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The upstream pseudoknots domain (UPD) of  Tobacco mosaic virus  (TMV) is located at the 3′-untranslated region (UTR). It plays an important role in virus replication and translation. To determine the importance of UPD and 3′-UTR, and the effects of introduced RNA elements in TMV 3′-UTR, a series of TMV mutants with internal poly(A) tract upstream of UPD was constructed for structural analysis by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). TMV(24A+UPD) and TMV(42A+UPD) formed a similar structure as that of TMV 3′-UTR, but TMV(62A+UPD) structures altered by the introduced poly(A) tract. In addition, TMV(24A+UPD) had a higher viral RNAs accumulation than TMV in  N. benthamiana  protoplasts, and induced lethal symptoms in the infected plants. TMV(62A+UPD) showed a drastically reduced accumulation, its coat protein was undetectable in protoplasts, and the inoculated plants remained symptomless. This study analyzed the structures of 3′-UTR of TMV and found that the longer poly(A) tract introduced upstream of UPD reduced viral RNAs accumulation and induced milder symptoms in  N. benthamiana . In conclusion, different lengths of the internal poly(A) tract introduced into the TMV 3′UTR lead to structural variations that affect virus accumulation and symptom expression.

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TMV mutants with poly(A) tracts of different lengths demonstrate structural variations in 3′UTR affecting viral RNAs accumulation and symptom expression