1.3.6
DEFINING THE FUNCTIONS OF COCKSFOOT MOTTLE SOBEMOVIRUS PROTEIN P1

E TRUVE, T TAMM, P MICHELSON and H PAVES

Institute for Chemistry, Physics & Biophysics, Tallinn, Estonia

Background and objectives
Cocksfoot mottle virus (CfMV) is a member of the plant sobemoviruses. The (+)-strand RNA genome of sobemoviruses is characterized by its small size and low number of independent open reading frames (ORFs). The function(s) of ORF1, present in all sequenced sobemoviruses, remain unknown. Most plant RNA viruses code for proteins, known as movement proteins (MPs), that enlarge the size exclusion limit of plasmodesmata and thus allow the movement of virus particles or non-virion forms of viral ribonucleoprotein complexes from the infected cell to neighbouring ones. In addition, several MPs cooperatively bind single-stranded nucleic acids, including viral RNA. According to sequence comparisons and functional studies, sobemoviral ORFs downstream from ORF1 seem not to encode proteins facilitating cell-to-cell transport of viruses. We have previously noted that CfMV P1, encoded by ORF1, shares some homology with plant RNA-binding proteins [1].

Results and conclusions
We have raised the hypothesis that CfMV P1 could function as the sobemoviral MP. To test the hypothesis, we have cloned CfMV ORF1 alone and in fusion with green fluorescent protein (GFP) under the control of the 35S promoter. We are currently electroporating CfMV ORF1::GFP fusion into protoplasts of dicot and monocot plants, or micro-injecting it into plant leaf trichomes. We are following the expression of the fusion construct and its movement from micro-injected cells to neighbouring cells.

We have also expressed CfMV P1 in bacteria and raised specific antisera against it. As some identified plant virus MPs interact or co-localize with the cytoskeleton (the function presumably needed for the intercellular transport and targeting to plasmodesmata), we are analysing the binding of CfMV P1 to actin and tubulin and its cellular co-localization with elements of the cytoskeleton. As previous in vitro studies have revealed, some plant virus MPs are also able to bind mammalian actin and/or tubulin. Therefore we have expressed CfMV ORF1::GFP under the control of the EF-1alpha promoter also in cos-7 fibroblasts. However, we could not detect any co-localization of CfMV P1 with mammalian microtubules or microfilaments under these conditions. Also, microtubule- and microfilament-disrupting drugs such as colchicine and cytochalasin did not cause redistribution of CfMV ORF1::GFP in cos-7 cells. In parallel, we are analysing the binding of CfMV P1 to different single- and double-stranded nucleic acids. The results of these experiments will be presented.

References
1. Mäkinen et al., 1995. Journal of General Virology 76, 2817-2825.