Abstract
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.
Highlights
42A and 62A) introduced before the upstream pseudoknotted domain (UPD)
Hibiscus latent Singapore virus (HLSV) is unable to replicate if the internal poly(A) tract is substituted by TMV UPD sequences, indicating that the internal poly(A) tract is not replaceable
Contained an internal poly(A) tract of 24 nt which was introduced upstream of TMV UPD at the 3′ -untranslated region (UTR) (Fig. 1). It replicated in N. benthamiana protoplasts and the accumulation of CP was higher than that of TMV in transfected protoplasts (Fig. 2)
Summary
42A and 62A) introduced before the upstream pseudoknotted domain (UPD). The genome of TMV contains a 5′ cap and a 3′ tRNA-like structure (TLS) with open reading frames of RNA-dependent RNA polymerase (RdRp), movement protein (MP) and coat protein (CP). Dulcamara mottle virus possesses an internal poly(A) tract in the 3′ -UTR which lacks a 3′ -terminal tRNA-like sequence (TLS). This is different from other tymoviruses[21]. HLSV is unable to replicate if the internal poly(A) tract is substituted by TMV UPD sequences, indicating that the internal poly(A) tract is not replaceable This is different from previous studies that the UPD can functionally substitute for a poly(A) tail in plant and animal cells[26]. Structural change in the 3′ -UTR was dependent on the length of the poly(A) tract that was introduced upstream of the UPD in TMV genomes, and the longer the tract, the lower the viral RNAs accumulation and its infectivity
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