Dynamics of coronavirus replication-transcription complexes

Abstract

Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.

FIG. 1.

Recruitment of MHV nsp2 to the RTCs. (A) LR7 cells transfected with pEGFP-nsp2 were mock infected (−MHV) or infected with MHV A59 (+MHV). Cells were fixed at 6 h p.i. and subsequently processed for immunofluorescence analysis using antibodies against nsp8. (B) Schematic representation of the C- and N-terminal truncations of MHV nsp2. The amino acids remaining are indicated. The EGFP tag at the C-terminal end of nsp2 is not indicated. (C and D) LR7 cells transfected with pEGFP-nsp2, pEGFP-nsp2AB, pEGFP-nsp2BC, or pEGFP-nsp2CD were fixed at 30 h posttransfection and processed for microscopic analysis (C); in addition, the mean arbitrary fluorescent intensities of 25 cells were determined using a DeltaVision RT microscope and software from Applied Precision (D). (E) LR7 cells transfected with pEGFP-nsp2AB, pEGFP-nsp2BC, or pEGFP-nsp2CD were mock infected or infected with MHV-A59. At 6 h p.i. the cells were fixed and processed for immunofluorescence analysis using nsp8 antibodies.

FIG. 2.

Characterization of recombinant MHV-nsp2GFP and subcellular localization of nsp2GFP. (A and B) LR7 cells were infected with MHV-nsp2GFP or MHV-WT (MOI of 10). (A) Culture medium was collected at different time points p.i., after which the viral infectivity was determined by a quantal assay on LR7 cells. The TCID₅₀ values are indicated. (B) Intracellular viral RNA (vRNA) levels were determined by a quantitative RT-PCR on the 1b and the N genes. The data are presented as relative vRNA levels. (C) LR7 cells infected with recombinant MHV-nsp2GFP were fixed and processed for immunofluorescence analysis using antibodies directed against nsp8 and dsRNA. (D and E) LR7 cells infected with recombinant MHV-nsp2GFP were fixed at the indicated time points and processed for immunofluorescence analysis using antibodies directed against nsp4. Images taken from the cells at the different time points were obtained at identical settings (D) while the settings were adjusted to demonstrate the colocalization between nsp2GFP and nsp4 at the 8-h time point (E). (F) Mock-, MHV-WT-, or MHV-nsp2GFP-infected cells were radiolabeled from 6 till 9 h p.i. Cells were lysed and processed for immunoprecipitation with antibodies directed against the nsp2 protein and analyzed by 12.5% SDS-PAGE. The filled triangle indicates the nsp2-GFP protein, the open triangle indicates the endogenous mature nsp2 protein, and the asterisk indicates an additional unidentified protein species.

FIG. 3.

nsp2-GFP localizes to DMVs and CMs. HeLa-CEACAM1a cells, infected with recombinant MHV-nsp2GFP, were fixed at 8 h p.i. and processed for ultrastructural analysis by chemical fixation and epon embedding (A). Alternatively, cryosections were prepared that were incubated with antibodies directed against the GFP tag, followed by immunogold labeling (B and C). Convoluted membranes are indicated by the asterisks. nsp2 labeling is indicated by the arrowheads. Scale bar, 200 nm.

FIG. 4.

nsp2 associates to the cytoplasmic face of the DMVs and CMs. (A) LR7 cells infected with MHV-nsp2RL or MHV-ERLM were processed for ultracentrifugation as described in the Materials and Methods section. The luciferase activity in the indicated fractions was determined, corrected for the volume of the fraction, and plotted as the percentage of the total amount of luciferase activity. (B) LR7 cells transfected with pER-Fluc were infected with MHV-nsp2RL. Membrane fractions, prepared as described in the Materials and Methods section, were mock treated with 20 μg/ml proteinase K in the presence or absence of 0.05% TX-100. Renilla and firefly luciferase activities in the differently treated samples were measured and are depicted relative to the mock-treated samples, which are set at 100%. (C) MHV-nsp2GFP-infected LR7 cells were fixed at 6 h p.i. and permeabilized with buffers containing either 0.5 μg/ml digitonin or 0.1% TX-100. Immunofluorescence was performed using antibodies directed against the N terminus (anti-M_N) or the C terminus (anti-M_C) of the MHV M protein or against the GFP tag (anti-GFP).

FIG. 5.

Trafficking of MHV replicative structures. Time-lapse recordings of MHV-nsp2GFP-infected LR7 cells were obtained using DeltaVision Core (API). Trafficking of selected nsp2-positive structures was determined. Tracks are indicated by white lines and numbered. (A) Tracks 1 to 4, 6, and 8 to 10 represent saltatory movements while tracks 5, 7, and 12 represent confined movements of small nsp2-GFP-positive structures. Track 11 represents confined movement followed by saltatory trafficking. Large, immobile nsp2-positive structures are indicated by the arrowheads. (B) The very long track taken by a small RTC demonstrating saltatory movement is shown. See also Videos S1 and S2 in the supplemental material.

FIG. 6.

The role of microtubules in transport. (A) Cells infected with MHV-nsp2mCherry were fixed at 6 h p.i. and processed for immunofluorescence analysis using the α-tubulin antibody to visualize microtubules. (B to D) LR7 cells were infected with MHV-nsp2RL or MHV-nsp2GFP either in the presence (+NOC) or absence (−NOC) of 1 μM nocodazole. Cells were lysed or fixed at the indicated time point, followed by determination of the luciferase expression levels (B); the TCID₅₀ value of the culture medium was determined (C), or cells were processed for microscopical analysis (D). The white lines in panel D indicate the contours of the cell. T, time.

FIG. 7.

Coronavirus RTCs are static entities. (A) FRAP was performed on MHV-nsp2GFP-infected cells at 7 h p.i. using the quantifiable laser module of the DeltaVision Core (API). A representative FRAP experiment is depicted, with the bleached area indicated by the white arrowheads in the magnification in the top right corners. (B to D) Fluorescence recovery graphs were generated of bleached ROI either in MHV-nsp2GFP-infected cells (B) or in cells transfected with nsp2-GFP which were subsequently mock infected with MHV-A59 (C) or infected with MHV-A59 (D). (E) Two LR7 cell cultures were infected with either MHV-nsp2GFP or MHV-nsp2mCherry, followed by incubation in the presence of HR2 peptide. At 6 h p.i., the HR2 peptide was removed; cells were trypsinized, mixed, and subsequently plated in the presence (+) or absence (−) of CHX. At 9 h p.i. the cells were processed for immunofluorescence analysis. Nuclear staining was obtained by TOPRO 3 iodide.