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. 2010 Oct 18;5(10):e13130.
doi: 10.1371/journal.pone.0013130.

Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal

Tyler M Sharp  1 , Susana Guix, Kazuhiko Katayama, Sue E Crawford, Mary K Estes
Affiliations

Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal

Tyler M Sharp et al. PLoS One. .

Abstract

Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. NV replication induces Golgi fragmentation.
Viral RNA, purified from the stool of a human volunteer infected with Norwalk virus, was transfected into Huh7 cells grown on coverslips. At 24 hours post-transfection (hpt), cells were fixed and stained for the viral protein VP1 (Alexa 488; green fluorescence) and either the cis (A) or trans (B) Golgi with antibody against GM130 or golgin-97 (Alexa 594; red fluorescence), respectively. Nuclei were stained with DAPI (blue fluorescence) and imaged by deconvolution microscopy. * indicate cells with disassembled Golgi.
Figure 2
Figure 2. NV p22 induces Golgi disassembly and inhibits protein secretion.
(A) Cells expressing GFP, GFP-tagged poliovirus (PV) 3A protein, or GFP-tagged Norwalk virus (NV) p22 were stained for the cis Golgi marker protein GM130 (Alexa 594-conjugated secondary antibody, red fluorescence) at the indicated times post-transfection. Nuclei were stained with DAPI (blue fluorescence), and cells were imaged by deconvolution microscopy. * indicates cells with dispersed Golgi; ∧ indicates cells with fragmented Golgi. (B) Quantitation of Golgi status in cells expressing the indicated proteins at 24 hours post-transfection (n = 3; minimum of 50 cells per experiment; ±SD). Differences between observed phenotypes are detailed in the text. Results are representative of four independent experiments. (C) Cells were transfected with the plasmid pCMV-UTR-SEAP expressing GFP, GFP-tagged PV 3A, or GFP-tagged NV p22. Secreted SEAP was quantified (see Materials and Methods section) as a representative indicator of cellular protein secretion at the indicated time points, and was defined by the equation: Secreted SEAP  =  (SEAPextracellular/(SEAPextracellular + SEAPintracellular)) ×100. Data are representative of three independent experiments (n = 3 for each time point; ±SD).
Figure 3
Figure 3. Expression of NV p22 induces alterations in secretory pathway ultrastructure.
At 24 hours post-transfection, cells expressing GFP (A) or GFP-p22 (B) were harvested and flow sorted for expression of GFP. Twenty-four hours after re-plating, cells were fixed and prepared for visualization by electron microscopy. The boxed regions represent the area magnified to the right. Left scale bars represent 2 µm, right scale bars represent 0.5 µm. N =  nucleus; black arrows indicate intact Golgi cisternae; black arrowheads indicate double-membrane vesicles; the asterisk indicates free membranes.
Figure 4
Figure 4. Amino acids 50–148 are sufficient to mediate Golgi localization of NV p22.
(A) GFP tagged N and C terminal deletion mutants of p22 were generated and, following expression in cells for 24 hours, were scored for their ability to localize to the cis Golgi. Amino acid numbering corresponds to NV sequence (NC_001959). (B) Cells expressing GFP-tagged p22(50–102), p22(103–148), and p22(50–148) were immuno-stained with antibody against the cis Golgi marker protein GM130 (Alexa 594-conjugated secondary antibody, red fluorescence), stained with DAPI (blue fluorescence), and imaged by deconvolution microscopy. (C) Cells expressing GFP, GFP-tagged PV 3A, GFP-tagged NV p22, or the indicated deletion mutants of p22 were harvested at 24 hpt. Cytosolic and membranous fractions of cells were collected and proteins were detected by western blot with monoclonal antibody against GFP.
Figure 5
Figure 5. A conserved noroviral ER export signal is necessary for p22 to inhibit protein secretion.
