Mutation of FMDV Lpro H138 residue drives viral attenuation in cell culture and in vivo in swine.

The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) is a papain like protease that cleaves the viral polyprotein and several host factors affecting host cell translation and induction of innate immunity. Introduction of Lpro mutations ablating catalytic activity is not tolerated by the virus, however, complete coding sequence deletion or introduction of targeted amino acid substitutions can render viable progeny. In proof-of-concept studies, we have previously identified and characterized FMDV Lpro mutants that are attenuated in cell culture and in animals, while retaining their capacity for inducing a strong adaptive immunity. By using molecular modeling, we have now identified a His residue (H138), that resides outside the substrate binding and catalytic domain, and is highly conserved across serotypes. Mutation of H138 renders possible FMDV variants of reduced virulence in vitro and in vivo. Kinetics studies showed that FMDV A12-LH138L mutant replicates similarly to FMDV A12-wild type (WT) virus in cells that do not offer immune selective pressure, but attenuation is observed upon infection of primary or low passage porcine epithelial cells. Western blot analysis on protein extracts from these cells, revealed that while processing of translation initiation factor eIF-4G was slightly delayed, no degradation of innate sensors or effector molecules such as NF-κB or G3BP2 was observed, and higher levels of interferon (IFN) and IFN-stimulated genes (ISGs) were induced after infection with A12-LH138L as compared to WT FMDV. Consistent with the results in porcine cells, inoculation of swine with this mutant resulted in a mild, or in some cases, no clinical disease but induction of a strong serological adaptive immune response. These results further support previous evidence that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.

Duration of protection and humoral immunity induced by an adenovirus-vectored subunit vaccine for foot-and-mouth disease (FMD) in Holstein steers.

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven hooved animals that continues to cause economic disruption in both endemic countries or when introduced into a formally FMD free country. Vaccines that protect against clinical disease and virus shedding are critical to control FMD. The replication deficient human adenovirus serotype 5 (Ad5) vaccine vector expressing empty FMD virus (FMDV) capsid, AdtFMD, is a promising new vaccine platform. With no shedding or spreading of viral vector detected in field trials, this vaccine is very safe to manufacture, as there is no requirement for high containment faciitites. Here, we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9 months following a single vaccination with the commercial AdtFMD vaccine, provisionally licensed for cattle in the United States. Further, we tested the effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome and humoral immunity. Results demonstrate that a single dose vaccination in cattle with the commercial vaccine vector expressing capsid proteins of the FMDV strain A24 Cruzeiro (Adt.A24), induced protection against clinical FMD at 6 months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9 months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Anti-FMDV antibody secreting cells are detected in peripheral blood early following vaccination, but are absent after 28 days post-vaccination. Thus, the decay in antibody mediated immunity over time is likely a function of FMDV-specific antibody half-life. These data reveal the short time span of anti-FMDV antibody secreting cells (ASCs) and important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.

Pathogenesis and micro-anatomic characterization of a cell-adapted mutant foot-and-mouth disease virus in cattle: Impact of the Jumonji C-domain containing protein 6 (JMJD6) and route of inoculation.

A companion study reported Jumonji-C domain containing protein 6 (JMJD6) is involved in an integrin- and HS-independent pathway of FMDV infection in CHO cells. JMJD6 localization was investigated in animal tissues from cattle infected with either wild type A24-FMDV (A24-WT) or mutant FMDV (JMJD6-FMDV) carrying E95K/S96L and RGD to KGE mutations in VP1. Additionally, pathogenesis of mutant JMJD6-FMDV was investigated in cattle through aerosol and intraepithelial lingual (IEL) inoculation. Interestingly, JMJD6-FMDV pathogenesis was equivalent to A24-WT administered by IEL route. In contrast, JMJD6-FMDV aerosol-infected cattle did not manifest signs of FMD and animals showed no detectable viremia. Immunofluorescent microscopy of post-mortem tissue revealed JMJD6-FMDV exclusively co-localized with JMJD6(+) cells while A24-WT was occasionally found in JMJD6(+) cells. In vitro, chemical uptake inhibitors demonstrated JMJD6-FMDV entered cells via clathrin-coated pit endocytosis. In vivo, JMJD6-FMDV exhibited preference for JMJD6(+) cells, but availability of this alternative receptor likely depends on route of inoculation.

