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Hibiscus chlorotic ringspot virus coat protein is essential for cell-to-cell and long-distance movement but not for viral RNA replication.

Niu S, Gil-Salas FM, Tewary SK, Samales AK, Johnson J, Swaminathan K, Wong SM - PLoS ONE (2014)

Bottom Line: 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.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.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 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore; Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Hainan, China.

ABSTRACT
Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus 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. In vitro 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 in vitro.

No MeSH data available.


Related in: MedlinePlus

Schematic representations of (A) HCRSV genome and (B) partial HCRSV CP sequence showing mutation sites and deletion.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.
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pone-0113347-g001: Schematic representations of (A) HCRSV genome and (B) partial HCRSV CP sequence showing mutation sites and deletion.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.

Mentions: To test effects of HCRSV CP on virus replication, movement and assembly, two CP mutants were constructed using HCRSV cDNA full-length clone p223 as template (Fig. 1A) based on the deduced CP amino acids sequence (Fig. 1B), and designated as p2590 (A to G) and p2776 (C to G) (Fig. 2A). In mutant p2590 (A to G), the CP start codon was replaced with GTG so that the translation of CP will presumably start at the second ATG (methionine 68), resulting in a truncated 30 kDa protein without N-terminal 67 amino acids. In mutant p2776 (C to G), the CCG codon encoding proline 63 was changed to GCG (encoding an alanine) in order to study its effects on virion assembly. Transcripts derived from these two mutants and the wt clones were transfected into kenaf protoplasts. Northern blot showed that there was no significant difference in viral RNA accumulation among them at 24 and 48 hpt, respectively (Fig. 2B). To investigate the CP expression from these 2 mutants, total proteins were extracted from transfected protoplasts at 72 hpt and corresponding western blot showed that the truncated CP was not detected in mutant 2590 (A to G), whereas CP expression of mutant 2776 (C to G) was reduced, compared to that of HCRSV wt (Fig. 2C). These results indicate that HCRSV CP is dispensable for viral RNA replication in kenaf protoplasts.


Hibiscus chlorotic ringspot virus coat protein is essential for cell-to-cell and long-distance movement but not for viral RNA replication.

Niu S, Gil-Salas FM, Tewary SK, Samales AK, Johnson J, Swaminathan K, Wong SM - PLoS ONE (2014)

Schematic representations of (A) HCRSV genome and (B) partial HCRSV CP sequence showing mutation sites and deletion.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234673&req=5

pone-0113347-g001: Schematic representations of (A) HCRSV genome and (B) partial HCRSV CP sequence showing mutation sites and deletion.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.
Mentions: To test effects of HCRSV CP on virus replication, movement and assembly, two CP mutants were constructed using HCRSV cDNA full-length clone p223 as template (Fig. 1A) based on the deduced CP amino acids sequence (Fig. 1B), and designated as p2590 (A to G) and p2776 (C to G) (Fig. 2A). In mutant p2590 (A to G), the CP start codon was replaced with GTG so that the translation of CP will presumably start at the second ATG (methionine 68), resulting in a truncated 30 kDa protein without N-terminal 67 amino acids. In mutant p2776 (C to G), the CCG codon encoding proline 63 was changed to GCG (encoding an alanine) in order to study its effects on virion assembly. Transcripts derived from these two mutants and the wt clones were transfected into kenaf protoplasts. Northern blot showed that there was no significant difference in viral RNA accumulation among them at 24 and 48 hpt, respectively (Fig. 2B). To investigate the CP expression from these 2 mutants, total proteins were extracted from transfected protoplasts at 72 hpt and corresponding western blot showed that the truncated CP was not detected in mutant 2590 (A to G), whereas CP expression of mutant 2776 (C to G) was reduced, compared to that of HCRSV wt (Fig. 2C). These results indicate that HCRSV CP is dispensable for viral RNA replication in kenaf protoplasts.

Bottom Line: 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.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.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 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore; Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Hainan, China.

ABSTRACT
Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus 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. In vitro 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 in vitro.

No MeSH data available.


Related in: MedlinePlus