Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission.

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the ß-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the ß-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the ß-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission.IMPORTANCECucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the ß-annulus at the icosahedral 3-fold axes. Therefore, the ß-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.

Atomic structure of Cucumber necrosis virus and the role of the capsid in vector transmission.

Cucumber Necrosis Virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome packaged in a T=3 icosahedral particle. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus. CNV undergoes a conformational change upon binding to the zoospore that is required for transmission, and specific polysaccharides on the zoospore surface have been implicated in binding. To better understand this transmission process, we have determined the atomic structure of CNV. As expected, being a member of the Tombusvirus genus, the core structure of CNV is highly similar to that of Tomato bushy stunt virus (TBSV), with major differences lying on the exposed loops. Also, as was seen with TBSV, CNV appears to have a calcium binding site between the subunits around the quasi-3-fold axes. However, unlike TBSV, there appears to be a novel zinc binding site within the ß annulus formed by the N termini of the three C subunits at the icosahedral 3-fold axes. Two of the mutations causing defective transmission map immediately around this zinc binding site. The other mutations causing defective transmission and particle formation are mapped onto the CNV structure, and it is likely that a number of the mutations affect zoospore transmission by affecting conformational transitions rather than directly affecting receptor binding.

Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

A comparison of Olpidium isolates from a range of host plants using internal transcribed spacer sequence analysis and host range studies.

Olpidium brassicae is a ubiquitous obligate root-infecting fungal pathogen. It is an important vector of a wide range of plant viruses. Olpidium isolates that infected brassica plants did not infect lettuce plants and vice-versa. Host range tests, PCR amplification and sequencing of the internal transcribed spacer (ITS) and 5.8S regions of 25 Olpidium isolates from brassica, carrot, cucumber and lettuce originating from four continents revealed differences between isolates. Based on their ability to infect lettuce and brassicas and the differences between their ITS1, 5.8S and ITS2 regions they could be separated into a number of distinct groups. Comparisons with other published sequences revealed two distinct genetic groups of brassica-infecting isolates, two distinct groups of lettuce-infecting isolates, one of which contained a carrot-infecting isolate and a distinct group comprising a cucumber-infecting isolate and a melon-infecting isolate. The possibility of the isolates belonging to three distinct species is discussed.

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus.

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.

Fungal transmission of plant viruses.

Fungal zoospores of Olpidium species transmit several viruses in the family Tombusviridae as well as in the Ophio- and Varicosavirus genera. This unit describes procedures for virus transmission by Olpidium sp. The method is useful for assessing fungal transmissibility of a given virus as well as for further studies on molecular and biological aspects of virus/vector interaction.

Evidence that binding of cucumber necrosis virus to vector zoospores involves recognition of oligosaccharides.

Despite the importance of vectors in natural dissemination of plant viruses, relatively little is known about the molecular features of viruses and vectors that permit their interaction in nature. Cucumber necrosis virus (CNV) is a small spherical virus whose transmission in nature is facilitated by zoospores of the fungus Olpidium bornovanus. Previous studies have shown that specific regions of the CNV capsid are involved in transmission and that transmission defects in several CNV transmission mutants are due to inefficient attachment of virions to the zoospore surface. In this study, we have undertaken to determine if zoospores contain specific receptors for CNV. We show that in vitro binding of CNV to zoospores is saturable and that vector zoospores bind CNV more efficiently than nonvector zoospores. Further studies show that treatment of zoospores with periodate and trypsin reduces CNV binding, suggesting the involvement of glycoproteins in zoospore attachment. In virus overlay assays, CNV binds to several proteins, whereas CNV transmission mutants either fail to bind or bind at significantly reduced levels. The possible involvement of specific sugars in attachment was investigated by incubating CNV with zoospores in the presence of various sugars. Two mannose derivatives (methyl alpha-D-mannopyranoside and D-mannosamine), as well as three mannose-containing oligosaccharides (mannotriose, alpha3,alpha6-mannopentaose, and yeast mannan) and L-(-)-fucose, all inhibited CNV binding at relatively low concentrations. Taken together, our studies suggest that binding of CNV to zoospores is mediated by specific mannose and/or fucose-containing oligosaccharides. This is the first time sugars have been implicated in transmission of a plant virus.

A cucumber necrosis virus variant deficient in fungal transmissibility contains an altered coat protein shell domain.

Little is currently known regarding the specific interactions that govern transmission of plant viruses by their vectors. A cucumber necrosis virus (CNV) variant (LL5) deficient in fungal transmissibility has been isolated from mechanically passaged CNV and characterized. Although LL5 accumulates to wild-type (WT) levels, is capable of rapid systemic infection, and produces stable, highly infectious particles, it is only inefficiently transmitted by Olpidium bornovanus zoospores. The LL5 coat protein (CP) gene was amplified by RT-PCR and cloned in place of the WT CNV CP gene in an infectious CNV cDNA clone. Particles derived from this construct also failed to be efficiently transmitted. The LL5 CP gene was sequenced and found to contain two amino acid substitutions relative to WT CNV CP. One substitution (Phe to Cys) occurred in the arm region and another (Glu to Lys) in the shell domain. These amino acid changes were separately introduced into the WT CNV genome through in vitro mutagenesis and it was found that the Glu to Lys change in the LL5 CP shell domain is largely responsible for the loss of transmissibility. In vitro binding assays were developed to determine if the defect in transmissibility was due to a defect in binding zoospores. LL5 particles were found to bind less efficiently than WT CNV. Furthermore, the nontransmissible tomato bushy stunt virus did not detectably bind zoospores. These binding studies suggest that the specificity of CNV transmission by O. bornovanus occurs through specific recognition of a putative zoospore receptor.

Involvement of the cucumber necrosis virus coat protein in the specificity of fungus transmission by Olpidium bornovanus.

Cucumber necrosis (CNV) and the cherry strain of tomato bushy stunt (TBSV-Ch) are tombusviruses which differ in transmissibility by the fungus Olpidium bornovanus (Sahtiyanci) Karling (= O. radicale Schwartz and Cook). Zoospores acquire and transmit CNV, but not TBSV-Ch, in the in vitro manner. To assess the role of the coat protein in the specificity of fungus transmission, reciprocal exchanges were made between the coat protein genes of these two viruses in full-length infectious cDNA clones. Virions containing a modified TBSV-Ch genome with the CNV coat protein gene were efficiently transmitted, but those containing a modified CNV genome with the TBSV-Ch coat protein gene were not. This is the first direct demonstration for the role of a viral coat protein in the specificity of transmission by a fungus.