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

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.

The Z Proteins of Pathogenic but Not Nonpathogenic Arenaviruses Inhibit RIG-i-Like Receptor-Dependent Interferon Production

“…As all arenaviruses encode a strong IFN antagonist NP, which inhibits the IFN induction through the conserved RNase domain (15)(16)(17)(18)(19)(20), we wondered what the respective roles are of pathogenic Z and NP in the IFN suppression during an authentic arenavirus infection. We thus generated rPICVs encoding a pathogenic Z protein (ZN LCMV ) in the backbone of an NP RNase catalytically inactive D380A mutant (NPm1).…”

“…Ebola virus (EBOV) VP35 blocks RLR signaling through multiple mechanisms such as sequestering the RIG-i cofactor PKR activator (PACT), preventing the interactions of TBK1 and IKKε with IRFs, and inhibiting IRF7 activity (10-14). Arenaviral nucleoprotein (NP) strongly inhibits the production of type I IFNs through its DEDDH exoribonuclease (RNase) activity, possibly by degrading the immunostimulatory dsRNA substrates (15)(16)(17)(18)(19)(20).Arenaviruses are a diverse family of negative-strand enveloped RNA viruses with a bisegmented RNA genome, which encodes two proteins on each segment in an ambisense orientation-glycoprotein GPC and nucleoprotein NP on the S segment and L polymerase protein and the small matrix protein Z on the L segment (21). Arenaviruses can cause a wide spectrum of diseases in humans, with limited preventive or therapeutic options (22, 23).…”

“…Ebola virus (EBOV) VP35 blocks RLR signaling through multiple mechanisms such as sequestering the RIG-i cofactor PKR activator (PACT), preventing the interactions of TBK1 and IKKε with IRFs, and inhibiting IRF7 activity (10-14). Arenaviral nucleoprotein (NP) strongly inhibits the production of type I IFNs through its DEDDH exoribonuclease (RNase) activity, possibly by degrading the immunostimulatory dsRNA substrates (15)(16)(17)(18)(19)(20).…”

Structural and Functional Diversity of Nairovirus-Encoded Nucleoproteins

“…The C-terminal domain of LASV NP is well known as a 3=-to-5= dsRNA-specific exonuclease (22,40,41). However, the function of the N-terminal domain is more complicated.…”

Structure of the Leanyer orthobunyavirus nucleoprotein–RNA complex reveals unique architecture for RNA encapsidation

“…For example, encapsidation of the entire genome of negative-sense single-stranded RNA (ssRNA) viruses by nucleoprotein (N) molecules prevents attacks by the host. Recently, N proteins have also been shown to possess secondary functions such as the exoribonuclease activity of the Lassa virus N protein that helps the virus evade host immune surveillance (1,2) or the endonuclease activity of the Crimean-Congo hemorrhagic fever virus (CCHFV) nucleoprotein (3). These additional roles for N protein were explained with the elucidation of the structure of the Lassa virus N protein that surprisingly revealed the presence of an exonuclease fold at the C terminus.…”