SUPPLEMENTAL MATERIAL UGA stop codon readthrough to translate intergenic region of Plautia stali intestine virus does not require RNA structures forming internal ribosomal entry site Nobuhiko Kamoshita and Shin-ichi Tominaga Supplemental Results and Discussion page 1 Materials and Methods page 8 References page 11 Tables S1–S3 page 13 Figures S1–S10 page 17 SUPPLEMENTAL RESULTS AND DISCUSSION I. SUPPLEMENTAL MATERIALS for the RESULTS section Deletion of intergenic region (IGR) Raw data of typical cell-free translation experiments are shown in Supplemental Figure S2. Uncapped dicistronic mRNAs were the template for deletion assays in cell-free systems and the amount of FLuc translated by internal initiation (IRES-FLuc) was higher than that of readthrough polypeptides (RT), as shown in Figures 4B and S2. Note that the unit for photo-stimulated value (PSL) for RT, as shown in the rightmost column in Supplemental Figure S2B, is two orders of magnitude lower than that for IRES-FLuc and RLuc. Through the measurement of luciferase enzymatic activities (Figure S2C), we found that firefly luciferase activity of IRES-FLuc in sample 10 [“170” in the column for sample 10 (column 10)], derived from monocistronic firefly luciferase protein with N-terminal nine-amino-acid and C-terminal 22-amino-acid extensions, was far higher than those from RT (columns 2–7, maximum of “9.4” in column 7), even after adjusting differences in the amounts of synthesized polypeptides using PSL values derived from 35S activity (Figure S2B). In addition, even within a series of 3ʹ deletion mutants, while relative 35S activities of readthrough polypeptides, shown as the RT proportion in Figure S2B, increased from “21” (column 2, 5th row) to “50” (column 7) as deletion proceeded, enzymatic firefly luciferase activity ratio (F/R ratio) in Figure S2C decreased from “2.7” (column 2, 4th row) to “1.3” (column 4) and then drastically increased to “15,” “18,” or “20” (columns 5–7). Since readthrough polypeptide is the sole source of firefly luciferase activity in these samples, the discrepancy between the L values in Figure S2C and the R values in Figure S2B indicates that specific firefly luciferase enzymatic activities of these fusion polypeptides differ according to the length of IGR inserted, as shown in Figure S2D (columns 2–7). If the specific enzymatic activity of each deletion mutant is different, it is impossible to use the enzymatic activity ratio to compare the readthrough level, unless the specific enzymatic activity of each fusion polypeptide is accurately determined in advance in different experiments. Therefore, we used radioactivity to evaluate the readthrough levels of deletion mutants in cell-free assay systems (Figures 4B and 4C) and chemiluminescence obtained from immunoblots for those expressed in cultured cells (Figures 4D and 4E). Nucleotide replacement assay See Supplemental Table S1 in page 13 of this Material. Kamoshita - Supplemental Material - 1 II. SUPPLEMENTAL MATERIALS for the DISCUSSION section Homology search of IGRp using PSI-BLAST In a typical search to obtain the results as shown in Supplemental Table S2, we obtained 17 hits after Iteration 1. The identified subjects were mostly prokaryotic, but one of them, XP_017087353.1 (subject 2 at the end of the search), was described as “PREDICTED: ATP-binding/permease protein CydC-like [Drosophila bipectinata],” as shown in Supplemental Table S2. This polypeptide sequence was derived from a DNA sequence obtained in the Bioproject analyzing Drosophila. Scrutiny of this sequence suggested us that this DNA sequence, which is polycistronic without any introns, is derived from prokaryotes rather than Drosophila. The sequence of XP_017087353.1 is actually essentially the same as WP_039143398.1 (subject 3), derived from Lactobacillus fructivorans. The sequence of XP_017087353.1 (subject 2) overlaps with the sequence of WP_039143398.1 after Met104. Conversely, a DNA sequence correspondent to amino acid sequence Met1 to Met104 of WP_039143398.1 is present in the data obtained in the Bioproject, which is the source of XP_017087353.1. In line with our criteria described in the Supplemental Materials and Methods, we never selected XP_017087353.1 as a sequence to construct PSSM (Position-specific scoring matrix) throughout the iterations of PSI-BLAST. Nonetheless, XP_017087353.1 had the second highest ranking at the end of the PSI-BLAST search. Identification of WP_039143398.1 (subject 3) and three more related sequences (subjects 9–11) implies a strong link between the two sequences of IGRp and CydD, a subunit of thiol reductant ABC exporter. According to the PSI-BLAST search, N-terminal residues of 4–47 in IGRp were aligned with the N-terminal transmembrane portion of CydD (122–165, Table S2 and Figure S5). Almost the same region of IGRp was also aligned with two other sequences, those of RadC and phage portal protein (Table S2 and Figure S5). Unfortunately, the