Cassava brown streak virus (Potyviridae) encodes a putative Maf/HAM1 pyrophosphatase implicated in reduction of mutations and a P1 proteinase that suppresses RNA silencing but contains no HC-Pro

Abstract

The complete positive-sense single-stranded RNA genome of Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae) was found to consist of 9,069 nucleotides and predicted to produce a polyprotein of 2,902 amino acids. It was lacking helper-component proteinase but contained a single P1 serine proteinase that strongly suppressed RNA silencing. Besides the exceptional structure of the 5'-proximal part of the genome, CBSV also contained a Maf/HAM1-like sequence (678 nucleotides, 226 amino acids) recombined between the replicase and coat protein domains in the 3'-proximal part of the genome, which is highly conserved in Potyviridae. HAM1 was flanked by consensus proteolytic cleavage sites for ipomovirus NIaPro cysteine proteinase. Homology of CBSV HAM1 with cellular Maf/HAM1 pyrophosphatases suggests that it may intercept noncanonical nucleoside triphosphates to reduce mutagenesis of viral RNA.

FIG. 1.

Comparison of the viral genomes in four genera of Potyviridae. The complete sequences of the type members of the genera Potyvirus (Potato virus Y, PVY; accession no. NC_001616), Rymovirus (Ryegrass mosaic virus, RGMV; AF035818), Tritimovirus (Wheat streak mosaic virus, WSMV; AF057533) and Ipomovirus (SPMMV; Z73124) are shown. Furthermore, genome structures of three additional ipomoviruses, SqVYV (EU259611), CVYV (NC_006941), and CBSV (FJ039520), whose genome sequence was determined in this study, are shown. Genera Macluravirus and Bymovirus were excluded because no complete viral genome sequence was available and because the genomes are bipartite, respectively. The box represents viral polyprotein translated from a large ORF. A small ORF created by +2 frameshifting is found in all the depicted viruses and shown in the drawing for CBSV below P3 as a black box (PIPO). The 5′UTR and 3′UTR are shown as bold lines. The numbers indicated after the 3′UTR refer to the genome size (nucleotides) and the size of the polyprotein (amino acids). The amino acids at the predicted proteolytic cleavage sites of the polyprotein are shown below the polyprotein. The estimated molecular weights of the mature proteins (in kilodaltons) are indicated in the box for each protein. The names of proteins above the polyprotein are as follows: P1, a serine proteinase, the first protein; P1a and P1b, two diversified serine proteinases; HC-Pro, helper component cysteine proteinase; P3, the third protein; 6K1 and 6K2, 6-kDa proteins; CI, cylindrical inclusion protein; VPg, viral genome-linked protein; NIa-Pro, the main viral proteinase; NIb, replicase. The primers used to amplify and sequence the CBSV genome in segments are shown at the bottom. Primers given at the bottom of the figure, from right to left, respectively (sequence and genomic position of the primers are in parentheses), are oligo(T)₂₅ and PVD4 (5′-CTCAATGTTCCTGATGATGA-3′, 6606 to 6625), HAM1R2 (5′-GGACTATTAGTCATCTTCACT-3′, 7292 to 7312) and CIF7 (5′-TGAACTACAAGGAACATCTG-3′, 2930 to 2949), CIR4 (5′-TGTCAACCAAATGAGCTGTG-3′, 3512 to 3531) and P3F3 (5′-ATGGAGTATGTAGGCAAACAT-3′, 1410 to 1430), and CBSV2R1 (5′-CATATGTCATGATTGTAATAG-3′, 2665 to 2685) and CBSV2F (5′-AAATAAACATGACATAAGAATAC-3′, 3 to 25). The primer GSP1 (5′-TGTTGCCCTCTCCGTCAAGC-3′, 191 to 210) was used for 5′ RACE.

FIG. 2.

Identities and phylogenetic relationships between mature proteins of CBSV, other ipomoviruses, and the type members of other genera in Potyviridae. (A) Percentage amino acid sequence identities between the mature proteins of CBSV and other viruses. In CVYV and SqVYV, P1a and P1b were compared to CBSV P1 (superscripts a and b, respectively). −, bymoviruses do not contain P1. (B) Phylogenetic analysis by the neighbor-joining method of three mature proteins of CBSV and other representative members of Potyviridae. AgMV, Agropyron mosaic virus, genus Rymovirus (sequence accession no. AY623626); ONMV, Oat necrotic mottle virus, genus Tritimovirus (AY377938); WMV, Watermelon mosaic virus, genus Potyvirus (EU660588); TuMV, Turnip mosaic virus, genus Potyvirus (D83184); BaYMV, Barley yellow mosaic virus, genus Bymovirus (NC_002990); OMV, Oat mosaic virus, genus Bymovirus (AJ306718); PVY, Potato virus Y; RGMV, Ryegrass mosaic virus; WSMV, Wheat streak mosaic virus; and EuRSV in the CP tree, genus Potyvirus (AY697300). Other acronyms are explained in the legend to Fig. 1. Only bootstrap values higher than 70% (of 1,000 replicates) are shown. Scale indicates Kimura units (38).

