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

Date Published: June 14, 2016

Publisher: Public Library of Science

Author(s): Yaiza Fernández-García, Juan Reguera, Carola Busch, Gregor Witte, Oliberto Sánchez-Ramos, Christian Betzel, Stephen Cusack, Stephan Günther, Sophia Reindl, Félix A. Rey.


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.

Partial Text

Andes virus (ANDV) belongs to the genus Hantavirus within the family Bunyaviridae. Hantaviruses can be pathogenic to humans and are distributed worldwide [1–3]. Their natural hosts are various rodent species [4]. ANDV is endemic in Argentina and Chile and its main reservoir host is the long-tailed pygmy rice rat (Oligoryzomys longicaudatus) [5]. The virus causes hantavirus cardiopulmonary syndrome (HCPS) that is associated with a case fatality of up to 40%. Related HCPS-causing hantaviruses are found throughout the Americas. However, in contrast to other hantaviruses, ANDV can be transmitted from human to human [6, 7].

This study provides structural and biochemical evidence for the existence of an endonuclease in the N terminus of ANDV L protein. In agreement with previous studies on expression of L protein in mammalian cells [36, 37], we have not been able to express the wild-type endonuclease domain in bacterial cells. Therefore, we took advantage of a range of mutations to facilitate expression of the domain. By using this approach, we expressed large amounts of various mutants and eventually solved the crystal structure of the ANDV L1–200 K127A mutant. The first structure of a hantavirus cap-snatching endonuclease reveals similarities to the related enzymes from LACV, LCMV, and IAV [16, 18, 20–23]. Besides the active site residues the sequence is hardly conserved. However there are obvious common structural features present: all cap-snatching endonucleases consist of two lobes with the conserved active site buried in a cavity in between. However, compared to the other enzymes, ANDV has positively charged patches surrounding the active site. The positively charged surface may increase the affinity of the enzyme for negatively charged RNA substrates. Whether or not this feature plays a role in the apparently strong endonuclease activity upon recombinant expression remains to be determined.




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