Complete Genome Sequence and Analyses of the Subgenomic RNAs of Sweet Potato Chlorotic Stunt Virus Reveal Several New Features for the Genus Crinivirus

Abstract

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3 terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5 ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.

Analysis of gene content in sweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNA silencing suppressor in only a few isolates: implications for viral evolution and synergism

“…2b, filled The complete sequence of RNA1 in SPCSV-Ug has been described in a previous study (Kreuze et al, 2002). The sequences corresponding to the region of RNA1 shown in the lower drawing were determined in isolates Tug2, Unj2, Mis1, m2-47 and Is in this study.…”

“…2b) and RdRp (data not shown). The youngest systemically infected leaves of I. setosa were tested, as a previous study on SPCSV-Ug showed that these tissues contained the highest amounts of the sgRNAs for RNase3 and p22 early in infection (Kreuze et al, 2002). Detection with the probe for RNase3 revealed a shift in the sizes of the genomic RNA1 and sgRNA for RNase3 in isolates Unj2, Mis1 and Is ( Fig.…”

“…An ORF for a putative 30 kDa protein would also be predicted for RNA1 of isolates Ug, Tug2, Unj2, Mis1 and m2-47 (but not Is) due to an AUG translation initiation codon at nt 7481 (according to the sequence of SPCSV-Ug; Kreuze et al, 2002). However, this codon is upstream from the determined 59 end of the sgRNA for the 27 kDa RNase3 (Kreuze et al, 2002) and most likely is not used. Hence, whilst it cannot be verified further due to unavailable sequence data, it seems that the Kenyan isolate (Hoyer et al, 1996a) encodes RNase3 but no p22.…”

“…The Hsp70h gene sequences can be used to place SPCSV isolates into two phylogenetically distinct groups (Fenby et al, 2002;IsHak et al, 2003; Tairo et al, 2005), which also correlate with serological differences (Hoyer et al, 1996b;Gibson et al, 1998;Alicai et al, 1999). According to these molecular criteria, SPCSV isolates Unj2 and Mis1 from distant locations in Tanzania (Tairo et al, 2005) and the Ugandan isolate SPCSV-Ug, for which the complete sequence is available (Kreuze et al, 2002), belong to the so-called East African (EA) strain whose distribution is largely confined to East Africa. Recently, a few SPCSV isolates serologically related to the EA strain were found in Peru (Gutiérrez et al, 2003).…”

“…In a Ugandan isolate of SPCSV (SPCSV-Ug), two proteins involved in RSS are encoded by genes at the 39 end of RNA1 (Kreuze et al, 2005). They are expressed from subgenomic RNAs (sgRNAs) that accumulate at high levels early in infection in the young, systemically infected leaves (Kreuze et al, 2002). One of these proteins (p22) has been shown to act alone in suppressing silencing of the green fluorescent protein reporter gene mRNA in leaves of Nicotiana benthamiana, but the suppression activity was further enhanced by co-expression of the other SPCSV RNA1-encoded protein, RNase3 (Kreuze et al, 2005).…”