FIG. 3.

Comparison of the amino acid sequences of HAM1h proteins in eight CBSV isolates and one isolate of EuRSV with the HAM1 proteins of prokaryotic and eukaryotic organisms, including genetic model species. (A) Alignment of the amino acid sequences. The highly conserved motifs are shown against a black background, whereas the somewhat less-conserved motifs are highlighted in gray. The predicted proteolytic cleavage sites flanking HAM1h in CBSV and EuRSV are boxed, and the last C-terminal and first N-terminal amino acids of NIb and CP, respectively, are shown. Prokaryotes, Escherichia coli (sequence accession 1K7K_A), Burkholderia multivorans (YP_001945326), plant endophytes Pseudomonas fluorescens (YP_262903) and an Azoarcus sp. (CAL96580), and the root nodule-inducing and -inhabiting Rhizobium leguminosarum (CAK05869). Fungi, unicellular fungus (yeast) S. cerevisiae (CAA89597) and filamentous fungi Neurospora crassa (XP_955963) and Aspergillus fumigatus (XP_754075). Plants, rice (Oryza sativa; NP_001064763) and Arabidopsis thaliana (NP_567410). Animals, nematode Caenorhabditis elegans (AAG00041), cilial protozoan Tetrahymena thermophila (XP_977249), aphid Acyrthosiphon pisum (XP_001952278), fly Drosophila ananassae (EDV32196), mosquito Culex pipiens (XP_001843604), flour beetle Tribolium castaneum (XP_974197), frog Xenopus laevis (AAI10772), zebrafish Danio rerio (NP_001093456), and mouse Mus musculus (CAM16449), and human (Homo sapiens; AAK21848). The isolates of CBSV (origin and accession number in parentheses) are NTG10 (Ntungamo, FJ026002), IGA8 (Iganga, FJ026000), LWR2 (Luwero, FJ025999), HAM9 (Hoima, FJ039521), BAS4 (Busia, FJ026001), and BAS2 (Busia, FJ039522) from Uganda and MLB9 (Muleba, FJ025998) and MLB3 (Muleba, FJ039520) from Tanzania. The sequence of EuRSV belongs to an isolate from the United States (AY697300). (B) A neighbor-joining tree of the deduced amino acid sequences of HAM1h proteins and the aforementioned related proteins. The N and C termini upstream and downstream from the first and last semiconserved (gray shading) amino acid residue, respectively (positions 25 and 212 in HAM1 of E. coli) (see panel A) were excluded from analysis. Scale indicates Kimura units (38). (C) Percent nucleotide (values above and to the right of the asterisks in the table) and amino acid (values below and to the left of asterisks in the table) identities of HAM1h-encoding sequences among isolates of CBSV.

FIG. 4.

Results of an agroinfiltration assay to test suppression of RNA silencing by the P1, P3, and HAM1h proteins of CBSV. (A) “Silencing on the spot” to induce “strong silencing” of the gfp gene for GFP (18) was achieved by coexpressing gfp from one A. tumefaciens strain and double-stranded (hairpin) RNA homologous to gfp from another strain in coinfiltrated leaf tissue of N. benthamiana, and (i) coinfiltration of a third strain expressing CBSV P1, P3, or HAM1h to suppress gfp silencing. Agrobacterium strains expressing HC-Pro of PVA (genus Potyvirus) or GUS were included as the positive or negative control, respectively. “Buffer” indicates that buffer instead of the third strain was used. In panel ii, the third infiltrated strain at the right side of the leaf expressed constructs in which translation of the CBSV protein was prevented by the introduced mutations (StopP1, StopP3, and StopHAM1h). Leaves were illuminated with UV light and photographed from the underside with a digital camera 5 days postinfiltration. (B) Cosuppression of gfp in transgenic N. benthamiana plants (line 16c) constitutively expressing gfp (note the green fluorescence in veins) (7). The spots were agroinfiltrated with a mixture of two Agrobacterium strains, one expressing gfp to achieve cosuppression (silencing) of gfp and another expressing P1, P3, or HAM1h or constructs in which translation of the CBSV protein was prevented (StopP1, StopP3, and StopHAM1h). Leaves were illuminated with UV light, photographed from the underside with a digital camera, and analyzed for gfp mRNA and siRNA accumulation 5 days postinfiltration. (C) Detection of gfp mRNA and siRNA in the leaf tissues illustrated in panel B by Northern blot analysis using a radiolabeled probe. M, leaf tissue of the 16c line mock infiltrated with buffer. Upper panel, accumulation of gfp mRNA in the respective infiltrated regions. Lower panel, accumulation of gfp-derived siRNA. Ethidium bromide-stained gels of 5S rRNA were used as loading controls.