Stability of Tobacco etch virus infectious clones in plasmid vectors

Abstract

Tobacco etch virus (TEV) has been traditionally used as a model to research many aspects of the molecular biology of plant RNA virus and, more recently, experimental evolution. However, the only plasmid of this virus species with an infectious clone that has been commonly available to research (pTEV7DA) is rather unstable when propagated in the bacterium Escherichia coli. Here, the TEV infectious clone contained in pTEV7DA is used to construct three new plasmids that allowed infecting the host plants from RNA transcripts synthesized in vitro (pMTEV), directly from plasmid DNA (p35TEV) and by agroinoculation (pGTEV). To increase stability of the three constructed plasmids in E. coli, superfluous vector sequences were removed and the virus expression cassettes were inserted between the plasmid replication origins and antibiotic selection markers in reverse orientation to the latter gene. Although the TEV cDNA in these three new plasmids is not interrupted by any exogenous sequence, they are more stable than the parental pTEV7DA during propagation in E. coli, indicating a major contribution of the plasmid context in virus cDNA stability. Using the different inocula produced from the three new plasmids the TEV infectivity was also compared. The results showed that agroinoculation is the most effective inoculation method and is where symptoms unfold earlier.

Introduction

Having virus infectious clones in plasmid vectors is an important landmark in the research of its molecular biology and interaction with hosts and transmission vectors. However, many times the resulting plasmids are unstable and degenerate during propagation in bacteria, limiting their usefulness (Boyer and Haenni, 1994). This problem is not infrequent in the long plus strand RNA viruses, like potyvirus, whose genome of about 9500 nt involves a long cDNA insert in a plasmid vector.

Tobacco etch virus (TEV) (Allison et al., 1986) infects numerous plant species and belongs to genus Potyvirus within the family Potyviridae and picorna-like virus supergroup (Koonin et al., 2008). The potyviral RNA genome is linked to a viral protein VPg at the 5′ end and contains a poly(A) tail at the 3′ end. It encodes an open reading frame that is translated to a polyprotein which is processed, in a regulated manner, by three viral proteases yielding a total of approximately ten mature proteins (Riechmann et al., 1992). Nonetheless, the RNA may encode more proteins like the recently proposed P3-PIPO which could be expressed through ribosomal frameshifting or transcriptional slippage mechanisms (Chung et al., 2008). TEV has traditionally been one of the model viruses used in the research of potyvirus molecular biology and with this virus species some pioneering findings have been accomplished including: the identification and characterization of the viral proteases (Carrington et al., 1989, Carrington et al., 1990, Dougherty et al., 1988), the subcellular localization of the viral proteins (Restrepo et al., 1990), important progresses in viral replication (Daròs et al., 1999, Haldeman-Cahill et al., 1998, Schaad et al., 1997), translation (Khan et al., 2008, Niepel and Gallie, 1999) and movement (Dolja et al., 1992), to go in depth into the virus–host interaction (Agudelo-Romero et al., 2008a, Chisholm et al., 2000, Lellis et al., 2002) and the transmission by aphids (Ruiz-Ferrer et al., 2005) or the description of the first viral suppressor of RNA silencing (Kasschau and Carrington, 1998). More recently, this virus species has also been the object of interest as a model organism in experimental evolution of plant RNA viruses (Agudelo-Romero et al., 2008b, Carrasco et al., 2007, de la Iglesia and Elena, 2007, Torres-Barceló et al., 2010). And, this is despite the only plasmid with a full-length clone of this virus commonly available to researchers (pTEV7DA), allowing production in vitro of infectious transcripts (Carrington et al., 1993, Dolja et al., 1993) is rather unstable when propagated in the bacterium Escherichia coli (Fig. 1A).

Several strategies have been proposed to solve the problem of instability of plasmid vectors containing RNA virus cDNAs during amplification in bacteria. Perhaps, the most accepted is the interruption of potentially toxic sequences in viral cDNA with introns, which are properly processed during viral RNA expression in the host. This strategy was proposed to stabilize a plasmid with a clone of the potyvirus Pea seedborne mosaic virus (Johansen, 1996) and has been successfully used with other potyviruses like Plum pox virus (López-Moya and García, 2000) or other RNA viruses including some from animal hosts (Gonzalez et al., 2002, Yamshchikov et al., 2001). This strategy requires, however, the interruption of the viral cDNA with exogenous sequences and the expression of the viral RNA form a DNA in the nucleus of the host cell which might not be desirable in some instances.

In the present work, the TEV infectious clone contained in plasmid pTEV7DA was taken as a starting point to construct three new plasmids that allowed infecting the host plants from RNA transcripts synthesized in vitro (pMTEV), directly from plasmid DNA (p35TEV) and by agroinoculation (pGTEV). The three new plasmids present substantially increased stability during propagation in E. coli compared to the parental pTEV7DA without the additional cost of interrupting the TEV cDNA by any exogenous sequence. This supports a major contribution of the plasmid context in virus cDNA stability. Finally, the infectivity of the three inocula that can be yielded by the three plasmids was compared showing superior performance of agroinoculation.