A Dominant-Negative Mutant of rab5 Inhibits Infection of Cells by Foot-and-Mouth Disease Virus: Implications for Virus Entry

Foot-and-mouth disease virus (FMDV) can use a number of different integrins (αvβ1, αvβ3, αvβ6, and αvβ8) as receptors to initiate infection. Infection mediated by αvβ6 is known to occur by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express αvβ8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by αvβ8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (∼35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus of the family Picornaviridae and the etiological agent responsible for FMD, an economically important and severe vesicular condition of cloven-hoofed animals, including cattle, pigs, sheep, and goats (2). The mature virus particle consists of a positive-sense single-stranded RNA genome (vRNA) enclosed within a nonenveloped icosahedral capsid formed from 60 copies each of four virus-encoded proteins, VP1 to VP4 (1).The initial stage of FMDV infection is virus binding to cell surface integrins via a highly conserved RGD motif located on the GH loop of VP1. A number of different species of RGD-binding integrins (αvβ1, αvβ3, αvβ6, and αvβ8) have been reported to serve as receptors for FMDV (5, 23-26). Using pharmacological and dominant-negative (DN) inhibitors of specific endocytic pathways in combination with immunofluorescence confocal microscopy, the cell entry pathway used by FMDV has been determined for αvβ6-expressing cells (6, 36). These studies established that infection occurs by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes, which serves as the trigger for capsid disassembly and translocation of the vRNA across the endosomal membrane into the cytosol. Internalized virus was detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomes (LE) or lysosomes (Lys) (the late endosomal compartments). Due to the extreme sensitivity of FMDV to acidic pH (15), it is thought that EE can provide a pH low enough for virus disassembly to occur; however, definitive proof that infection takes place from within EE is still lacking. For example, the possibility cannot be excluded that a productive infection requires virus transport to late endosomal compartments, where, following capsid disassembly and viral genome transfer into the cytosol, the capsid proteins are rapidly degraded.rab proteins control multiple membrane trafficking events in the cell. They are members of the ras superfamily of small GTP-binding proteins and cycle between active GTP- and inactive GDP-bound states (22, 38, 39, 47, 50). Conversion between these states is regulated by guanine nucleotide exchange factors, which stimulate the binding of GTP, and GTPase-activating proteins that which accelerate GTP hydrolysis. Activated rab proteins are recruited onto membrane-bounded compartments where they regulate many steps of vesicle trafficking, including vesicle budding, movement, tethering, and fusion (35, 61). Each rab is recruited to a specific compartment and functions through interactions with specific effectors that mediate the downstream rab-associated functions (39). In mammalian cells, at least 12 rab proteins that regulate trafficking through the endosomal pathway have been identified (27). Of these, rab4, rab5, rab7, and rab11 play major roles in endocytic vesicle trafficking. rab5 is present on EE and regulates transport of incoming endocytic vesicles from the plasma membrane (PM) to EE and homotypic EE fusion events (3, 8, 10, 20, 30, 44, 52). Both rab4 and rab11 are regulators of receptor recycling from EE back to the PM (34); rab4 is localized primarily to EE and regulates rapid recycling directly back to the PM (16, 45, 48, 51, 56), and rab11 is localized primarily to the PNRE and regulates a slower recycling pathway through these compartments (21, 43, 54, 60). In addition rab11 also regulates membrane traffic from endocytic recycling compartments to the trans-Golgi network (55). rab7 is located primarily on LE and regulates traffic from EE to LE and between LE and Lys (7, 9, 18, 32, 40, 58, 59). The unique targeting of rab proteins to distinct cellular compartments and their specificity as regulators of vesicular trafficking has made them important tools for studying endocytosis. For example, expression of DN or constitutively active mutants of rab proteins that regulate endosomal traffic has been used to identify the intracellular transport steps that are required for infection by a number of different viruses (13, 14, 28, 31, 41, 42, 49, 53, 57, 59).Here we have investigated the intracellular transport steps required for FMDV infection using porcine IBRS-2 cells, which are derived from a natural host of FMDV. IBRS-2 cells use αvβ8, and not αvβ6, as the major FMDV receptor (11). Our initial experiments confirmed that FMDV infection mediated by αvβ8 is dependent on clathrin-mediated endocytosis and on an acidic pH within endosomes. The effect on FMDV infection within IBRS-2 cells of DN mutants of cellular rab proteins that regulate endosomal traffic was examined. These experiments show that rab5 is needed for FMDV infection, as expression of DN rab5 reduced the number of FMDV-infected cells by ∼80%. In contrast, expression of either DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (∼35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.