Novel Function(s) of Arenavirus NP Exoribonuclease

PROJECT SUMMARY/ABSTRACT Several mammalian arenaviruses cause severe and fatal zoonotic diseases in humans, for which vaccines and treatments are very limited. Lassa virus (LASV) is the causative agent for Lassa fever (LF) that is currently endemic in Western Africa. Despite its importance to public health, important knowledge gaps still exist in the basic biology for LASV and other highly pathogenic arenaviruses, partly due to the limitation of high containment BSL4 facilities required for infection experiments. The RNA synthesis of RNA virus is generally error prone as viral RNA-dependent RNA polymerase lacks proofreading activity. As negative-sense RNA virus, how arenavirus ensures proper RNA synthesis is largely unclear. The LASV nucleoprotein has a DEDDH 3' to 5' exoribonuclease motif (ExoN), of which its function in virus life cycle is still a puzzle. The current paradigm in the field is that the NP ExoN activity efficaciously degrades virus-derived double-stranded RNA and is the key to LASV evasion of innate immunity. Intriguingly, the ExoN motif is highly conserved in NPs of all arenaviruses, regardless of pathogenicity. Therefore, it is very likely that the NP ExoN has important but yet-to-be-identified function(s) in arenavirus replication in addition to immune evasion. In this project, we aim to define the important role(s) of arenavirus NP ExoN in virus replication. Our preliminary data indicated that: 1). Loss of NP ExoN activity resulted in aberrant genomic RNA production and a drastic reduction in LASV RNA level in IFN-deficient cells; and 2). Loss of NP ExoN activity increased LASV sensitivity to mutagenic nucleoside analogue treatment. We propose that LASV NP ExoN promotes proper viral RNA synthesis, controls aberrant viral genomic RNA formation and/or ensures the fidelity of viral RNA replication. To define the impact of NP ExoN on the integrity and functionality of viral genomic RNA, we will explore to utilize an innovative long-read sequencing technology to systematically investigate the sequence and abundancy of genomic RNAs at single molecule level. Arenavirus has been known to form defective interfering particles, which regulates virus infection in vivo and in vitro. The molecular basis for DI genome remains elusive due to technical obstacle. The long-read sequencing technology may enable us to identify DI candidates and the potential role of NP ExoN in regulating DI formation. We will also investigate whether arenavirus NP ExoN has proofreading activity that ensures the fidelity of viral RNA replication. At the end of this project, we may discover novel and important function(s) of arenavirus NP ExoN in virus life cycle and move the field forward. Using LASV as a model, we may better understand how arenavirus virus ensures proper viral RNA synthesis. With the novel long-read sequencing technology, this study may overcome technical barrier and increase our knowledge on basic virology of pathogenic arenaviruses. The data may open up new directions. For instance, future studies on the mechanisms underlying the important roles of NP ExoN are warranted. This project may also facilitate antiviral development by targeting NP ExoN activity.