Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles.Arenaviruses are the causative agents of a number of human diseases, including lymphocytic choriomeningitis, Lassa fever, and Bolivian hemorrhagic fever. These viruses are carried asymptomatically in wild rodents and are transmitted through contact with contaminated excretions. Endemic infection in these rodents maintains a reservoir of virus that can reemerge in human populations as outbreaks (1,18,30) or as novel pathogenic species (6). No FDA-licensed vaccines exist for these viruses, and current antiarenaviral therapy is limited to the off-label use of the nucleoside analogue ribavirin, which has had mixed success in treating cases of arenaviral hemorrhagic fever (HF) disease, while being associated with significant side effects (5).The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is ubiquitous in wild rodents and can infect domesticated mice and hamsters. These infected hamsters have most recently been responsible for reported disease in humans (2, 34). Additionally, reports of transplant-associated LCMV disease and fatality highlight the clinical relevance of arenaviral infection in immunocompromised individuals (15). In addition, congenital transmission of LCMV has been implicated in a variety of clinical conditions that affect mainly the retina and brain (3, 4).Arenaviruses are enveloped viruses bearing a bi-segmented, negative-sense RNA genome. Each segment contains two open reading frames (ORFs) in the ambisense orientation that are separated by a noncoding intergenic region (IGR) and bounded at their 5Ј and 3Ј ends by untranslated regions (UTRs) that are critical for the control of viral R...