Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.
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.
Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.|Department of Pharmacology, University of Concepción, Concepción, Chile.
Yaiza
Y
Fernández-García
European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.
Juan
J
Reguera
Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
Carola
C
Busch
Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany.
Gregor
G
Witte
Department of Pharmacology, University of Concepción, Concepción, Chile.
Oliberto
O
Sánchez-Ramos
Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany.
Christian
C
Betzel
European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.
Stephen
S
Cusack
Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
Stephan
S
Günther
Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
Sophia
S
Reindl
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004720:Endonucleases / Q000737:chemistry* / Q000378:metabolism*; D006476:Orthohantavirus / Q000737:chemistry / Q000201:enzymology; D011487:Protein Conformation; D055550:Protein Stability; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D014764:Viral Proteins; C500570:L protein, hantavirus; D012324:RNA-Dependent RNA Polymerase; D004720:Endonucleases