Movement-associated proteins of Potato virus A : attachment to virus particles and phosphorylation

The particles of Potato virus A (PVA; genus Potyvirus) are helically constructed filaments that contain multiple copies of a single type of coat-protein (CP) subunit and a single copy of genome-linked protein (VPg), attached to one end of the virion. Examination of negatively-stained virions by electron microscopy revealed flexuous, rod-shaped particles with no obvious terminal structures. It is known that particles of several filamentous plant viruses incorporate additional minor protein components, forming stable complexes that mediate particle disassembly, movement or transmission by insect vectors. The first objective of this work was to study the interaction of PVA movement-associated proteins with virus particles and how these interactions contribute to the morphology and function of the virus particles. Purified particles of PVA were examined by atomic force microscopy (AFM) and immuno-gold electron microscopy. A protrusion was found at one end of some of the potyvirus particles, associated with the 5' end of the viral RNA. The tip contained two virus-encoded proteins, the genome-linked protein (VPg) and the helper-component proteinase (HC-Pro). Both are required for cell-to-cell movement of the virus. Biochemical and electron microscopy studies of purified PVA samples also revealed the presence of another protein required for cell-to-cell movement the cylindrical inclusion protein (CI), which is also an RNA helicase/ATPase. Centrifugation through a 5-40% sucrose gradient separated virus particles with no detectable CI to a fraction that remained in the gradient, from the CI-associated particles that went to the pellet. Both types of particles were infectious. AFM and translation experiments demonstrated that when the viral CI was not present in the sample, PVA virions had a beads-on-a-string phenotype, and RNA within the virus particles was more accessible to translation. The second objective of this work was to study phosphorylation of PVA movement-associated and structural proteins (CP and VPg) in vitro and, if possible, in vivo. PVA virion structural protein CP is necessary for virus cell-to-cell movement. The tobacco protein kinase CK2 was identified as a kinase phosphorylating PVA CP. A major site of CK2 phosphorylation in PVA CP was identified as a single threonine within a CK2 consensus sequence. Amino acid substitutions affecting the CK2 consensus sequence in CP resulted in viruses that were defective in cell-to-cell and long-distance movement. The CK2 regulation of virion assembly and cell-to-cell movement by phosphorylation of CP was possibly due to the inhibition of CP binding to viral RNA. Four putative phosphorylation sites were identified from an in vitro phosphorylated recombinant VPg. All four were mutated and the spread of mutant viruses in two different host plants was studied. Two putative phosphorylation site mutants (Thr45 and Thr49) had phenotypes identical to that of a wild type (WT) virus infection in both Nicotiana benthamiana and N. tabacum plants. The other two mutant viruses (Thr132/Ser133 and Thr168) showed different phenotypes with increased or decreased accumulation rates, respectively, in inoculated and the first two systemically infected leaves of N. benthamiana. The same mutants were occasionally restricted to single cells in N. tabacum plants, suggesting the importance of these amino acids in the PVA infection cycle in N. tabacum.Perunavirus A (PVA) on kasvivirus. Se kuuluu suureen virusten sukuun, joka infektoi laajalti viljelykasveija, kuten vihanneskasveija (peruna, tomaatti, makea peruna, sipuli), joitakin hedelmäpuulajeija (luumu, sitruuna), tupakkaa, ohraa, mausteita (kardemumma, vanilja) sekä koristekasveija (tulppaani, narsissi). Tartunnan saaneet kasvit kasvavat paljon hitaammin ja sato jää jopa 40% niukemmaksi. Viruksen leviämisen ja tartunnan kierteen ymmärtäminen sekä mahdollisten torjuntamenetelmien löytäminen on ensisijainen tavoite kasvivirustieteessä. Tämä työ yrittää vastata kysymykseen, miten PVA viruksen proteiinit osallistuvat viruksen leviämiseen tartunnan saaneessa kasvissa. PVA viruksen partikkeli on notkean sauvan muotoinen, ja se koostuu noin 2000 kuoriproteiinin (CP) molekyylistä. Sauvan sisällä on ribonukleiinihappomolekyyli (RNA-molekyyli), jonka toisessa päässä on kiinnittyneenä VPg-proteiini. Kummatkin, CP ja VPg, yhdessä muutaman muun virusproteiinin kanssa osallistuvat viruksen leviämiseen yhdestä kasvin solusta toiseen ja viruksen siirtymiseen kirvojen avulla kasvista toiseen. Tämä työ pohjautuu kahteen julkaistuun artikkeliin sekä kahteen toimitukseen lähetettyyn artikkeliluonnokseen, jotka kuvaavat liikkumiseen liittyvien proteiinien CI ja HC-Pro kiinnittymistä virusyksikköihin, ja mahdollista viruksen solusta toiseen liikkumisen rajoitusta CP:n ja VPg:n kemiallisella muunnoksella (fosforylaatio)

Cylindrical inclusion protein of potato virus A is associated with a subpopulation of particles isolated from infected plants

Potato virus A (PVA) particles were purified by centrifugation through a 30% sucrose cushion and the pellet (P1) was resuspended and sedimented through a 5 - 40% sucrose gradient. The gradient separation resulted in two different virus particle populations: a virus fraction (F) that formed a band in the gradient and one that formed a pellet (P2) at the bottom of the gradient. All three preparations contained infectious particles that retained their integrity when visualized by electron microscopy (EM). Western blotting of the P1 particles revealed that the viral RNA helicase, cylindrical inclusion protein (CI), co-purified with virus particles. This result was confirmed with co-immunoprecipitation experiments. Cl was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of Cl. ATPase activity was detected in all three preparations with the greatest amount in P2. Results from immunogold-labelling EM experiments suggested that a fraction of the Cl present in the preparations was localized to one end of the virion. Atomic force microscopy (AFM) studies showed that P1 and P2 contained intact particles, some of which had a protruding tip structure at one end, whilst F virions were less stable and mostly appeared as beaded structures under the conditions of AFM. The RNA of the particles in F was translated five to ten times more efficiently than RNA from P2 particles when these preparations were subjected to translation in wheat-germ extracts. The results are discussed in the context of a model for Cl-mediated functions.</p

Cylindrical inclusion protein of potato virus A is associated with a subpopulation of particles isolated from infected plants

Potato virus A (PVA) particles were purified by centrifugation through a 30% sucrose cushion and the pellet (P1) was resuspended and sedimented through a 5 - 40% sucrose gradient. The gradient separation resulted in two different virus particle populations: a virus fraction (F) that formed a band in the gradient and one that formed a pellet (P2) at the bottom of the gradient. All three preparations contained infectious particles that retained their integrity when visualized by electron microscopy (EM). Western blotting of the P1 particles revealed that the viral RNA helicase, cylindrical inclusion protein (CI), co-purified with virus particles. This result was confirmed with co-immunoprecipitation experiments. Cl was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of Cl. ATPase activity was detected in all three preparations with the greatest amount in P2. Results from immunogold-labelling EM experiments suggested that a fraction of the Cl present in the preparations was localized to one end of the virion. Atomic force microscopy (AFM) studies showed that P1 and P2 contained intact particles, some of which had a protruding tip structure at one end, whilst F virions were less stable and mostly appeared as beaded structures under the conditions of AFM. The RNA of the particles in F was translated five to ten times more efficiently than RNA from P2 particles when these preparations were subjected to translation in wheat-germ extracts. The results are discussed in the context of a model for Cl-mediated functions.</p