Teresa de los Santos

The Leader Proteinase of Foot-and-Mouth Disease Virus Inhibits the Induction of Beta Interferon mRNA and Blocks the Host Innate Immune Response

ABSTRACT We have previously shown that the leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) blocks cap-dependent mRNA translation and that a genetically engineered FMDV lacking the leader proteinase coding region (A12-LLV2) is attenuated in cell culture and susceptible animals. The attenuated phenotype apparently is a consequence of the inability of A12-LLV2 to block the expression of type I interferon (IFN-α/β) protein, resulting in IFN-induced inhibition of FMDV replication. Here we show that in addition to preventing IFN-α/β protein synthesis, Lpro reduces the level of immediate-early induction of IFN-β mRNA and IFN-stimulated gene products such as double-stranded RNA-dependent protein kinase R (PKR), 2′,5′-oligoadenylate synthetase, and Mx1 mRNAs in swine cells. Down-regulation of cellular PKR by RNA interference did not affect wild-type virus yield but resulted in a higher yield of A12-LLV2, indicating a direct role of PKR in controlling FMDV replication in the natural host. The observation that Lpro controls the transcription of genes involved in innate immunity reveals a novel role of this protein in antagonizing the cellular response to viral infection.

Degradation of Nuclear Factor Kappa B during Foot-and-Mouth Disease Virus Infection

ABSTRACT We have previously shown that the leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) interferes with the innate immune response by blocking the translation of interferon (IFN) protein and by reducing the immediate-early induction of beta IFN mRNA and IFN-stimulated genes. Here, we report that Lpro regulates the activity of nuclear factor κB (NF-κB). Analysis of NF-κB-dependent reporter gene expression in BHK-21 cells demonstrated that infection with wild-type (WT) virus has an inhibitory effect compared to infection with a genetically engineered mutant lacking the leader coding region. The expression of endogenous NF-κB-dependent genes tumor necrosis factor alpha and RANTES is also reduced in WT virus-infected primary porcine cells. This inhibitory effect is neither the result of a decrease in the level of the mRNA of p65/RelA, a subunit of NF-κB, nor a block on the nuclear translocation of p65/RelA, but instead appears to be a consequence of the degradation of accumulated p65/RelA. Viral Lpro is localized to the nucleus of infected cells, and there is a correlation between the translocation of Lpro and the decrease in the amount of nuclear p65/RelA. By using a recombinant cardiovirus expressing Lpro, we demonstrate that the disappearance of p65/RelA takes place in the absence of any other FMDV product. The observation that Lpro disrupts the integrity of NF-κB suggests a global mechanism by which FMDV antagonizes the cellular innate immune and inflammatory responses to viral infection.

Enhanced Antiviral Activity against Foot-and-Mouth Disease Virus by a Combination of Type I and II Porcine Interferons

ABSTRACT Previously, we showed that type I interferon (alpha/beta interferon [IFN-α/β]) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture, and swine inoculated with 109 PFU of human adenovirus type 5 expressing porcine IFN-α (Ad5-pIFN-α) were protected when challenged 1 day later. In this study, we found that type II pIFN (pIFN-γ) also has antiviral activity against FMDV in cell culture and that, in combination with pIFN-α, it has a synergistic antiviral effect. We also observed that while each IFN alone induced a number of IFN-stimulated genes (ISGs), the combination resulted in a synergistic induction of some ISGs. To extend these studies to susceptible animals, we inoculated groups of swine with a control Ad5, 108 PFU of Ad5-pIFN-α, low- or high-dose Ad5-pIFN-γ, or a combination of Ad5-pIFN-α and low- or high-dose Ad5-pIFN-γ and challenged all groups with FMDV 1 day later. The control group and the groups inoculated with either Ad5-pIFN-α or a low dose of Ad5-pIFN-γ developed clinical disease and viremia. However, the group that received the combination of both Ad5-IFNs with the low dose of Ad5-pIFN-γ was completely protected from challenge and had no viremia. Similarly the groups inoculated with the combination of Ad5s with the higher dose of Ad5-pIFN-γ or with only high-dose Ad5-pIFN-γ were protected. The protected animals did not develop antibodies against viral nonstructural (NS) proteins, while all infected animals were NS protein seropositive. No antiviral activity or significant levels of IFNs were detected in the protected groups, but there was an induction of some ISGs. The results indicate that the combination of type I and II IFNs act synergistically to inhibit FMDV replication in vitro and in vivo.

Evading the host immune response: how foot‐and‐mouth disease virus has become an effective pathogen

Foot-and-mouth disease virus (FMDV) causes an economically devastating disease of cloven-hoofed animals. In this review, we discuss the mechanisms FMDV has evolved to counteract the host innate and adaptive immune responses and the role of viral proteins in this process. The viral leader proteinase, L pro, limits the host innate response by inhibiting the induction of interferon beta (IFN beta) mRNA and blocking host cell translation. A second viral proteinase, 3C pro, may affect host cell transcription because it cleaves histone H3. Viral protein 2B in conjunction with 2C or their precursor 2BC inhibits protein trafficking through the endoplasmic reticulum and Golgi apparatus. A decrease in surface expression of major histocompatibility class I molecules during FMDV infection suggests that 2B, 2C and/or 2BC may be involved in delaying the initiation of the host adaptive immune response and also adversely affect the secretion of induced signaling molecules. FMDV also causes a transient lymphopenia in swine, but the mechanism involved is not understood nor have any viral protein(s) been implicated. Furthermore, the interaction of FMDV with various cells in the immune system including lymphocytes and dendritic cells and the possible role of apoptosis and autophagy in these interactions are discussed.

