Functional regulation of an arenavirus polymerase complex

Abstract

Emerging RNA viruses present recurring challenges to human, livestock, and plant healthcare efforts worldwide. With compact genomes copied by an inherently error-prone RNA-dependent RNA polymerase (RdRP), RNA viruses are remarkably adept at exploring the functional sequence space of their genomic capacity and navigating diverse cellular landscapes. Viruses with negative-strand RNA genomes (NSVs) are some of the most medically, socially, and economically important pathogens throughout human history, including influenza, measles, Ebola, rabies, and Lassa virus. The large (L) NSV polymerase uniquely harbors all enzymatic machinery necessary for viral genome replication and gene expression and is an essential component of mature virion particles. The central functions of NSV L proteins in the virus infection cycle highlight these enzymes as key druggable targets for antiviral intervention strategies. Here, we study the functional regulation of the L protein of the arenavirus Machupo virus (MACV), the cause of Bolivian hemorrhagic fever in humans. We have identified short, structured RNA ligands from the 5’ ends of the genome and antigenome RNA segments which bind, activate, and license MACV L for activity at discrete promoter regions. We also developed a system for isolating active L bound to its nucleoprotein-encapsidated RNA template (RNP) from mammalian cells and provide biochemical and structural analyses of these complexes. Moreover, we explored natural and synthetic inhibitors of the RdRP catalytic site for biochemically capturing an inactive arenavirus L bound to its RNA template. These findings uncover new conserved mechanisms of polymerase activation for viruses with multiple negative-strand genomic RNA segments and provide a framework for high-throughput development of antiviral compounds targeting arenavirus L in the context of its RNP template.