Protein composition of 6K2-induced membrane structures formed during Potato virus A infection

Abstract

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein-induced membranous structures from Potato virus A (PVA)-infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N-terminal Twin-Strep-tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tagged Cerulean-6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep-tag-based affinity chromatography was developed. Both (+)- and (-)-strand PVA RNA and viral protein VPg were co-purified specifically with the affinity tagged PVA-SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA-SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2-induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co-purified with PVA-derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.

Keywords: 6K2 protein; Potato virus A; potyvirus; proteome; viral replication complex.

Figure 1

Schematic representation of the constructs. The PVA‐SC6K construct allows the expression of 2×Strep‐Cerulean fluorescent protein (CFP)‐6K2 (SC6K2) fusion protein in the context of Potato virus A (PVA) infection. The PVA‐C6K construct is similar, except that it lacks the 2×Strep‐tag. SC6K2 fusion is expressed from the MC‐SC6K construct (MC, membrane control) in a non‐infected background. The PVA‐6KY construct allows the expression of the 6K2‐yellow fluorescent protein (6KY) fusion in the context of PVA infection. Hatched rectangles flanking the fluorescent protein‐6K2 cassettes denote NIa protease cleavage sites.

Figure 2

PVA‐C6K and PVA‐6KY are both infectious and the N‐terminal 6K2 tag is accessible for affinity purification. (a) Comparison of fluorescence derived from C6K (green) and 6KY (green) during Potato virus A (PVA) infection by confocal microscopy. The C6K protein localized mostly to scattered vesicles, whereas the 6KY signal was detected mostly in association with chloroplasts (chl., red). Magnified sections show 6K2 vesicle association with chloroplasts. (b) Electron microscopic images of the infected tissues. Both PVA‐C6K and PVA‐6KY produced cytoplasmic cylindrical inclusions which are indicated with arrowheads. (c) Affinity chromatography purification of the 6K2‐fusion protein from the infection context, revealing N‐terminally fused Cerulean fluorescent protein (CFP) to be better accessible for the green fluorescent protein (GFP)‐trap matrix compared with the C‐terminally fused yellow fluorescent protein (YFP). (d) Western blot analysis verified the presence of PVA coat protein (CP) in the upper leaves at 10 days post‐infiltration (DPI), indicating that PVA‐SC6K, PVA‐C6K and PVA‐6KY are all able to cause systemic infection.

Figure 3

Purification of 6K2‐associated membranes from Potato virus A (PVA) infection. (a) A schematic representation of the purification protocol. PVA‐SC6K‐ and PVA‐C6K‐infected and SC6K‐expressing leaf tissues were homogenized and the cleared lysates were subjected to sucrose gradient centrifugation. Fraction 5 collected from the gradient was subjected to affinity purification via the 2×Strep‐tag. (b) The sucrose gradient fractions were analysed by Western blot analysis. SC6K concentrated to fraction 5 in the infection context and to fractions 5–7 when SC6K was expressed alone. (c) SC6K protein and its binding partners were subjected to 2×Strep‐tag affinity purification. SC6K protein was significantly enriched in the eluate. The 2× Strep‐tag‐specific purification was controlled with tag‐less C6K protein (F.T., flow through). (d) The outcome of the purification procedure was assessed by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), followed by silver staining, which revealed clear differences in the protein content between the purified PVA‐SC6K sample in the left panel and the controls: PVA‐C6K in the left panel and MC‐SC6K in the right panel.

Figure 4

Potato virus A (PVA) RNA and viral genome‐linked protein (VPg) content in the purified 6K2‐associated membrane samples. (a) The percentages of recovered viral RNA (vRNA) indicate significant enrichment of PVA RNA in the PVA‐SC6K sample when compared with the PVA‐C6K sample. Mean values from three independent biological replicates are given. The error bars indicate the standard deviation. *P < 0.05. (b) The presence of viral (+) and (−)RNA in the column eluates of PVA‐SC6K, PVA‐C6K and MC‐SC6K samples was analysed by reverse transcriptase‐polymerase chain reaction (RT‐PCR). RNA samples from 2×Strep‐tag purifications were incubated prior to PCR with (+) or without (−) reverse transcriptase in the presence of either a (−)‐strand‐ or (+)‐strand‐specific primer. (c) Replication protein VPg was detected by Western blotting with anti‐VPg antibody in PVA‐SC6K samples, but not in the control PVA‐C6K.

Figure 5

Morphological characterization of the purified 6K2‐associated membranes. Affinity‐purified PVA‐SC6K, PVA‐C6K and MC‐SC6K samples were negatively stained with uranyl acetate and examined by electron microscopy (EM). Two types of membrane structure were observed: individual vesicles (left panels; shown by arrowheads) and membrane clusters (middle panels). Strep‐Tactin matrix captured PVA particles non‐specifically from infected samples (right panels). The sizes of the purified individual vesicles vary between 50 and 100 nm, with the median size being 56 nm (n = 40). Scale bar, 500 nm.