Suppress Host Innate Immune Responses Research Articles

The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKα and IKKβ through the recruitment of TOLLIP.

The multigene family genes (MGFs) in the left variable region (LVR) of the African swine fever virus (ASFV) genome have been reported to be involved in viral replication in primary porcine alveolar macrophages (PAMs) and virulence in pigs. However, the exact functions of key MGFs in the LVR that regulate the replication and virulence of ASFV remain unclear. In this study, we identified the MGF300-2R gene to be critical for viral replication in PAMs by deleting different sets of MGFs in the LVR from the highly virulent strain ASFV HLJ/18 (ASFV-WT). The ASFV mutant lacking the MGF300-2R gene (Del2R) showed a 1-log reduction in viral titer, and induced higher IL-1β and TNF-α production in PAMs than did ASFV-WT. Mechanistically, the MGF300-2R protein was found to interact with and degrade IKKα and IKKβ via the selective autophagy pathway. Furthermore, we showed that MGF300-2R promoted the K27-linked polyubiquitination of IKKα and IKKβ, which subsequently served as a recognition signal for the cargo receptor TOLLIP-mediated selective autophagic degradation. Importantly, Del2R exhibited a significant reduction in both replication and virulence compared with ASFV-WT in pigs, likely due to the increased IL-1β and TNF-α, indicating that MGF300-2R is a virulence determinant. These findings reveal that MGF300-2R suppresses host innate immune responses by mediating the degradation of IKKα and IKKβ, which provides clues to paving the way for the rational design of live attenuated vaccines to control ASF.

Pulmonary Expression of Interleukin-17 Contributes to Neutrophil Infiltration into the Lungs during Pneumonic Plague.

Inhalation of respiratory droplets infected with Yersinia pestis results in a rapidly progressing and lethal necrotic pneumonia called primary pneumonic plague. Disease manifests as biphasic, with an initial preinflammatory phase with rapid bacterial replication in the lungs absent readily detectable host immune responses. This is followed by the onset of a proinflammatory phase that sees the dramatic upregulation of proinflammatory cytokines and extensive neutrophil accumulation in the lungs. The plasminogen activator protease (Pla) is an essential virulence factor that is responsible for survival of Y. pestis in the lungs. Our lab recently showed that Pla functions as an adhesin that promotes binding to alveolar macrophages to facilitate translocation of effector proteins called Yops into the cytosol of target host cells via a type 3 secretion system (T3SS). Loss of Pla-mediated adherence disrupted the preinflammatory phase of disease and resulted in early neutrophil migration to the lungs. While it is established that Yersinia broadly suppresses host innate immune responses, it is not clear precisely which signals need to be inhibited to establish a preinflammatory stage of infection. Here, we show that early Pla-mediated suppression of Interleukin-17 (IL-17) expression in alveolar macrophages and pulmonary neutrophils limits neutrophil migration to the lungs and aids in establishing a preinflammatory phase of disease. In addition, IL-17 ultimately contributes to neutrophil migration to the airways that defines the later proinflammatory phase of infection. These results suggest that the pattern of IL-17 expression contributes to the progression of primary pneumonic plague.

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease.

As the pathogen of COVID‐19, SARS‐CoV‐2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain‐like protease or PLPro. It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS‐CoV‐2 infection. To repurpose drugs for PLPro, we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLPro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PLPro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2‐043, TCID, DUB‐IN‐1, DUB‐IN‐3, PR‐619, and S130 with an IC50 value below 40 μM and four inhibitors GRL0617, SJB2‐043, TCID, and PR‐619 with an IC50 value below 10 μM. Among four inhibitors with an IC50 value below 10 μM, SJB2‐043 is the most unique in that it does not fully inhibit PLPro but has a noteworthy IC50 value of 0.56 μM. SJB2‐043 likely binds to an allosteric site of PLPro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID‐19 drug repurposing by targeting PLPro holds promise, but in‐depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.

The Phytophthora effector Avh241 interacts with host NDR1-like proteins to manipulate plant immunity.

