Host Target Proteins Research Articles

Devastating intimacy: the cell biology of plant-Phytophthora interactions.

SummaryAn understanding of the cell biology underlying the burgeoning molecular genetic and genomic knowledge of oomycete pathogenicity is essential to gain the full context of how these pathogens cause disease on plants. An intense research focus on secreted Phytophthora effector proteins, especially those containing a conserved N‐terminal RXLR motif, has meant that most cell biological studies into Phytophthora diseases have focussed on the effectors and their host target proteins. While these effector studies have provided novel insights into effector secretion and host defence mechanisms, there remain many unanswered questions about fundamental processes involved in spore biology, host penetration and haustorium formation and function.

DDX3X inhibitors, an effective way to overcome HIV-1 resistance targeting host proteins

The huge resources that had gone into Human Immunodeficiency virus (HIV) research led to the development of potent antivirals able to suppress viral load in the majority of treated patients, thus dramatically increasing the life expectancy of people living with HIV. However, life-long treatments could result in the emergence of drug-resistant viruses that can progressively reduce the number of therapeutic options, facilitating the progression of the disease. In this scenario, we previously demonstrated that inhibitors of the human DDX3X helicase can represent an innovative approach for the simultaneous treatment of HIV and other viral infections such as Hepatitis c virus (HCV). We reported herein 6b, a novel DDX3X inhibitor that thanks to its distinct target of action is effective against HIV-1 strains resistant to currently approved drugs. Its improved in vitro ADME properties allowed us to perform preliminary in vivo studies in mice, which highlighted optimal biocompatibility and an improved bioavailability. These results represent a significant advancement in the development of DDX3X inhibitors as a novel class of broad spectrum and safe anti-HIV-1 drugs.

Protein Kinase D: A new target in the fight against flu?

Abstract Summary Antivirals targeting host proteins may slow development of viral resistance and work against multiple viruses. The serine/threonine kinase protein kinase D (PKD) is one promising target as others have shown inhibiting PKD restricts rhinovirus and hepatitis in cell culture. We show inhibiting PKD restricts Influenza A virus (IAV) in cell culture and in mice. Methods The competitive PKD inhibitor, CRT, was given to A549, 16HBE cells and mice, prior to or during stimulation with dsRNA or IAV. Pulmonary barrier integrity was quantified by assaying total protein leak into the lumen (inside/out leak), and loss of 4kDa FITC-dextran out of the lumen (outside/in leak). Inflammatory cytokine levels were determined via ELISA/multiplex and mRNA levels via RT-PCR. Transcription factor activity was assayed via luciferase reporter system. Viral load was determined via RT-PCR of viral M protein. For in vivo IAV experiments, mice were given CRT/vehicle post-infection with lethal dose IAV (PR/8) and morbidity assessed by weight loss. Results Conclusion PKD inhibition promotes barrier integrity, lowers transcription of potentially pathogenic pro-inflammatory cytokines, and restricts viral replication, highlighting the potential of CRT as a novel anti-viral therapeutic.

The Multifarious Role of 14-3-3 Family of Proteins in Viral Replication

The 14-3-3 proteins are a family of ubiquitous and exclusively eukaryotic proteins with an astoundingly significant number of binding partners. Their binding alters the activity, stability, localization, and phosphorylation state of a target protein. The association of 14-3-3 proteins with the regulation of a wide range of general and specific signaling pathways suggests their crucial role in health and disease. Recent studies have linked 14-3-3 to several RNA and DNA viruses that may contribute to the pathogenesis and progression of infections. Therefore, comprehensive knowledge of host–virus interactions is vital for understanding the viral life cycle and developing effective therapeutic strategies. Moreover, pharmaceutical research is already moving towards targeting host proteins in the control of virus pathogenesis. As such, targeting the right host protein to interrupt host–virus interactions could be an effective therapeutic strategy. In this review, we generated a 14-3-3 protein interactions roadmap in viruses, using the freely available Virusmentha network, an online virus–virus or virus–host interaction tool. Furthermore, we summarize the role of the 14-3-3 family in RNA and DNA viruses. The participation of 14-3-3 in viral infections underlines its significance as a key regulator for the expression of host and viral proteins.

