PMC

Sequence changes in the I-shaped 3′CITE and PTE acquired during serial passage in plants. (A) Predicted secondary structure of the I-shaped 3′CITE in CIRV-M. The two guanine residues shaded in gray, present in the original 3′CITE of CIRV-M, were substituted with adenine residues during serial passage, as depicted by the arrows. (B) Predicted secondary structure of the PTE in CIRV-C. Changes to the base pair highlighted in gray were observed in two separate serial passage experiments. The single-base substitutions that arose during serial passage in isolate 1 and isolate 2 are shown underlined in the resulting predicted base pairs, indicated above the structure. (C) List of constructs engineered to contain full-length passaged genomes or 3′CITEs containing mutations acquired during passage. See the text for details.

Infection of N. benthamiana plants by wt and hybrid CIRV genomes. (A) Upper panel: inoculated leaves 6 days after inoculation with in vitro-generated transcripts of the genomes indicated at the top. Lower panel: Northern blot detecting viral RNA extracted 6 days postinoculation (dpi) from leaves inoculated with the genomic RNAs indicated above the blot. The two lanes for each genome contain samples from separate plants. The positions of the genomic (g) and subgenomic (sg1 and sg2) RNAs are shown to the right of the blot. (B) Symptoms (upper panel) and RNA accumulation (lower panel) in upper noninoculated leaves at either 6 dpi (for CIRV and CIRV-C) or 11 dpi (for mock treatment, CIRV-M, and CIRV-T). Leaves shown are representative of all plants inoculated with the indicated genome over three separate experiments. In each trial, two plants were inoculated with each genome.

CIRV 3′CITE activity in eIF-depleted wheat germ extract supplemented with eIFs. CIRV genomic RNA was generated in vitro and then incubated at a concentration of 25 nM in wge reaction mixtures containing [³⁵S]met for 1 h at 25°C. After separation of proteins by SDS-PAGE, bands corresponding to p36 were quantified by radioanalytical scanning of the gel and normalized to the level of p36 produced in nondepleted wge, which was set at 100. (A) Translation of p36 from CIRV genome RNA in factor-depleted wge supplemented with the indicated amounts of eIF4F or eIFiso4F. Error bars indicate standard errors of the means (SEM) of the results from three separate experiments. (B) SDS-PAGE gel showing p36 accumulation (± SEM) in factor-depleted wge supplemented with 20 nM eIF4F or its subunits as indicated above the gel.

In vitro translation and in vivo replication of CIRV genomes containing heterologous 3′CITEs. (A) p36 production in wheat germ extract (wge) from wt and hybrid viral genomes. CIRV-M is the hybrid CIRV genome in which the wt Y-shaped 3′CITE was replaced with the I-shaped 3′CITE of MNeSV. Similarly, CIRV-C contains the PTE of CLSV, and CIRV-T contains the BTE of TNV-D. p36 levels are shown below the gel (± SEM) relative to CIRV-ΔTE, which was set to 1. (B) Northern blot analysis of viral RNA accumulation in N. benthamiana (upper panel) or barley (lower panel) protoplasts inoculated with the RNAs identified at the top. The positions of the genomic (g) and subgenomic (sg1 and sg2) RNAs are indicated to the right of the blots. Relative accumulation of genomic RNA, normalized to wt CIRV (set to 100), is shown below each blot with SEM of the results from three separate experiments.

In vitro and in vivo activities of hybrid CIRV genomes containing 3′CITE mutations obtained during serial passage. (A) Graphical representation of p36 accumulation in wge translation reactions. The relative levels of p36 produced from the RNAs identified below the graph were normalized to the level seen with CIRV-ΔTE (set to 1). (B) Accumulation of viral genomic RNAs in N. benthamiana and barley protoplasts (analyzed by Northern blotting). (C) Upper panel: inoculated leaves at 6 dpi and upper noninoculated leaves at 6 dpi (for CIRV, CIRV-C, CIRV-Cp, and CIRV-CpTE) or 10 dpi (for mock treatment, CIRV-M, CIRV-Mp, and CIRV-MpTE). Lower panel: Northern blot showing viral RNA accumulation in leaves from two plants inoculated with the RNAs indicated above the photographs. Leaves shown are representative of all plants inoculated with the indicated genome over three separate experiments. In each trial, two plants were inoculated with each genome.

