Establishment of cell lines persistently infected with foot-and-mouth disease virus

Cell lines persistently infected with foot-and-mouth disease virus (FMDV) have been established by growth of BHK-21 (c-13) or IBRS-2 (c-26) that survived standard cytolytic infections with FMDV. They maintain cytoplasmic FMDV RNA sequences, as shown by dot blot hybridization tests, using cloned FMDV cDNA as probes. Cell line C1-BHK-Rc1 was derived by infection of cloned BHK-21 c1 cells and plaque-purified FMDV C-S8 c1. Indirect immunofluorescence assays indicated the presence of FMDV antigens. It was resistant to superinfection by FMDV C-S8 c1, O-S7, or A5, but not by encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or Semliki forest virus (SFV). Infectious FMDV was detected in the culture medium only up to cell passage 65. The virus isolated from C1-BHK-Rc1 cells showed decreased plaque size and diminished yield in infections at 42 degrees. Multiple mutations in the intracellular FMDV RNA have been detected by T1 oligonucleotide fingerprinting of genomic RNA segments hybridized to FMDV cDNA fragments. At late cell passages, when no infectious FMDV is detected, cells continue to express viral antigens and FMDV RNAs with deletions of up to 3 kb have been identified by Northern blot analysis. We conclude that persistent infections of cell cultures with FMDV are readily established and that multiple genetic and phenotypic variations occur in the virus during persistence.     

Arenavirus entry occurs through a cholesterol-dependent, non-caveolar, clathrin-mediated endocytic mechanism

Arenaviruses are important causes of viral hemorrhagic fevers in humans. Arenavirus infection of cells occurs via a pH-dependent endocytic route, but detailed studies of entry pathways have not been done. We investigated the role of cell membrane cholesterol, caveolae, and clathrin coated pits in infection by Lassa virus (LASV), which utilizes alpha-dystroglycan (alpha-DG) as a receptor, and Pichindé virus (PICV), which does not. Depletion of cellular cholesterol by treatment with methyl betacyclodextrin (MbetaCD) or nystatin/progesterone inhibited PICV replication and transfer of packaged marker gene by LASV or PICV pseudotyped retroviral particles. In cells lacking caveolae due to silencing of the caveolin-1 gene, no inhibition of PICV infection or LASV pseudotype transduction was observed. However, PICV infection and LASV and PICV pseudotype transduction was inhibited when an Eps15 dominant negative mutant was used to inhibit clathrin-mediated endocytosis. Altogether, the results indicate that diverse arenaviruses have a common requirement for cell membrane cholesterol and clathrin mediated endocytosis in establishing infection.     

Inhibition of foot and mouth disease virus (FMDV) uncoating by a plant-derived peptide isolated from Melia azedarach L leaves

Summary  Meliacine (MA), a peptide isolated from leaves of the high plant Melia azedarach L inhibited the multiplication of foot and mouth disease virus (FMDV) in BHK-21 cells. In this report, we establish that the MA-inhibitable process takes place within the first hour of the viral reproductive cycle. MA had no virucidal effect and did not affect adsorption and penetration of the virus in cells. In experiments with neutral red-labeled virus, it was found that MA significantly suppressed the development of photoresistance of the virus in infected cells. In untreated cultures nearly all virus which adsorbed to cells was uncoated within 1 h at 37 °C, whereas in treated cultures, even after 3 h only 3% of the virus was uncoated. Labeling of BHK-21 cells with acridine orange showed that MA affects the pH of intracellular acidic vesicles. Therefore, it is concluded that MA prevents the process of uncoating of FMDV in BHK-21 cells by inhibiting vacuolar acidification. Accepted October 15, 1997 Received April 21, 1997     

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.     

Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture

The genetic heterogeneity generated upon passage of foot-and-mouth disease virus (FMDV) in cell culture has been evaluated by T1-oligonucleotide fingerprinting of genomic RNA. Plaque-purified FMDV O-S7 and C-S8 were propagated by serial low multiplicity infections of BHK-21 (c-13) or IBRS-2 (c-26) cells. In independent parallel passage of the same virus, different oligonucleotide variations were fixed in the RNAs. T1-oligonucleotide fingerprinting of RNA from 34 individual viral clones derived from two passaged populations shows an extensive heterogeneity, with some mutations present in only one of the cloned genomes analyzed. Some FMDV variants are phenotypically distinct in that they yield increased progeny in infections of cell monolayers. From the number of variant sequences it can be estimated that each infectious RNA in the population differs in two to eight mutations from the average parental sequence. Thus, passaged FMDV populations consist of a pool of variants, an observation previously made with phage Q beta (E. Domingo, D. Sabo, T. Taniguchi, and C. Weissmann, Cell 13, 735-744, 1978). The FMDV genome must be described as a fluctuating distribution of sequences due to its high mutability. This may be the basis of the extensive genetic and antigenic diversity of this virus in nature.     

Antiviral activity of human lactoferrin: inhibition of alphavirus interaction with heparan sulfate

Human lactoferrin is a component of the non-specific immune system with distinct antiviral properties. We used alphaviruses, adapted to interaction with heparan sulfate (HS), as a tool to investigate the mechanism of lactoferrin's antiviral activity. Lactoferrin inhibited infection of BHK-21 cells by HS-adapted, but not by non-adapted, Sindbis virus (SIN) or Semliki Forest virus (SFV). Lactoferrin also inhibited binding of radiolabeled HS-adapted viruses to BHK-21 cells or liposomes containing lipid-conjugated heparin as a receptor analog. On the other hand, low-pH-induced fusion of the viruses with liposomes, which occurs independently of virus-receptor interaction, was unaffected. Studies involving preincubation of virus or cells with lactoferrin suggested that the protein does not bind to the virus, but rather blocks HS-moieties on the cell surface. Charge-modified human serum albumin, with a net positive charge, had a similar antiviral effect against HS-adapted SIN and SFV, suggesting that the antiviral activity of lactoferrin is related to its positive charge. It is concluded that human lactoferrin inhibits viral infection by interfering with virus-receptor interaction rather than by affecting subsequent steps in the viral cell entry or replication processes.     

Vesicular stomatitis virus induces apoptosis at early stages in the viral cycle and does not depend on virus replication

We detected apoptosis induction in the vesicular stomatitis virus (VSV) infected mammalian cell lines Vero-76, Cos-7, and BHK-21. Cell lines were analyzed by chromosomal DNA fragmentation and nuclear morphology. In order to determine the step in the viral cycle at which apoptosis of infected cells is triggered, chemical and physical agents were used to block viral infection at different times and then the apoptotic response of infected cells was examined. The treatment of Vero-76 infected cells with a lysosomotrophic agent, such as NH4Cl, was shown to abrogate virus apoptosis induction. On the other hand, VSV-induced apoptosis was not blocked by the presence of cycloheximide, suggesting that the de novo viral protein synthesis is not required for this process. UV-inactivated viruses were also capable of inducing apoptosis in Vero-76 cells, indicating that the activation of a programmed cell death process by VSV does not require viral replication. We conclude from these findings that VSV induces apoptosis at early stages of infection.     

T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19–20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.     

A segmented form of foot-and-mouth disease virus interferes with standard virus: a link between interference and competitive fitness

Serial passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells at high multiplicity of infection resulted in dominance of particles containing defective RNAs that were infectious by complementation in the absence of standard viral RNA. In the present study, we show that the defective FMDV particles interfere with replication of the cognate standard virus. Coinfections of defective FMDV with standard FMDV mutants that differ up to 151-fold in relative fitness have documented that the degree of interference is higher for low fitness than for high fitness standard virus. These comparisons suggest a likely overlap between those mechanisms of intracellular competition that underlie viral interference and those expressed as fitness differences between two viruses when they coinfect the same cells. Interference may contribute to the selective pressures that help maintain dominance of segmented defective RNAs over the standard FMDV genome.     

