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. CI was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of CI. 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 CI 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 CI-mediated functions.
INTRODUCTIONPotato virus A (PVA) belongs to the genus Potyvirus in the family Potyviridae. A distinct feature of this virus family is the ability to induce formation of laminate or pinwheelshaped cytoplasmic inclusion bodies, which consist of accumulated cylindrical inclusion protein (CI; reviewed by Edwardson, 1992;Hammond, 1992). The first indication of possible involvement of CI in virus movement came when CIs of several potyviruses were shown to form cone-shaped structures anchored to the cell wall or plasma membrane in close proximity to plasmodesmata (Lawson & Hearon, 1971;Langenberg, 1986; Rodríguez-Cerezo et al., 1997;Roberts et al., 1998). Genetic evidence that potyviral CI plays an essential role in virus movement was obtained when it was shown that several replication-competent tobacco etch virus CI mutants possessed cell-to-cell or long-distance movement defects in tobacco plants (Carrington et al., 1998). Observations from infected cells led to the suggestion that CI may be functional only for a short period of time, in the first five to seven cells of the infection front (Roberts et al., 1998). The ability of CI to form pinwheel structures and to assist in virus movement depends on its ability to self-interact, and the selfinteraction domain has been located in the N-terminal part of the protein (Lopez et al., 2001). Mutations abolishing CI self-interaction inhibit potyvirus cell-to-cell movement (Gó mez de Cedró n et al., 2006) and it has been propo...