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. 2019 Jan 4;11(1):27.
doi: 10.3390/v11010027.

KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport

Vishakha Tiwarekar  1 , Markus Fehrholz  2 , Jürgen Schneider-Schaulies  3
Affiliations

KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport

Vishakha Tiwarekar et al. Viruses. .

Abstract

Recently, we found that the cytidine deaminase APOBEC3G (A3G) inhibits measles (MV) replication. Using a microarray, we identified differential regulation of several host genes upon ectopic expression of A3G. One of the up-regulated genes, the endoplasmic reticulum (ER) protein retention receptor KDELR2, reduced MV replication ~5 fold when it was over-expressed individually in Vero and CEM-SS T cells. Silencing of KDELR2 in A3G-expressing Vero cells abrogated the antiviral activity induced by A3G, confirming its role as an A3G-regulated antiviral host factor. Recognition of the KDEL (Lys-Asp-Glu-Leu) motif by KDEL receptors initiates the retrograde transport of soluble proteins that have escaped the ER and play an important role in ER quality control. Although KDELR2 over-expression reduced MV titers in cell cultures, we observed no interaction between KDELR2 and the MV hemagglutinin (H) protein. Instead, KDELR2 retained chaperones in the ER, which are required for the correct folding and transport of the MV envelope glycoproteins H and fusion protein (F) to the cell surface. Our data indicate that KDELR2 competes with MV envelope proteins for binding to calnexin and GRP78/Bip, and that this interaction limits the availability of the chaperones for MV proteins, causing the reduction of virus spread and titers.

Keywords: GRP78; KDELR2; calnexin; measles virus; surface transport.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The A3G upregulated gene KDELR2 reduces MV replication in Vero cells. (A) Total RNA from Vero 023 and Vero A3G was isolated and reverse transcribed into cDNA. KDELR2-specific cDNA was then amplified using SYBR-Green Real-Time qPCR (n = 3). (B) The protein expression of KDELR2 was analyzed using Western blot. Equal amounts of cell lysates were separated on 12% SDS-PAGE and transferred on a nitrocellulose (NC) membrane. Target proteins were probed with primary KDELR2 antibody and HRP conjugated secondary antibody then developed using ECL (lane 1: Vero 023, lane 2: Vero A3G, lane 3: Vero KDELR2, lane 4: Vero A3G + KDELR2shRNA). (C) Transduced Vero cells were infected with MV eGFP at MOI of 0.1. The titer of newly synthesized virus in these cells was determined 48 h post infection on Vero cells (n = 3). Significance was calculated using the Student’s t test (** p < 0.01). (D) Representative micrograph of MV syncytium based on eGFP fluorescence 72 h post infection (magnification × 100, size bar: 150 µm).
Figure 2
Figure 2
KDELR2 reduces MV replication in CEM-SS T cells. Transduced CEM-SS T cells were infected with MV-eGFP at a MOI of 0.1. The titer of newly synthesized virus in these cells was determined 48 h post infection on Vero cells (n = 3). Significance was calculated using the Student’s t test (** p < 0.01).
Figure 3
Figure 3
KDELR2 over-expression leads to a reduction of MV-H and -F surface expression. Vero-023, Vero-A3G, and KDELR2 over-expressing Vero cells (KDELR2) were infected with MV-eGFP at a MOI of 0.1 for 48 h. Cells were fixed and stained (surface expression), or fixed, permeabilized, and stained (total expression) with monoclonal antibodies to MV-H (A,B) and MV-F (C,D) and secondary antibodies. The MV-H and –F expression was analyzed by flow cytometry. Examples of representative experiments with isotype controls (left signals) and surface and total H (A) and F (C) expression (dark signals) are shown. The results of three experiments are summarized in bar graphs (B,D). Data of mean fluorescence intensities (MFI) were presented as a percent of control normalized to values of Vero-023 cells. Significances were determined using the Student’s t-test (* p < 0.05; n.s. = not significant).
Figure 4
Figure 4
No detectable interaction was seen between KDELR2 and MV-H, but of KDELR2 with GRP78. (A) Transduced Vero cells over-expressing the KDELR2 (Vero-KDELR2-Flag) were infected with MV (MV-Edmonston not expressing eGFP) at a MOI of 0.1. After 8, 16, 20, and 24 h, cells were fixed and stained with antibodies to Flag and Alexa-488-conjugated secondary antibodies (green), to MV-H and Alexa-594-conjugated secondary antibodies (red), and DAPI (blue). Photomicrographs were taken using a confocal microscope (enlargement 400×; size bar = 20 µm). For co-immunoprecipitations, lysates of MV-infected and uninfected (as indicated) Vero-023 and Vero-KDELR2-Flag (KD) cells remained untreated (input, lanes 2 to 5) or were treated with anti-Flag antibody-conjugated beads for precipitation (IP: anti-Flag, lanes 6 to 9). Control precipitation was with unconjugated beads (BC, lane 10). Proteins were size separated by 10% SDS-PAGE, blotted onto nitrocellulose, and in (B) detected with antibodies to MV-H (polyclonal rabbit H45, upper panel), or to Flag detecting KDELR2 and secondary antibodies as indicated (lower panel). In (C), Western blots were treated with antibodies to GRP78 (upper panel), or to Flag detecting KDELR2 and secondary antibodies as indicated (lower panel).
Figure 5
Figure 5
KDELR2 over-expression reduces the surface expression of calnexin and GRP78. Surface (A) and total (B) chaperone expression were quantified by flow cytometry. Vero-023 and KDELR2 over-expressing Vero cells (KDELR2) remained uninfected (open columns) or were infected with MV-eGFP for 48 h at a MOI of 0.1 (grey columns). Cells were stained after fixation (surface, A), and after fixation and permeabilization (total, B) with antibodies to calnexin, GRP78, and calreticulin as indicated, and the fluorescence intensities were evaluated by flow cytometry. Exclusively, eGFP-positive cells were evaluated as infected cells and eGFP-negative cells as uninfected cells. Data representative of three independent experiments were normalized to values of uninfected Vero-023 cells, and are presented as relative mean fluorescent intensities (MFI). Significances were determined using the Student’s t-test. The subcellular localization of the chaperones calnexin (C), GRP78 (D), and calreticulin (E) in comparison to KDELR2-Flag and nuclei (DAPI) was analyzed using a confocal microscope (enlargement 1000×; size bar = 20 µm).
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