Foot-and-mouth disease virus replication sites form next to the nucleus and close to the Golgi apparatus, but exclude marker proteins associated with host membrane compartments

Abstract

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.

Guinea Pig-Adapted Foot-and-Mouth Disease Virus with Altered Receptor Recognition Can Productively Infect a Natural Host

“…A single open reading frame encodes all of the capsid, as well as a total of nine additional mature, nonstructural (NS) proteins, including two proteases (L and 3C) and an RNA-dependent RNA polymerase (3D) (14,69,73). As shown for a number of different picornaviruses, the mature NS proteins, as well as some of their protein precursors, are involved in multiple functions needed for virus multiplication and the host cell membrane rearrangements associated with viral RNA replication (3,25,35,46,55,60,65,82).FMDV can initiate the infection of cultured cells via different ␣v integrins (␣v␤1,␣v␤3, ␣v␤6, and ␣v␤8) (15,31,41,44,45), and viruses that are infectious in vivo have been reported to use integrins ␣v␤3 and ␣v␤6 as receptors (45,58). This latter integrin is expressed constitutively on the epithelial cells targeted by FMDV in cattle and is most likely the major in vivo receptor for this virus (56).…”

“…A single open reading frame encodes all of the capsid, as well as a total of nine additional mature, nonstructural (NS) proteins, including two proteases (L and 3C) and an RNA-dependent RNA polymerase (3D) (14,69,73). As shown for a number of different picornaviruses, the mature NS proteins, as well as some of their protein precursors, are involved in multiple functions needed for virus multiplication and the host cell membrane rearrangements associated with viral RNA replication (3,25,35,46,55,60,65,82).…”

Identification of Cellular Genes Affecting the Infectivity of Foot-and-Mouth Disease Virus

“…NTPDase6 belongs to a family of enzymes known to modulate a variety of important cellular responses by controlling extracellular nucleotide concentrations; these include blood clotting and neural signaling transmission (29,55,56; for a review, see reference 27). However, NTPDase6, unlike other members of the NTPDase family, exists as both a soluble form and a membrane-bound form, raising the possibility that this enzyme may be implicated in membrane rearrangement events that occur during normal FMDV replication (32,37,39). The cell clone containing an antisense NTPDase6 EST was further characterized.…”

“…In the case of FMDV, the replication complex recently has been described, and the involvement of the Golgi apparatus or the endoplasmic reticulum has been reported (32,37,39,41). Preliminary electromicroscopy analysis of NTPDase6-expressing cell clone 30 infected with FMDV at an MOI of 10 showed a reduced number of replication complexes at 4 hpi compared to infected LF-BK tTA cells.…”

Differentiation of Foot-and-Mouth Disease Virus-Infected Pigs from Vaccinated Pigs using a Western Blotting Assay Based on Baculovirus-Expressed Nonstructural Proteins 2C and 3D

“…The protein 2C is responsible for various aspects of virus replication [4,6,8]. Because the molecular mass of 2C protein is relatively small, it is highly possible that this protein is removed from the vaccine material in the process of vaccine production.…”