Poly(A) introduced upstream of the upstream pseudoknot domain of Tobacco mosaic virus led to sequence deletion after serial passaging in host plants

Abstract

TMV (24A + UPD) is a mutant virus that a 24 nt internal poly(A) tract was introduced downstream of the coat protein (CP) gene in TMV genome. TMV (24A + UPD) induced more severe necrosis in Nicotiana benthamiana and its RNA level accumulated at higher level than that of TMV, indicating that the introduced 24 nt poly(A) tract enhanced virus replication and virulence. In this study, TMV (24A + UPD) was serially passaged 10 times in N. benthamiana to analyze the predominant sequence changes of the introduced internal poly(A) tract and the upstream pseudoknot domain (UPD) in its genome. Results showed that the introduced 24 nt of the poly(A) tract was extended from first to seventh rounds of serial passaging, but shortened from the eighth passage, and reduced to only three adenines at the tenth passage. Also, the nucleotide sequences downstream of the introduced poly(A) tract were deleted gradually during the 10 cycles of passaging. There were mutant viruses generated with partial deletion of CP gene during serial passaging, indicating that extension of the introduced internal poly(A) tract also led to deletion of coding gene sequence of TMV (24A + UPD). These results provided valuable information for our understanding of the dynamics in viral sequence changes to reach a tipping point in its host plants in order to maintain a co-existence relationship during virus evolution. In addition, the introduction of an internal poly(A) tract may be applied to other viruses to study virus evolution and natural selection in host plants.