Coronavirus envelope (E) protein remains at the site of assembly

Abstract

Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly.

Keywords: CLEM; Coronavirus; Envelope protein; FRAP; Live-cell imaging; Protein transport/localization; Virus assembly.

Fig. 1

MHV E localizes in the ERGIC and Golgi. Mouse 17Cl1 cells were infected with MHV A59 at a MOI of 1 and analyzed at 6 h p.i. (A) Cells were processed for dual-label immunofluorescence detection of E and protein disulfide isomerase (PDI), ERGIC-53, or mannosidase II (Mann-II), as ER, ERGIC, and Golgi markers, respectively. The bottom panels were probed for M and E proteins using rabbit polyclonal anti-E 9410 and mouse monoclonal J1.3 and J2.7 antibodies, respectively. Alexa-Fluor tagged secondary antibodies were used to counter stain the primary antibodies. Merged images are shown in the far right column, with enlarged insets of selected cells. Epifluorescence images were taken using a 60× objective. (B) Cells were processed for dual-label immunofluorescence detection using antibodies specific for E (9410) and cis Golgi GM 130, medial Golgi Mann-II or trans Golgi p230. Confocal images were taken with a 100× objective. Merged images are shown in the far right column, with enlarged insets of selected cells. Scale bar, 5 μm (A, B).

Fig. 2

MHV E does not traffic beyond the site of assembly. 17Cl1 mouse cells were infected with MHV A59 at a MOI of 1 and fixed at the specified times after infection. Indirect immunofluorescence was used to probe for E (green) and M (red) proteins. In the merged images yellow signifies where the two proteins colocalize at different times during infection. Confocal images were taken with a 63× objective. Scale bars: 10 μm (8, 12, and 16 h p.i.) and 20 μm (24 h p.i.).

Fig. 3

MHV E does not traffic to the cell surface. (A) 17Cl1 cells were infected with MHV at an MOI of 0.1. At 16 & 18 h p.i., surface proteins were biotinylated. Surface (S) and intracellular (I) proteins were analyzed for spike (S) and envelope (E) proteins by Western blotting. For the analysis 10% and 20% of the intracellular and surface fractions, respectively were loaded. The Western blot for the E protein was exposed 15× longer than that for the S protein. (B) 17Cl1 mouse cells were infected with MHV at an MOI of 0.1 (top three panels) and probed with antibodies specific for S, M and E after cells were permeabilized with digitonin or TX-100 to reveal the cytoplasmic or both luminal and cytoplasmic epitope of the protein. (C) BHK-21 cells were transfected with pCAGGS vector expressing MHV E with a Strep- tag at the amino end. The amino and carboxy ends of E were detected with an anti-Strep monoclonal, StrepMAB-Classic and rabbit anti-E 9410 antibodies, respectively. Scale bar, 5 μm.

Fig. 4

Characterization of TC-tagged E. The schematic illustrates the location of TC tag at the carboxy ends of WT E. 293T cells were transfected with pCAGGS E-TC, fixed at 8 h posttransfection and processed for immunofluorescence to detect E using Lumio™ Green (left images), and ER, ERGIC or Golgi (red) markers (middle images). Yellow in the merged images indicates colocalization of signals. Enlarged inserts of selected cells are shown at the far right. Scale bar, 5 μm.

Fig. 5

MHV recombinant with TC-tagged E. (A) 17Cl1 mouse cells were infected with WT or MHV E-TC viruses at a MOI of 1. Cells were labeled with Lumio Green™ at 8 h p.i., fixed and stained for E with antibody 9410. Yellow in the merged panels indicates colocalization of TC-Lumio™ Green and E. (B) Growth kinetics and plaque characteristics of WT and MHV E-TC viruses were analyzed in 17Cl1 cells infected at a MOI of 0.01. Titers were determined by plaque assay on L2 cells at the indicated times. Growth kinetic titers represent the average of two independent experiments from plaque assays performed in duplicate. Scale bar, 5 μm.

Fig. 6

Dynamics of TC-tagged E in infected cells. (A) Mouse 17Cl1 cells were infected with MHV E-TC at a MOI of 1. At 8 h p.i. cells were treated with Lumio™ Green. (A) Live cells were examined by confocal microscopy and images were collected at 20 min intervals over a 180 min time course with a Z-section setting of 0.5 μm. (B) FRAP was performed after treatment of infected cells with Lumio™ Green. Selected regions of interest were bleached and fluorescence recovery was monitored every 10 s over a ~300 s time course. Representative images from four cells are shown before and after photobleaching, bleached area indicated by arrows. (C) Fluorescence intensities (normalized to prebleach values) are shown plotted against time and the calculated mobile fractions (M_f) are indicated for each cell (n=14). Error bars indicate standard deviations from the mean. (D) Recovery fractions for the cells shown in B are plotted over the 300 s following photobleaching. (E) Recovery time constants for individual cells are shown. Asterisks in panels C and D indicate results from corresponding cells in panel B. FRAP images from 14 cells were analyzed from three independent experiments. Scale bar in A, 5 μm.

Fig. 7

CLEM of TC-tagged E in infected cells. Mouse 17Cl1 cells were infected with MHV E-TC virus at a MOI of 1. (A) At 8 h p.i. cells were stained with Lumio™ Red (left), followed by photoconversion (PC, right). (B) A low-magnification electron micrograph of the boxed cell in A is shown after photoconversion. The electron dense region corresponding to the Golgi region is boxed. A higher magnification shows electron density in Golgi stacks indicated by the arrows adjacent to the nucleus (N). Scale bars, 1 μm and 50 nm, respectively, for images in B.