bPotyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.