Role of Jumonji C-domain containing protein 6 (JMJD6) in infectivity of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) utilizes four integrins (αvß1, αvß3, αvß6, and αvß8) as its primary cell receptor. During cell culture propagation, FMDV frequently adapts to use heparan sulfate (HS), and rarely utilizes an unidentified third receptor. Capsid mutations acquired by a soluble integrin resistant FMDV cause (i) adaptation to CHO-677 cells (ii) increased affinity to membrane-bound Jumonji C-domain containing protein 6 (JMJD6) (iii) induced JMJD6 re-localization from the cell surface and cytoplasm to the nucleus. Interestingly, pre-treatment of cells with N- and C-terminal JMJD6 antibodies or by simultaneous incubation of mutant virus with soluble JMJD6 (but not by treatment with HS or αvß6) impaired virus infectivity in cultured cells. JMJD6 and mutant virus co-purified by reciprocal co-immunoprecipitation. Molecular docking predictions suggested JMJD6 C-terminus interacts with mutated VP1 capsid protein. We conclude when specific VP1 mutations are displayed, JMJD6 contributes to FMDV infectivity and may be a previously unidentified FMDV receptor.

Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

BACKGROUND: The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3C(pro), and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated. METHODS: Sam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored. RESULTS: FMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3D(pol) and 3C(pro) consistent with early observations of FMDV 3C(pro)-induced cleavage of Sam68. CONCLUSION: We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3C(pro) and 3D(pol) and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3C(pro) and 3D(pol).

Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1 G-H loop as a new tool for studying FMDV pathogenesis.

Foot-and-mouth disease virus (FMDV) VP1 G-H loop contains the major antigenic site. By replacing the sequence upstream of the RGD motif with a FLAG epitope, a marker virus for pathogenesis studies was generated. In cell culture, the recombinant virus containing FLAG (A24-FLAG) exhibited similar plaque phenotypes and growth kinetics to parental virus. A24-FLAG was distinguished, neutralized, and immunoprecipitated by FLAG anti-sera. A24-FLAG infected cattle exhibited FMD and an antibody response similar to parental virus. FLAG epitope stability was confirmed both in vitro and in vivo. Interestingly, no anti-FLAG antibodies were detectable in cattle up to 21 days post-inoculation. A24-FLAG G-H loop modeling suggested FLAG was rendered a cryptic site, inaccessible to the host immune system. These studies demonstrate the FMDV VP1 G-H loop tolerance to substitutions without detriment to pathogenesis and antigenicity. Finally, A24-FLAG manifested virulence in cattle as parental virus, and could be distinguished and tracked by tag-specific anti-sera.

Homology modeling and analysis of structure predictions of the bovine rhinitis B virus RNA dependent RNA polymerase (RdRp).

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D(pol)). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D(pol) using a combination of different computational tools. Model structures of BRBV 3D(pol) were verified for their stereochemical quality and accuracy. The BRBV 3D(pol) structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3D(pol) (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3D(pol) as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3D(pols) is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3D(pol) compared to FMDV 3D(pol), indicate that despite a very high homology to FMDV 3D(pol), BRBV 3D(pol) may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.

The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C(pro) induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5' non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C(pro).

Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase.

BACKGROUND: Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections. METHODOLOGY/PRINCIPAL FINDINGS: Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC(50)s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC(50) = 12 µM and EC(90) = 20 µM). SIGNIFICANCE: We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.

The region between the two polyprotein initiation codons of foot-and-mouth disease virus is critical for virulence in cattle.

