J Bacteriol Virol.  2012 Mar;42(1):49-55. 10.4167/jbv.2012.42.1.49.

Characterization of a Replication Element in the Coat Protein ORF of Turnip Yellow Mosaic Virus

• Shin HI
• Cho TJ

• KMID: 1434772
• DOI: http://doi.org/10.4167/jbv.2012.42.1.49

Abstract

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus that has a single 6.3 kb positive-strand RNA as a genome. Previously, it was observed that the recombinant construct TY-eGFP2, where an eGFP gene was inserted at the position downstream of the coat protein (CP) ORF of TYMV genome, barely replicated. The inhibition of replication was relieved by insertion of an additional copy of the 3' quarter of the CP ORF after the foreign sequence. In this study, we have examined if the 3' quarter of the CP ORF contains any replication elements. M-fold analysis predicted three stem-loop structures in this region. Analysis of the TY-eGFP2 constructs containing one or two of these stem-loop structures indicates that the secondary structure predicted in the region between nt-6139 and nt-6181, termed SL2, is essential for TYMV replication. The critical role of SL2 was confirmed by the observation that deletion of the 3' quarter of the CP ORF from the wild-type TYMV genome nearly abolished replication and that insertion of SL2 into the deletion mutant restored the replication. Mutations disrupting the stem of SL2 greatly reduced viral RNA replication, indicating that the secondary structure is essential for the enhancing activity.

Keyword

TYMV; Replication element; Plant RNA virus

MeSH Terms

Animals
Brassica napus
Coat Protein Complex I
Ecthyma, Contagious
Genome
RNA
RNA, Viral
Tymovirus
Viruses
Coat Protein Complex I
RNA
RNA, Viral

Figure

• 

  Figure 1 TYMV constructs. (A) Wild-type and recombinant TYMV constructs. TYW represents a wild-type genome. TY-eGFP2 construct has the enhanced green fluorescent protein gene between the CP ORF and the 3'-terminal TLS. TY-eGFP2+CP6203, where the TYMV sequence downstream of the foreign gene begins with the nt-6203 of the wild type, was observed to show little viral replication (13). (B) Secondary structures predicted at the 3'-end proximal region of the CP ORF. The three stem-loop structures were designated SL1, SL2, and SL3. The two constructs shown below were designed to have all or part of the stem-loop structures. TY-eGFP2+CP6139 and TY-eGFP2+CP6186 have additional CP ORF sequence starting with the nt-6139 and nt-6186, respectively. All these constructs have a 3'-terminal ribozyme sequence derived from hepatitis delta virus (HDV).

• 

  Figure 2 Replication of the TY-eGFP2 constructs having extra CP sequences. (A) Northern and Western analysis of the replication. Seven days after agroinfiltration of N. benthamiana leaf with various TYMV constructs, total RNA was extracted from the leaf. One µg or 0.1 µg (10-1) of total RNA was size-fractionated in the 1% agarose gel and examined by Northern blot analysis, using the DIG-labeled probe representing the CP ORF. The blots were developed by chemiluminescent immunodetection of DIG. The panel below the Northern blot represents a gel stained with ethidium bromide. In Western analysis of coat protein expression, 1 µl of 1:10 diluted (10-1) or undiluted leaf extract was loaded and electrophoresed in 12.5% SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane. Coat protein was detected using anti-TYMV coat protein rabbit antibody and anti-rabbit HRP conjugate. The membrane was developed by a Luminata™ Forte (Millipore) using luminol as the substrate. (B) Northern analysis using DIG-labeled eGFP sequence as a probe. Note that the smaller sgRNA is evident.

• 

  Figure 3 Examination of the role of SL2 in wild-type TYMV. (A) TYMV constructs for dissecting the role of SL2. In TYΔCP2, the nucleotides between #6068 and #6202 were deleted and several cloning sites were added. In TYΔCP2+SL2, the sequence between nt-6137 and nt-6183 was added back to the SmaI and EcoRI sites of the TYΔCP2. (B) Northern and Western analyses of the replication of TYΔCP2 and TY-CP2+SL2 constructs. The analyses were carried out as described in Fig. 2.

• 

  Figure 4 Efficiency of SL2 variants. (A) Constructs containing modified SL2 sequences. The nucleotides in the three TYΔCP2+mSL2 constructs that differ from the wild-type SL2 are highlighted by arrows and grey boxes. Here, only the sequences representing the stem-loop structures are shown. (B) Northern and Western analyses of the replication of various SL2 constructs. The analyses were carried out as described.

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