Complete nucleotide sequence of a South African isolate of grapevine leafroll-associated virus 3 reveals a 5′UTR of 737 nucleotides

Abstract

Grapevine leafroll-associated virus 3 (GLRaV-3) is the type species of the genus Ampelovirus (family Closteroviridae) [1]. It is an economically important virus that is known to only infect Vitis spp. and that has a negative impact on the wine and table grape industries worldwide. To date, there has been only one report that claims the complete nucleotide sequence of GLRaV-3, by Ling et al. in 2004 (isolate NY-1, AF037268) [2]. In that report the 50 untranslated region (UTR) was found to be 158 nt in length. Here we report that the sequence of GLRaV-3, isolate GP18, has a 50UTR of 737 nt. This extended UTR was found in all other South African isolates of GLRaV-3 that were analysed. Grapevine material (Cabernet Sauvignon) was harvested in the Somerset West wine-producing region in South Africa from a monitored vineyard. RNA of isolate GP18 was extracted from a vine that displayed symptoms for the first time (Pietersen, pers comm). GP18 double-stranded RNA (dsRNA) was extracted from phloem tissue of wooded canes using an adapted cellulose (CF11) column method first described by Hu et al. [3]. RT-PCR was performed with primer sets designed to cover a large portion of the genome (nucleotides 1,835–17,905 of AF037268) in 10 overlapping clones (Fig. 1). Amplicons were cloned and sequenced and a consensus sequence was generated using BioEdit [4]. Primers were not included in the consensus sequence assembly of these overlapping sequences. Poly(A) tailing was performed on dsRNA in an attempt to reach the 50and 30terminal ends (Fig. 1) [5]. The 30-terminus of GP18 was found to be similar to that of the NY-1 isolate. However, we were unable to reach the reported 50-end of the NY-1 isolate and consistently found amplicons that started at +50 nt. After further experimentation with PCR conditions, a range of amplicons were generated that extended beyond the NY-1 sequence’s 50-end. A possible explanation for this inconsistency, using poly(A) tailing, might be the high uracil content in the 50-region upstream of the +50 site in the NY-1 strain that leads to stretches of adenines on the negative strand, which could act as priming sites for the oligo(dT) primer and lead to fragments shorter than the true genomic size. To determine the 50 end of the GP18 genome, total RNA was extracted from grapevine phloem tissue and subjected to RNA ligase-mediated rapid amplification of cDNA Ends (RLM-RACE) using the FirstChoice RLM-RACE kit (Ambion, USA) as per the manufacturer’s instructions. The amplicon generated for the 50-end of the GP18 genome was larger than expected (Fig. 1). It was cloned and four clones sequenced. The 50UTR was found to extend beyond the sequence reported for the NY-1 isolate [2]. The adapter ligation reaction was repeated on the same total RNA treated with calf intestine alkaline phosphatase (CIP) and tobacco acid pyrophosphatase (TAP), and a further five clones were sequenced. All nine clones showed the first 365 nucleotides of the NY-1 sequence and an additional 579 nucleotides upstream of the 50-end. The efficacy of RLM-RACE to determine the 50-termini of multiple ssRNA viruses from total RNA in a single reaction was also investigated. RLM-RACE was performed on total RNA extracted from three grapevine plants (K1, K5, K6) that contained a mixture of viruses (GLRaV-2, -3 and grapevine rupestris stem pitting-associated virus H. J. Maree M.-J. Freeborough J. T. Burger (&) Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa e-mail: jtb@sun.ac.za 1 The GenBank accession number for the sequence reported in this paper is EU259806.