Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Abstract

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3'-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected.

Fig. 1.. FHV constructs. (A) pFHV-wHDV construct. In this construct, the cDNA of FHV RNA1 is contained in a plasmid vector. The 3´-terminal portion, the sequence between SacI and SpeI sites, was replaced with a synthetic DNA that contains several restriction recognition sites (grey box,) and a ribozyme sequence derived from hepatitis delta virus (HDV). In the HDV ribozyme sequence, the 75^th nucleotide C was replaced with A, to make it have weaker ribozyme activity . The sgRNA transcription start site is indicated by +1. Downstream of the B2 stop codon, an AflII site was introduced by replacing one nucleotide (capital letter). (B) TY-FHV construct. The DNA between SnaBI and SpeI sites of the pFHV-wHDV was used to replace the eGFP gene of TY-eGFP . The FHV sequence is placed downstream of the tymobox (black box), a subgenomic RNA (sgRNA) promoter. The two sgRNAs produced from this construct are depicted by arrows.

Fig. 2.. Replication and RNA packaging of TY-FHV constructs. Seven days after agroinfiltration of N. benthamiana leaf with various TY-FHV constructs, total RNA was extracted from the leaf. The leaf extract was treated with RNase A, and then the viral RNA protected from the RNase action was prepared by phenol extraction. 1 μg of total RNA and equivalent amounts of encapsidated RNA were size-fractionated in the 1% agarose gel and examined by Northern blot analysis, using the DIG-labeled probe representing the (A) TYMV CP ORF, or (B) FHV sgRNA. The blots were developed by chemiluminescent immunodetection of DIG. The panel below the Northern blot represents a gel stained with ethidium bromide. In panel A, gRNA (8.1 kb), sgRNA1 (2.5 kb), and sgRNA2 (0.7 kb) of TY-GUS are marked on the left. The recombinant sgRNAs from TY-FHV constructs are indicated by black arrow-heads. In panel B, putative FHV RNA1 (3.1 kb) and sgRNA (0.4 kb or 1.1 kb) are indicated by white arrow-heads. The RNA bands marked by black arrow heads are the same as in panel A.

Fig. 3.. Northern analysis, using a mixture of TYMV CP and FHV probes. (A) Northern blot hybridization was carried out, as described in Fig. 2, except that the probe used was a mixture of TYMV CP and FHV sgRNA sequences. The RNAs produced from TY-GUS, i.e. gRNA (8.1 kb), sgRNA1 (2.5 kb), and sgRNA2 (0.7 kb), are marked on the left. Eight RNA species from TY-FHV are marked 1 to 8. Four RNA species from TY-FHV(eGFP) are marked a to d. The rightmost sample TY-GG(2.4), which contains a 2.4 kb insert sequence (two copies of partial GUS sequences), was included as a blotting control. (B) Schematic representation of the RNA species from TY-FHV. The sgRNA promoter tymobox is represented as a black box. Five RNA species, which are identified by the size and the results of hybridization to two different probes, are depicted below the construct. (C) Schematic representation of the RNA species from TY-FHV(eGFP). Four RNA species are schematically drawn below the construct. The FHV sequences are indicated in thick lines.

Fig. 4.. Western analysis of eGFP expression in TY-FHV(eGFP). 10 μl of leaf extract was loaded, and electrophoresed in 12.5% SDS-polyacrylamide gel. TY-eGFP was included as a control. The proteins were transferred to a nitrocellulose membrane. eGFP was detected using anti-GFP rabbit antibody and anti-rabbit HRP conjugate. The membrane was developed by a Luminata^TM Forte (Millipore), using luminol as the substrate.