Biologically active transcripts of cloned DNA of the coat protein messenger of two plant viruses

Abstract

To initiate infection, a mixture of the three genomic RNAs of alfalfa mosaic virus (AIMV) has to be supplemented with a small amount of coat protein or RNA 4, the subgenomic messenger for coat protein. The possibility to replace RNA 4 in the inoculum by in vitro synthesized transcripts of a cloned DNA copy of the coat protein cistron was investigated using the SP6 transcription system. Transcripts with or without the cap structure m(7)G(5')ppp(5')G were both translated in vitro in viral coat protein, but only capped transcripts yielded an infectious mixture when added to the AIMV genomic RNAs. This indicates that the cap structure is essential to the in vivo translatin of RNA 4. Similar results were obtained with RNAs transcribed in vitro from a DNA copy of the putative coat protein cistron of tobacco streak virus (TSV). re]19850822 rv]19851203 ac]19860114.

Biologically Active Transcripts of Beet Necrotic Yellow Vein Virus RNA-3 and RNA-4

“…Incorporation of a cap structure at the 5' terminus of a synthetic transcript (Contreras et al, 1982) usually results in a marked enhancement of its biological activity (Ahlquist et al, 1984;Loesch-Fries et al, 1985;Dawson et al, 1986;Langereis et al, 1986). Consequently, the transcripts used in this study were routinely synthesized in the presence of 500 ~tM-mTGpppG and a 25 p.i initial concentration of GTP, with additional aliquots of GTP provided during the course of the reaction.…”

“…Development of efficient in vitro run-off transcription systems based on cloned bacteriophage promoters and purified Escherichia coli or bacteriophage RNA polymerase has led to the production of biologically active RNA transcripts from eDNA clones of a number of plant and animal viruses (Ahlquist et al, 1984;Mitzutani & Colonno, 1985;Loesch-Fries et al, 1985;Dasmahapatra et al, 1986;Dawson et al, 1986;van der Werf et al, 1986;Meshi et al, 1986;Langereis et al, 1986;Rice et al, 1987). Such transcripts represent well-defined and stable inocula which are, furthermore, easily manipulated at the eDNA level by recombinant eDNA techniques.…”

The 3′-untranslated region of alfalfa mosaic virus RNA 3 contains at least two independent binding sites for viral coat protein

“…The cloning of plasmids pT72-42 , pTE6 and pTE7 with inserts that can be transcribed with T7 RNA polymerase has been described previously (27,28). pT72-42 contains a full-length copy of RNA 4, pTE6 contains the 5'-terminal 681 nucleotides (nt) of RNA 4 and pTE7 contains the 3'-terminal 208 nt downstream of a pBR322 sequence of 783 nt (Fig.…”

The 3′‐terminal untranslated region of alfalfa mosaic virus RNA 4 facilitates the RNA entry into translation in a cell‐free system

“…The pT7-2-42 plasmid containing a DNA copy of AIMV RNA 4 under the control of T7 promoter was kindly provided by Dr. K. Langereis [7,8].…”