Targeting of cucumber necrosis virus coat protein to the chloroplast stroma attenuates host defense response

ABSTRACT The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T=3 particles, but other particle forms have not been observed. Particle polymorphism in several T=3 icosahedral viruses has been observed in vitro following the removal of an extended N-terminal region of the CP subunit. In the case of CNV, we have recently described the structure of T=1 particles that accumulate in planta during infection by a CNV mutant (R1+2) in which a large portion of the N-terminal RNA binding domain (R-domain) has been deleted. In this report we further describe properties of this mutant and other CP mutants that produce polymorphic particles. The T=1 particles produced by R1+2 mutants were found to encapsidate a 1.9-kb RNA species as well as smaller RNA species that are similar to previously described CNV defective interfering RNAs. Other R-domain mutants were found to encapsidate a range of specifically sized less-than-full-length CNV RNAs. Mutation of a conserved proline residue in the arm domain near its junction with the shell domain also influenced T=1 particle formation. The proportion of polymorphic particles increased when the mutation was incorporated into R-domain deletion mutants. Our results suggest that both the R-domain and the arm play important roles in the formation of T=3 particles. In addition, the encapsidation of specific CNV RNA species by individual mutants indicates that the R-domain plays a role in the nature of CNV RNA encapsidated in particles.

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Identification of Specific Cucumber Necrosis Virus Coat Protein Amino Acids Affecting Fungus Transmission and Zoospore Attachment

Journal of Virology ◽

10.1128/jvi.75.12.5576-5583.2001 ◽

2001 ◽

Vol 75 (12) ◽

pp. 5576-5583 ◽

Cited By ~ 37

Author(s):

Kishore Kakani ◽

Jean-Yves Sgro ◽

D'Ann Rochon

Keyword(s):

Amino Acids ◽

Coat Protein ◽

Binding Assays ◽

Protein Subunit ◽

Necrosis Virus ◽

Threefold Axis ◽

Cucumber Necrosis Virus ◽

Virus Coat ◽

The Individual

ABSTRACT Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector Olpidium bornovanus. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138–146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.

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A 38-Amino-Acid Sequence Encompassing the Arm Domain of the Cucumber Necrosis Virus Coat Protein Functions as a Chloroplast Transit Peptide in Infected Plants

Journal of Virology ◽

10.1128/jvi.00153-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 7952-7964 ◽

Cited By ~ 30

Author(s):

Yu Xiang ◽

Kishore Kakani ◽

Ron Reade ◽

Elizabeth Hui ◽

D'Ann Rochon

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Coat Protein ◽

Transit Peptide ◽

Proteolytic Cleavage ◽

Binding Motif ◽

Necrosis Virus ◽

Chloroplast Transit Peptide ◽

Cucumber Necrosis Virus ◽

Chloroplast Stroma

ABSTRACT Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed.

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Evaluation of the Roles of Specific Regions of the Cucumber Necrosis Virus Coat Protein Arm in Particle Accumulation and Fungus Transmission

Journal of Virology ◽

10.1128/jvi.02485-05 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5968-5975 ◽

Cited By ~ 19

Author(s):

Elizabeth Hui ◽

D'Ann Rochon

Keyword(s):

Coat Protein ◽

Rna Binding ◽

Virus Transmission ◽

Wild Type ◽

Necrosis Virus ◽

Particle Accumulation ◽

Olpidium Bornovanus ◽

Particle Stability ◽

Cucumber Necrosis Virus ◽

Virus Coat

ABSTRACT The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron composed of 180 identical coat protein (CP) subunits. Each CP subunit includes a 34-amino-acid (aa) arm which connects the RNA binding and shell domains. The arm is comprised of an 18-aa “β” region and a 16-aa “ε” region, with the former contributing to a β-annular structure involved in particle stability and the latter contributing to quasiequivalence and virion RNA binding. Previous work has shown that specific regions of the CNV capsid play important roles in transmission by zoospores of the fungal vector Olpidium bornovanus and that particle expansion is essential for this process. To assess the importance of the two arm regions in particle accumulation, stability, and virus transmission, five CP arm deletion mutants were constructed. Our findings indicate that β(−) mutants are capable of producing particles in plants; however, the arm(−) and ε(−) mutants are not. In addition, β(−) particles bind zoospores less efficiently than wild-type CNV and are not fungally transmissible. β(−) particles are also less thermally stable and disassemble under swelling conditions. Our finding that β(−) mutants can accumulate in plants suggests that other features of the virion, such as RNA/CP interactions, may also be important for particle stability.

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