We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.Citrus tristeza virus (CTV) is distributed worldwide and is the causal agent of one of the most economically important diseases of citrus. CTV, a member of the genus Closterovirus within the family Closteroviridae, is phloem limited and is transmitted by aphids in a semipersistent manner. CTV virions are filamentous flexuous particles about 2,000 nm long, with two coat proteins (CP and CPm) covering 95 and 5% of the particle length, respectively (8). The CTV genome is a single-stranded, positive-sense RNA of 19,226 to 19,296 nucleotides (nt) (18,27,48,51) organized in 12 open reading frames encoding at least 19 proteins. These include two papain-like proteases, replication-associated proteins (RNA polymerase, helicase, and methyltransferase), a homologue of the HSP70 protein, two coat proteins (CP and CPm), RNA-binding protein p23 (23), a p20 protein that accumulates in the amorphous inclusion bodies (14), and other proteins of so far unknown function (p61, p13, and p18) (Fig. 1). CTV-infected plants contain, in addition to the genomic RNA, 3Ј-coterminal subgenomic RNAs (15) and defective RNAs (D RNAs), the latter resulting from extensive internal deletions of the genomic RNA (2,26,28,50).CTV isolates differing in the type and intensity of symptoms induced in different citrus species and cultivars and in their aphid transmissibility have been reported worldwide (38). In the last two decades, efforts have been taken to develop molecular techniques for rapid differentiation of CTV isolates and identification of molecular markers related to CTV-induced symptoms. Variation in serological reactivity, peptide maps of the coat protein, double-stranded RNA (dsRNA) patterns, hybridization with cDNA probes, restriction fragment length polymorphism, and single-strand conformation polymorphism (SSCP) have been described in attempts to differentiate CTV isolates (29).Nucleotide sequence analysis is the most accurate procedure for CTV differentiation and estimation of molecular or genetic variation. To date, the complete g...

The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown. The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2. We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions. Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis. No mutations affected the RNA binding capacity of the CI protein. These mutations were also introduced in the PPV genome making use of a full-length cDNA clone. Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type. These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell.

The plum pox potyvirus (PPV) cylindrical inclusion (CI) protein fused to the maltose binding protein (MBP) has been synthesized in Escherichia coli and purified by affinity chromatography in amylose resin. In the absence of any other viral factors, the fusion product had NTPase, RNA binding and RNA helicase activities. These in vitro activities were not affected by removal of the last 103 amino acids of the CI protein. However, other deletions in the C-terminal part of the protein, although leaving intact all the region conserved in RNA helicases, drastically impaired the ability to unwind dsRNA and to hydrolyze NTPs. A mutant protein lacking the last 225 residues retained the competence to interact with RNA. Further deletions mapped boundaries of the RNA binding domain within residues 350 and 402 of the PPV CI protein. This region includes the arginine-rich motif VI, the most carboxy terminal conserved domain of RNA helicases of the superfamily SF2. These results indicate that NTP hydrolysis is not an essential component for RNA binding of the PPV CI protein.