Plant pathogens rely on effector proteins to suppress host innate immune responses and facilitate colonization. Although the Phytophthora sojae RxLR effector Avh241 promotes Phytophthora infection, the molecular basis of Avh241 virulence remains poorly understood. Here we identified non-race specific disease resistance 1 (NDR1)-like proteins, the critical components in plant effector-triggered immunity (ETI) responses, as host targets of Avh241. Avh241 interacts with NDR1 in the plasma membrane and suppresses NDR1-participated ETI responses. Silencing of GmNDR1s increases the susceptibility of soybean to P. sojae infection, and overexpression of GmNDR1s reduces infection, which supports its positive role in plant immunity against P. sojae. Furthermore, we demonstrate that GmNDR1 interacts with itself, and Avh241 probably disrupts the self-association of GmNDR1. These data highlight an effective counter-defense mechanism by which a Phytophthora effector suppresses plant immune responses, likely by disturbing the function of NDR1 during infection.

An Evaluation of the Fasciola hepatica miRnome Predicts a Targeted Regulation of Mammalian Innate Immune Responses.

Understanding mechanisms by which parasitic worms (helminths) control their hosts’ immune responses is critical to the development of effective new disease interventions. Fasciola hepatica, a global scourge of humans and their livestock, suppresses host innate immune responses within hours of infection, ensuring that host protective responses are quickly incapacitated. This allows the parasite to freely migrate from the intestine, through the liver to ultimately reside in the bile duct, where the parasite establishes a chronic infection that is largely tolerated by the host. The recent identification of micro(mi)RNA, small RNAs that regulate gene expression, within the extracellular vesicles secreted by helminths suggest that these non-coding RNAs may have a role in the parasite-host interplay. To date, 77 miRNAs have been identified in F. hepatica comprising primarily of ancient conserved species of miRNAs. We hypothesized that many of these miRNAs are utilized by the parasite to regulate host immune signaling pathways. To test this theory, we first compiled all of the known published F. hepatica miRNAs and critically curated their sequences and annotations. Then with a focus on the miRNAs expressed by the juvenile worms, we predicted gene targets within human innate immune cells. This approach revealed the existence of targets within every immune cell, providing evidence for the universal management of host immunology by this parasite. Notably, there was a high degree of redundancy in the potential for the parasite to regulate the activation of dendritic cells, eosinophils and neutrophils, with multiple miRNAs predicted to act on singular gene targets within these cells. This original exploration of the Fasciola miRnome offers the first molecular insight into mechanisms by which F. hepatica can regulate the host protective immune response.

Mce1C and Mce1D facilitate N. farcinica invasion of host cells and suppress immune responses by inhibiting innate signaling pathways

The mammalian cell entry (Mce) family of proteins consists of invasin-like membrane-associated proteins. The roles of Mce1C and Mce1D proteins in host–pathogen interactions have not been investigated. In this study, we demonstrate that Mce1C and Mce1D protein is localized in the cell wall fraction of N. farcinica. Both N. farcinica Mce1C and Mce1D proteins are expressed at the level of protein and mRNA and elicit antibody responses during infection. Mce1C and Mce1D facilitate the internalization of Escherichia coli expressing Mce1C protein or latex beads coated with Mce1D protein by HeLa cells, respectively. We further demonstrate that Mce1C and Mce1D can suppress the secretion of the proinflammatory factors TNF-α and IL-6 in macrophages infected with Mycobacterium smegmatis expressing Mce1C or Mce1D and promote the survival of M. smegmatis expressing Mce1C or Mce1D in macrophages. In addition, Mce1C and Mce1D supress the activation of the NF-κB and MAPK signaling pathways by blocking the phosphorylation of AKT, P65, ERK1/2, JNK, or P38 in macrophages. These findings suggest that Mce1C and Mce1D proteins facilitate N. farcinica invasion of HeLa cells and suppress host innate immune responses by manipulating NF-κB and MAPK signaling pathways, which may provide a target for N. farcinica treatment.

Effect of the Viral Hemorrhagic Septicemia Virus Nonvirion Protein on Translation via PERK-eIF2α Pathway.

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.

Blood-stage malaria parasites manipulate host innate immune responses through the induction of sFGL2.

Malaria parasites suppress host immune responses to facilitate their survival, but the underlying mechanism remains elusive. Here, we found that blood-stage malaria parasites predominantly induced CD4+Foxp3+CD25+ regulatory T cells to release soluble fibrinogen-like protein 2 (sFGL2), which substantially enhanced the infection. This was attributed to the capacity of sFGL2 to inhibit macrophages from releasing monocyte chemoattractant protein-1 (MCP-1) and to sequentially reduce the recruitment of natural killer/natural killer T cells to the spleen and the production of interferon-γ. sFGL2 inhibited c-Jun N-terminal kinase phosphorylation in the Toll-like receptor 2 signaling pathway of macrophages dependent on FcγRIIB receptor to release MCP-1. Notably, sFGL2 were markedly elevated in the sera of patients with malaria, and recombinant FGL2 substantially suppressed Plasmodium falciparum from inducing macrophages to release MCP-1. Therefore, we highlight a previously unrecognized immune suppression strategy of malaria parasites and uncover the fundamental mechanism of sFGL2 to suppress host innate immune responses.

