Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses

Abstract

In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of "virus factories" in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).

Keywords: RNA virus replication; endoplasmic reticulum modification; intracellular traffic; membrane vesicles; virus replication factory.

FIGURE 1

(A) Schematic representation of the BYV genome and the encoded proteins. 1a polyprotein encompasses the leader papain-like cysteine proteinase (L-PCP), methyltransferase (MTR), unique central region (CR), and NTPase/helicase (HEL). Vertical solid bar, established cleavage site of the L-PCP; broken bars, arbitrary cleavage sites within the 1a polyprotein calculated from the apparent molecular masses of the MTR- and HEL-containing proteins. Enlarged map of hydrophobic domains (black) and hydrophilic hinges (gray) in the 1a CR is shown below. The portions used for cloning and expression as GFP C-terminal fusions (CR-1 and CR-2) are indicated by bars. (B) Electron microscopy of the BYV-induced ultrastructures in leaf parenchyma cells of Tetragonia expansa (21 days p.i.). Double-membrane vesiles (DMVs, small arrows) and vesicle packets (VPs, large arrows) on a tissue section embedded in Epon after fixation with glutaraldehyde and OsO₄. Scale bar, 1 μm.

FIGURE 2

Localization of GFP-fused CR-1 and CR-2 in epidermal cells of N. benthamiana leaves. Proteins were expressed by agroinfiltration and visualized at 48 h post infiltration by confocal laser scanning microscopy. (A) Co-expression of GFP:CR-1 with the red fluorescent marker protein mCherry, which localizes to the cytoplasm and the nucleoplasm in plant cells (Lee et al., 2008). (B) and (C) Co-expression of GFP:CR-2 with mCherry in two individual cells. Arrows indicate the motile CR-2 globules revealed in frame captures. (D) Co-expression of GFP-CR-2 with YFP-Tal (red channel), a fluorescent marker for actin cytoskeleton (Shemyakina et al., 2011). (E) Expression of ER-mRFP, the protein targeted to the ER lumen by N-terminal signal peptide and C-terminal ER-retention signal (Haseloff et al., 1997), in the perinuclear region of a plant cell. (F–H) Co-expression of GFP:CR-2 with ER-mRFP. (F) Perinuclear groups of GFP:CR-2-containing globules. (G) Modified perinuclear ER representing diffuse membrane reservoirs. (H) Overlap of images (F) and (G). All images represent the superpositions of series of confocal optical sections. N, nucleus. Scale bar, 10 μm.