Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein

Abstract

Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1–200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure–function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target

“…Here, we report the identification of the N-terminal endonuclease domain of the L polymerase of SFTSV, which is crucial for cap-dependent viral mRNA transcription. We solved the X-ray crystal structure of the endonuclease domain at 2.4-Å resolution, which revealed features that are unique to SFTSV as well as those features that are shared among other cap-snatching viral endonuclease domains (Ferná ndez-García et al, 2016;Jones et al, 2019;Reguera et al, 2010Reguera et al, , 2016Song et al, 2016). We also demonstrated the inhibition of SFTSV and HRTV endonuclease activities by using IAV polymerase acidic protein (PA) inhibitors, including Baloxavir acid (BXA) derived from a recently FDA-approved drug, Baloxavir marboxil (BXM), suggesting that the banyangvirus endonuclease domain is a major therapeutic target that can guide the development of broad-spectrum antivirals against sNSVs.…”

Transcription and replication mechanisms of Bunyaviridae and Arenaviridae L proteins

“…This motif defines this family of ENs, and can be found in the amino terminus of near all sNSV L proteins with the notable exception of nairovirus, including Crimean Congo Hemorrhagic Fever Virus (CCHFV), which after the EN motif have an additional extension of ≈600 aa at N-terminus harbouring the OTU domain (see below). Additional structures of cap-snatching ENs were reported for hantaviruses and Lassa virus (Fernández-García et al, 2016;Reguera et al, 2016b;Wallat et al, 2014), which together with Orthomyxoviridae family ENs (Dias et al, 2009;Guilligay et al, 2014;Yuan et al, 2009) have generated a broad perspective of their structure and function. The PD.…”

Hantavirus RdRp Requires a Host Cell Factor for Cap Snatching

“…N protein binds to the mRNA caps and protects capped mRNA fragments up to 180 nucleotides inside P-bodies that were efficiently processed by an unknown mechanism to generate 14-nucleotide-long RNA primers with a 3= G residue (18). Since bacterially expressed and purified endonuclease domain nonspecifically cleaved the RNA in vitro (11), it was not clear how capped primers of appropriate length and defined 3= terminus are generated during cap snatching. Our recently published results demonstrating the interaction between the N protein and C-terminal uncharacterized domain of RdRp provided insights about a possible mechanism for the generation of capped primers of appropriate length and defined 3= terminus from the protected capped mRNA fragments inside P-bodies.…”

“…The structure-based sequence alignments revealed that the cap-snatching endonuclease domain almost certainly exists at the N terminus of almost all known negative-strand and capsnatching segmented RNA viruses, including bunyaviruses, tentiviruses, arenaviruses, and orthomyxoviruses. The X-ray crystal structures of cap-snatching endonuclease domain from La Crosse orthobunyavirus (LCV), Andes hantavirus, and influenza virus reveal a similar overall fold and two identical metal binding active sites (8,9,11,25). Interestingly, the purified N-terminal endonuclease domain from LCV, hantavirus, and influenza virus nonspecifically cleaved RNA in vitro, and the enzymatic activity was metal ion dependent (8,9,11,25).…”

“…The X-ray crystal structures of cap-snatching endonuclease domain from La Crosse orthobunyavirus (LCV), Andes hantavirus, and influenza virus reveal a similar overall fold and two identical metal binding active sites (8,9,11,25). Interestingly, the purified N-terminal endonuclease domain from LCV, hantavirus, and influenza virus nonspecifically cleaved RNA in vitro, and the enzymatic activity was metal ion dependent (8,9,11,25). The LCV, hantavirus, and influenza virus use capped primers of 10 to 14 nucleotides in length, containing 3= U, G, and C or A residues (18,26,27).…”