Complete nucleotide sequence and genome organization of hibiscus chlorotic ringspot virus, a new member of the genus Carmovirus: evidence for the presence and expression of two novel open reading frames

Abstract

The complete nucleotide sequence of hibiscus chlorotic ringspot virus (HCRSV) was determined. The genomic RNA (gRNA) is 3,911 nucleotides long and has the potential to encode seven viral proteins in the order of 28 (p28), 23 (p23), 81 (p81), 8 (p8), 9 (p9), 38 (p38), and 25 (p25) kDa. Excluding two unique open reading frames (ORFs) encoding p23 and p25, the ORFs encode proteins with high amino acid similarity to those of carmoviruses. In addition to gRNA, two 3'-coterminated subgenomic RNA (sgRNA) species were identified. Full-length cDNA clones derived from gRNA and sgRNA were constructed under the control of a T7 promoter. Both capped and uncapped transcripts derived from the full-length genomic cDNA clone were infectious. In vitro translation and mutagenesis assays confirmed that all the predicted ORFs except the ORF encoding p8 are translatable, and the two novel ORFs (those encoding p23 and p25) may be functionally indispensable for the viral infection cycle. Based on virion morphology and genome organization, we propose that HCRSV be classified as a new member of the genus Carmovirus in family Tombusviridae.

FIG. 1

Comparison of genome organization between HCRSV (this study), CarMV (9), and CPMoV (42). Open rectangles, predicted ORFs in each viral genome; shaded rectangles, novel ORFs. The potential readthrough and in-frame UAG amber termination codons for the first ORF of each virus and the locations of various HCRSV ORFs, the full-length gRNA, and sgRNA1 and 2 are shown. Numbers represent nucleotide positions.

FIG. 2

(A) Northern blot analysis of single-stranded HCRSV RNAs isolated from virion particles obtained from infected kenaf leaves. The lanes represent hybridization of probes to the 5′, central, and 3′ regions of the viral RNA. Also shown is a determination of the 5′-terminal ends of HCRSV gRNA (B), sgRNA1 (C), and sgRNA2 (D) by primer extension assay. The size of the primer extension product was analyzed on 8% polyacrylamide denaturing gel. Lane M, molecular weight marker provided by the primer extension kit. Positions of primer extension products (lane E) are indicated by arrowheads, and accurate sizes were determined through alignment with a pSK(+) sequence ladder encompassing the beta-galactosidase gene.

FIG. 3

Analysis of the in vitro translation products of RNAs cotranscribed from pHCRSV223 (gRNA), pHCRSV129 (sgRNA1), and pHCRSV80 (sgRNA2) in wheat germ extracts with the TnT coupled translation system. [³⁵S]methionine-labeled translation products were separated on SDS–17.5% polyacrylamide gel and visualized by autoradiography. Lane M, molecular mass markers in kilodaltons.

FIG. 4

Analysis of the in vitro translation products of RNAs cotranscribed from mutants pHCRSV223/M28 (A), pHCRSV223/M81 (B), and pHCRSV129/M8 and pHCRSV223/M9 (C) and controls pHCRSV223 and pHCRSV129 in wheat germ extracts with the TnT coupled translation system. [³⁵S]methionine-labeled translation products were separated on SDS–12.5% (A), –8.5% (B), and –17.5% (C) polyacrylamide gels and visualized by autoradiography. The molecular mass markers in kilodaltons are indicated at the right of each panel.

FIG. 5

Analysis of the in vitro translation products of RNAs cotranscribed from pHCRSV223, pHCRSV223/M23, and pHCRSV223/M25 (A) and from pHCRSV/M25 and pHCRSV80 (B) in wheat germ extracts with the TnT coupled translation system. [³⁵S]methionine-labeled translation products were separated on SDS–12.5% polyacrylamide gel and were visualized by autoradiography. The molecular mass markers in kilodaltons are indicated at the right of each panel.

FIG. 6

Analysis of the in vitro translation products from total viral RNA (lane 1) and gel-purified gRNA (lane 2) in wheat germ extracts separated on SDS–12% polyacrylamide gel. The radiolabeled in vitro translation products from total RNA were immunoprecipitated with either preimmune serum (lane 3) or anti-HCRSV (lane 4), separated on SDS–12.5% polyacrylamide gels, and visualized by autoradiography. The coat protein was also detected by electrophoresis of the purified virus particles on SDS–12.5% polyacrylamide gel and visualized by staining with Coomassie brilliant blue (lane 5). Numbers indicate molecular markers in kilodaltons (lane 6).