Biochemical and genetic evidence for interactions between potato A potyvirus-encoded proteins P1 and P3 and proteins of the putative replication complex

Interactions of the first and third proteins (P1 and P3) of the potato A po tyvirus (PVA) with the other six main proteins of PVA were studied using Es cherichia coil-expressed recombinant proteins in two in vitro interaction a ssays and a genetic assay yeast two-hybrid system (YTHS). In overlay blotti ng and binding assays in liquid, P1 and P3 interacted with each other and w ith proteins of the putative replication complex of potyvirus: RNA-helicase (CI), viral protein genome-linked (VPg), NIa proteinase part (NIaPro), and RNA-dependent-RNA-polymerase (NIb). In addition, P1 self-interaction and i nteraction with helper-component proteinase (HC-Pro) also were detected. Ne ither P1 nor P3 interact with coat protein (CP) or with various control pro teins. In the YTHS, P1 interacted only with CI and P3 with Nib. The differe nt results obtained using the two test systems may reflect changes in inter actions at different stages of potyvirus infection: in the virus genome rep lication and the virion accumulation stages when nonstructural proteins for m inclusions. Our data are consistent with previous functional data, indica ting that P1 and P3 proteins are involved in potyvirus genome amplification and provide the first direct evidence that these proteins interact with th e proteins that have been shown to be part of the replication complex. (C) 1999 Academic Press.