed, replicated, and assembled with other viral components; and a global view of the cellular machinery hijacked by the viral nucleoprotein. This work improves our basic understanding of the arenavirus life cycle and may suggest novel antiviral therapeutic targets that could be exploited in the future.