Cell density-dependent expression of viral antigens during persistence of foot-and-mouth disease virus in cell culture.

Immunofluorescence analyses of FMDV persistently infected BHK-21 cells showed that in cultures from early stages of the persistence (passage 15) only about 10% of cells displayed viral antigens, while at late stages (passage 100) no FMDV antigen-positive cells were found. Positive cells at passage 15 displayed a number of structural alterations that did not differ from those observed in lytically infected cells. In these monolayers, and remarkably, clusters of cells that exceeded confluence were associated with an enhancement of cells positive for FMDV antigens, suggesting cell density-dependent expression of viral antigens. Inhibition of virus spread by blocking endosomal acidification, or addition of neutralizing antibodies to the culture medium reduced the number of FMDV antigen-positive cells within the monolayers. These results suggest that extracellular virus transmission plays an important role during FMDV persistence in cell culture and that this process fits the characteristics of a carrier culture model.

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid lability and confer resistance to acid-dependent uncoating inhibition.

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH(4)Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH(4)Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH(4)Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.

Internalization of swine vesicular disease virus into cultured cells: a comparative study with foot-and-mouth disease virus.

We performed a comparative analysis of the internalization mechanisms used by three viruses causing important vesicular diseases in animals. Swine vesicular disease virus (SVDV) internalization was inhibited by treatments that affected clathrin-mediated endocytosis and required traffic through an endosomal compartment. SVDV particles were found in clathrin-coated pits by electron microscopy and colocalized with markers of early endosomes by confocal microscopy. SVDV infectivity was significantly inhibited by drugs that raised endosomal pH. When compared to foot-and-mouth disease virus (FMDV), which uses clathrin-mediated endocytosis, the early step of SVDV was dependent on the integrity of microtubules. SVDV-productive endocytosis was more sensitive to plasma membrane cholesterol extraction than that of FMDV, and differential cell signaling requirements for virus infection were also found. Vesicular stomatitis virus, a model virus internalized by clathrin-mediated endocytosis, was included as a control of drug treatments. These results suggest that different clathrin-mediated routes are responsible for the internalization of these viruses.

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus.

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing either 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle.

Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: a comparative study with foot-and-mouth disease virus and vesicular stomatitis virus.

The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV (approximately 5 log) and VSV (approximately 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.

Productive entry of type C foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol.

We have characterized the entry leading to productive infection of a type C FMDV in two cell lines widely used for virus growth, BHK-21 and IBRS-2. Inhibition of clathrin-mediated endocytosis by sucrose treatment decreased both cell entry and virus multiplication. Evidence of a direct requirement of clathrin for productive viral entry was obtained using BHK21-tTA/anti-CHC cells, which showed a significant reduction of viral entry and infection when the synthesis and functionality of clathrin heavy chain was inhibited (Tet- cells). This was also observed for vesicular stomatitis virus (VSV) productive entry. The effect of NH(4)Cl and concanamycin A on FMDV entry and infection was consistent with the requirement of acidic compartments for decapsidation and virus replication. As expected from its higher stability at acidic pH, this requirement was higher for VSV. Since BHK-21 and IBRS-2 cells expressed caveolin-1, we explored the effect on productive virus entry of drugs that interfere with caveolae-mediated endocytosis. Treatment with nystatin did not reduce entry and infection of FMDV or VSV, while cholesterol depletion with MbetaCD significantly inhibited both steps of the FMDV cycle, indicating that plasma membrane cholesterol is required for virus productive entry.

Differential distribution of non-structural proteins of foot-and-mouth disease virus in BHK-21 cells.

Differences in the kinetics of expression and cell distribution among FMDV non-structural proteins (NSPs) have been observed in BHK-21-infected cells. 3D(pol) was the first protein detected by immunofluorescence (1.5 h p.i.), showing a perinuclear distribution. At 2-2.5 h p.i., 2B, 2C, 3B and 3C were detected, mostly exhibiting a punctuated, scattered pattern, while 3A and 3D(pol) appeared concentrated at one side of the nucleus. This distribution was exhibited by all the NSPs from 3 h p.i., being 2C and, to a lesser extent, precursors 2BC and 3ABBB, the only proteins detected by Western blotting at that infection time. From 4 h p.i., all mature NSPs as well as precursors 2BC, 3ABBB, 3ABB, 3AB and 3CD(pol) were detected by this technique. In spite of their similar immunofluorescence patterns, 2C and 3A co-localized partially by confocal microscopy at 3.5 h p.i., and 3A, but not 2C, co-localized with the ER marker calreticulin, suggesting differences in the distribution of these proteins and/or their precursors as infection proceeded. Transient expression of 2C and 3AB resulted in punctuated fluorescence patterns similar to those found in early infected cells, while 3A showed a more diffuse distribution. A shift towards a fibrous pattern was noticed for 3ABB, while a major change was observed in cells expressing 3ABBB, which displayed a perinuclear fibrous distribution. Interestingly, when co-expressed with 3D(pol), the pattern observed for 3ABBB fluorescence was altered, resembling that exhibited by cells transfected with 3AB. Transient expression of 3D(pol) showed a homogeneous cell distribution that included, as determined by confocal microscopy, the nucleus. This was confirmed by the detection of 3D(pol) in nuclear fractions of transfected cells. 3D(pol) and its precursor 3CD(pol) were also detected in nuclear fractions of infected cells, suggesting that these proteins can directly interact with the nucleus during FMDV infection.

Hepatocyte growth factor and its receptor are required for malaria infection.

Plasmodium, the causative agent of malaria, must first infect hepatocytes to initiate a mammalian infection. Sporozoites migrate through several hepatocytes, by breaching their plasma membranes, before infection is finally established in one of them. Here we show that wounding of hepatocytes by sporozoite migration induces the secretion of hepatocyte growth factor (HGF), which renders hepatocytes susceptible to infection. Infection depends on activation of the HGF receptor, MET, by secreted HGF. The malaria parasite exploits MET not as a primary binding site, but as a mediator of signals that make the host cell susceptible to infection. HGF/MET signaling induces rearrangements of the host-cell actin cytoskeleton that are required for the early development of the parasites within hepatocytes. Our findings identify HGF and MET as potential targets for new approaches to malaria prevention.