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Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.

ABSTRACT: Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2'-O-methyltransferase (2'O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2'O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

SUBMITTER: Lugari A

PROVIDER: S-EPMC2963367 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledThe 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2'-O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2'-O-MTase activity (E. Decroly et al., J Virol 82:8071-8084, 2008, http://dx.doi.org/10.1128/JVI.00407-08; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010, http://dx.doi.org/10.1371/journal.ppat.1000863; E. Decroly et al., PLoS Pathog 7:e1002059, 2011, http://dx.doi.org/10.1371/journal.ppat.1002059; Y. Chen et al., PLoS Pathog 7:e1002294, 2011, http://dx.doi.org/10.1371/journal.ppat.1002294) . In this study, we demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2'-O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2'-O-MTase activity can be targeted in vitro and in vivo.ImportanceCoronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2'-O-MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2'-O-MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2'-O-MTase of coronaviruses.

Project description:The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase) that is thought to methylate the ribose 2'-OH of the first transcribed nucleotide (N1) of viral RNA cap structures. This 2'-O-MTase activity is regulated by nsp10. The 2'-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2'-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7mGpppG (cap-0) into 7mGpppG2'Om (cap-1) RNA by 2'-OH methylation of N1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2'-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2'-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2'-O methylation and to restore the host antiviral response. IMPORTANCE MERS-CoV codes for a cap 2'-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2'O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2'-O-methyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2'-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.

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Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.

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