Mutational Analysis of the Replication Signals in the 3′-Nontranslated Region of Citrus Tristeza Virus

AbstractCitrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb messenger-sense RNA genome consisting of 12 open reading frames with nontranslated regions (NTR) at the 5′ and 3′ termini. The 273 nucleotide (nt) 3′-NTR is highly conserved (∼95%) among the sequenced CTV isolates in contrast to the highly diverse 5′-NTR sequences. The 3′ replication signals were mapped to the 3′ 234 nts within the NTR. This region of CTV does not contain a poly-A tract nor does it appear to fold as a tRNA-mimic. Instead, a computer-predicted thermodynamically stable secondary structure comprised of 10 stem-and-loop (SL) structures, referred to as SL1 to SL10 (5′ to 3′), was common to all CTV isolates. This putative structure was used as a guide to examine the 3′ requirements for replication in vivo. The resulting data suggest that a complex 3′ structure is required for those functions that provide for efficient replication of CTV in vivo such as minus-strand initiation, regulation of strand asymmetry, effective translation of the myriad of viral mRNAs, or stability of RNAs. Deletions into the 3′-NTR, up to 66 nts from the 5′ direction and 11 nts from the 3′ direction, deleting or disrupting putative SL1, SL2 and SL3, or SL10, resulted in continued replication, suggesting that these sequences are not essential for basal-level replication, but are required for efficient replication. Predicted stem loops 3 through 10 were examined by mutations designed to alter the primary structures while preserving the secondary structures. Mutations designed to disrupt the predicted stems of SL3, SL5, SL7, SL9, or SL10 resulted in substantially reduced levels of replication, while compensatory mutations resulted in partial restorations of replication, suggesting that these predicted secondary structures are involved in replication. Also, the putative loop sequences of SL5, SL6, SL7, and SL9 tolerated mutagenesis with continued but reduced levels of replication. In contrast, all mutations introduced into putative SL4, SL8, and the stem of SL6 prevented replication, suggesting that the primary structure of these regions make up the core of the 3′ replication signal. The 3′ triplet, CCA, was shown to be necessary for efficient replication, but deletion of eleven nts to expose an internal CCA resulted in continued replication