A Dual-Functioning 5ʹ-PPP-NS1shRNA that Activates a RIG-I Antiviral Pathway and Suppresses Influenza NS1

Retinoic acid-inducible gene-I (RIG-I) is a cytosolic pathogen sensor that is crucial against a number of viral infections. Many viruses have evolved to inhibit pathogen sensors to suppress host innate immune responses. In the case of influenza, nonstructural protein 1 (NS1) suppresses RIG-I function, leading to viral replication, morbidity, and mortality. We show that silencing NS1 with in-vitro-transcribed 5′-triphosphate containing NS1 short hairpin RNA (shRNA) (5′-PPP-NS1shRNA), designed using the conserved region of a number of influenza viruses, not only prevented NS1 expression but also induced RIG-I activation and type I interferon (IFN) expression, resulting in an antiviral state leading to inhibition of influenza virus replication in vitro. In addition, administration of 5′-PPP-NS1shRNA in prophylactic and therapeutic settings resulted in significant inhibition of viral replication following viral challenge in vivo in mice with corresponding increases of RIG-I, IFN-β, and IFN-λ, as well as a decrease in NS1 expression.

MicroRNA transcriptome analysis of poly I:C-stimulated and PRRSV-infected porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe reproductive failure in sows, respiratory diseases, and high mortality in piglets, which results in serious economic losses to the swine industry worldwide. Previous studies have described that PRRSV could suppress the host immune system and had antiapoptotic activity in its initial phase of infection. Polyinosinic-polycytidylic acid (poly I:C), a synthesized analogue of viral double-strand RNA, activates innate immunity responses and induces apoptosis in cells. Therefore, we performed miRNA transcriptome analysis of poly I:C-stimulated and PRRSV-infected porcine alveolar macrophages (PAMs) using deep sequencing technology, to compare the different miRNA profiles between the statuses of innate immune activation and inactivation. After sequencing, 267 known mature miRNAs and 64 novel miRNAs were observed in PAMs, and a total of 197 miRNAs were significantly differently expressed in poly I:C-stimulated PAMs, compared with mock control cells. Thirty-three of them were also significantly alerted in PRRSV-infected PAMs. This indicated that PRRSV only slightly alerted the miRNA expression profile of host cells compared with poly I:C-stimulated PAMs, which confirmed that PRRSV could suppress host innate immune responses during the early stages of infection. Among the differentially expressed miRNAs, we found that ssc-miR-27b-3p could significantly inhibit PRRSV RNA and protein replication in MARC-145 cells and PAMs. Its antiviral mechanism needs further research in the future.

Residues F103 and M106 within the influenza A virus NS1 CPSF4-binding region regulate interferon-stimulated gene translation initiation

Influenza A virus (IAV) non-structural protein 1 (NS1) suppresses host innate immune responses by inhibiting type I interferon (IFN) production. We provide evidence that residues F103 and M106 in the CPSF4-binding domain of A/HK/1/68 [H3N2] NS1 contribute to post-transcriptional inhibition of antiviral IFN-stimulated genes (ISGs), thereby suppressing an antiviral type I IFN response. Recombinant (r) IAVs encoding F103L and M106I mutations in NS1 replicate to significantly lower viral titers in human A549 lung epithelial cells and primary type II alveolar cells. In A549 cells, rIAVs encoding these mutant NS1s induce higher levels of IFN-β production and are more sensitive to the antiviral effects of IFN-β treatment. qPCR characterization of polysomal mRNA, in the presence or absence of IFN-β treatment, identified a greater proportion of heavy polysome-associated ISGs including EIF2AK2, OAS1, and MxA in A549 cells infected with rIAVs encoding these CPSF4-binding mutant NS1s, in contrast to rIAV encoding wildtype NS1.

