Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells.
The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.
van Bokhoven
H
H
Department of Molecular Biology, Agricultural University, Dreyenlaan, Wageningen, The Netherlands.
van Lent
J W
JW
Custers
R
R
Vlak
J M
JM
Wellink
J
J
van Kammen
A
A
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D007887:Fabaceae / Q000382:microbiology*; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics / Q000378:metabolism*; D009036:Moths / Q000166:cytology / Q000648:ultrastructure; D010946:Plants, Medicinal; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011498:Protein Precursors; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase