Hibiscus Chlorotic Ringspot Virus Coat Protein Is Essential for Cell-to-Cell and Long-Distance Movement but Not for Viral RNA Replication

<div><p><i>Hibiscus chlorotic ringspot virus</i> (HCRSV) is a member of the genus <i>Carmovirus</i> in the family <i>Tombusviridae</i>. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (<i>Hibiscus cannabinus</i> L.), two HCRSV mutants, designated as p2590 (A to G) in which the first start codon ATG was replaced with GTG and p2776 (C to G) in which proline 63 was replaced with alanine, were constructed. <i>In vitro</i> transcripts of p2590 (A to G) were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for β-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G) was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G) inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the β-annulus domain is required in T = 3 assembly <i>in vitro</i>.</p></div

HCRSV virion assembly in inoculated kenaf cotyledons at 5 dpi.

<p>(A) Observation of virions under transmission electron microscope. Virus particles from inoculated cotyledons at 5 dpi were partially purified and negatively stained with 2% uranyl acetate. Partially purified virions were only obtained in HCRSV wt extract, regardless of using Tris pH 7.3 (top two panels) or sodium acetate pH 5.2 (bottom two panels) in sucrose cushion and resuspension buffer. Arrow heads point to virions. Each bar represents 100 nm. (B) Western blot analysis of HCRSV CP from the same extracts with or without dilution.</p

Oligonucleotides used in this study.

a<p>Upper case, upper case with underlined and lower case refer to HCRSV cDNA nucleotides, mutated HCRSV cDNA nucleotides and added sequences (T7 promoter or restriction enzyme sites), respectively.</p>b<p>Numbers correspond to HCRSV nucleotide positions.</p><p>Oligonucleotides used in this study.</p

Translation of HCRSV CP and its mutants in wheat germ extract.

<p>(A) Schematic representation of HCRSV sgRNA2 mutants. p30 is an ORF encoding a putative 30 kDa protein. Mutant 2590 (A to G)-ACG/ACA represents two individual sgRNA2 mutations at ACG or ACA, respectively. The TCG in mutant 2590 (A to G) before the second in-frame ATG of CP was substituted with Kozak sequence ACG or ACA. ΔN (1–67) represents HCRSV sgRNA2 with an N-terminal deletion of CP amino acids between the first and the second ATG of the CP gene ORF and the dotted line represents the deleted nucleotides. (B & C) <i>In vitro</i> translation of HCRSV sgRNA2 and its mutants. The PCR products of the sgRNA2 of HCRSV and its mutants were used for <i>in vitro</i> transcription, followed by <i>in vitro</i> translation and the products were labeled with biotinylated lysine.</p

HCRSV viral RNA and CP accumulation in inoculated kenaf cotyledons.

<p><i>In vitro</i> transcripts (0.5 µg for each cotyledon) were inoculated onto kenaf cotyledons and the inoculated cotyledons were collected at different time points. (A) Northern blot analysis of viral RNA. Total RNA (5 µg each) extracted from cotyledons at 1, 2 and 3 days post inoculation (dpi), respectively, was used for viral RNA detection. (B) Western blot analysis of viral CP. Total protein extracted from cotyledons at 4 dpi was used for HCRSV CP detection by western blot. (C) Observation for local lesions in inoculated kenaf cotyledons at 4 dpi.</p

Re-assembly of HCRSV particles after trypsin digestion.

<p>(A) Time-course trypsin digestion of HCRSV virions at room temperature (RT). HCRSV swollen virions were digested with trypsin at RT and harvested at different time points (0, 5, 25 and 60 min, respectively), followed by separation of the digested proteins on 15% SDS-PAGE gel. (B) Limited trypsin digestion. To optimize the digestion in order to obtain the only one smaller size protein band, samples were differentially treated. Symbols “+” and “–” above each lane represent treatment with or without corresponding reagents or temperature conditions. Samples were collected immediately after each denaturation and stored in freezer, followed by separation on 12% SDS-PAGE gel, immediately after the last sample was collected. (C) Observation of trypsin digested HCRSV particles. The limited trypsin-digested swollen HCRSV virions were dialyzed by a two-step method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113347#pone.0113347-Garmann1" target="_blank">[33]</a>, followed by concentration and observation under TEM. Each bar represents 40 nm. (D) Detection of HCRSV RNA in reassembled particles by RT-PCR with primers HC-R3, HC-F5 and HC-R5, HC-F8 and HC-R3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113347#pone-0113347-t001" target="_blank">Table 1</a>) for detection of its genomic and sgRNA.</p

HCRSV RNA and CP accumulation in transfected kenaf protoplasts.

<p>(A) Schematic representation of mutant 2590 (A to G) and mutant 2776 (C to G). Only region covering sgRNA2 is shown to indicate the mutation sites. p30 is an ORF encoding a putative 30 kDa protein. (B) Northern blot analysis of viral RNA accumulation. <i>In vitro</i> transcripts (10 µg each) were transfected into 9×10⁵ protoplasts and harvested at different time points. Total RNA (2.5 µg each) extracted from protoplasts collected at 24 and 48 h post transfection (hpt), respectively, was used for viral RNA detection. DIG-labeled 425 bp HCRSV PCR product located in the 3′ region of the genome was used as the probe for hybridization. (C) Western blot analysis of HCRSV CP. Total protein was extracted from protoplasts collected at 72 hpt. CBB denotes Coomassie blue staining.</p

Detection of HCRSV RNA and its CP accumulation in upper leaves at 25 dpi.

<p>(A) Detection of viral RNA in upper leaves by RT-PCR using primer HC-R3, followed by PCR using primers HC-F8 and HC-R3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113347#pone-0113347-t001" target="_blank">Table 1</a>). (B) Detection of HCRSV CP in upper leaves by western blot. (C) Symptoms of upper leaves of inoculated kenaf plants at 25 dpi.</p

Schematic representations of (A) HCRSV genome and (B) partial HCRSV CP sequence showing mutation sites and deletion.

<p>Rectangles in (A) represent open reading frames. Underlined amino acids in (B) indicate the non-expressed portion as the CP start codon ATG was replaced with GTG in mutant p2590 (A to G) and the boxed proline (CCG) was substituted with alanine (GCG) to remove the kink of CP in mutant p2776 (C to G). The circled methionine residues are the translation initiation sites of CP in wt HCRSV (p223) and mutant p2590 (A to G). Symbol slash (/) represents the cleavage site when swollen HCRSV virions were digested with trypsin.</p