Type III secretion systems impact Mesorhizobium amorphae CCNWGS0123 compatibility with Robinia pseudoacacia.

Rhizobia and legume plants are famous mutualistic symbiosis partners who provide nitrogen nutrition to the natural environment. Rhizobial type III secretion systems (T3SSs) deliver effectors that manipulate the metabolism of eukaryotic host cells. Mesorhizobium amorphae CCNWGS0123 (GS0123) contains two T3SS gene clusters, T3SS-I and T3SS-II. T3SS-I contains all the basal components for an integrated T3SS, and the expression of T3SS-I genes is up-regulated in the presence of flavonoids. In contrast, T3SS-II lacks the primary extracellular elements of T3SSs, and the expression of T3SS-II genes is down-regulated in the presence of flavonoids. Inoculation tests on Robinia pseudoacacia displayed considerable differences in gene expression patterns and levels among roots inoculated with GS0123 and T3SS-deficient mutant (GS0123ΔrhcN1 (GS0123ΔT1), GS0123ΔrhcN2 (GS0123ΔT2) and GS0123ΔrhcN1ΔrhcN2 (GS0123ΔS)). Compared with the GS0123-inoculated plants, GS0123ΔT1-inoculated roots formed very few infection threads and effective nodules, while GS0123ΔT2-inoculated roots formed a little fewer infection threads and effective nodules with increased numbers of bacteroids enclosed in one symbiosome. Moreover, almost no infection threads or effective nodules were observed in GS0123ΔS-inoculated roots. In addition to evaluations of plant immunity signals, we observed that the coexistence of T3SS-I and T3SS-II promoted infection by suppressing host defense response in the reactive oxygen species defense response pathway. Future studies should focus on identifying rhizobial T3SS effectors and their host target proteins.

Identification of AIDS-Associated Kaposi Sarcoma: A Functional Genomics Approach.

BackgroundKaposi sarcoma-associated herpes virus (KSHV) is one of the most common causal agents of Kaposi Sarcoma (KS) in individuals with HIV-infections. The virus has gained attention over the past few decades due to its remarkable pathogenic mechanisms. A group of genes, ORF71, ORF72, and ORF73, are expressed as polycistronic mRNAs and the functions of ORF71 and ORF72 in KSHV are already reported in the literature. However, the function of ORF73 has remained a mystery. The aim of this study is to conduct comprehensive exploratory experiments to clarify the role of ORF73 in KSHV pathology and discover markers of AIDS-associated KSHV-induced KS by bioinformatic approaches.Methods and ResultsWe searched for homologues of ORF-73 and attempted to predict protein-protein interactions (PPI) based on GeneCards and UniProtKB, utilizing Position-Specific Iterated BLAST (PSI-BLAST). We applied Gene Ontology (GO) and KEGG pathway analyses to identify highly conserved regions between ORF-73 and p53to help us identify potential markers with predominant hits and interactions in the KEGG pathway associated with host apoptosis and cell arrest. The protein p53 is selected because it is an important tumor suppressor antigen. To identify the potential roles of the candidate markers at the molecular level, we used PSIPRED keeping the conserved domains as the major parameters to predict secondary structures. We based the FUGE interpretation consolidations of the sequence-structure comparisons on distance homology, where the score for the amino acids matching the insertion/deletion (indels) detected were based on structures compared to the FUGE database of structural profiles. We also calculated the compatibility scores of sequence alignments accordingly. Based on the PSI-BLAST homologues, we checked the disordered structures predicted using PSI-Pred and DISO-Pred for developing a hidden Markov model (HMM). We further applied these HMMs models based on the alignment of constructed 3D models between the known structure and the HMM of our sequence. Moreover, stable homology and structurally conserved domains confirmed that ORF-73 maybe an important prognostic marker for AIDS-associated KS.ConclusionCollectively, similar variants of ORF-73 markers involved in the immune response may interact with targeted host proteins as predicted by our computational analysis. This work also suggests the existence of potential conformational changes that need to be further explored to help elucidate the role of immune signaling during KS towards the development of therapeutic applications.

