Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase

We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-alpha/beta). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-alpha or -beta individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-dependent protein kinase (PKR). In support of these findings, treatment of porcine and bovine cells with 2-aminopurine, an inhibitor of PKR, increased the yield of virus 8.8- and 11.2-fold, respectively, compared to that in untreated infected cells. In addition, results of FMDV infection in mouse embryonic fibroblast cells derived from gene knockout mice lacking the gene for RNase L(-/-) or PKR(-/-) or both indicated an important role for PKR in the inhibition of FMDV replication.     

The foot-and-mouth disease virus leader proteinase gene is not required for viral replication.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase has only two known functions: (i) autocatalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. Cleavage of p220 appears to be important for picornavirus replication, since rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220. To explore the role of L in FMDV replication, we generated synthetic FMDV genomes lacking the L gene and tested their viability in cells. Genomes were constructed with the N-terminal Gly codon of VP4 positioned directly following either the first (Lab) or second (Lb) Met codon of the L protein. Cells transfected with synthetic RNAs lacking L and initiating with the Lab Met codon failed to produce viable virus, but cells transfected with RNAs that utilized the second AUG to drive translation of the viral polyprotein produced viable viruses. These leader-deleted viruses produced plaques on BHK cells that were slightly smaller than those produced by wild-type (WT) virus, grew to slightly lower titers than WT virus in BHK cells, shut off host protein synthesis more slowly than WT virus, and were slightly attenuated in mice. These studies indicate that the L proteinase is not essential for FMDV replication and show that in the cells and animals tested the L gene has a limited effect on virus replication.     

The leader proteinase of foot-and-mouth disease virus negatively regulates the type I interferon pathway by acting as a viral deubiquitinase

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is a papain-like proteinase that plays an important role in FMDV pathogenesis. Previously, it has been shown that L(pro) is involved in the inhibition of the type I interferon (IFN) response by FMDV. However, the underlying mechanisms remain unclear. Here we demonstrate that FMDV Lb(pro), a shorter form of L(pro), has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His148) are highly conserved in FMDV Lb(pro) of all seven serotypes and that the topology of FMDV Lb(pro) is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and to that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral DUB. Both purified Lb(pro) protein and in vivo ectopically expressed Lb(pro) removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. Furthermore, Lb(pro) significantly inhibited ubiquitination of retinoic acid-inducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6), and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lb(pro) that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lb(pro) as well as its ability to block signaling to the IFN-β promoter. Collectively, these results demonstrate that FMDV Lb(pro) possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.     

Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.     

Residue L143 of the foot-and-mouth disease virus leader proteinase is a determinant of cleavage specificity

The foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) self-processes inefficiently at the L(pro)/VP4 cleavage site LysLeuLys*GlyAlaGly (* indicates cleaved peptide bond) when the leucine at position P2 is replaced by phenylalanine. Molecular modeling and energy minimization identified the L(pro) residue L143 as being responsible for this discrimination. The variant L(pro) L143A self-processed efficiently at the L(pro)/VP4 cleavage site containing P2 phenylalanine, whereas the L143M variant did not. L(pro) L143A self-processing at the eIF4GII sequence AspPheGly*ArgGlnThr was improved but showed more-extensive aberrant processing. Residue 143 in L(pro) is occupied only by leucine and methionine in all sequenced FMDV serotypes, implying that these bulky side chains are one determinant of the restricted specificity of L(pro).     

Eukaryotic initiation factor 4GI is a poor substrate for HIV-1 proteinase

Eukaryotic initiation factor (eIF) 4GI is efficiently cleaved during picornaviral replication. eIF4GI processing has also recently been observed during HIV-1 replication. We have compared the efficiency of eIF4GI proteolysis in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (L(pro)) or the HIV-1 proteinase (HIV-1(pro)). L(pro) cleaved 50% eIF4GI within 12 min whereas HIV-1(pro) required 4 h; the concentrations were 2 pg/microl (0.1 nM) for L(pro) and 60 pg/microl (2.66 nM) for HIV-1(pro). HIV-1(pro) processing of eIF4GI is therefore not quantitatively analogous to that of L(pro), suggesting that the primary function of eIF4GI cleavage in HIV-1 replication may not be protein synthesis inhibition.     

The binding of foot-and-mouth disease virus leader proteinase to eIF4GI involves conserved ionic interactions

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) initially cleaves itself from the polyprotein. Subsequently, L(pro) cleaves the host proteins eukaryotic initiation factor (eIF) 4GI and 4GII. This prevents protein synthesis from capped cellular mRNAs; the viral RNA is still translated, initiating from an internal ribosome entry site. L(pro) cleaves eIF4GI between residues G674 and R675. We showed previously, however, that L(pro) binds to residues 640-669 of eIF4GI. Binding was substantially improved when the eIF4GI fragment contained the eIF4E binding site and eIF4E was present in the binding assay. L(pro) interacts with eIF4GI via residue C133 and residues 183-195 of the C-terminal extension. This binding domain lies about 25 A from the active site. Here, we examined the binding of L(pro) to eIF4GI fragments generated by in vitro translation to narrow the binding site down to residues 645-657 of human eIF4GI. Comparison of these amino acids with those in human eIF4GII as well as with sequences of eIF4GI from other organisms allowed us to identify two conserved basic residues (K646 and R650). Mutation of these residues was severely detrimental to L(pro) binding. Similarly, comparison of the sequence between residues 183 and 195 of L(pro) with those of other FMDV serotypes and equine rhinitis A virus showed that acidic residues D184 and E186 were highly conserved. Substitution of these residues in L(pro) significantly reduced eIF4GI binding and cleavage without affecting self-processing. Thus, FMDV L(pro) has evolved a domain that specifically recognizes a host cell protein.     

Identification and characterization of interferon-induced proteins that inhibit alphavirus replication

Alpha/beta interferon (IFN-alpha/beta) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-alpha/beta can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized had potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.     

Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice

We previously showed that the intrahepatic induction of cytokines such as alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibits hepatitis B virus (HBV) replication noncytopathically in the livers of transgenic mice. The intracellular pathway(s) responsible for this effect is still poorly understood. To identify interferon (IFN)-inducible intracellular genes that could play a role in our system, we crossed HBV transgenic mice with mice deficient in IFN regulatory factor 1 (IRF-1), the double-stranded RNA-activated protein kinase (PKR), or RNase L (RNase L) (IRF-1(-/-), PKR(-/-), or RNase L(-/-) mice, respectively), three well-characterized IFN-inducible genes that mediate antiviral activity. We showed that unmanipulated IRF-1(-/-) or PKR(-/-) transgenic mice replicate HBV in the liver at slightly higher levels than the respective controls, suggesting that both IRF-1 and PKR individually appear to mediate signals that modulate HBV replication under basal conditions. These same animals were responsive to the antiviral effects of the IFN-alpha/beta inducer poly(I-C) or recombinant murine IFN-gamma, suggesting that under these conditions, either the IRF-1 or the PKR genes can mediate the antiviral activity of the IFNs or other IFN-inducible genes mediate the antiviral effects. Finally, RNase L(-/-) transgenic mice were undistinguishable from controls under basal conditions and after poly(I-C) or IFN-gamma administration, suggesting that RNase L does not modulate HBV replication in this model.     

PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.     

Constitutive expression of alpha interferon by skin dendritic cells confers resistance to infection by foot-and-mouth disease virus

The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-beta) mRNA but not IFN-alpha mRNA. However, these DC secreted both IFN-alpha and IFN-beta proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-alpha/beta, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-alpha protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-alpha in uninfected animals. This is the first demonstration of the constitutive expression of IFN-alpha in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.     

Sindbis virus translation is inhibited by a PKR/RNase L-independent effector induced by alpha/beta interferon priming of dendritic cells

The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.     

Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro

Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.