(A) Amino acids 50–148 from NV p22 were aligned with homologues [“p22-like (p22L) proteins”] from representative genogroup 1 (GI) and genogroup 2 (GII) human noroviruses of various genotypes. The figure illustrates six of 72 sequences analyzed. Conserved residues are shown in bold; the blue box indicates conservation of the membrane association domain (MAD); the red box indicates conservation of the YXΦESDG motif. NV is Norwalk virus (NC_001959), CV is Chiba virus (AB042808), HV is Hawaii virus (U07611), SMV is Snow Mountain virus (AY134748), U201 is Saitama U201 virus (AB039782), and MD145 is MD145 virus (AY032605). (B) Alignment of NV p22 with various cellular and viral proteins that contain an ER export signal. VSV G is the vesicular stomatitis virus glycoprotein, LAP is lysosomal acid phosphatase, VZV GPI is varicella zoster virus glycoprotein I, and CD3γ is a component of the T cell receptor. Adapted from Nishimura and Balch, 1997 . (C) Cells were transfected with the plasmid pCMV-UTR-SEAP expressing GFP, GFP-tagged NV p22, or the indicated mutants within the ER export signal of p22. Secreted SEAP was quantified (see Materials and Methods) as a representative indicator of cellular protein secretion at the indicated time points and was defined by the equation: Secreted SEAP  =  (SEAPextracellular)/(SEAPextracellular + SEAPintracellular) ×100. Data are representative of three independent experiments (n = 3 for each time point; ±SD). (D and E) p22 with the YXΦESDG motif mutated to AXΦASDG (D; E, bottom panels) or wildtype NV p22 (E, top panels) were expressed as GFP fusion proteins. Cells were fixed at 24 hpt and stained with antibody against the cis Golgi marker protein GM130 (D) or the endoplasmic reticulum marker protein calnexin (E) (Alexa 594-conjugated secondary antibody, red fluorescence). Nuclei were stained with DAPI (blue fluorescence) and cells were imaged by deconvolution microscopy. The inset in E is an 8X magnification of the boxed region.
Figure 6
Figure 6. The ER export signal mimic of p22 can substitute for the signal of VSV G.
(A) Summary of the sequence of the wildtype, mutant, and chimeric VSV G proteins used. Critical residues of the VSV G ER export signal and homologous regions of p22 are shown in bold. (B) Representative individual samples from EndoH sensitivity assay of VSV G proteins. Wildtype (G), mutant [G(6xA)] and chimeric (G/p22 and G/AXΦA) VSV G proteins were metabolically labeled with 35S-Methionine at 24 hours post-transfection and incubated for the indicated period of time, harvested in lysis buffer, immuno-precipitated with monoclonal antibody against the luminal domain of VSV G and digested with endoglycosidase H (EndoH). R =  EndoH resistant; S =  EndoH sensitive. (C) Cells were transfected with plasmids encoding the indicated constructs of VSV G. At 24 hours-post-transfection, cells were labeled with 35S-Met and at various times post-pulse cells were harvested, immuno-precipitated with antibody against the luminal domain of VSV G and assayed for their sensitivity to EndoH. Data are composite (mean ± SD) of six individual samples (n = 6) for each time point from two independent experiments.
Figure 7
Figure 7. SEAP is differentially retained in the presence of PV 3A and NV p22.
Cells expressing GFP alone (A), or GFP-tagged 3A (B) or NV p22 (C) were fixed at 24 hpt and immuno-stainined with antibody against SEAP (Alexa 594-conjugated secondary antibody) and the COPI marker protein β-COP (Alexa 647-conjugated secondary antibody), stained with DAPI (blue fluorescence), and imaged by deconvolution microscopy. Channels were pseudo-colored as indicated for merged images. Insets in B and C represent a 6X zoom of the boxed region.
Figure 8
Figure 8. Secretory pathway cargo is retained in COPII vesicles in the presence of p22, but not p22(AXΦA).
Cells expressing GFP alone (A), or GFP-tagged NV p22 (B) or p22(AXΦA) (C) were fixed at 24 hpt and immuno-stained with antibody against SEAP (Alexa 594-conjugated secondary antibody) and the COPII marker protein Sec24C (Alexa 647-conjugated secondary antibody), stained with DAPI (blue fluorescence), and imaged by deconvolution microscopy. Channels were pseudo-colored as indicated for merged images. Inset in B represents an 8X zoom of the boxed region.
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