Role of clathrin-mediated endocytosis during vesicular stomatitis virus entry into host cells

Vesicular stomatitis virus (VSV) is well established to enter cells by pH-dependent endocytosis, but mechanistic aspects of its internalization have remained unclear. Here, we examined the functional role of clathrin in VSV entry by expression of a dominant-negative mutant of Eps15 (GFP-Eps15Delta95/295), a protein essential for clathrin-mediated endocytosis. Whereas expression of GFP alone had no effect on VSV infection, expression of GFP-Eps15Delta95/295 severely limited infection. As independent ways to examine clathrin function, we also examined cells that had been treated with chlorpromazine and utilized small interfering RNA (siRNA) techniques. Inhibition of clathrin-mediated endocytosis by chlorpromazine treatment, as well as clathrin knock-down using siRNA duplexes directed against the clathrin heavy chain, also prevented VSV infection. In combination with previous morphological approaches, these experiments establish clathrin as an essential component needed for endocytosis of VSV.     

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.     

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5'NCR, VP4, VPg, POL, and 3'NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by approximately 10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.     

The adsorption and degradation of foot-and-mouth disease virus by isolated BHK-21 cell plasma membranes

Foot-and-mouth disease virus (FMDV) was examined for its ability to adsorb specifically to plasma membranes isolated from BHK-21 cells. The membranes were prepared by the polyethylene glycol-dextran method, and characterized by increases in specific activity of ouabain-sensitive Na⁺K⁺-ATPase and 5′-nucleotidase, and by enrichment in ³H-fucose over unfractionated homogenates. The membranes adsorbed purified radiolabeled FMDV type A₁₂119 with kinetics characteristic of intact cells. Plasma membranes prepared from cells pretreated with trypsin were unable to adsorb virus. The adsorption of labeled FMDV was inhibited by unlabeled virus. Treatment of virus with trypsin, which cleaves capsid protein 3, greatly reduced its ability to adsorb to both plasma membranes and intact cells. After adsorption of virus to membranes at 4°, subsequent incubation at 37° under physiological conditions resulted in a rapid elution of bound virus in an unmodified form which reached approximately 80% by 1 hr. Incubation of the membrane-virus complex at 33° under low-salt conditions degraded the virus particles to intact and fragmented viral RNA and 12 S protein subunits. Membrane-induced viral degradation did not occur at 4° but was observed within 5 min after shifting to 33°. Thus, isolated plasma membranes from BHK-21 cells retain receptors for FMDV possessing uncleaved capsid protein 3. In addition, the eclipse and uncoating of FMDV in intact cells probably occurs at the plasma membrane, and in confirmation of previously reported results, the postadsorptive degradation, unlike that of other picornaviruses, occurs in a single step without the production of intermediate subviral particles.     

Foot-and-mouth disease virus (FMDV) leader proteinase negatively regulates the porcine interferon-λ1 pathway

Foot-and-mouth disease is a highly contagious viral disease caused by foot-and-mouth disease virus (FMDV) of wild and domestic cloven-hoofed animals, and causes an economically important disease in the swine industry. In this study, we found that the replication of FMDV in IBRS-2 cells could be significantly inhibited after treatment with the purified recombinant porcine interferon lambda 1 (IFN-λ1), a newly identified type III interferon. However, FMDV could not activate the IFN-λ1 promoter and IFN-λ1 mRNA expression in infected IBRS-2 cells, suggesting that FMDV has evolved mechanisms to interrupt the antiviral function of IFN-λ1. The cause of this inhibition was determined by screening all structural and non-structural proteins of FMDV, and the leader proteinase (L(pro)) was found to exhibit the highest potential to inhibit poly(I:C)-induced IFN-λ1 promoter activity. Further study revealed that the catalytic activity and a SAP (SAF-A/B, Acinus, and PIAS) domain of L(pro) were required for suppressing poly(I:C)-induced IFN-λ1 production. These data suggest that FMDV replication could be inhibited by porcine IFN-λ1, but that the virus has evolved specific mechanisms to inhibit this action.     

