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. 2018 Sep 6;46(15):7902-7912.
doi: 10.1093/nar/gky637.

The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure

Baptiste Martin  1 , Bruno Coutard  1 , Théo Guez  2 , Guido C Paesen  3 , Bruno Canard  1 , Françoise Debart  2 , Jean-Jacques Vasseur  2 , Jonathan M Grimes  3   4 , Etienne Decroly  1
Affiliations

The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure

Baptiste Martin et al. Nucleic Acids Res. .

Abstract

Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2'O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2'O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2'O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting internal adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2'O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA internal methylations in the viral replication.

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Figures

Figure 1.
Figure 1.
The MTase+CTD domain of Sudan ebolavirus L protein. (A) Sequence analysis of the mononegavirus L protein revealed six conserved regions (CRI to CRVI, yellow boxes) that contain motifs responsible for the different activities of the L (motifs mapped with asterisks) (Poch et al., 1990). Additionally, the recently published VSV L structure resolved by cryo-EM shows that the L protein is organized in 4 domains: the RNA-dependent RNA polymerase (RdRp) intricated with the polyribonucleotidyltransferase (PRNTase or Cap), a connector domain, the methyltransferase domain (MTase) and a poorly conserved C-terminal domain (CTD). (B) Based on alignments with the VSV L protein, the MTase+CTD domain in SUDV L protein was defined as the fragment covering amino acids 1713–2211. SAM-binding site motifs (GxGxG) and the 2’O catalytic tetrad K-D-K-E have also been identified (asterisks). (C) SDS-PAGE of purified, recombinant SUDV MTase+CTD containing an N-terminal oligohistidine-tag (58.4 kDa).
Figure 2.
Figure 2.
SUDV MTase+CTD has a cap-independent MTase activity. The Sudan ebolavirus (SUDV) methyltransferase domain MTase+CTD domain exhibits methyltransferase activity. (A) SUDV MTase activity measurements on a set of synthetic RNAs (13-mers) corresponding to the 5′end of SUDV mRNAs, some of which are pre-methylated at specific positions (Gppp: cap, mGppp: N7-methylated cap, Gm: 2’O-methylated nucleotide). Results have been normalized to those obtained with an unmethylated control (n = 6). Values represent normalized mean ± standard deviation. (B) MTase activity measurements of SUDV catalytic wild-type (WT) and mutants with altered catalytic K-D-K-E tetrad residues on a capped synthetic SUDV sequence-specific RNA (Gppp-13 mers). Mutant activities were normalized to those of a WT control (n = 3). Values represent normalized mean ± standard deviation. (C) SUDV MTase activity measurements on a set of synthetic SUDV-sequence-based RNAs (13 mers) with differing 5′ ends (p: monophosphate, ppp: triphosphate, Gppp: cap). Results have been normalized to those obtained with an unmethylated control (n = 6). Values represent normalized mean ± standard deviation. (D) Fluorescence polarization measurements of the SUDV MTase+CTD domain for synthetic SUDV sequence-specific RNAs (13 mers) with or without a cap structure (Gppp: cap, ppp: uncapped) (n = 3). Kd values were very similar, 0.64 μM compared to 0.68 μM, respectively. Values were normalized to the highest signal for each RNA (n = 3) and are given as normalized mean ± standard deviation.
Figure 3.
Figure 3.
SUDV MTase+CTD methylates internal adenosine residues of synthetic RNAs. The Sudan ebolavirus (SUDV) L protein MTase with the C-terminal domain (MTase+CTD) catalyses internal methylations. (A) MTase activity measurements on synthetic, 27-nucleotide-long homopolymeric RNAs (HO-(G/C/U/A)27). Groups have been normalized versus the activity on HO-(A)27 (n = 6). Values represent normalized mean ± standard deviation. (B) MTase activity evaluation on synthetic HO-(A)27 RNAs with 2’O-methylated residues (Am) or all residues 2’O methylated ((Am)). Groups have been normalized versus the activity on HO-(A)27 (n = 6). Data represent normalized mean ± standard deviation. (C) MTase activity evaluation on a set of synthetic SUDV sequence-specific RNAs (13 mers) with cap-1 (mGpppGm) and internal 2’O-methylated residues (Gm/Um/Am)). Groups have been normalized versus the activity on SUDV sequence-specific RNA with a cap-1 and no internal methylation (n = 6). Data represent normalized mean ± standard deviation. (D) RNA affinity evaluation by fluorescence polarization of synthetic SUDV sequence-specific capped RNAs (13-mers) with or without a cap-1 (mGpppGm) and internal 2’O-methylated adenosine residues (Am) and polyadenosine RNAs with or without 2’O methylations for the SUDV MTase+CTD domain (n = 3). Respective Kd are estimated at 0.88, 0.80, 0.58 and 0.61 μM. Results have been normalized versus the highest signal for each RNA (n = 3). Data represent normalized mean ± standard deviation. (E) HPLC profile of SUDV MTase+CTD methylated HO-(A)27 following treatment with snake venom phosphodiesterase and alkaline phosphatase. Controls identify adenosine (2’OH-A) at a retention point of 11.2 min and 2’O-methylated adenosine (2’OCH3-A) at a retention point of 14.6 min. These compounds were detected in a 98:2 ratio in the methylated HO-(A)27 RNA.
Figure 4.
Figure 4.
SUDV methylates its cap structure on the N7 position. The Sudan ebolavirus (SUDV) L protein MTase with the C-terminal domain (MTase+CTD) catalyses internal methylations and also methylates the cap structure. (A) MTase activity measurement at pH 7.0 and 8.0 on a set of synthetic SUDV sequence-specific RNAs (13-mers) that are uncapped (pppG) or capped (GpppX, mGpppX, GpppXm, mGpppXm), and carry internal 2’O-methylated adenosine residues ((Am)). Results have been normalized versus the activity on GpppG(Am)-SUDV12 RNA (n = 3). Values represent normalized mean ± standard deviation. (B) Thin layer chromatography of cap structures of control RNAs and a synthetic SUDV sequence-specific RNA (13-mers) with a 2’O-methylated cap (GpppGm) and internal 2’O methylated adenosines (Am) incubated with SUDV MTase+CTD domain for 0h, 3h and overnight (O/N). (C) Thin layer chromatography of cap structures from control RNAs and from synthetic SUDV VP40 sequence-specific RNAs (13-, 31-mers) with an unmethylated cap (GpppG). All RNAs were incubated with the SUDV MTase+CTD domain for 6h.
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