Effects of picornavirus 3A Proteins on Protein Transport and GBF1-dependent COP-I recruitment

Abstract

The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence, to inhibit protein transport. These results show that the N terminus of the CVB3 3A protein is important for binding of GBF1 and its transport-inhibiting function. Taken together, our data demonstrate that the activity of the enterovirus 3A protein to inhibit GBF1-dependent COP-I recruitment is unique among the picornaviruses.

FIG. 1.

Effects of picornavirus 3A proteins on COP-I recruitment to membranes and VSVG trafficking. (A) COP-I distribution in 3A-expressing cells. The bars show the means ± standard errors of the means (SEM) of the percentage of cells in which COP-I was redistributed to the cytosol, or localized at intact Golgi or dispersed membrane structures, as calculated from at least 400 cells, which were counted in groups of ∼100 cells in two independent experiments. As a control, we determined COP-I localization in cells expressing the CVB3 2B protein (no 3A). (B) BGM cells expressing Myc-tagged CVB3, PV1, HRV14, HRV2, EMCV, FMDV, or HAV 3A proteins were stained for the Myc tag and COP-I (α/γ-COP). Asterisks indicate 3A-positive cells. (C) The percentage of cells in which the VSVG protein was localized to the plasma membrane. At 40°C, VSVG is improperly folded and as a consequence retained in the ER. Upon shifting to the permissive temperature (32°C), VSVG is correctly folded and transported to the plasma membrane. The graph shows the means ± SEM of the percentage of cells in which VSVG localized at the plasma membrane, calculated from at least 200 cells, which were counted in groups of ∼50 cells in two independent experiments. “a” indicates result significantly different from control cells without 3A (calculated by using the Student t test; P < 0.05).

FIG. 2.

The HRV 3A proteins have a reduced ability to bind GBF1 and inhibit its function. (A) Alignment of enterovirus and HRV 3A proteins. Gray boxes indicate the hydrophobic regions of the 3A proteins, as predicted using the Kyte and Doolittle method. (B) FRAP traces of cells expressing YFP-GBF1 either alone (no 3A) or together with CFP fusion proteins of the 3A proteins of CVB3, PV, HRV14, or HRV2. The traces in the graphs show the average recovery (n > 10 cells) from at least two independent experiments. The fluorescence intensity before bleaching was normalized to 1, and the fluorescence intensity directly after bleaching was normalized to 0. The fluorescence intensity was corrected for bleaching of the cell during imaging and for background fluorescence. (C) Interaction of the 3A proteins of CVB3, PV1, HRV14, and HRV2 with the N-terminal part of GBF1 as assayed by yeast two-hybrid analysis. Upper, middle, and lower panels show growth of yeast on nonselective medium (leucine- and tryptophane-deficient medium), selective medium lacking histidine (-His), and selective medium lacking adenine (-Ade), respectively.

FIG. 3.

The N terminus of 3A is very important for its transport-inhibiting function. (A) The percentage of cells in which the VSVG protein was localized at the plasma membrane. The percentages were calculated as described in the legend to Fig. 1. (B) COP-I localization in cells expressing HRV14 3A-E8K, C3H14 3A, C3H2 3A, or C3PV 3A. The percentages were calculated as described in the legend to Fig. 1. (C) BGM cells expressing Myc-tagged mutant or chimeric 3A proteins (HRV14 3A-E8K, C3H14, and C3H2) were stained for the Myc tag to show their localization. (D) Interaction of the mutant or chimeric 3A proteins (HRV14 3A-E8K, C3H14, and C3H2) with the N-terminal part of GBF1 as assayed by yeast two-hybrid analysis. The results are depicted as described in the legend to Fig. 2. (E) FRAP traces of cells coexpressing YFP-GBF1 and CFP fusion proteins of HRV14 3A (wild-type or E8K). Traces were calculated as described in the legend for Fig. 2. C3H14, CVB3/HRV14 3A chimeric protein; C3H2, CVB3/HRV2 3A chimeric protein; C3PV, CVB3/PV1 3A chimeric protein. a, significantly different from HRV14 3A wt-expressing cells; b, significantly different from HRV2 3A wt-expressing cells; c, significantly different from PV1 3A wt-expressing cells (calculated with Student's t test; P < 0.05).