Salmonella spv locus suppresses host innate immune responses to bacterial infection

Salmonella enterica serovar typhimurium (S. typhimurium) is globally distributed and causes massive morbidity and mortality in humans and animals. S. typhimurium carries Salmonella plasmid virulence (spv) locus, which is highly conserved and closely related to bacterial pathogenicity, while its exact role in host immune responses during infection remains to be elucidated. To counteract the invaders, the host has evolved numerous strategies, among which the innate immunity and autophagy act as the first defense. Recently, zebrafish has been universally accepted as a valuable and powerful vertebrate model in analyzing bacteria-host interactions. To investigate whether spv locus enhances the virulence of Salmonella by exerting an effect on the host early defense, zebrafish larvae were employed in this study. LD50 of S. typhimurium to zebrafish larvae and bacterial dissemination were analyzed. Sudan black B and neutral red staining were performed to detect the responses of neutrophils and macrophages to Salmonella infection. Autophagy agonist Torin1 and inhibitor Chloroquine were used to interfere in autophagic flux, and the protein level of Lc3 and p62 were measured by western blotting. Results indicated that spv locus could decrease the LD50 of S. typhimurium to zebrafish larvae, accelerate the reproduction and dissemination of bacteria by inhibiting the function of neutrophils and macrophages. Moreover, spv locus restrained the formation of autophagosomes in the earlier stage of autophagy. These findings suggested the virulence of spv locus involving in suppressing host innate immune responses for the first time, which shed new light on the role of spv operon in Salmonella pathogenicity.

Mycobacterium tuberculosis Mce3E suppresses host innate immune responses by targeting ERK1/2 signaling.

Crucial to the pathogenesis of the tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis is its ability to subvert host immune defenses to promote its intracellular survival. The mammalian cell entry protein 3E (Mce3E), located in the region of difference 15 of the M. tuberculosis genome and absent in Mycobacterium bovis bacillus Calmette-Guérin, has an essential role in facilitating the internalization of mammalian cells by mycobacteria. However, relatively little is known about the role of Mce3E in modulation of host innate immune responses. In this study, we demonstrate that Mce3E inhibits the activation of the ERK1/2 signaling pathway, leading to the suppression of Tnf and Il6 expression, and the promotion of mycobacterial survival within macrophages. Mce3E interacts and colocalizes with ERK1/2 at the endoplasmic reticulum in a DEF motif (an ERK-docking motif)-dependent manner, relocates ERK1/2 from cytoplasm to the endoplasmic reticulum, and finally reduces the association of ERK1/2 with MEK1 and blocks the nuclear translocation of phospho-ERK1/2. A DEF motif mutant form of Mce3E (F294A) loses its ability to suppress Tnf and Il6 expression and to promote intracellular survival of mycobacteria. Inhibition of the ERK1/2 pathway in macrophages using U0126, a specific inhibitor of the ERK pathway, also leads to the suppressed Tnf and Il6 expression and the enhanced intracellular survival of mycobacteria. Taken together, these results suggest that M. tuberculosis Mce3E exploits the ERK1/2 signaling pathway to suppress host innate immune responses, providing a potential Mce3E-ERK1/2 interface-based drug target against M. tuberculosis.

The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.

All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase.

HHQ and PQS, two Pseudomonas aeruginosa quorum‐sensing molecules, down‐regulate the innate immune responses through the nuclear factor‐κB pathway

To explore whether bacterial secreted 4-hydroxy-2-alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild-type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor-alpha and interleukin-6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild-type did not. The addition of 4-hydroxy-2-heptylquinoline (HHQ) or 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild-type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor-kappaB (NF-kappaB) binding to its binding sites and the expression of NF-kappaB target genes, and PQS delayed inhibitor of kappaB degradation, indicating that the effect of HHQ and PQS was mediated through the NF-kappaB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

Subversion of Innate Immune Responses by Brucella through the Targeted Degradation of the TLR Signaling Adapter, MAL

Gram-negative bacteria belonging to the Brucella species cause chronic infections that can result in undulant fever, arthritis, and osteomyelitis in humans. Remarkably, Brucella sp. genomes encode a protein, named TcpB, that bears significant homology with mammalian Toll/IL-1 receptor domains and whose expression causes degradation of the phosphorylated, signal competent form of the adapter MyD88-adapter-like (MAL). This effect of TcpB is mediated through its box 1 region and has no effect on other TLR adapter proteins such as MyD88 or TIR-domain containing adapter protein-inducing IFNbeta. TcpB also does not affect a mutant, signal-incompetent form of MAL that cannot be phosphorylated. Interestingly, the presence of TcpB leads to enhanced polyubiquitination of MAL, which is likely responsible for its accelerated degradation. A Brucella abortus mutant lacking TcpB fails to reduce levels of MAL in infected macrophages. Therefore, TcpB represents a unique pathogen-derived molecule that suppresses host innate-immune responses by specifically targeting an individual adapter molecule in the TLR signaling pathway for degradation.

A Shared Interface Mediates Paramyxovirus Interference with Antiviral RNA Helicases MDA5 and LGP2

Diverse members of the Paramyxovirus family of negative-strand RNA viruses effectively suppress host innate immune responses through the actions of their V proteins. The V protein mediates interference with the interferon regulatory RNA helicase MDA5 to avoid cellular antiviral responses. Analysis of the interaction interface revealed the MDA5 helicase C domain as necessary and sufficient for association with V proteins from human parainfluenza virus type 2, parainfluenza virus type 5, measles virus, mumps virus, Hendra virus, and Nipah virus. The identified approximately 130-residue region is highly homologous between MDA5 and the related antiviral helicase LGP2, but not RIG-I. Results indicate that the paramyxovirus V proteins can also associate with LGP2. The V protein interaction was found to disrupt ATP hydrolysis mediated by both MDA5 and LGP2. These findings provide a potential mechanistic basis for V protein-mediated helicase interference and identify LGP2 as a second cellular RNA helicase targeted by paramyxovirus V proteins.

Epstein-Barr Virus BGLF4 Kinase Suppresses the Interferon Regulatory Factor 3 Signaling Pathway

The BGLF4 protein kinase of Epstein-Barr virus (EBV) is a member of the conserved family of herpesvirus protein kinases which, to some extent, have a function similar to that of the cellular cyclin-dependent kinase in regulating multiple cellular and viral substrates. In a yeast two-hybrid screening assay, a splicing variant of interferon (IFN) regulatory factor 3 (IRF3) was found to interact with the BGLF4 protein. This interaction was defined further by coimmunoprecipitation in transfected cells and glutathione S-transferase (GST) pull-down in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I:C)-stimulated IFN-beta promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN-beta mRNA and the phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 was shown not to affect the dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C) treatment. Notably, BGLF4 reduces the amount of active IRF3 recruited to the IRF3-responsive element containing the IFN-beta promoter region in a chromatin immunoprecipitation assay. BGLF4 phosphorylates GST-IRF3 in vitro, but Ser339-Pro340 phosphorylation-dependent, Pin1-mediated downregulation is not responsible for the repression. Most importantly, we found that three proline-dependent phosphorylation sites at Ser123, Ser173, and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contribute additively to the BGLF4-mediated repression of IRF3(5D) transactivation activity. IRF3 signaling is activated in reactivated EBV-positive NA cells, and the knockdown of BGLF4 further stimulates IRF3-responsive reporter activity. The data presented here thus suggest a novel mechanism by which herpesviral protein kinases suppress host innate immune responses and facilitate virus replication.

Salmonella evades host innate immunity via AvrA mediated inhibition of cytokine production and pro‐apoptotic pathways

The enteric pathogen Salmonella typhimurium suppresses host innate immune responses via secretion of preformed effector proteins into host cells during invasion. One of these proteins, AvrA, has potent immunosuppressive and anti‐apoptotic effects in mammalian cultured cells and in a Drosophila animal model via inhibition of the JNK pathway. To study the role of AvrA in a murine model of natural infection, wild type Salmonella harboring AvrA or an isogenic AvrA null mutant Salmonella were orally administered to mice pretreated with streptomycin and infectious phenotypes compared. Infection with the AvrA null Salmonella caused increased levels of apoptosis in the cecal epithelium and in lymphoid resident monocytic cells at 6h post infection. Additionally, the AvrA null Salmonella induced increased serum cytokine levels, including KC, IL‐1beta and TNFalpha. Furthermore, AvrA null Salmonella caused increased hepatosplenomegaly by 7 days post infection. This coincided with increased bacterial load in the liver, spleen and mesenteric lymph node, and more severe weight loss. We conclude that Salmonella evades host innate immunity and gains systemic access/dissemination via AvrA mediated inhibition of cytokine production and apoptosis. Research funding provided by NIH AI 64462.