function of RadC is currently obscure. However, the crystal structure from Chlorobium tepidum (pdb 2QLC) is available, in which similar pattern of secondary structures, as predicted by PSIPRED, is present (helix–sheet–sheet–helix), along the alignment with IGRp (6–48). Note that, in this region, different structures of helix–helix–helix and α-transmembrane domain (see below) are predicted by JPRED and three different algorithms for transmembrane helix prediction, respectively. Currently, we have no explanation for the different prediction in this region. Phage portal protein from Blastopirellula marina is a protein predicted from a DNA sequence obtained in the BioProject. Amino acid sequences 7–50 in the N-terminal region of IGRp was aligned with amino acid Kamoshita - Supplemental Material - 2 residues spanning in 217–261 in the phage portal protein, which is located in the middle of phage portal superfamily domain of lambda-type. Since the length of the query is only 64 amino acids, there is a limit to the prediction. Nonetheless, the alignments with portions of three different proteins suggest that IGRp can be integrated into proteins and modulate their functions. Role of IGRp in viral replication (1) C-terminal extension of 3D In most RNA viruses, RNA replicase is the largest polypeptide in terms of amino acid length. Unique motifs conserved among other polymerases (motifs A–E) are encoded in addition to motif F, which is specific to RdRp (GDD motif C is shown with a purple box in Figure S8). While the polymerase reaction itself is carried out by RdRp in vitro, a single enzyme is insufficient for RNA synthesis in infected cells, or needs to be regulated, and in general, RdRp forms a complex with other viral and cellular proteins and associates with specific structures in the cytoplasm (Flint et al. 2015). PSIV 3D is the largest polypeptide in PSIV, although it is shorter than that of CrPV 3D by 21 amino acids (Figure S8). It is actually the smallest among the 3D replicases in 15 dicistroviruses listed in Figure S6. Picornavirus 3D is smaller than dicistrovirus 3D and 3D from poliovirus (PV) lacks any motifs that associates with a membrane. PV 3D recruits the viral protein 3AB for association with a membrane (Flint et al., 2015). In an effort to prove the motif which can associate with a membrane, transmembrane motif on viral polypeptide was investigated using several transmembrane search tools. With the usage of PHDhtm (Rost et al., 1995), we could not obtain any hits on PV 3D, while foot-and-mouth disease virus (FMDV) 3D gave a hit overlapping with an α11 helix (304Ser–319Leu, Figure S8A). When CrPV and PSIV sequences are investigated, in addition to the sequence overlapping with FMDV α11 helix (340Ile–351Tyr in PSIV and 360Asn–369Arg in CrPV, Figure S8A), IGRp 18Phe–41Leu was predicted (Figures S5 and S8B). As mentioned earlier, sheet and helix structures are predicted between Ile20 and His33 in IGRp, by the different algorithms PSIPRED and JPRED, respectively (Figure S5). When a helix is formed, it may acquire membrane affinity and 3D–IGRp protein can be localized to membrane components with different affinity from 3D, which then modulate the RNA replication of PSIV. However, future study Kamoshita - Supplemental Material - 3 using biochemical and viral replication system is necessary to prove this hypothesis. It is also necessary to determine whether 3D–IGRp can be cleaved from VP2 somewhere within or around IGRp sequence. PSI-BLAST search using PSIV 3D–IGRp as a query detects homology of IGRp with the C-termini of picornaviral 3D (Figure S8B) and RdRps from some plant viruses including strawberry mottle virus (data not shown) in the family Secoviridae. (2) N-terminal extension of VP2 With the knowledge of the crystal structures for the virion of CrPV and three other dicistroviruses (BQCV, TrV and IAPV), N-terminal addition of an extra 594 residues (PSIV 3D–IGRp, Figure S7) at the very end of VP2 will most likely interfere with the progress of virion assembly, especially after the formation of the pentamer. With additional cleavage somewhere within or around IGRp, efficient assembly into a virion will proceed. Prediction of cleavage by PSIV 3C/3CD To predict cleavage sites, we first compared the sequence of PSIV 3C protease with those of picornaviruses, crystal structures of which have been studied (Figure S9A). Then, information on the cleavage site was extracted from the reference peptide sequence of each virus and aligned as a dodecapeptide sequence surrounding the cleavage site (red triangles in Figure S9B). (1) Comparison with picornavirus 3C In a PSI-BLAST search using the 3C region of PSIV or CrPV as a query, hepatitis A virus (HAV) and FMDV 3C gave high scores among 3C proteases with known crystal structures. 3C of poliovirus 1, now classified as species Human enterovirus C, was included because detailed information about the specificity towards peptide substrate is available.