Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease

Kathryn M Hastie,Liam B King,Erica Ollmann Saphire,Michelle A Zandonatti,Luis Menéndez-Arias

doi:10.1371/journal.pone.0044211

Abstract

Lassa virus causes hemorrhagic fever characterized by immunosuppression. The nucleoprotein of Lassa virus, termed NP, binds the viral genome. It also has an additional enzymatic activity as an exonuclease that specifically digests double-stranded RNA (dsRNA). dsRNA is a strong signal to the innate immune system of viral infection. Digestion of dsRNA by the NP exonuclease activity appears to cause suppression of innate immune signaling in the infected cell. Although the fold of the NP enzyme is conserved and the active site completely conserved with other exonucleases in its DEDDh family, NP is atypical among exonucleases in its preference for dsRNA and its strict specificity for one substrate. Here, we present the crystal structure of Lassa virus NP in complex with dsRNA. We find that unlike the exonuclease in Klenow fragment, the double-stranded nucleic acid in complex with Lassa NP remains base-paired instead of splitting, and that binding of the paired complementary strand is achieved by “relocation” of a basic loop motif from its typical exonuclease position. Further, we find that just one single glycine that contacts the substrate strand and one single tyrosine that stacks with a base of the complementary, non-substrate strand are responsible for the unique substrate specificity. This work thus provides templates for development of antiviral drugs that would be specific for viral, rather than host exonucleases of similar fold and active site, and illustrates how a very few amino acid changes confer alternate specificity and biological phenotype to an enzyme.

Highlights

The causative agent of Lassa Fever, an arenavirus called Lassa, is endemic in Western Africa, causes hundreds of thousands of infections per year, and is the viral hemorrhagic fever most frequently transported to the United States and Europe
Lassa NP has a tertiary fold that is similar to, and an exonuclease active site that is completely conserved with other DEDDh exonucleases like interferonstimulated gene-20 (ISG-20) [5], E. coli DNA polymerase IIIe (e186) [6], and Trex 1 [7,8] and Trex 2 [9]
Structure of the exonuclease domain of Lassa NP bound to doublestranded RNA (dsRNA)

Summary

Introduction

The causative agent of Lassa Fever, an arenavirus called Lassa, is endemic in Western Africa, causes hundreds of thousands of infections per year, and is the viral hemorrhagic fever most frequently transported to the United States and Europe. Lassa NP has a tertiary fold that is similar to, and an exonuclease active site that is completely conserved with other DEDDh exonucleases like interferonstimulated gene-20 (ISG-20) [5], E. coli DNA polymerase IIIe (e186) [6], and Trex 1 [7,8] and Trex 2 [9]. Among this family of enzymes, Lassa NP is the only one known to have a strict specificity for a certain substrate, and the only one known to efficiently digest double-stranded RNA [3]. For example ISG-20 can digest both ssRNA and DNA [10] while e186 and Trex 1 and 2 can digest ssDNA or dsDNA [11,12]

Methods

Results

Conclusion