Evidence that Binding of Cucumber Necrosis Virus to Vector Zoospores Involves Recognition of Oligosaccharides

Self Cite

The Cucumber necrosis virus (CNV) particle is a T‫3؍‬ 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 "␤" region and a 16-aa "" region, with the former contributing to a ␤-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 ␤(؊) mutants are capable of producing particles in plants; however, the arm(؊) and (؊) mutants are not. In addition, ␤(؊) particles bind zoospores less efficiently than wild-type CNV and are not fungally transmissible. ␤(؊) particles are also less thermally stable and disassemble under swelling conditions. Our finding that ␤(؊) mutants can accumulate in plants suggests that other features of the virion, such as RNA/CP interactions, may also be important for particle stability.The capsids of many plant and animal viruses are multifunctional (2), having roles in genome protection, cell-to-cell and long-distance movement within plants (4), vector transmission (9, 16, 21, 25), replication (1), and suppression of gene silencing (22,34). Structural studies of several plant viruses have revealed that the overall architecture of the Tϭ3 capsid can be highly conserved between otherwise divergent virus groups, including those of several animal viruses (14). The structures of many plant virus particles have been obtained, and several in vitro studies have been conducted to assess the roles of the different structural domains in particle integrity and assembly, but few in vivo studies have been conducted. Moreover, fewer studies that relate the various structural domains to other possible functions of viral capsids have been reported.Cucumber necrosis virus (CNV), a member of the Tombusviridae family, is a 33-nm spherical virus that encapsidates a monopartite, positive-sense RNA genome (28). In nature, transmission of CNV occurs via zoospores of the fungus Olpidium bornovanus, in which zoospore-bound particles are transmitted to cucumber following zoospore entry into root cells (3,25). Based on structural homology to Tomato bushy stunt virus (12, 15), CNV is a Tϭ3 icosahedron consisting of 180 identical 41-kDa coat protein (CP) subunits. Each subunit consists of three major structural domains: the R domain, which extends interiorly in the capsid; the S domain, which forms the shell of the capsid; and the P domain, which projects outward from the capsid. The P and S domains are joined by a 5-amino-acid (aa) hinge, and the R and S domains are connected by a 34-aa arm (Fig. 1A). The CP subunit exists i...

mentioning

confidence: 99%

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

Hui

Rochon

2006

Self Cite

“…1) exists near the junction of the arm and S-domains of several related small spherical plant RNA viruses (4). It has recently been suggested that this residue may be involved in the ability of the arm to adopt the two arm conformations required for quasiequivalent interactions among the subunits in Tϭ3 particles (19). The amino-terminal portion of three C-subunit arms interact at the particle threefold axis to form a ␤-annulus which contributes to, but is not necessary for, the integrity of the capsid and overall particle stability (14,16,21).…”

mentioning

confidence: 99%

Induction of Particle Polymorphism by Cucumber Necrosis Virus Coat Protein Mutants In Vivo

Kakani

Reade

Katpally

et al. 2008

Self Cite

The Cucumber necrosis virus (CNV) particle is a T‫3؍‬ icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T‫3؍‬ particles, but other particle forms have not been observed. Particle polymorphism in several T‫3؍‬ 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 T‫1؍‬ 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 T‫1؍‬ 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 T‫1؍‬ 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 T‫3؍‬ 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.

“…CNV particles are transmitted in nature through the recognition of specific amino acids in the shell and the protruding domains of the CP by glycoprotein receptors present on the surface of zoospores of the fungus Olpidium bornovanus (37,38). Receptor binding initiates a conformational change that is essential for vector transmission (33,(38)(39)(40).…”

mentioning

confidence: 99%

“…Receptor binding initiates a conformational change that is essential for vector transmission (33,(38)(39)(40). CNV particles have been found to interact with Hsp70 of fungal zoospores as determined by Western blotting and virus overlay assays (unpublished observations).…”

mentioning

confidence: 99%

Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

Alam

Rochon

2017

Self Cite

Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of Cucumber necrosis virus (CNV), a nonenveloped positive-sense single-stranded RNA [(ϩ)ssRNA] virus having a Tϭ3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog Hsc70-2 copurifies with CNV particles. Virus overlay and immunogold labeling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus, indicating an intimate association with particles. In investigating a possible role of Hsc70-2 in particle disassembly, we showed that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions than those incubated with prebleed control antibody on Chenopodium quinoa. In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant Nicotiana benthamiana Hsc70-2 with virus particles in vitro leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy, and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus.IMPORTANCE Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of nonenveloped spherical RNA viruses. Previous work has shown that host HSP70 homologs play multiple roles in the CNV infection cycle. We therefore examined the potential role of these cellular components in the CNV disassembly process. We show that the HSP70 family member Hsc70-2 is physically associated with CNV virions and that HSP70 antibody reduces the ability of CNV to establish infection. Statistically significantly fewer lesions are produced when virions having undetectable HSc70-2 are used as an inoculum. Finally incubation of Hsc70-2 with CNV particles results in conformational changes in particles. Taken together, our data point to an important role of the host factor Hsc70-2 in CNV disassembly.