Isolation of the glycoprotein of vesicular stomatitis virus and its binding to cell surfaces

The glycoprotein (G) of vesicular stomatitis virus (VSV) was radiolabelled, extracted and purified so that its potential interaction with host cell surfaces could be studied. When BHK-21 cells were incubated with the radiolabelled virus glycoprotein, the virus component rapidly attached to the cell surface. The attachment was shown to be temperature-dependent adn saturated at approx. 3 X 10(5) molecules/cell. The omission of Mg2+ or Ca2+ from the incubation medium had little effect on the glycoprotein binding. Treating the isolated G protein and intact virions with neuraminidase did not significantly decrease their binding to BHK-21 cells. Pre-incubating cells with trypsin did not decrease the attachment of VSV virions nor the binding of purified G protein. Treating cells with phospholipase A or phospholipase C suggested that the binding of the glycoprotein and the intact virion might have been dissimilar. Unlabelled glycoprotein competitively inhibited binding of the labelled molecules although the presence of intact virions did not inhibit attachment of the G protein. Likewise, saturating amounts of the glycoprotein did not decrease binding of VSV to BHK-21 cells. These observations suggested that either the isolated glycoprotein bound to cell surface components that were distinct from the virion receptor or that the manner of the purified glycoprotein attachment differed from the G protein still associated with the intact virion. Chemical crosslinking and diagonal two-dimensional gel electrophoresis were used to identify and to compare the cell surface components responsible for glycoprotein and virion attachment.     

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.     

Biochemical studies on the entry of sindbis virus into BHK-21 cells and the effect of NH4Cl

The importance of a lysosomal pathway for the infection of cells by Sindbis virus was studied. BHK-21 cells were infected with Sindbis virus radiolabeled with l-[³⁵S]methionine and the fate of viral proteins was followed up to 120 min postinfection. Virus was transiently associated with a subcellular fraction enriched in lysosomes (identified by four-to to efold enrichment in the enzyme marker acid phosphatase). Capsid proteins were preferentially released into the cytoplasm after 60 min of infection and significant degradation of viral proteins occurred between 1 and 2 hr after infection. NH₄Cl, a weak base thought to increase the intralysosomal pH and impair lysosomal function, blocked an early event between the binding of virus on the cell surface and penetration into lysosomes. This contradicts the presumed lysosomal site of action of this chemical on Sindbis virus infection and is consistent with an effect on receptor clustering into coated pits or the delivery of endocytic vesicle content to lysosomes. The results of these studies indicate the involvement of intracellular vesicles, either lysosomes or vesicles of similar size, and thus a receptor-mediated endocytic pathway (viropexis) as the primary mechanism for entry of Sindbis virus into BHK-21 cells.     

A second RGD motif in the 1D capsid protein of a SAT1 type foot-and-mouth disease virus field isolate is not essential for attachment to target cells

The amino acid sequence motif Arg-Gly-Asp (RGD), located in the surface-exposed betaG-betaH loop of the 1D protein of different serotypes and subtypes of foot-and-mouth disease virus (FMDV), is highly conserved and participates in binding of FMDV to susceptible cells. Previous sequence analyses of the 1D-encoding region of a FMDV serotype SAT1 field isolate from Namibia (NAM/307/98) indicated the presence of a second RGD motif upstream of the conserved betaG-betaH loop RGD. The role of these RGD sequences in virus infection was investigated by mutating the betaG-betaH loop RGD to a KGE tripeptide, using a genome-length infectious chimeric cDNA clone. Although the infectivity of the derived mutant viruses for baby hamster kidney cells (BHK-21) was lost, subsequent replacement of the KGE sequence with RGD in the mutant cDNA clone led to recovery of infectious viruses. Furthermore, viral RNA replication could be demonstrated with the genetically engineered mutant and non-mutant viruses. The presence of virus particles in the transfected cells could be also demonstrated by electron microscopy. These results demonstrate that, in contrast to the betaG-betaH loop RGD motif, the second RGD sequence in the capsid protein 1D of NAM/307/98 does not function as a ligand for receptor binding in BHK-21 cells.