Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis

Abstract

Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER.

Fragmentation of the Golgi apparatus provides replication membranes for human rhinovirus 1A

“…The effect of picornavirus 3A proteins on the Golgi is distinct depending on the viral origin of the protein. When expressed in isolation, poliovirus 3A localizes to the ER and induces swelling and distension of that organelle, but no effects on the Golgi complex were reported (Doedens et al, 1997;Suhy et al, 2000). Another group demonstrated that expression of poliovirus 3A is capable of dispersing the Golgi.…”

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…Poliovirus 3A protein ectopically expressed in cell culture can inhibit the vesicular traYcking of secretory proteins from the ER to the Golgi complex (Doedens et al, 1997;Doedens and Kirkegaard, 1995). The 3A protein remains associated with ER membranes but can be delivered into vesicles, similar to those found in infected cells and by expression of 2BC (Dodd et al, 2001).…”

A Guide to Viral Inclusions, Membrane Rearrangements, Factories, and Viroplasm Produced During Virus Replication

“…Poliovirus and coxsackievirus slow protein movement through the secretory pathway (Doedens and Kirkegaard, 1995;Wessels et al, 2005). Expression of 2B, 2BC, and 3A individually were all able to slow secretion (Cornell et al, 2006;Doedens and Kirkegaard, 1995;Doedens et al, 1997;van Kuppeveld et al, 1997;Wessels et al, 2005Wessels et al, , 2006a, but for both viruses the 3A protein was found to have the greatest impact on ER-to-Golgi transport. Poliovirus infection, and the 3A protein expressed alone in cells, reduces surface expression of MHC class I, the TNF receptor, and secretion of b-IFN, IL-6, and IL-8 (Choe et al, 2005;Deitz et al, 2000;Dodd et al, 2001;Neznanov et al, 2001), and this may offer an immune evasion strategy to the picornaviruses.…”

“…In Poliovirus, Lys9 appears important, and in the triple-proline motif (positions 16-18), only the Pro18 is indispensable for inhibition of protein secretion (Choe et al, 2005). A serine insertion in 3A protein between Thr14 and Ser15, creating the 3A-2 mutant virus (Berstein and Baltimore, 1988), was found to abolish the ER-to-Golgi inhibition of protein trafficking but has little effect on virus replication or membrane rearrangements (Dodd et al, 2001;Doedens et al, 1997). This important observation shows that the ability of 3A to inhibit protein secretion is separate from its role in membrane rearrangements and viral replication.…”