A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport

Abstract

The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro 19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro 14 , Pro 17 , or Pro 20 also impaired this ability, but to a lesser extent. The mutation of Pro 18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

“…The plasmid p3A-myc and the p53CB3/T7 3A mutant full length infectious clones have been described previously [12,21]. To construct the plasmids p3A-V45A-myc and p3A-H57Y-myc, the 3A gene containing the V45A or H57Y mutation was amplified using the mutant full-length clones as a template.…”

“…CVB3 and CVB3-Rluc, which contains the Renilla luciferase gene upstream of the capsid coding region, were obtained by transfection of RNA transcripts derived from the full length infectious clones p53CB3/T7 and pRLuc-53CB3/T7 in BGM cells as described before [21,62]. CVB3(-Rluc) carrying the V45A or H57Y mutation in the 3A protein were obtained in a similar manner using plasmids p53CB3/T7-3A-V45A or -H57Y described elsewhere [12] or pRluc-53CB3/T7-3A-V45A or -H57Y.…”

“…Activated Arf1 is responsible for the recruitment of the COP-I complex, a coatomer protein complex that induces membrane curvature and initiates budding of transport vesicles [15][16][17]. When expressed in isolation, 3A disrupts ER-to-Golgi transport by blocking the formation of COP-I coat complexes on membranes [18][19][20][21]. The 3A protein most likely exerts this effect by directly interacting with GBF1 [22,23].…”

Structure-Function Analysis of the Coxsackievirus Protein 3A

“…pACT-3A and pBIND-3A were described previously (5). To obtain pACT and pBIND-3A mutant plasmids, the wt 3A sequence was replaced by mutant 3A sequences using the enzymes Bst1107I and BamHI.…”

“…3A inhibits protein transport between the endoplasmic reticulum and Golgi (4,5), whereas the step inhibited by the 2B protein is presently unknown. The inhibition of protein transport by 3A is not essential for viral replication in cell culture (4,5) but has been proposed to have a function in evasion of innate and acquired immune responses and the extrinsic apoptotic pathway (6 -8).…”