Infection of cells by picornaviruses leads to the generation of intracellular membrane vesicles. The expression of poliovirus (PV) 3A protein causes swelling of the endoplasmic reticulum (ER) and inhibition of protein trafficking between the ER and the Golgi apparatus. Here, we report that the nonstructural proteins of a second picornavirus, foot-and-mouth disease virus (FMDV), also perturb the secretory pathway. FMDV proteins 3A, 2B, 2C, and 2BC expressed alone in cells were recovered from crude membrane fractions, indicating membrane association. Immunofluorescence microscopy showed that 3A was located in a reticular structure and 2B was located in the ER, while 2C was located in both the ER and the bright punctate structures within the Golgi apparatus. 2BC gave punctate cytoplasmic staining and also caused accumulation of ER proteins in large vesicular structures located around the nuclei. The effect of the FMDV proteins on the trafficking of the vesicular stomatitis virus glycoprotein (G protein) from the ER to the cell surface was determined. Unlike its PV counterpart, the 3A protein of FMDV did not prevent trafficking of the G protein to the cell surface. Instead, surface expression of the G protein was blocked by 2BC, with retention of the G protein in a modified ER compartment staining for 2BC. The results suggest that the nonstructural proteins of different picornaviruses may vary in their ability to perturb the secretory pathway. Since FMDV 2BC can block the delivery of proteins to the cell surface, it may, as shown for PV 3A, play a role in immune evasion and contribute to the persistent infections observed in ruminants.It has been known for several years that the secretory pathway is disrupted in cells infected with picornaviruses (3, 11, 13-15, 31, 38, 48). This is characterized by the appearance of large numbers of membrane vesicles in the cytoplasm. In the case of poliovirus (PV) infection, the membrane vesicles are thought to originate from the endoplasmic reticulum (ER), either from COPII-coated vesicles that move proteins from the ER to the Golgi apparatus or from double-membraned vacuoles that extend from the ER during autophagy (6,37,43). Several studies suggest that the rearranged membranes are utilized during virus replication (7,10,40,43). Viral proteins responsible for replication and newly synthesized viral RNA are, for example, associated with these membranes, and membrane fractions isolated from infected cells can synthesize viral RNA in vitro (6,44,47). A link between a functioning secretory pathway and virus replication has also been provided by the observation that brefeldin A (BFA), a drug that blocks ER-to-Golgi transport by preventing the formation of transport vesicles, blocks replication of PV (23, 28). The membrane rearrangements seen within infected cells are caused by the nonstructural proteins encoded by the P2 and P3 regions of the genome. Studies on the activity of individual PV proteins and the membranes they modify have implicated a role for the nonstructural proteins ...

A single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae. All fumarase translation products are targeted and processed in mitochondria before distribution. Here we show that targeting of fumarase is coupled to translation and initially involves insertion of the protein across the mitochondrial membranes and processing by the matrix protease. Rapid folding of fumarase may determine its requirement for coupling of its translocation with translation and unique route of distribution. The amino termini of most fumarase molecules are translocated across the mitochondrial membranes and processed. Unlike the in vivo situation where these molecules are released into the cytosol, in vitro they remain externally attached to the mitochondria, thereby positioned for release from the organelle. Our model suggests that fumarase displays a unique mechanism of targeting and distribution, which occurs cotranslationally and involves folding and retrograde movement of the processed protein back through the translocation pore.Cytosolic and mitochondrial fumarase isoenzymes are encoded by the same gene (FUM1) in Saccharomyces cerevisiae (1). We have shown previously that these proteins follow a unique mechanism of subcellular localization and distribution in vivo. First, there is only one translation product of FUM1, and it is targeted to mitochondria by a characteristic NH 2 -terminal peptide presequence, which is subsequently removed by the mitochondrial matrix peptidase. Second, it appears that a subset of the processed fumarase molecules are fully imported into the matrix, whereas the majority (70 -80%) are released back into the cytosol as soluble active enzyme by an unknown mechanism (2).The question as to whether in vivo initiation of protein import into mitochondria must occur during (cotranslational) or can, with equal efficiency, occur after completion of protein synthesis (posttranslational) has been addressed previously. It has been proposed that in vivo the normal mode of import of such proteins is co-rather than posttranslational, as suggested by the observations that ribosomes synthesizing mitochondrial proteins were found associated with mitochondria, only minute amounts of some precursors of mitochondrial proteins were detected in yeast cells in vivo, and inhibition of translation inhibits import of mitochondrial proteins (3-7). On the other hand, in vivo accumulated precursors were observed to be chased into mitochondria. Thus, practically all naturally occurring proteins that have been studied could be imported posttranslationally in vivo, suggesting that translation and import are not necessarily coupled. In addition, in vitro, virtually all mitochondrial precursor proteins so far investigated are, under appropriate conditions, successfully imported posttranslationally. In the unique case of fumarase, translocation into the mitochondrial matrix in vivo appears to be strictly cotranslational (2). When fumarase precursors are accu...

Infection of cells with picornaviruses can lead to a block in protein secretion. For poliovirus this is achieved by the 3A protein, and the consequent reduction in secretion of proinflammatory cytokines and surface expression of major histocompatibility complex class I proteins may inhibit host immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2BC is processed to 2B and 2C during infection and that individual 2B and 2C proteins are unable to block secretion stimulated us to study the effects of 2BC processing on the secretory pathway. Even though 2BC was processed rapidly to 2B and 2C, protein transport to the plasma membrane was still blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block.

Picornavirus infection of cells generally results in the production of membranous vesicles containing the viral proteins necessary for viral RNA synthesis. To determine whether foot-and-mouth disease virus (FMDV) infection induced similar structures, and which cellular components were involved, the subcellular distribution of FMDV proteins was compared with protein markers of cellular membrane compartments. Using immunofluorescence analysis and digital deconvolution, it was shown that FMDV structural and non-structural proteins co-localize to punctate structures in juxtanuclear virus assembly sites close to the Golgi complex. Significantly, viral protein 2C did not co-localize with marker proteins of the cis-or medial-Golgi compartments or trans-Golgi network. Furthermore, incubation of infected cells with brefeldin A caused a redistribution of Golgi proteins to the endoplasmic reticulum, but did not affect the distribution of 2C and, by inference, the integrity of the virus assembly site. Taken with the observation that 2C was membrane-associated, but failed to fractionate with Golgi markers on density gradients, it was possible to conclude that Golgi membranes were not a source of structures containing 2C. Further immunofluorescence analysis showed that 2C was also separate from marker proteins of the endoplasmic reticulum, endoplasmic reticulum intermediate compartment, endosomes and lysosomes. The results suggest that the membranes generated at FMDV assembly sites are able to exclude organelle-specific marker proteins, or that FMDV uses an alternative source of membranes as a platform for assembly and replication.

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