A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex

Yuri Kusov; Jinzhi Tan; Enrique Alvarez; Luis Enjuanes; Rolf Hilgenfeld

doi:10.1016/j.virol.2015.06.016

A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex

Yuri Kusov, Jinzhi Tan, Enrique Alvarez, Luis Enjuanes, Rolf Hilgenfeld^*

^*Corresponding author for this work

Institute of Biochemistry

98 Citations (Scopus)

Abstract

The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

Original language	English
Journal	Virology
Volume	484
Pages (from-to)	313-322
Number of pages	10
ISSN	0042-6822
DOIs	https://doi.org/10.1016/j.virol.2015.06.016
Publication status	Published - 04.07.2015

Funding

We thank Doris Mutschall for excellent technical assistance, Liliya Grin and Luisa Stroeh for help with the preparation and analysis of several constructs, and Dr. Jian Lei for discussion. This work was supported by the German Center for Infection Research (DZIF) , the European Commission (through its projects SILVER (Contract no. 260644 ) and EMPERIE (Contract no. 223498 )), the Ministry of Science and Innovation of Spain ( BIO2010-16705 ), the U.S. National Institutes of Health (Grant nos. 2P01AI060699-06A1 and CRIP-HHSN266200700010C ), and the Sino-German Center for the Promotion of Research , Beijing, China ( GZ 236 (202/9 )). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Appendix A

Research Areas and Centers

Academic Focus: Center for Infection and Inflammation Research (ZIEL)

Coronavirus related work

Research on SARS-CoV-2 / COVID-19

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.virol.2015.06.016

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title = "A G-quadruplex-binding macrodomain within the {"}SARS-unique domain{"} is essential for the activity of the SARS-coronavirus replication-transcription complex",

abstract = "The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the {"}SARS-unique domain{"}. The X domain was proposed to be an ADP-ribose-1{"}-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.",

author = "Yuri Kusov and Jinzhi Tan and Enrique Alvarez and Luis Enjuanes and Rolf Hilgenfeld",

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doi = "10.1016/j.virol.2015.06.016",

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journal = "Virology",

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T1 - A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex

AU - Kusov, Yuri

AU - Tan, Jinzhi

AU - Alvarez, Enrique

AU - Enjuanes, Luis

AU - Hilgenfeld, Rolf

PY - 2015/7/4

Y1 - 2015/7/4

N2 - The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

AB - The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

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DO - 10.1016/j.virol.2015.06.016

M3 - Journal articles

C2 - 26149721

AN - SCOPUS:84947236129

SN - 0042-6822

VL - 484

SP - 313

EP - 322

JO - Virology

JF - Virology

ER -

A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex

Abstract

Funding

Research Areas and Centers

Coronavirus related work

UN SDGs

Access to Document

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