Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector

Abstract

Interactions between Cauliflower mosaic virus (CaMV) and its aphid vector are regulated by the viral protein P2, which binds to the aphid stylets, and protein P3, which bridges P2 and virions. By using baculovirus expression of P2 and P3, electron microscopy, surface plasmon resonance, affinity chromatography, and transmission assays, we demonstrate that P3 must be previously bound to virions in order that attachment to P2 will allow aphid transmission of CaMV. We also show that a P2:P3 complex exists in the absence of virions but is nonfunctional in transmission. Hence, unlike P2, P3 and virions cannot be sequentially acquired by the vector. Immunogold labeling revealed the predominance of spatially separated P2:P3 and P3:virion complexes in infected plant cells. This specific distribution indicates that the transmissible complex, P2:P3:virion, does not form primarily in infected plants but in aphids. A model, describing the regulating role of P3 in the formation of the transmissible CaMV complex in planta and during acquisition by aphids, is presented, and its consequences are discussed.

Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

“…From studies implicating virionbound P3 in aphid transmission and cell-to-cell movement, it was proposed that P3 is an auxiliary protein whose role is to "functionalize" the preformed virus particle (43). This possibility is consistent with experimental data showing that P3 readily associates, with very high affinity, with previously formed and purified virions (7,37). Once associated with virions as trimeric assemblies through the formation of an antiparallel coiled-coil network (Tv in Fig.…”

“…Such P3-decorated virions can thus move to and traverse the plasmodesmata through the lumen of the tubules formed by the movement protein P1 (43). Alternatively, for plant-to-plant transmission, they can be taken up by aphids, where they attach to P2 already associated with the insect receptor (7). A prominent feature of the P3 structure relies on the presence of the CXC ring motif conserved in all members of the genus Caulimovirus.…”

“…P3 is produced initially in the viral factories (30), where replication, encapsidation, and accumulation of around 95% of virions also occur (7,17). From studies implicating virionbound P3 in aphid transmission and cell-to-cell movement, it was proposed that P3 is an auxiliary protein whose role is to "functionalize" the preformed virus particle (43).…”

“…In an earlier study, we showed that, when a TB is ingested by an aphid vector, P2 is efficiently retained within its mouthparts whereas P3 is set free and lost. The preformed P3-virion complexes are acquired later by the aphid, during subsequent intracellular probing, and attach to the aphid-bound P2 (7). A way to understand this switch from P2/P3 coaggregation within the TB and their mutual release in the aphid mouthparts would be to imagine that the cysteine residues in P3 act as a redox-sensitive cluster, reminiscent of the property of a comparable cystine ring described in the COMP protein referred to above (36).…”

“…It is constituted of three concentric shells, built from 420 capsid protein subunits, surrounding a large inner cavity with a diameter of approximately 250 Å (4,37). The physical association between P3 and viral particles has been consistently demonstrated in mature CaMV virions by copurification, immunolabeling experiments, and in vitro interactions (7,(23)(24)(25). Recently, cryoelectron microscopy (cryo-EM) image reconstruction further showed that P3 molecules decorate virions, with their N-terminal ectodomains forming an antiparallel ␣-helical coiled-coil network at the surface and their C-terminal domains interacting with the coat protein and reaching inside the virus particle.…”