| Arenaviruses are one of the most neglected tropical pathogens. The most prominent human pathogen belonging to this group is Lassa Fever virus (LASV), which is responsible for ∼5,000 - 10,000 deaths and ∼2 million infections annually. Mutational analysis of arenaviral proteins may provide insight into the pathological mechanisms of these viruses.;A related arenavirus, Pichinde virus (PICV), is capable of causing a disease in guinea pigs that mimics LASV infection in humans. We have sequenced two strains of PICV with different disease phenotypes in infected guinea pigs. The P2 strain causes a mild disease from which the animals quickly recover. The P18 strain results in a highly virulent disease, which results in death. While the phenotypes are vastly different, relatively few sequence changes exist between them.;We have developed reverse genetics systems for both the P2 and P18 strains of PICV, which have allowed us to generate recombinant viruses. Through mutational analysis, we have identified sequence changes in the L polymerase that contribute to the increased pathogenesis of P18. Three amino acid changes in the C terminus of L are required for increased virulence of the P18 strain by increasing the rate of viral genome replication, indicating that the rate of viral genome replication is an important factor in arenavirus pathogenesis.;We have also developed a LASV minireplicon system in order to perform mutational analysis of the LASV nucleoprotein (NP). We have shown that LASV NP contains a cap-binding domain in its N terminus that is required to initiate viral RNA transcription. We have also shown that LASV NP contains a 3'-5' exoribonuclease domain in its C terminus that is required to mediate type-I interferon inhibition. We therefore propose a novel mechanism of immune evasion in which the NP protein degrades viral RNAs that would otherwise be detected by innate immune sensors, therefore allowing these viruses to replicate to high levels in infected patients.;Collectively, this work characterizes two important viral factors (NP and L polymerase) required for arenaviral replication, transcription and immune evasion. This information will aid in the development of vaccines and therapeutics against arenaviral hemorrhagic fever. |
