Site-specific cleavage and religation of viral RNAs I. Infectivity of barley stripe mosaic virus RNA religated from functionally active segments and restoration of the internal poly(A) tract in progeny

Abstract

The genomic RNAs of barley stripe mosaic virus (BSMV) were used in experiments involving site-specific cleavage to yield noninfective but functionally active fragments and subsequent restoration of infectious viral RNA by means of religation. The specific fragments were obtained after site-specific cleavage of BSMV RNAs with RNase H in the presence of oligo(dT) 10 complementary to the internal poly(A) track in BSMV genomic RNAs: (i) long (L) 5′-terminal fragments, lacking the tyrosine-accepting activity, but capable of directing in vitro a full set of BSMV-specific polypeptides ( A. A. Agranovsky, V. V. Dolja, and J. G. Atabekov, 1982, Virology 119, 51–58) and (ii) short (Sh) fragments containing a tRNA-like structure accepting tyrosine. L- and Sh-fragments of BSMV RNAs were religated by T4 RNA ligase producing infectious BSMV RNA. The religated BSMV RNA was shown to lack the internal poly(A) track present in authentic BSMV RNA. Nevertheless the typical poly(A) spectra were restored in the progeny RNA from religated, poly(A) - deficient BSMV RNA.