A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA

Abstract

The 5 untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of CC and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5 UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops. Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants. Different symptoms were observed for the various mutants, pointing to a functional role of the CC and C-A mismatches in the hairpins of the 5 UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5 UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants. Remarkably, in all cases CC or C-A mismatches were introduced. The internal loop of the 5-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin. A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5 UTR in viral packaging is proposed.

A novel, multipartite, negative-strand RNA virus is associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.)

“…They seem to function as promoters for transcription and replication (Fodor et al, 1994(Fodor et al, , 1995Prehaud et al, 1997;Flick et al, 2002;Barr et al, 2003; Barr & Wertz, 2004; Kohl et al, 2006). Normally, these cis-acting sequences contain a few nucleotides that do not base pair and affect short loops that may have regulatory functions (Hellendoorn et al, 1997;Ito & Lai, 1997;Koev et al, 2002;Satyanarayana et al, 2002;Qi et al, 2003;Kohl et al, 2006).…”

Restoration of a Stem-Loop Structure Required for Potato Virus X RNA Accumulation Indicates Selection for a Mismatch and a GNRA Tetraloop

“…Although we have not looked for secondsite reversions that may also affect the fitness of progeny RNAs, our data indicate that preferred RNA sequence and structural elements of SL1 are selected for during PVX replication. Similar approaches have been used to identify functionally important structural features of several other viral RNAs, including HIV-1 (Berkhout and Klaver, 1995;Harrich et al, 1995), TCV (Carpenter et al, 1995;Carpenter and Simon, 1998), the bacteriophage MS2 (Olsthoorn et al, 1994), Q␤ (Klovins et al, 1998), turnip yellow mosaic virus (Hellendoorn et al, 1997), and cauliflower mosaic virus (Pooggin et al, 1998).…”

“…It is possible that PVX CP recognition sites have been altered by SL1 mutations, and revertant RNAs that restore CP binding may allow for efficient assembly of the genomic RNA and thus are selected for during encapsidation. Serial passage experiments with turnip yellow mosaic virus transcripts containing mutations in a 5Ј NTR stem-loop structure indicated that C-C or C-A mismatches were important for viral infection and appeared to function in viral packaging (Hellendoorn et al, 1997). For Rous sarcoma virus, Miller et al (1997) have shown that stem and loop elements within the U5-IR hairpin contribute to multiple biological roles, including the efficiency of reverse transcription and RNA packaging.…”

Nemesia ring necrosis virus: a new tymovirus with a genomic RNA having a histidylatable tobamovirus-like 3′ end

“…However, U-C mismatches, which occur in HP 2 and 3 of NeRNV RNA, have previously been found only in HP 2 of the 59-UTR of OYMV RNA (Hellendoorn et al, 1996b) and in the RNAs of the related Anagyris vein clearing virus, Plantago mottle virus and Scrophularia mottle virus (Koenig et al, 2005). For TYMV, it has been shown that the C-C (and also C-A) pairs in the internal loops of the hairpins of the 59-UTR form protonated base pairs at low pH and play an important role in the initiation of RNA encapsidation (Hellendoorn et al, 1997;Bink et al, 2002).…”