Fig 1.
Schematic representation of the unconventional mechanism of mRNA cap formation by VSV L Protein.
Possible mechanism described for the vesicular stomatitis virus (VSV) L protein where first hydrolyzes the γ-phosphate of guanosine-5’-triphosphate (GTP; Gppp) to yield guanosine diphosphate (GDP; Gpp) and inorganic phosphate (Pi) [31]. Then, nascent mRNA transcripts that carry a triphosphate group at their 5’ end form a covalent adduct with the PRNTase histidine residue present in the conserved ‘HR’ motif of L, releasing pyrophosphate (PPi). The transfer of pNp-RNA to the GDP (in which N denotes the first transcribed nucleotide) forms the capped structure (GpppNp-RNA) that is subsequently methylated by S-adenosyl-L-methionine (AdoMet or SAM)-dependent methyltransferases (MTases) present in the C-terminal domain of L. First, the (nucleoside-2’-O)-methyltransferase (2’-O-MTase) transfers the methyl group from SAM to the first nucleotide of the nascent RNA, forming GpppNmp-RNA and releasing S-adenosyl homocysteine (SAH). Then, the cap structure is methylated on the guanine N7 position by the (guanine-N7-) methyltransferase (N7-MTase) to generate cap-1 RNA (m7GpppNmp-RNA).
Fig 2.
The recombinant RSV MTase-CTD protein.
(A) Schematic representation of the domain organization of the RSV L protein. Blue, RNA-dependent RNA-polymerase (RdRp) domain; green, polyribonucleotidyl-transferase (PRNTase) domain; yellow, connector domain (CD); orange, methyltransferase (MTase) domain; red, C-terminal domain (CTD). Amino acid residue numbers indicate the functional domain boundaries. The conserved regions (CR) within the L proteins of non-segmented negative-strand RNA viruses are indicated. Arrows denote the position of active site residues required for function, and dots indicate conserved motifs with the starting amino acid residue number in gray. (B) The RSV MTase-CTD fragment was defined based on the alignment with the VSV L protein. Arrow indicates the K1831-D1936-K1973-E2004 catalytic tetrad, typical of 2’-O-MTases. Dots indicate conserved motifs with the starting amino acid residue number in gray: the GxGxGx SAM/SAH binding site motif in the MTase domain, and the K-K-G motif, reminiscent of eukaryotic GTases, in the CTD domain. (C) SDS-PAGE of the purified recombinant RSV MTase-CTD protein containing an N-terminal histidine tag (49.3 kDa). Molecular weights (in kilodaltons) of the ladder are shown on the left, and the MTase-CTD band is labeled on the right.
Fig 3.
Cap-dependent MTase activity of the recombinant RSV MTase-CTD protein.
(A) The transfer of tritiated methyl groups from S-adenosylmethionine (SAM) molecules to synthetic capped RNAs of different lengths (5-, 9-, 11- and 15- nucleotide-long), which mimic the 5’ end of RSV mRNA, was assessed by filter-binding assay. The RSV MTase-CTD was used at the concentration of 25 nM. Data are the mean values ± standard error of the mean (SEM) of three independent measurements. (B) MTase activity measurements using capped and uncapped RSV9 RNAs (GGG ACA AAA) methylated at specific positions. Values represent the mean ± SEM (n = 3). (C) MTase activity evaluation on synthetic, 27-nucleotide-long homopolymeric RNAs (HO-(G/C/U/A)27). Values were normalized to the activity on Gppp GGG-RSV9, and are the mean ± SEM (n = 3). CPM, count per minute.
Fig 4.
RSV MTase activity is sequence-specific.
Substrate specificity was determined by filter-binding assay, using the synthetic capped RNA substrates with different 5’ end sequences and lengths (numbers after virus abbreviation) described in the table on the right. hMPV, human Metapneumovirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; MERS, Middle East Respiratory Syndrome; SUDV, Sudan Ebolavirus; RABV, Rabies virus; WNV, West Nile virus; DV, Dengue virus. The RSV MTase-CTD was used at 25 nM. Values were normalized to the activity on Gppp GGG RSV9, and are the mean ± SEM (n = 3).
Fig 5.
N7- and 2’-O-methylation activity of the RSV MTase-CTD protein.
(A) MTase activity measurements by filter-binding assay using capped RSV9 RNAs (GGG ACA AAA). Values were normalized to the activity on Gppp GGG-RSV9 and are the mean ± SEM (n = 3). (B) Time-course of N7- and 2’-O-methylation activities on GpppGm and mGpppG RSV9 RNAs, respectively, by the RSV MTase-CTD protein (25 nM) measured by filter-binding assay at different pH (from 6.0 to 9.0). The plotted values were obtained after 3 h incubation at 30°C. Values were normalized to the maximum activity on each substrate and are the mean ± SEM (n = 3). (C, D) Thin-layer chromatography analysis of cap structures of control RNAs and substrates (G*pppG (C left), mG*pppG (C right), G*pppGm (D)) incubated with the RSV MTase-CTD (* indicates G(32P)-labeled). Nucleotides 2–9 were removed by nuclease P1 digestion and caps were separated using 0.65 M LiCl as mobile phase. Controls (G*pppG, G*pppGm, mG*pppG, mG*pppGm) were obtained using the pppG-RSV9 substrate and vaccinia virus MTases that specifically methylate caps at the N7 or 2’-O positions. The RSV MTase-CTD was used at the concentration of 25 nM. O/N:overnight. (E) Densitometry quantitations of TLC analysis expressed as a ratio of the substrate consumption (G*pppG (left), mG*pppG (middle), G*pppGm (right)) and product formation over time. Values are the mean ± SEM (n = 3).
Fig 6.
MTase activity of the RSV L-P complex.
(A) SDS-PAGE of the purified RSV L-P complex. Molecular weights (in kilodaltons) of the ladder are shown on the left, and the L and P bands are labeled on the right. (B) Time-course analysis of methylation by thin-layer chromatography of the cap structures of control RNAs and substrates (G*pppG and mG*pppG) incubated with RSV L-P (* indicates G(32P)-labeled) for up to 6 h at 30°C. (C) After overnight (O/N) incubation, nucleotides 2–9 were removed by nuclease P1 digestion and caps were separated using 0.65 M LiCl as mobile phase. Controls (G*pppG, G*pppGm, mG*pppG, mG*pppGm) were obtained using the pppG-RSV9 substrate and vaccinia virus MTases that specifically methylate caps at the N7 or 2’-O positions. The RSV L-P complex was used at the concentration of 100 nM. (D) Densitometry quantitations of TLC analysis expressed as a ratio of the substrate consumption (G*pppG (left), mG*pppG (middle), G*pppGm (right)) and product formation over time. Values are the mean ± SEM (n = 3).
Fig 7.
Inhibition of the MTase activity by SAM analogues.
Increasing concentrations of SAH or sinefungin (previously dissolved in water) were incubated with 25 nM RSV MTase-CTD in a reaction mixture (40 mM Tris-HCl, pH 7.5, 2 μM SAM and 0.1 μM 3H-SAM) in the presence of 0.7 μM of GpppG-RSV9 synthetic RNA, and with 60 nM RSV L-P complex in a reaction mixture (40 mM Tris-HCl, pH 7.5, 0.17 μM SAM and 0.8 μM 3H-SAM) in the presence of 1.8 μM synthetic RNA (GpppG-RSV9). Reactions were incubated at 30°C for 3 h. Values were normalized and fitted with Prism (GraphPad software) using the following equation: Y = 100/(1+((X/IC50)^Hillslope)) (n = 3; mean value ± SEM).