To explore the role in viral pathogenesis of the region located between the two functional AUG (inter-AUG) in foot-and-mouth disease virus (FMDV), we derived viruses containing transposon (tn) inserts from a mutagenized cDNA infectious clone of FMDV (pA24-WT). Mutant viruses containing an in-frame 57-nt transposon insertion grew at a slower rate and had a smaller plaque size phenotype than the parental virus (A24-WT). A mutant virus containing a 51-nt deletion in inter-AUG had a similar phenotype in cell culture to that of A24-WT. When tested by aerosol inoculation in cattle (3 animals per virus), the deletion mutant was fully virulent as was A24-WT. Mutant viruses containing insertions in inter-AUG did not cause clinical disease or viremia. However, viruses that partially or totally removed the tn insertion during animal infection reverted to virulence in 2 inoculated steers. Therefore, this study identified inter-AUG as an FMDV viral virulence determinant in cattle infected by aerosol route.

Molecular and phylogenetic analyses of bovine rhinovirus type 2 shows it is closely related to foot-and-mouth disease virus.

Bovine rhinovirus 2 (BRV2), a causative agent of respiratory disease in cattle, is tentatively assigned to the genus Rhinovirus in the family Picornaviridae. A nearly full-length cDNA of the BRV2 genome was cloned and the nucleotide sequence determined. BRV2 possesses a putative leader proteinase, a small 2A protein and a poly(C) tract, which are characteristic of aphthoviruses. Alignment of BRV-2 and FMDV polyproteins showed that 41% of amino acids were identical within the P1 region. Furthermore, 2A, 2C, 3B(3), 3C and 3D proteins are as much as 67%, 52%, 52%, 50%, and 64% identical, respectively. BRV2 leader protein is rapidly released from the viral polyprotein and cleaves eIF4G at a rate similar to FMDV leader proteinase, suggesting a functional relationship between the leader protein in these viruses. The results suggest that BRV2 is closely related to FMDV and should therefore be considered as a new species within the genus Aphthovirus.

Analysis of a foot-and-mouth disease virus type A24 isolate containing an SGD receptor recognition site in vitro and its pathogenesis in cattle.

Foot-and-mouth disease virus (FMDV) initiates infection by binding to integrin receptors via an Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. Following serial passages of a type A(24) Cruzeiro virus (A(24)Cru) in bovine, via tongue inoculation, a virus was generated which contained an SGD sequence in the cell receptor-binding site and expressed a turbid plaque phenotype in BHK-21 cells. Propagation of this virus in these cells resulted in the rapid selection of viruses that grew to higher titers, produced clear plaques, and now contained an RGD sequence in place of the original SGD. To study the role of the SGD sequence in FMDV receptor recognition and bovine virulence, we assembled an infectious cDNA clone of an RGD-containing A(24)Cru and derived mutant clones containing either SGD with a single nucleotide substitution in the R(144) codon or double substitutions at this position to prevent mutation of the S to an R. The SGD viruses grew poorly in BHK-21 cells and stably maintained the sequence during propagation in BHK-21 cells expressing the bovine alpha(V)beta(6) integrin (BHK3-alpha(V)beta(6)), as well as in experimentally infected and contact steers. While all the SGD-containing viruses used only the bovine alpha(V)beta(6) integrin as a cellular receptor with relatively high efficiency, the revertant RGD viruses utilized either the alpha(V)beta(1) or alpha(V)beta(3) bovine integrins with higher efficiency than alpha(V)beta(6) and grew well in BHK-21 cells. Replacing the R at the -1 SGD position with either K or E showed that this residue did not contribute to integrin utilization in vitro. These results illustrate the rapid evolution of FMDV with alteration in receptor specificity and suggest that viruses with sequences other than RGD, but closely related to it, can still infect via integrin receptors and induce and transmit the disease to susceptible animals.

Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells.