Interaction of the CIRV 3′CITE with eIF4F and eIFiso4F. (A) SDS-PAGE analysis of p36 production in wge. CIRV-ΔTE is a 3′-truncated version of CIRV that does not contain a 3′CITE; CIRV-Ym1 contains a mutated 3′CITE (mutation shown in ). (B) Northern blot showing genomic (g) and subgenomic (sg1 and sg2) viral RNA isolated from cucumber protoplasts 22 h after inoculation with the genomic RNAs indicated above the blot. N. benth, N. benthamiana. (C) Western blots showing the detection of the eIFs indicated to the right. Blots were performed on eluates from streptomycin-conjugated Sepharose columns containing StreptoTagged versions of the RNAs indicated above. Prior to elution, columns loaded with RNA were incubated with wge and then washed. Lane 1 contained 2 pmol of each purified eIF; lanes 2 and 3 contained eluate from the wt Y-shaped 3′CITEs of TBSV and CIRV, respectively; lane 4 contained eluate from the CIRV 3′CITE mutant Ym1; lane 5 contained eluate from RNA corresponding to the CIRV 5′UTR; lane 6 contained eluate from the I-shaped 3′CITE of MNeSV, which was used as a positive control.

Predicted secondary structures of the CIRV wt and heterologous 3′CITEs used in this study. (A) Schematic representation of the CIRV genome. The thick horizontal line represents the RNA genome, with boxes depicting encoded proteins. Viral protein p36 and its readthrough product p95 are translated directly from the genome, as indicated by the dotted lines below. The start sites of two subgenomic RNAs produced during infection (sg1 and sg2) are shown by arrows below the genome. Sg1 is the template for p41 translation, while p22 and p19 are translated from sg2. The relevant RNA secondary structures in the 5′- and 3′UTRs are depicted schematically, with complementary adapter loops shown in white. The nucleotide sequences of the 5′- and 3′-adapter loops are shown adjacent to the UTRs with black characters in gray boxes, with complementary bases in white. (B) Mfold-predicted Y-shaped secondary structure of the wt CIRV 3′CITE. The 3′-adapter loop is shaded in gray, and bases that are complementary to the 5′ adapter are shown in white. The dashed boxes denote the nucleotide substitutions made in mutant Ym1. (C to E) Mfold-predicted secondary structures of the I-shaped-, PTE-, and BTE-class 3′CITEs that were engineered to replace the wt element in the CIRV genome. The virus that naturally contains each 3′CITE is specified alongside the predicted structure. In the PTE (D) and BTE (E), the wt adapter loops (shown in gray boxes) were replaced with the sequence indicated by the arrows to increase base-pairing potential with the CIRV 5′-adapter loop. (F) List of wt and hybrid CIRV genomes used in this study and the wt and heterologous 3′CITEs they contain.

A possible alternative consensus secondary structure for the I-shaped 3′CITE. (A) Comparison of the I-shaped 3′CITEs of different viruses. The Mfold-predicted [1] and alternative [2] secondary structures of the wt MNeSV 3′CITE are depicted in the left-hand box. The small arrowheads indicate residues that were previously found to be highly flexible by solution structure mapping (). The two guanine residues that were substituted with adenines during serial passage (present in CIRV-MpTE and depicted in structure [3]) are shown in white text on a black background. I-shaped 3′CITEs naturally occurring in other viruses are shown in the right-hand box (structures [4] to [7]) and are depicted in the alternative secondary structure conformation. Asterisks denote a conserved adenine residue that is not maintained in MNeSV (compare to structure [2]). The gray rectangle highlights the previously defined core consensus region of the I-shaped 3′CITE, with the consensus sequence specified in structure [8]. Nucleotides represented by a white “X” are not defined in the consensus sequence. (B) Mutations made to the alternative structure four-base central stem in CIRV-M and CIRV-MpTE, as indicated. For each mutant, the targeted base pair is indicated by an arrow, and the mutations made are shown with mutant names above in bold. (C) Relative p36 accumulation in wge translation reaction mixtures containing RNA transcripts of the mutants named below the graph. p36 levels were normalized to CIRV-M, which was set to 100.