Molecular Variability of the 5′- and 3′-Terminal Regions of Citrus Tristeza Virus RNA

Abstract

Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative "zinc-finger" domain adjacent to a basic region in p23, the predicted product of ORF 11, suggests that this protein might act as a regulatory factor during virus replication.

The Complex Genetics of Citrus tristeza virus

“…In fact, a T36 CTV replicon consisting in only these genomic regions is able to selfreplicate in protoplasts of N. benthamiana [6]. As indicated previously, the CTV 5´NTR predicted secondary structure folded into two SL separated by a short spacer region [25]. Directed mutations disrupting this predicted secondary structure were shown to abolish replication, whereas compensatory mutations resumed replication, suggesting that the secondary structure of the 5´ NTR is more important than the primary structure for CTV replication [23].…”

Molecular Virology and Pathogenicity of Citrus tristeza virus

“…The 5´ termini of the CTV genome is protected with a cap structure . CTV 5´ NTR is the most variable genomic region with nucleotide identities as low as 42% among some CTV isolates ( 3) (Albiach-Marti et al, 2000b;López et al, 1998). Remarkably, the CTV 5´NTR secondary structure is similar even for divergent genotypes and folded in two stem-loops separated by a short spacer region (Gowda et al, 2003b(Gowda et al, , 2009López et al, 1998).…”

“…CTV 5´ NTR is the most variable genomic region with nucleotide identities as low as 42% among some CTV isolates ( 3) (Albiach-Marti et al, 2000b;López et al, 1998). Remarkably, the CTV 5´NTR secondary structure is similar even for divergent genotypes and folded in two stem-loops separated by a short spacer region (Gowda et al, 2003b(Gowda et al, , 2009López et al, 1998). This secondary structure contains the sequences necessary for both replication and particle assembly (Gowda et al, 2003b;Satyanarayana et al, 2004;Tatineni et al, 2010).…”

“…This secondary structure contains the sequences necessary for both replication and particle assembly (Gowda et al, 2003b;Satyanarayana et al, 2004;Tatineni et al, 2010). Opposite to the sequence divergence of the CTV 5´NTRs, the 3´ NTR sequences are almost identical (Figure 3) (Harper et al, 2010;López et al, 1998). The 3´NTR lacks a poly-A tract and does not appear to fold in a tRNAlike structure but instead consist in a secondary structure of 10 stemloop structures, which contain the sequences necessary for minus-strand initiation for the CTV gRNA and the sgRNAs (Satyanarayana et al, 2002a) 2.2 Viral RNA species generated during Citrus tristeza virus replication CTV replication is an extraordinary process that generates at least 35 different species of viral RNA in CTV-infected cells ) plus a myriad of defective RNAs (DRNAs) (Albiach-Marti et al, 2000a;Ayllon et al, 1999a;Mawassi et al, 1995).…”

“…Many attempts have been made to develop rapid diagnostics for specific CTV syndromes. Their application has led to the establishment of some correlations between various serological and molecular markers with CTV pathotypes López et al, 1998;Permar et al, 1990;Sambade et al, 2003). Although these molecular markers are a valuable tool for genotyping a particular CTV population, recombinants between genotypes affecting the disease determinants could be present, thus invalidating these methodologies for severe/mild strain differentiation.…”