A host target of a bacterial cysteine protease virulence effector plays a key role in convergent evolution of plant innate immune system receptors.

Some virulence effectors secreted from pathogens target host proteins and induce biochemical modifications that are monitored by nucleotide-binding and leucine-rich repeat (NLR) immune receptors. Arabidopsis RIN4 protein (AtRIN4: RPM1-interacting protein 4) homologs are present in diverse plant species and targeted by several bacterial type III effector proteins including the cysteine protease AvrRpt2. RIN4 is 'guarded' by several independently evolved NLRs from various plant species, including Arabidopsis RPS2. Recently, it was shown that the MR5 NLR from a wild apple relative can recognize the AvrRpt2 effector from Erwinia amylovora, but the details of this recognition remained unclear. The present contribution reports the mechanism of AvrRpt2 recognition by independently evolved NLRs, MR5 from apple and RPS2, both of which require proteolytically processed RIN4 for activation. It shows that the C-terminal cleaved product of apple RIN4 (MdRIN4) but not AtRIN4 is necessary and sufficient for MR5 activation. Additionally, two polymorphic residues in AtRIN4 and MdRIN4 are identified that are crucial in the regulation of and physical association with NLRs. It is proposed that polymorphisms in RIN4 from distantly related plant species allow it to remain an effector target while maintaining compatibility with multiple NLRs.

Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein.

Plant pathogens secrete proteins called effectors into the cells of their host to modulate the host immune response against colonization. Effectors can either modify or arrest host target proteins to sabotage the signaling pathway, and therefore are considered potential drug targets for crop disease control. In earlier research, the Xanthomonas type III effector XopAI was predicted to be a member of the arginine-specific mono-ADP-ribosyltransferase family. However, the crystal structure of XopAI revealed an altered active site that is unsuitable to bind the cofactor NAD+, but with the capability to capture an arginine-containing peptide from XopAI itself. The arginine peptide consists of residues 60 through 69 of XopAI, and residue 62 (R62) is key to determining the protein–peptide interaction. The crystal structure and the molecular dynamics simulation results indicate that specific arginine recognition is mediated by hydrogen bonds provided by the backbone oxygen atoms from residues W154, T155, and T156, and a salt bridge provided by the E265 sidechain. In addition, a protruding loop of XopAI adopts dynamic conformations in response to arginine peptide binding and is probably involved in target protein recognition. These data suggest that XopAI binds to its target protein by the peptide-binding ability, and therefore, it promotes disease progression. Our findings reveal an unexpected and intriguing function of XopAI and pave the way for further investigation on the role of XopAI in pathogen invasion.

Candidate Effectors From Uromyces appendiculatus, the Causal Agent of Rust on Common Bean, Can Be Discriminated Based on Suppression of Immune Responses.

Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean.

CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infection.

Salmonella presents a global public health concern. Central to Salmonella pathogenicity is an ability to subvert host defences through strategically targeting host proteins implicated in restricting infection. Therefore, to gain insight into the host-pathogen interactions governing Salmonella infection, we performed an in vivo genome-wide mutagenesis screen to uncover key host defence proteins. This revealed an uncharacterized role of CYRI (FAM49B) in conferring host resistance to Salmonella infection. We show that CYRI binds to the small GTPase RAC1 through a conserved domain present in CYFIP proteins, which are known RAC1 effectors that stimulate actin polymerization. However, unlike CYFIP proteins, CYRI negatively regulates RAC1 signalling, thereby attenuating processes such as macropinocytosis, phagocytosis and cell migration. This enables CYRI to counteract Salmonella at various stages of infection, including bacterial entry into non-phagocytic and phagocytic cells as well as phagocyte-mediated bacterial dissemination. Intriguingly, to dampen its effects, the bacterial effector SopE, a RAC1 activator, selectively targets CYRI following infection. Together, this outlines an intricate host-pathogen signalling interplay that is crucial for determining bacterial fate. Notably, our study also outlines a role for CYRI in restricting infection mediated by Mycobacterium tuberculosis and Listeria monocytogenes. This provides evidence implicating CYRI cellular functions in host defence beyond Salmonella infection.

