Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

Abstract

Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.Poliovirus (PV), the prototype member of the family Picornaviridae, contains an RNA genome of plus polarity with a single large open reading frame (ORF) coding for a polyprotein of 265 kDa. The translation product is cleaved by intrinsic viral proteases into about 20 proteins, of which intermediate as well as end products are functional (38, 83, 88; reviewed in reference 35). The proteins encoded by the P1 genomic region form the capsid, whereas most of the nonstructural proteins, encoded by the P2 and P3 genomic region, are involved in viral RNA replication (2,27,28,37,48,55,58,82). The viral genome replicates asymmetrically with an excess of plus strands produced in multistranded replicative intermediates (2,17,31,49). PV genome replication depends on the template RNA molecule carrying a cis-acting replicative element (see references 32 and 54), a primer (54), viral and cellular proteins and their proper interactions (2,29,30,53), and the presence of membranes (10,12,14,26,74).Genome replication of all plus-strand RNA viruses investigated so far takes place in membrane-bound replication complexes (see references 19 and 65 and references therein). However, the morphology of such replication complexes of different viruses is diverse, and different cellular compartments provide membranes for and are involved in viral replication (44,56,65). PV replication complexes are built up from individual membranous vesicles (PV vesicles) which are assembled into specific higher-order structures (11,12,15).The PV replicat...

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