Cauliflower mosaic virus major inclusion body protein interacts with the aphid transmission factor, the virion-associated protein, and gene VII product

Abstract

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral infection. In order to perform its various tasks, P6 interacts with both viral and host factors, as well as forming electron-dense cytoplasmic inclusion bodies. Here we investigate the interactions of P6 with three CaMV proteins: P2 (aphid transmission factor), P3 (virion-associated protein), and P7 (protein of unknown function). Based on yeast two-hybrid and maltose-binding protein pull-down experiments, P6 interacted with all three of these CaMV proteins. P2 helps to stabilize P6 inclusion bodies. Although the P2s from two CaMV isolates (W260 and CM1841) differ in the ability to stabilize inclusion bodies, both interacted similarly with P6. This suggests that inclusion body stability may not be dependent on the efficiency of P2-P6 interaction. However, neither P2 nor P3 interacted with P7 in yeast two-hybrid assays.

Fig. 1

Interaction of the gene III (P3) and gene VII products (P7) with the gene VI product (P6) of Cauliflower mosaic virus. (A) Diagram of constructs tested for interaction with the yeast two-hybrid system. Constructs are not drawn to scale. Black box, LexA DNA-binding domain (from pEG202); hatched box, B42 transcription activation domain (from pJG4-5); white boxes, full-length CaMV proteins; P3 (129 amino acids long), P6 (520 amino acids), and P7 (96 amino acids). Numbers in bold to the left of each pair of constructs correspond to β-galactosidase assay data shown in B and yeast growth in (C). (B) β-Galactosidase activity of yeast transformants expressing constructs shown in (A). The bar graph indicates average β-galactosidase activity (on abscissa) for β independent yeast colonies along with the standard deviation. Numbers at the bottom (ordinate) correspond to the transformants in C. (C) Growth of yeast transformants on media with (left) and without (right) leucine. The streaks are numbered (key in (D)) to correspond to the constructs shown in (A) and the β-galactosidase activities shown in (B). (D) Key for the plates in (C). (E) Maltose binding protein pull-downs of P3 or P7 with P6. Load; amount of protein initially loaded onto column; flow-through, proteins that do not bind to the amylose column and are washed off; Elution, proteins eluting off the amylose resin. P3/P6, P3-maltose binding protein fusion polypeptide expressed in Escherichia coli, was mixed with P6 expressed in E. coli, loaded on an amylose column, eluted with maltose and probed with a P6 antibody. P7/P6, P7-maltose binding protein fusion polypeptide expressed in E. coli, was mixed with P6 expressed in E. coli, loaded on an amylose column, eluted with maltose and probed with a P6 antibody. pMAL/P6, maltose binding protein expressed in E. coli, was mixed with P6 expressed in E. coli, loaded on an amylose column, eluted with maltose and probed with a P6 antibody.

Fig. 2

Interaction of the gene II (P2) and gene VI products (P6) of Cauliflower mosaic virus. (A) Diagram of constructs tested for interaction with the yeast two-hybrid system. Black box, LexA DNA-binding domain; hatched box, B42 transcription activation domain; white (CM1841 proteins) and gray (W260 proteins) boxes, full-length CaMV genes; P2 (159 amino acids long), P6 (520 amino acids), P7 (96 amino acids). Numbers in bold to the left of each pair of constructs correspond to β-galactosidase assay data shown in (B) and yeast growth in (C). (B) β-Galactosidase activity of yeast transformants expressing constructs shown in (A). (C) Growth of yeast transformants on media with (left) and without (right) leucine. (D) Key for the plates in (C). (E) Maltose binding protein pull-downs of P2 with P6. P2/P6, P2-maltose binding protein fusion polypeptide expressed in Escherichia coli, was mixed with P6 expressed in E. coli, loaded on an amylose column, eluted with maltose and probed with a P6 antibody. pMAL/P6, maltose binding protein, was mixed with P6, loaded on an amylose column, eluted with maltose and probed with a P6 antibody.