Type III secretion systems impact Mesorhizobium amorphae CCNWGS0123 compatibility with Robinia pseudoacacia

Rhizobia and legume plants are famous mutualistic symbiosis partners who provide nitrogen nutrition to the natural environment. Rhizobial type III secretion systems (T3SSs) deliver effectors that manipulate the metabolism of eukaryotic host cells. Mesorhizobium amorphae CCNWGS0123 (GS0123) contains two T3SS gene clusters, T3SS-I and T3SS-II. T3SS-I contains all the basal components for an integrated T3SS, and the expression of T3SS-I genes is up-regulated in the presence of flavonoids. In contrast, T3SS-II lacks the primary extracellular elements of T3SSs, and the expression of T3SS-II genes is down-regulated in the presence of flavonoids. Inoculation tests on Robinia pseudoacacia displayed considerable differences in gene expression patterns and levels among roots inoculated with GS0123 and T3SS-deficient mutant (GS0123ΔrhcN1 (GS0123ΔT1), GS0123ΔrhcN2 (GS0123ΔT2) and GS0123ΔrhcN1ΔrhcN2 (GS0123ΔS)). Compared with the GS0123-inoculated plants, GS0123ΔT1-inoculated roots formed very few infection threads and effective nodules, while GS0123ΔT2-inoculated roots formed a little fewer infection threads and effective nodules with increased numbers of bacteroids enclosed in one symbiosome. Moreover, almost no infection threads or effective nodules were observed in GS0123ΔS-inoculated roots. In addition to evaluations of plant immunity signals, we observed that the coexistence of T3SS-I and T3SS-II promoted infection by suppressing host defense response in the reactive oxygen species defense response pathway. Future studies should focus on identifying rhizobial T3SS effectors and their host target proteins.

Prediction of bacterial E3 ubiquitin ligase effectors using reduced amino acid peptide fingerprinting.

BackgroundAlthough pathogenic Gram-negative bacteria lack their own ubiquitination machinery, they have evolved or acquired virulence effectors that can manipulate the host ubiquitination process through structural and/or functional mimicry of host machinery. Many such effectors have been identified in a wide variety of bacterial pathogens that share little sequence similarity amongst themselves or with eukaryotic ubiquitin E3 ligases.MethodsTo allow identification of novel bacterial E3 ubiquitin ligase effectors from protein sequences we have developed a machine learning approach, the SVM-based Identification and Evaluation of Virulence Effector Ubiquitin ligases (SIEVE-Ub). We extend the string kernel approach used previously to sequence classification by introducing reduced amino acid (RED) alphabet encoding for protein sequences.ResultsWe found that 14mer peptides with amino acids represented as simply either hydrophobic or hydrophilic provided the best models for discrimination of E3 ligases from other effector proteins with a receiver-operator characteristic area under the curve (AUC) of 0.90. When considering a subset of E3 ubiquitin ligase effectors that do not fall into known sequence based families we found that the AUC was 0.82, demonstrating the effectiveness of our method at identifying novel functional family members. Feature selection was used to identify a parsimonious set of 10 RED peptides that provided good discrimination, and these peptides were found to be located in functionally important regions of the proteins involved in E2 and host target protein binding. Our general approach enables construction of models based on other effector functions. We used SIEVE-Ub to predict nine potential novel E3 ligases from a large set of bacterial genomes. SIEVE-Ub is available for download at https://doi.org/10.6084/m9.figshare.7766984.v1 or https://github.com/biodataganache/SIEVE-Ub for the most current version.

