Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.

Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNA...

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Autores principales:	Juan Reguera, Friedemann Weber, Stephen Cusack
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/6e7d5a3c8d1d400a834e726757968f7a
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spelling	oai:doaj.org-article:6e7d5a3c8d1d400a834e726757968f7a2021-11-18T06:01:37ZBunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.1553-73661553-737410.1371/journal.ppat.1001101https://doaj.org/article/6e7d5a3c8d1d400a834e726757968f7a2010-09-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20862319/pdf/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 A resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4-6 segments), and orthomyxoviruses (6-8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation.Juan RegueraFriedemann WeberStephen CusackPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 6, Iss 9, p e1001101 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Juan Reguera Friedemann Weber Stephen Cusack Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
description	Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 A resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4-6 segments), and orthomyxoviruses (6-8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation.
format	article
author	Juan Reguera Friedemann Weber Stephen Cusack
author_facet	Juan Reguera Friedemann Weber Stephen Cusack
author_sort	Juan Reguera
title	Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
title_short	Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
title_full	Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
title_fullStr	Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
title_full_unstemmed	Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
title_sort	bunyaviridae rna polymerases (l-protein) have an n-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/6e7d5a3c8d1d400a834e726757968f7a
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Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.

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