Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid–base catalysis and substrate-dependent metal ion activation

Huang, Tong; Snell, Kimberly C.; Kalia, Nidhi; Gardezi, Shahbaz; Guo, Lily; Harris, Michael E.

doi:10.1016/j.jbc.2023.104787

Cited by 5 publications

(3 citation statements)

References 60 publications

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“…Previous studies reported that nsp15 required Mn 2+ for optimal EndoU activity ( 26 , 27 , 40 , 41 ) and a Mn 2+ concentration of 5 mM was commonly used. As 5 mM Mn 2+ is physiologically irrelevant ( 42 , 43 ), we conducted the cleavage reaction at Mn 2+ concentrations of 0.5 mM and zero in addition to 5 mM.…”

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity

Wang,

Zhu

2024

Nucleic Acids Research

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It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However, how nsp15 processes viral dsRNA, commonly considered as a genome replication intermediate, remains elusive. Previous research has mainly focused on short single-stranded RNA as substrates, and whether nsp15 prefers single-stranded or double-stranded RNA for cleavage is controversial. In the present work, we prepared numerous RNA substrates, including both long substrates mimicking the viral genome and short defined RNA, to clarify the substrate preference and cleavage pattern of SARS-CoV-2 nsp15. We demonstrated that SARS-CoV-2 nsp15 preferentially cleaved pyrimidine nucleotides located in less thermodynamically stable areas in dsRNA, such as AU-rich areas and mismatch-containing areas, in a nicking manner. Because coronavirus genomes generally have a high AU content, our work supported the mechanism that coronaviruses evade the antiviral response mediated by host cell dsRNA sensors by using nsp15 dsRNA nickase to directly cleave dsRNA intermediates formed during genome replication and transcription.

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Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity

Wang,

Zhu

2024

Nucleic Acids Research

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“…Furthermore, to exclude the effect of potential nuclease contamination, we purified both the wild-type SARS-CoV-2 nsp15 with a 6× His tag at the N-terminus and its active-site mutant, H234A, as a control for the cleavage reaction, by metal affinity chromatography and gel filtration chromatography (Figure 1B). Previous studies reported that nsp15 required Mn 2+ for optimal EndoU activity (Bhardwaj et al, 2004; Huang et al, 2023; Ivanov et al, 2004; Zhang et al, 2018); therefore, we initially conducted the cleavage reaction at a Mn 2+ concentration of 5 mM, which was commonly used in previous studies. We observed that nsp15 efficiently cleaved both ssRNA and dsRNA substrates under this condition (Figures S1A and S1B).…”

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity

Wang,

Zhu

2023

Preprint

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SUMMARYIt has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However, how nsp15 processes viral dsRNA, commonly considered as a genome replication intermediate, remains elusive. Previous research has mainly focused on short single-stranded RNA as substrates, and whether nsp15 prefers single-stranded or double-stranded RNA for cleavage is controversial. In the present work, we prepared numerous RNA substrates, including both long substrates mimicking the viral genome and short defined RNA, to clarify the substrate preference and cleavage pattern of SARS-CoV-2 nsp15. We demonstrated that SARS-CoV-2 nsp15 preferentially cleaved flexible pyrimidine nucleotides located in AU-rich areas and mismatch-containing areas in dsRNA via a nicking manner. The AU content and distribution in dsRNA along with the RNA length affected cleavage by SARS-CoV-2 nsp15. Because coronavirus genomes generally have a high AU content, our work supported the mechanism that coronaviruses evade the antiviral response mediated by host cell dsRNA sensors by using nsp15 dsRNA nickase to directly cleave dsRNA intermediates formed during genome replication and transcription.

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“…Nsp13 is an RNA helicase and is essential for transcription and translation [101,102]. Nsp16 is a 2′-O-methyltransferase, whilst Nsp15 has endoribonuclease activity [103][104][105][106] (these and other functions, with additional relevant references, are summarized in Table 1).…”

Section: Sars-cov-2mentioning

confidence: 99%

SARS-CoV-2 and the DNA damage response

Grand

2023

Journal of General Virology

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The recent coronavirus disease 2019 (COVID-19) pandemic was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is characterized by respiratory distress, multiorgan dysfunction and, in some cases, death. The virus is also responsible for post-COVID-19 condition (commonly referred to as ‘long COVID’). SARS-CoV-2 is a single-stranded, positive-sense RNA virus with a genome of approximately 30 kb, which encodes 26 proteins. It has been reported to affect multiple pathways in infected cells, resulting, in many cases, in the induction of a ‘cytokine storm’ and cellular senescence. Perhaps because it is an RNA virus, replicating largely in the cytoplasm, the effect of SARS-Cov-2 on genome stability and DNA damage responses (DDRs) has received relatively little attention. However, it is now becoming clear that the virus causes damage to cellular DNA, as shown by the presence of micronuclei, DNA repair foci and increased comet tails in infected cells. This review considers recent evidence indicating how SARS-CoV-2 causes genome instability, deregulates the cell cycle and targets specific components of DDR pathways. The significance of the virus’s ability to cause cellular senescence is also considered, as are the implications of genome instability for patients suffering from long COVID.

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Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid–base catalysis and substrate-dependent metal ion activation

Cited by 5 publications

References 60 publications

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity

SARS-CoV-2 and the DNA damage response

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