Genotype 1a is a most prevalent genotype of hepatitis C virus in North America yet HCV replication has been studied predominantly with genotype 1b subgenomic replicons under neomycin selection in Huh-7 cells. Development of 1a-related dicistronic replicons under neo selection proved difficult and required either "conditioned" Huh-7 cells and/or chimeric genomes harboring pre-engineered adaptive mutations. We report the construction of a novel dicistronic genotype 1a(H77C) replicon expressing the puromycin N-acetyltransferase (PAC) gene as a selectable marker that, without prior introduction of adaptive mutations, allows establishment of puromycin-resistant Huh-7 colonies after transfection of naive Huh-7 cells. The large majority of HCV1a/PAC replicons did not reveal any adaptive mutations on short-term passage of Huh-7 cells. Continued passage led to mutations in the non-structural genes although these mutations did not significantly enhance replication of the original replicon. Transfection with total cellular RNA isolated from HCV1a/PAC replicon-containing cells led to a significant increase in colony-forming ability. The data identify PAC as an efficient selectable marker for studies of HCV replication, which may be useful with different genotypes in different host cell systems.

A "slide-back" mechanism for the initiation of protein-primed RNA synthesis by the RNA polymerase of poliovirus.

Poliovirus RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction can be reproduced in vitro with an assay that utilizes two purified viral proteins, RNA polymerase 3Dpol and viral protein 3CDpro, synthetic VPg, UTP, and Mg2+. The template for the reaction is either poliovirus RNA or transcripts of a small RNA hairpin, termed cre(2C), located in the coding sequence of protein 2CATPase. The products of the reaction are VPgpU and VPgpUpU, the primers used by 3Dpol for RNA synthesis. With mutant template RNAs in this assay we determined the precise initiation site. Our results indicate that 1) 3Dpol does not possess strict specificity toward the nucleotide it links to VPg, 2) A-5 of the conserved 1GXXXAAAXXXXXXA14 sequence in the loop is the template nucleotide for the linkage of both the first and second UMPs to VPg, 3) VPgpUpU is synthesized by a "slide-back" mechanism, and 4) A-6 provides specificity to the reaction during the slide-back step and also modulates the uridylylation reaction. In additional experiments we determined the effect of mutations in the 5AAA7 sequence of cre(2C) on viral growth, RNA replication, and on the activity of the 2CATPase protein. Furthermore, we observed that the spacing between G-1 and A-5 and the size of the loop affect the yield but not the nature of the VPg-linked products.

Functional dissection of a poliovirus cis-acting replication element [PV-cre(2C)]: analysis of single- and dual-cre viral genomes and proteins that bind specifically to PV-cre RNA.

The role of the cis replication element (cre) in the 2C(ATPase) coding region of the poliovirus (PV) genome has been studied with a series of mutants derived from either a PV1 full-length genome or a replicon (P/L) containing the firefly luciferase reporter gene in place of the capsid region. Using the P/L replicon we have inserted cre elements at three different locations in the genome including the 5' nontranslated region and within the open reading frame. The successful recovery of replication of a nonviable P/L (A(5)C) mutant replicon with an artificial cre element as "rescuer," in addition to the results of site-directed mutagenesis and experiments with truncated forms of PV-cre(2C), indicated that (i) the sequence within the upper stem and loop regions contains the minimal cre RNA required for VPg uridylylation in vitro, (ii) the location of the cre RNA in the poliovirus genome is not relevant to RNA infectivity, and (iii) specific binding of 3CD(pro) to PV-cre(2C) occurs within the upper stem region and probably involves several contact residues. The role of a 14-nucleotide conserved "core" sequence among known cre structures in picornaviruses was examined by site-directed mutagenesis of individual nucleotides. In addition to a conserved AAA (4472 to 4474) triplet previously shown to be the primary RNA template for VPg uridylylation by the PV RNA polymerase 3D(pol) (E. Rieder, A. V. Paul, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10371-10380, 2000), we have now shown that important residues (G(4468) and A(4481)) are contained in a predicted internal bulge at the upper stem-loop of PV-cre(2C). We have further demonstrated that the viral proteins 3CD(pro) and 3C(pro) form stable complexes with a transcript PV-cre(2C) RNA that can be considered critical for VPg uridylylation.