Lack of selective resistance of influenza A virus in presence of host-targeted antiviral, UV-4B

Development of antiviral drug resistance is a continuous concern for viruses with high mutation rates such as influenza. The use of antiviral drugs targeting host proteins required for viral replication is less likely to result in the selection of resistant viruses than treating with direct-acting antivirals. The iminosugar UV-4B is a host-targeted glucomimetic that inhibits endoplasmic reticulum α-glucosidase I and II enzymes resulting in improper glycosylation and misfolding of viral glycoproteins. UV-4B has broad-spectrum antiviral activity against diverse viruses including dengue and influenza. To examine the ability of influenza virus to develop resistance against UV-4B, mouse-adapted influenza virus was passaged in mice in the presence or absence of UV-4B and virus isolated from lungs was used to infect the next cohort of mice, for five successive passages. Deep sequencing was performed to identify changes in the viral genome during passaging in the presence or absence of UV-4B. Relatively few minor variants were identified within each virus and the ratio of nonsynonymous to synonymous (dN/dS) substitutions of minor variants confirmed no apparent positive selection following sustained exposure to UV-4B. Three substitutions (one synonymous in PB2, one nonsynonymous in M and PA each) were specifically enriched (>3%) in UV-4B-treated groups at passage five. Recombinant viruses containing each individual or combinations of these nonsynonymous mutations remained sensitive to UV-4B treatment in mice. Overall, these data provide evidence that there is a high genetic barrier to the generation and selection of escape mutants following exposure to host-targeted iminosugar antivirals.

Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence.

Banana bunchy top virus (BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO (RbcL) and elongation factor 2 (EF2), involved in protein synthesis. Therefore, the peptide released from B4 (also a precursor) may act as a non-canonical modifier to influence host-pathogen interactions involving BBTV and plants.

Post‐translational modifications in effectors and plant proteins involved in host–pathogen conflicts

Molecular interplay between two species is largely driven by protein–protein interactions and protein modifications that set the pace of co‐evolution in these species. During host–pathogen interactions, proteins involved in virulence and defence impart tempospatial dynamic post‐translational modifications (PTMs) to gain advantage for the causative species. Pathogens mainly cause disease in plant hosts by secreting elicitors (peptides and small molecules) or proteins in the inter‐ and intracellular space of host cells. These pathogen proteins have evolved a wide array of sophisticated mechanisms to manipulate host responses, including resistance. Through a set of diverse events ranging from PTMs to post‐translational oligomerization, these proteins are able to enhance virulence and suppress the otherwise elaborate plant immune system. Similarly, PTMs adapted by host proteins often lead to the activation of a robust defence response. Insights into the PTMs of pathogen and host proteins are therefore germane to the understanding of the co‐evolutionary arms race. This review summarizes the characterization of PTMs in pathogen effectors and their target host proteins. Based on this, a metaphorical view of host–pathogen conflicts is proposed, where PTMs act as molecular pivots in a 3D combinatorial game model – a novel abstraction of the arms race, where these molecular pivots restore the balance of competition between the two organisms.

The ubiquitin ligase SspH1 from Salmonella uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation

SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D∼Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D∼Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3∼Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D∼Ub and targets the E2∼Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2∼Ub-binding region is dynamic but stabilized in the E3∼Ub intermediate. Our results support a model in which both subunits of an Ube2D∼Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3∼Ub to substrate.

