Translation of Tobacco Ringspot Virus RNA in Reticulocyte Lysate: Proteolytic Processing of the Primary Translation Products

Abstract

SUMMARYTobacco ringspot virus (TobRV) RNA was translated efficiently in rabbit reticulocyte lysate and directed the synthesis of two principal polypeptides, Mr 207 × 103 (207K) and l16K, corresponding to the translation products of RNA-1 and RNA-2 respectively. In addition, a l12K RNA-2-encoded polypeptide was sometimes detected. The l16K polypeptide was immunoprecipitated with anti-TobRV serum, suggesting that it was a precursor to coat protein. When translations were performed in the presence of dithiothreitol, the 207K polyprotein was apparently cleaved to yield 37K and 180K polypeptides, with additional processing into 65K and 128K polypeptides. Cleavage of the RNA-2-encoded polyprotein also occurred, although to a much lesser extent than that of the 207K polyprotein; polypeptides of 88K and 54K, both immunoprecipitated with antiviral serum, were identified as RNA-2-encoded cleavage products.

Synthesis and proteolytic processing of arabis mosaic nepovirus, cherry leaf roll nepovirus, and strawberry latent ringspot nepovirus proteins in reticulocyte lysate

“…The sizes of the RNA-1 components of ArMV, CLRV, and SLRV are typical of the nepovirus group: each encoded a polyprotein of Mr 250k that corresponds closely to the potential coding capacity of the single large ORFs of TBRV [11] and Hungarian grapevine chrome mosaic nepovirus (GCMV; [23]) RNA-1 components (Mr 254k and 250k, respectively). However, the primary products of translation of nepovirus RNA-1 components have consistently been reported to be smaller than this: for example, translation of TBRV RNA-1 [7] and GFLV RNA-1 [32] in vitro yielded proteins of M r 220k, and translation of TobRV RNA-1 has variously been reported to yield a protein of M r 225k [5] or 207k [18]. It is only recently that a TBRV RNA-l-encoded translation product of Mr 250k has been identified [3].…”

“…They have bipartite single-stranded RNA genomes, which are 3'-polyadenylated and whose 5'-termini are covalently linked to a protein (VPg) [ 16, and references therein]. The genomic components of three nepoviruses (grapevine fanleaf: GFLV; tobacco ringspot: TobRV; tomato black ring: TBRV) have been translated in vitro to large polypeptides [3,5,7,18,32]. This has led to the generalization that nepovirus expression involves proteolytic processing of polyproteins: the primary translation products of were fixed, dried and autoradiographed.…”

Proteases involved in the processing of viral polyproteins

“…Both RNAs contain a VPg (4K) at their 5' end a poly (A) tail at their 3' end. In vitro TobRV RNA 1 is translated into a 207K protein which upon prolonged incubation in the presence of DTT is cleaved into 37K and 180K proteins where upon the 180K protein is further processed into 128K and 65K proteins [34]. RNA2 has been translated into a 116K protein, which was processed upon addition of the translation products of R N A 2 into smaller products among which the viral coat protein [t8, 34].…”

In vitro processing of the RNA-2-encoded polyprotein of two nepoviruses: tomato black ring virus and grapevine chrome mosaic virus

“…In the same translation conditions, the TBRV RNA-2-encoded polyprotein remained stable. Earlier results based on in vitro translation of the RNAs of two nepoviruses, grapevine fan leaf virus (GFLV) (Morris-Krsinich et al, 1983) and tobacco ringspot virus (TobRV) (Forster & MorrisKrsinich, 1985;Jobling & Wood, 1985) have shown that the RNA-2-encoded polyprotein is probably processed by a protease activity present in the in vitro translation products of RNA-1 but the cleavage products could not be located within the polyproteins.…”