Epidemiological and molecular characteristics of the PB1-F2 proteins in H7N9 influenza viruses, Jiangsu.
ABSTRACT: The recent sporadic infections of humans in China with previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern. The aim is to find out the epidemiological and molecular analysis of the PB1-F2 proteins in H7N9 influenza viruses, in Jiangsu province. Sequences were obtained from GISAID database. Data were analyzed by using Molecular Evolutionary Genetics Analysis software and Bayesian Markov chain Monte Carlo method. From March 1, 2013, to May 31, 2014, 53 patients were confirmed to be infected with the H7N9 virus; one was a retrospective case in Jiangsu province. 38 sequences of PB1 in H7N9 of Jiangsu were obtained from the GISAID online and were then divided into three lineages. Of these sequences, 4 sequences and 3 sequences encode an N-terminally truncated PB1-F2 (52aa)polypeptide and C-terminally truncated PB1-F2 (76aa) polypeptide, respectively. The remaining sequences encode a full-length PB1-F2 (90aa). We estimated a mean evolutionary rate of 3.053×10(-3)?subs/site/year (95% HPD: 2.021×10(-3)-4.051×10(-3)). The site-by-site analysis of selection pressure analysis revealed positively and negatively (12, 3), respectively, selected sites. Influenza A (H7N9) virus adapting into new host, PB1-F2 of H7N9, might be faced with higher selection pressures.
Project description:Human infection with avian influenza A (H5N1) and (H7N9) viruses causes severe respiratory diseases. PB1-F2 protein is a critical virulence factor that suppresses early type I interferon response, but the mechanism of its action in relation to high pathogenicity is not well understood. Here we show that PB1-F2 protein of H7N9 virus is a particularly potent suppressor of antiviral signaling through formation of protein aggregates on mitochondria and inhibition of TRIM31-MAVS interaction, leading to prevention of K63-polyubiquitination and aggregation of MAVS. Unaggregated MAVS accumulated on fragmented mitochondria is prone to degradation by both proteasomal and lysosomal pathways. These properties are proprietary to PB1-F2 of H7N9 virus but not shared by its counterpart in WSN virus. A recombinant virus deficient of PB1-F2 of H7N9 induces more interferon ? in infected cells. Our findings reveal a subtype-specific mechanism for destabilization of MAVS and suppression of interferon response by PB1-F2 of H7N9 virus.
Project description:The emergence of avian H7N9 influenza A virus in humans with associated high mortality has highlighted the threat of a potential pandemic. Fatal H7N9 infections are characterized by hyperinflammation and increased cellular infiltrates in the lung. Currently there are limited therapies to address the pathologies associated with H7N9 infection and the virulence factors that contribute to these pathologies. We have found that PB1-F2 derived from H7N9 activates the NLRP3 inflammasome and induces lung inflammation and cellular recruitment that is NLRP3-dependent. We have also shown that H7N9 and A/Puerto Rico/H1N1 (PR8)PB1-F2 peptide treatment induces significant mitochondrial reactive oxygen production, which contributes to NLRP3 activation. Importantly, treatment of cells or mice with the specific NLRP3 inhibitor MCC950 significantly reduces IL-1? maturation, lung cellular recruitment, and cytokine production. Together, these results suggest that PB1-F2 from H7N9 avian influenza A virus may be a major contributory factor to disease pathophysiology and excessive inflammation characteristic of clinical infections and that targeting the NLRP3 inflammasome may be an effective means to reduce the inflammatory burden associated with H7N9 infections.
Project description:<h4>Background</h4>In China since the first human infection of avian influenza A (H7N9) virus was identified in 2013, it has caused serious public health concerns due to its wide spread and high mortality rate. Evidence shows that bird migration plays an essential role in global spread of avian influenza viruses. Accordingly, in this paper, we aim to identify key bird species and geographical hotspots that are relevant to the transmission of avian influenza A (H7N9) virus in China.<h4>Methods</h4>We first conducted phylogenetic analysis on 626 viral sequences of avian influenza A (H7N9) virus isolated in chicken, which were collected from the Global Initiative on Sharing All Influenza Data (GISAID), to reveal geographical spread and molecular evolution of the virus in China. Then, we adopted the cross correlation function (CCF) to explore the relationship between the identified influenza A (H7N9) cases and the spatiotemporal distribution of migratory birds. Here, the spatiotemporal distribution of bird species was generated based on bird observation data collected from China Bird Reports, which consists of 157?272 observation records about 1145 bird species. Finally, we employed a kernel density estimator to identify geographical hotspots of bird habitat/stopover that are relevant to the influenza A (H7N9) infections.<h4>Results</h4>Phylogenetic analysis reveals the evolutionary and geographical patterns of influenza A (H7N9) infections, where cases in the same or nearby municipality/provinces are clustered together with small evolutionary differences. Moreover, three epidemic waves in chicken along the East Asian-Australasian flyway in China are distinguished from the phylogenetic tree. The CCF analysis identifies possible migratory bird species that are relevant to the influenza A(H7N9) infections in Shanghai, Jiangsu, Zhejiang, Fujian, Jiangxi, and Guangdong in China, where the six municipality/provinces account for 91.2% of the total number of isolated H7N9 cases in chicken in GISAID. Based on the spatial distribution of identified bird species, geographical hotspots are further estimated and illustrated within these typical municipality/provinces.<h4>Conclusions</h4>In this paper, we have identified key bird species and geographical hotspots that are relevant to the spread of influenza A (H7N9) virus. The results and findings could provide sentinel signal and evidence for active surveillance, as well as strategic control of influenza A (H7N9) transmission in China.
