Biochemical and mutational analysis of a plant virus polyprotein cleavage site.

Abstract

The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346,000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus‐encoded proteinase at five different sites between the dipeptides Gln‐Ser or Gln‐Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence…Glu‐Xaa‐Xaa‐Tyr‐Xaa‐Gln‐Ser or Gly…. We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site‐directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell‐free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.

The Complete Nucleotide Sequence of Plum Pox Virus RNA

“…It has been proposed that cowpea mosaic virus (CPMV) polyproteins are cleaved at selected Gln-Gly, GinMet and Gln-Ser sites (van Wezenbeek et al, 1983). Only Gln-Ser and Gln-Gly sites were used to cleave the polyprotein of TEV by the viral protease (Dougherty et al, 1988). In addition to these sites a Gln-Ala site is used for protease cleavage in the TVMV polyprotein (Domier et al, 1986).…”

“…Dougherty et al (1988) have used in vitro mutagenesis of selected protease cleavage sites in the polyprotein of TEV to demonstrate strong inhibition, to the point of elimination of cleavage, on substitution of Gin residues by other amino acids. Therefore, the failure of aphid transmission of PPV-NAT may be due to the position of the cleavage site between these two proteins.…”

Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide.

“…In an effort to accumulate multiple proteins in transgenic plants we developed an expression cassette based on the nuclear inclusion (NIa) proteinase from tobacco etch potyvirus (TEV) (Marcos & Beachy, 1994). The NIa protein is one of three proteinases in TEV that are responsible for processing the viral polyprotein (Carrington & Dougherty, 1987 ;Riechmann et al, 1992), by cleaving at conserved heptapeptide sequences located in the viral polyprotein Dougherty et al, 1988). NIa is also capable of proper cleavage of a portion of the TEV polyprotein in transgenic plants (Restrepo-Hartwig & Carrington, 1992).…”

Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1

“…Mutational analysis (46) showed that the cleavage site can be modified in its second position without extensive loss of efficiency and that pairs such as Q/I, Q/R or Q/N are recognized about as well as the wild type Q/S site in TEV. It is thus possible that the Q/I at position 1428 is the cleavage site between the protease and the polymerase, since its position corresponds almost exactly to that observed in CPMV.…”