Engineering Infectious cDNA Clones of Two Pepino mosaic virus Strains in Arizona
Pepino mosaic virus (PepMV), a member of Potexvirus genus, is a significant pathogen in greenhouse tomato production worldwide. To better understand the virus and the disease it causes, we initiated the molecular characterization of an Arizona isolate (PepMV-AZ). Cloning and sequencing analysis of selected genomic regions revealed the presence in this isolate of two distinct strains that share a sequence identity of only 85%. One strain was similar to several isolates found in Europe with sequence identities of ~99%, another strain was similar to the US1 isolate with a sequence identity of 99%. Only one of 96 clones sequenced was found to be a recombinant between the two strains, indicating a very low frequency of recombination in PepMV. To clone high-fidelity infectious cDNA, purified viral RNA used as a template to synthesize full-length infectious cDNA. The first strand cDNA synthesis was primed by an oligo(dT)40 primer with a BamHI site at its 5 end. The second strand cDNA synthesis was carried out with the Pfusion Taq DNA polymerase, primed with an oligonucleotide that contain a T7 promoter sequence at the 5 end and 32 nucleotides identical to the 5 terminal sequences of a specific PepMV strain. The full-length infectious cDNA was cloned into the puc18 plasmid. Infectious transcripts were synthesized readily in vitro by T7 RNA polymerase using linearized plasmids as templates.
PepMV is an emerging pathogen that has rapidly become endemic in greenhouse tomatoes1,2. Since the first report of the virus on tomatoes3, PepMV has spread across the globe. Symptoms of PepMV on tomatoes vary from yellow or chlorotic spots, leaf beaching, to fruit discoloration (Fig. 1).
Fig. 1. PepMV symptoms. Yellow blotches on tomatoes in a commercial greenhouse (A), chlorotic spots on experimental tomatoes (B); a bleached patch on a hybrid tomato (C); bleach-ing of tomato leaves under low lights (D); and mosaic & bubbles on N. clevelandii (E).
As a member of Potexvirus, PepMV possesses a genome organization typical of potexviruses. The (+)-sense, polyadenylated RNA genome of 6.4 kb contains 5 open reading frames (ORFs) for a replicase subunit with methyl transferase, helicase, and polymerase domains; a triple gene block consisting of 26k, 14k, and 9k proteins; and a capsid protein1 (Fig. 2A).
Fig. 2. A. Schematic of PepMV genome organization and locations of primers used in cloning and PCR amplification; B. PepMV genomic fragments amplified with the primer pair P47 and Pc690 (lane 2) and the primer pair P4250 and Pc5210 (lane 1); C. Viral RNA extracted from partially purified (lane 1) and purified (lane 2) PepMV preparations; D. The RNaseH digested (lane 1) and undigested (lane 2) full-length cDNA:RNA hybrid made by Superscript III with primer BamdT and ds cDNA subsequently produced by Phusion DNA polymerase with primer EuroT7 (lane 3). E. BamHI-linearized pUC18 plasmid containing a full-length infectious PepMV cDNA (lane 1) and in vitro transcripts synthesized by T7 RNA polymerase from the plasmid. M, Fermentas 1kb plus DNA ladder.
Phylogenetic analysis grouped the 26 PepMV genomic sequences available in Genbank into five distinct, well supported clades (Fig. 3), with the exception of the apparently recombinant US2 genome4. The sequence identities are greater than 99% within each clade except Ch2 where sequence identities ranges from 97 to 99%. Each clade possibly represents a strain with two or more member isolates. This classification breaks the original Ch2 clade1 into a new Ch2 clade and a new Polish clade (PL).
Two PepMV genomic fragments from an Arizona PepMV isolate were cloned by RT-PCR with two pairs of primers conserved among PepMV isolates: A 650 bp fragment near the 5 terminus and an internal 1 kb fragment over the p26 ORF (Fig. 2A, 2B). Sequencing of over 80 random clones revealed the presence of two distinct populations of sequences: those of the EU strain and of the US1 strain (Fig. 4). Only one recombinant sequence was found, indicating a low recombination frequency in PepMV.
Fig. 4. The Arizona isolate contains two PepMV strains. Sequences from 13 randomly selected RT-PCR clones of the 1 kb region containing the p26 ORF of PepMV were aligned and analyzed phylogenetically as described in Fig. 3. Cymbidium mosaic virus (CymMV, EF125180) was included as an outgroup. Two populations of PepMV sequences are evident, one similar to the EU strain and another to the US1 strain. Clone Pep4250-11 is a recombinant consisting of about 850 nucleotides from the US1 strain and at the 5 end and about 100 nucleotides from the EU strain at the 3 end.
In order to engineer PepMV infectious cDNA clones with high fidelity, virions were purified from PepMV-infected Nicotiana clevelandii (Fig. 1E). viral RNA was then isolated (Fig. 2C) and reverse-transcribed into cDNA using Superscript III and BamdT, an oligo(dT) primer with a BamHI site. The cDNA:RNA hybrid migrated similarly as dsDNA of the same size (Fig. 2D). After digestion with RNaseH, the second strand cDNA was primed with oligonucleotides containing the T7 promoter sequence and sequences specific to the two PepMV strains found in the Arizona isolate, and then extended with the high fidelity Phusion DNA polymerase. Because of the large amount of starting viral RNA, the finished dsDNA of the expected size was easily visualized (Fig. 2D). Full-length cDNA was then ligated into the puc18 plasmid for propagation. BamHI-linearized plasmids directed the in vitro synthesis of full-length PepMV transcripts (Fig. 2E).
The Arizona PepMV isolate contained a mixture of two PepMV strains with infrequent recombination. A protocol was developed that allows one step high fidelity cloning and engineering of infectious cDNA from large RNA viruses.
1. I. M. Hanssen and B. Thomma. 2010. Mol. Plant Pathology 11, 179-189.
2. K. S. Ling et al. 2008. Plant Disease 92, 1683-1688.
3. R. A. A. van der Vlugt et al. 2000. Plant Disease 84, 103-103.
4. C. J. Maroon-Lango et al. 2005. Archives of Virology 150, 1187-1201.