D'Ann Rochon (1955-2022), a life of passion for plant virology.

D'Ann Rochon passed away on November 29th 2022. She is remembered for her outstanding contributions to the field of plant virology, her strong commitment to high quality science and her dedication to the training and mentorship of the next generation of scientists. She was a research scientist for Agriculture and Agri-Food Canada and an Adjunct Professor for the University of British Columbia. Her research program provided new insights on the infection cycle of tombusviruses and related viruses, including ground-breaking research on the structure of virus particles, the mechanisms of virus transmission by fungal zoospores, and the complexity of plant-virus interactions. She also developed diagnostic antibodies for plum pox virus and little cherry virus 2 that have had a significant impact on the management of these viruses.

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.

A highly basic KGKKGK sequence in the RNA-binding domain of the Cucumber necrosis virus coat protein is associated with encapsidation of full-length CNV RNA during infection.

The Cucumber necrosis virus particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. The N-terminal 58 aa residue segment of the CP R domain is believed to bind viral RNA within virions and during assembly. We report results of in vivo experiments that examine the role of the R domain in assembly. Deletion analyses identified 3 conserved 5-10 aa regions as playing critical roles. A highly basic KGKKGK sequence was found to be both necessary and sufficient for encapsidation of the full-length genome and polymorphic virions were produced in mutants lacking the KGKKGK sequence. The amount of full-length RNA present in virions was substantially reduced in R domain mutants where 2 of the 4 lysine residues were substituted with alanine, whereas substitution of 4 lysines by arginine had only a modest effect. The potential role of the R domain in formation of a scaffold for particle assembly is discussed.

Induction of particle polymorphism by cucumber necrosis virus coat protein mutants in vivo.

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.

Interactions between the West Nile virus capsid protein and the host cell-encoded phosphatase inhibitor, I2PP2A.

The West Nile virus (WNV) capsid protein functions in virus assembly to package genomic RNA into nucleocapsid structures. It is becoming clear, that in addition to their structural roles, capsid proteins of RNA viruses have non-structural functions. For example, the WNV capsid protein has been implicated as a pathogenic determinant. Presumably, many, if not all, of the non-structural functions of this protein involve interactions with host cell-encoded proteins. In the present study, we used affinity purification to isolate human proteins that bind to the WNV capsid protein. One of the capsid binding proteins is I(2)(PP2A), a previously characterized inhibitor of the serine/threonine phosphatase PP2A. Mapping studies revealed that capsid binding site overlaps with the region of I(2)(PP2A) that is required for inhibition of PP2A activity. Moreover, expression of the WNV capsid protein resulted in significantly increased PP2A activity and expected downstream events, such as inhibition of AP1-dependent transcription. Infected cells treated with I(2)(PP2A)-specific siRNAs produced less infectious virus than control siRNA-transfected cells, but this difference was minimal. Together, our data indicate that interactions between WNV capsid and I(2)(PP2A) result in increased PP2A activity. Given the central role of this phosphatase in cellular physiology, capsid/I(2)(PP2A) interactions may yet prove to be important for viral pathogenesis.

Structures of T=1 and T=3 particles of cucumber necrosis virus: evidence of internal scaffolding.

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus, of which tomato bushy stunt virus (TBSV) is the type member. The capsid protein for this group of viruses is composed of three major domains: the R domain, which interacts with the RNA genome: the S domain, which forms the tight capsid shell: and the protruding P domain, which extends approximately 40 Angstrom from the surface. Here, we present the cryo-transmission electron microscopy structures of both the T=1 and T=3 capsids to a resolution of approximately 12 Angstrom. The T=3 capsid is essentially identical with that of TBSV, and the T=1 particles are well described by the A subunit pentons from TBSV. Perhaps most notable is the fact that the T=3 particles have an articulated internal structure with two major internal shells, while the internal core of the T=1 particle is essentially disordered. These internal shells of the T=3 capsid agree extremely well in both dimension and character with published neutron-scattering results. This structure, combined with mutagenesis results in the accompanying article, suggests that the R domain forms an internal icosahedral scaffold that may play a role in T=3 capsid assembly. In addition, the N-terminal region has been shown to be involved in chloroplast targeting. Therefore, this region apparently has remarkably diverse functions that may be distributed unevenly among the quasi-equivalent A, B, and C subunits.

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.

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.

Molecular aspects of plant virus transmission by olpidium and plasmodiophorid vectors.

The genome structures of a large number of viruses transmitted by olpidium and plasmodiophorid vectors have been determined. The viruses are highly diverse, belonging to 12 genera in at least 4 families. Plasmodiophorids are now classified as protists rather than true fungi. This finding, along with the recognition of the great variety of viruses transmitted by olpidium and plasmodiophorid vectors, will likely lead to an elaboration of the details of in vitro and in vivo transmission mechanisms. Recent progress in elucidating the interaction between Cucumber necrosis virus (CNV) and its zoospore vector suggests that specific sites on the capsid as well as on the zoospore are involved in transmission. Moreover, some features of CNV/zoospore attachment are similar to poliovirus/host cell interactions, suggesting evolutionary conservation of functional features of plant and animal virus capsids.

Evidence that vector transmission of a plant virus requires conformational change in virus particles.

Transmission of Cucumber necrosis virus (CNV) by zoospores of its fungal vector, Olpidium bornovanus, involves specific adsorption of virus particles onto the zoospore plasmalemma prior to infestation of cucumber roots by virus-bound zoospores. Previous work has shown that specific components of both CNV and zoospores are required for successful CNV/zoospore recognition. Here, we show that limited trypsin digestion of CNV following in vitro CNV/zoospore binding assays, results in the production of specific proteolytic digestion products under conditions where native CNV is resistant. The proteolytic digestion pattern of zoospore-bound CNV was found to be similar to that of swollen CNV particles produced in vitro, suggesting that zoospore-bound CNV is in an altered conformational state, perhaps similar to that of swollen CNV. We show that an engineered CNV mutant (Pro73Gly) in which a conserved proline residue (Pro73) in the beta-annulus of the CP arm is changed to glycine is resistant to proteolysis following in vitro zoospore binding assays. Moreover, Pro73Gly particles are transmitted only poorly by O.bornovanus. Together, the results of these studies suggest that CNV undergoes conformational change upon zoospore binding and that the conformational change is important for CNV transmissibility.

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.