Insight into the Human Pathodegradome of the V8 Protease From  <i>Staphylococcus aureus</i>

Staphylococcus aureus possesses ten extracellular proteases, with mostly unknown targets in the human proteome. To assist with bacterial protease target discovery, we appliedand compared two N-terminomics methods to investigate cleavage of human serum proteins by S. aureus V8 protease, discovering 85 host-protein targets. Amongst these were virulence-relevant complement, iron sequestration, clotting cascade, and host protease inhibitor proteins. Protein cleavage sites were identified, providing insight into the disruption of host protein function by V8. Complement proteins were cleaved within peptidase and sushi domains, and host protease inhibitors were cleaved outside their protease-trapping motifs. Our data highlight the potential for further application of N-terminomics in discovery of bacterial protease substrates in other host niches, and provide omics-scale insight into the role of the V8 protease in S. aureus pathogenesis.

Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3)

Staphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein–protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3–TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2–SSL3 interaction which adds to our understanding of staphylococcal host specificity.

234 - Lung epithelial PDIA3 plays a critical role in influenza infection

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and that this interaction is required for efficient oxidative folding of HA in vitro. However it is unknown whether these host-viral protein interactions occur during active infection and whether this interaction represent a putative target for the treatment of influenza infection. Current therapies targeting viral proteins are often limited in their effectiveness due to rapid development of viral resistance; targeting host proteins may avoid these limitations. Here we show that PDIA3 is specifically upregulated in IAV infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Among PDI inhibitors tested, we found LOC14 inhibits PDIA3 at an IC50 of 5μM. Treatment with LOC14 inhibits PDIA3 activity in lung epithelial cells and subsequently decreases disulfide bonds, and oligomerization (maturation) of HA in both pandemic H1N1 and seasonal H3N2 infected lung epithelial cells. Disulfide labeling analysis indicated that inhibition of PDIA3 activity also decreased disulfide bonds in IAV-neuraminidase (NA) and these differences in oxidative folding correspond to a subsequent decrease in NA activity. Furthermore, these decreases in disulfide bonds significantly decrease viral load, and pro-inflammatory response from primary lung epithelial cells. Lung epithelial specific deletion of PDIA3 in mice results in a significant decreases in viral burden and levels of inflammatory-immune markers in mouse lung, as well as significantly improved airway mechanics (function). Taken together, these results suggest PDIA3 is required for effective influenza pathogenesis in vivo, and inhibition of PDIA3 may aid future development anti-influenza therapies based on inhibition of host PDIs to circumvent antigenic drifts and shifts of influenza and may lend critically needed help during pandemic and severe influenza seasons.

Specific recognition of two MAX effectors by integrated HMA domains in plant immune receptors involves distinct binding surfaces

The structurally conserved but sequence-unrelated MAX (Magnaporthe oryzae avirulence and ToxB-like) effectors AVR1-CO39 and AVR-PikD from the blast fungus M. oryzae are recognized by the rice nucleotide-binding domain and leucine-rich repeat proteins (NLRs) RGA5 and Pikp-1, respectively. This involves, in both cases, direct interaction of the effector with a heavy metal-associated (HMA) integrated domain (ID) in the NLR. Here, we solved the crystal structures of a C-terminal fragment of RGA5 carrying the HMA ID (RGA5_S), alone, and in complex with AVR1-CO39 and compared it to the structure of the Pikp1HMA/AVR-PikD complex. In both complexes, HMA ID/MAX effector interactions involve antiparallel alignment of β-sheets from each partner. However, effector-binding occurs at different surfaces in Pikp1HMA and RGA5HMA, indicating that these interactions evolved independently by convergence of these two MAX effectors to the same type of plant target proteins. Interestingly, the effector-binding surface in RGA5HMA overlaps with the surface that mediates RGA5HMA self-interaction. Mutations in the HMA-binding interface of AVR1-CO39 perturb RGA5HMA-binding, in vitro and in vivo, and affect the recognition of M. oryzae in a rice cultivar containing Pi-CO39 Our study provides detailed insight into the mechanisms of effector recognition by NLRs, which has substantial implications for future engineering of NLRs to expand their recognition specificities. In addition, we propose, as a hypothesis for the understanding of effector diversity, that in the structurally conserved MAX effectors the molecular mechanism of host target protein-binding is conserved rather than the host target proteins themselves.