Interferon-Induced Protection against Foot-and-Mouth Disease Virus Infection Correlates with Enhanced Tissue-Specific Innate Immune Cell Infiltration and Interferon-Stimulated Gene Expression

ABSTRACT Previously, we demonstrated that type I interferon (IFN-α/β) or a combination of IFN-α/β and type II IFN (IFN-γ) delivered by a replication-defective human adenovirus 5 (Ad5) vector protected swine when challenged 1 day later with foot-and-mouth disease virus (FMDV). To gain a more comprehensive understanding of the mechanism of protection induced by IFNs, we inoculated groups of six swine with Ad5-vectors containing these genes, challenged 1 day later and euthanized 2 animals from each group prior to (1 day postinoculation [dpi]) and at 1 (2 dpi) and 6 days postchallenge (7 dpi). Blood, skin, and lymphoid tissues were examined for IFN-stimulated gene (ISG) induction and infiltration by innate immune cells. All IFN-inoculated animals had delayed and decreased clinical signs and viremia compared to the controls, and one animal in the IFN-α treated group did not develop disease. At 1 and 2 dpi the groups inoculated with the IFNs had increased numbers of dendritic cells and natural killer cells in the skin and lymph nodes, respectively, as well as increased levels of several ISGs compared to the controls. In particular, all tissues examined from IFN-treated groups had significant upregulation of the chemokine 10-kDa IFN-γ-inducible protein 10, and preferential upregulation of 2′,5′-oligoadenylate synthetase, Mx1, and indoleamine 2,3-dioxygenase. There was also upregulation of monocyte chemotactic protein 1 and macrophage inflammatory protein 3α in the skin. These data suggest that there is a complex interplay between IFN-induced immunomodulatory and antiviral activities in protection of swine against FMDV.

Bovine Type III Interferon Significantly Delays and Reduces the Severity of Foot-and-Mouth Disease in Cattle

ABSTRACT Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-λ, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-λ3 (boIFN-λ3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-λ3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-λ3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-λ3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle.

A Conserved Domain in the Leader Proteinase of Foot-and-Mouth Disease Virus Is Required for Proper Subcellular Localization and Function

ABSTRACT The leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) is involved in antagonizing the innate immune response by blocking the expression of interferon (IFN) and by reducing the immediate-early induction of IFN-β mRNA and IFN-stimulated genes. In addition to its role in shutting off cap-dependent host mRNA translation, Lpro is associated with the degradation of the p65/RelA subunit of nuclear factor κB (NF-κB). Bioinformatics analysis suggests that Lpro contains a SAP (for SAF-A/B, Acinus, and PIAS) domain, a protein structure associated in some cases with the nuclear retention of molecules involved in transcriptional control. We have introduced a single or a double mutation in conserved amino acid residues contained within this domain of Lpro. Although three stable mutant viruses were obtained, only the double mutant displayed an attenuated phenotype in cell culture. Indirect immunofluorescence analysis showed that Lpro subcellular distribution is altered in cells infected with the double mutant virus. Interestingly, nuclear p65/RelA staining disappeared from wild-type (WT) FMDV-infected cells but not from double mutant virus-infected cells. Consistent with these results, NF-κB-dependent transcription was not inhibited in cells infected with double mutant virus in contrast to cells infected with WT virus. However, degradation of the translation initiation factor eIF-4G was very similar for both the WT and the double mutant viruses. Since Lpro catalytic activity was demonstrated to be a requirement for p65/RelA degradation, our results indicate that mutation of the SAP domain reveals a novel separation-of-function activity for FMDV Lpro.

Porcine type I interferon rapidly protects swine against challenge with multiple serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Current inactivated vaccines require approximately 7 days to induce protection, but before this time vaccinated animals remain susceptible to disease. Previously, we demonstrated that intramuscular (IM) inoculation of a replication-defective human adenovirus type 5 (Ad5) vector containing a porcine interferon α gene (pIFNα) can protect swine challenged 1 day later by intradermal (ID) injection with FMDV A24 Cruzeiro from both clinical disease and virus replication. To extend these studies to other FMDV serotypes, we demonstrated the effectiveness of Ad5-pIFNα against ID challenge with O1 Manisa and Asia-1 and against A24 Cruzeiro in a direct contact challenge model. We also showed that an Ad5 vector containing the pIFNβ gene can protect swine against ID challenge with A24 Cruzeiro. Further, IM inoculation of a 10-fold lower dose of Ad5-pIFNα at 4 sites in the neck compared with 1 site in the hind limb can protect swine against ID challenge. These studies demonstrate the ability of Ad5-delivered type I IFN to rapidly protect swine against several FMDV serotypes and suggest that various modifications of this approach may enable this strategy to be successfully used in other FMD susceptible species.

Antiviral Activity of Bovine Type III Interferon Against Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is one of the most serious threats to the livestock industry. Despite the availability of a vaccine, recent outbreaks in disease-free countries have demonstrated that development of novel FMD control strategies is imperative. Here we report the identification and characterization of bovine (bo) interferon lambda 3 (IFN-λ3), a member of the type III IFN family. Expression of boIFN-λ3 using a replication-defective human adenovirus type 5 vector (Ad5-boIFN-λ3) yielded a glycosylated secreted protein with antiviral activity against FMD virus (FMDV) and vesicular stomatitis virus in bovine cell culture. Inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and up-regulation of IFN stimulated gene expression in multiple tissues susceptible to FMDV infection. Our results demonstrate that the type III IFN family is conserved in bovines and boIFN-λ3 has potential for further development as a biotherapeutic candidate to inhibit FMDV or other viruses in cattle.

Novel Antiviral Therapeutics to Control Foot-and-Mouth Disease

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8 mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8 mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24 h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.