Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo

Abstract

The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of Mr 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-l-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein.The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.

Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo

“…In general all movement proteins, or those proteins presumed to be so, including those of nepo-and comoviruses described to date, have been shown to be associated with these fractions (Demangeat et al, 1992;Hibrand et al, 1992;Shanks et al, 1989;Wellink et al, 1987). However, according to our data, the G F L V P38 protein is also highly cytosolic since it is found in large quantities in the S-30 soluble protein fraction.…”

“…grapevine chrome mosaic virus (GCMV) 46K protein (Hibrand et al, 1992), tomato black ring virus (TBRV) 46K protein (Demangeat et al, 1992) and TomRSV 45K protein (Wieczorek & Sanfagon, 1993)] and comoviruses [CPMV 48K protein (Wellink et al, 1987) and red clover mottle virus (RCMV) 43K protein (Shanks et al, 1989)]. Many of the aforesaid proteins, some of which have been shown to be viral movement proteins, do not accumulate in vivo late in infection (Demangeat et al, 1992;Hibrand et al, 1992). In addition, CPMV 48K protein displays low stability and is almost completely degraded after 8 h of pulse-chase (Rezelman et al, 1989).…”

Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase.

“…et al, 1992). In contrast, the VPg-Pro-Pol precursor of tomato black ring nepovirus was found to accumulate in vitro and in vivo, indicating inefficient cleavage at both the VPg-Pro and Pro-Pol cleavage sites (Demangeat et al, 1992 ;Hemmer et al, 1995). TomRSV VPg-Pro contains protease activity on a P2 substrate recognized in trans .…”

“…This process has been extensively studied for many picornavirus superfamily members including poliovirus (Jore et al, 1988 ;Ypma-Wong et al, 1988 a ;Baum et al, 1991 ;Ha$ mmerle et al, 1991 ;Kean et al, 1991), potyviruses (Carrington et al, 1989Dougherty & Parks, 1991 ;Parks et al, 1995 ;Verchot et al, 1991), comoviruses (Dessens & Lomonossoff, 1991 ;Peters et al, b, 1995 and, to a lesser extent, nepoviruses (Demangeat et al, 1992 ;Margis et al, 1994 ; Author for correspondence : He! le ' ne Sanfaçon (at the Pacific AgriFood Research Centre).…”

Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes

“…We are currently raising polyclonal antibodies against the Pro and Pol domains in an attempt to detect such intermediates in infected plant cells. It should be noted that the VPg-Pro-Pol intermediate was detected in plants infected by Tomato black ring virus (genus Nepovirus, subgroup II) (13) and that several large polyproteins containing VPg were also detected in ERenriched fractions from GFLV-infected plant extracts (42). Finally, while it is possible that the luminal orientation of the VPg in at least some of the membrane-associated proteins may play an important (as yet unknown) biological role, it may also represent a means to dispose of excess VPg and regulate the replication of the virus.…”