Project description:Influenza A virus segment 2 is known to encode two polypeptides in overlapping open reading frames: PB1, the polymerase, and PB1-F2, a proapoptotic virulence factor. We show that a third major polypeptide is synthesized from PB1 mRNA via differential AUG codon usage. PB1 codon 40 directs translation of an N-terminally truncated version of the polypeptide (N40) that lacks transcriptase function but nevertheless interacts with PB2 and the polymerase complex in the cellular environment. Importantly, the expression of N40, PB1-F2, and PB1 are interdependent, and certain mutations previously used to ablate PB1-F2 production affected N40 accumulation. Removal of the PB1-F2 AUG upregulated N40 synthesis, while truncating PB1-F2 after codon 8 (with a concomitant M40I change in PB1) abolished N40 expression. A virus lacking both N40 and PB1-F2 replicated normally. However, viruses that did not express N40 but retained an intact PB1-F2 gene overexpressed PB1 early in infection and replicated slowly in tissue culture. Thus, the influenza A virus proteome includes a 12th primary translation product that (similarly to PB1-F2) is nonessential for virus viability but whose loss, in particular genetic backgrounds, is detrimental to virus replication.
Project description:Highly pathogenic influenza A viruses (IAV) from avian hosts were first reported to directly infect humans 20 years ago. However, such infections are rare events, and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This short (90-amino-acid) peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1-associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammal-adapted H1N1 IAV is resistant to HAX-1 imposed restriction, while the PA of an avian-origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian-origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2-deficient H5N1 IAV is attenuated in the presence of large amounts of HAX-1. Mammal-adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in the presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans.IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species, including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear what restricts these interspecies jumps on the host side or what promotes them on the virus side. Here we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction factor of the viral polymerase complex HAX-1. Interestingly, we found that human influenza A virus polymerase complexes are already adapted to HAX-1 and do not require this function of PB1-F2. We thus propose that a functional full-length PB1-F2 supports direct transmission of bird viruses into humans.
Project description:The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics.
Project description:The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses, and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type-I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, towards proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics.
Project description:PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses' pathogenic phenotypes. Non-human influenza strains may act as donors of virulent PB1-F2 proteins. Previously, avian influenza strains were identified as a potential source of inflammatory, but not cytotoxic, PB1-F2 residues. Here, we analyze the frequency of virulence-associated residues in PB1-F2 sequences from IAVs circulating in mammalian species in close contact with humans: pigs, horses, and dogs. All four inflammatory residues were found in PB1-F2 proteins from these viruses. Among cytotoxic residues, I68 was the most common and was especially prevalent in equine and canine IAVs. Historically, PB1-F2 from equine (about 75%) and canine (about 20%) IAVs were most likely to have combinations of the highest numbers of residues associated with inflammation and cytotoxicity, compared to about 7% of swine IAVs. Our analyses show that, in addition to birds, pigs, horses, and dogs are potentially important sources of pathogenic PB1-F2 variants. There is a need for surveillance of IAVs with genetic markers of virulence that may be emerging from these reservoirs in order to improve pandemic preparedness and response.
Project description:PB1-F2 is a multifunctional protein and contributes to the pathogenicity of influenza A viruses. PB1-F2 is known to have strain and cell specific functions. In this study we have investigated the apoptotic and inflammatory responses of PB1-F2 protein from influenza viruses of diverse pathogenicities in A549 lung epithelial cells. Overexpression of PB1-F2 resulted in apoptosis and heightened inflammatory response in A549 cells. Comparison revealed that the response varied with each subtype. PB1-F2 protein from highly pathogenic H5N1 virus induced least apoptosis but maximum inflammatory response. Results indicated that apoptosis was mediated through death receptor ligands TNF? and TRAIL via Caspase 8 activation. Significant induction of cytokines/chemokines CXCL10, CCL5, CCL2, IFN?, and IL-6 was noted in A549 cells transfected with PB1-F2 gene construct of 2008 West Bengal H5N1 virus (H5N1-WB). On the contrary, PB1-F2 construct from 2007 highly pathogenic H5N1 isolate (H5N1-M) with truncated N-terminal region did not evoke as exuberant inflammatory response as the other H5N1-WB with full length PB1-F2, signifying the importance of N-terminal region of PB1-F2. Sequence analysis revealed that PB1-F2 proteins derived from different influenza viruses varied at multiple amino acid positions. The secondary structure prediction showed each of the PB1-F2 proteins had distinct helix-loop-helix structure. Thus, our data substantiate the notion that the contribution of PB1-F2 to influenza pathogenicity is greatly strain specific and involves multiple host factors. This data demonstrates that PB1-F2 protein of influenza A virus, when expressed independently is minimally apoptotic and strongly influences the early host response in A549 cells.
Project description:The 11th influenza A virus gene product is an 87-amino-acid protein provisionally named PB1-F2 (because it is encoded by an open reading frame overlapping the PB1 open reading frame). A significant fraction of PB1-F2 localizes to the inner mitochondrial membrane in influenza A virus-infected cells. PB1-F2 appears to enhance virus-induced cell death in a cell type-dependent manner. For the present communication we have identified and characterized a region near the COOH terminus of PB1-F2 that is necessary and sufficient for its inner mitochondrial membrane localization, as determined by transient expression of chimeric proteins consisting of elements of PB1-F2 genetically fused to enhanced green fluorescent protein (EGFP) in HeLa cells. Targeting of EGFP to mitochondria by this sequence resulted in the loss of the inner mitochondrial membrane potential, leading to cell death. The mitochondrial targeting sequence (MTS) is predicted to form a positively charged amphipathic alpha-helix and, as such, is similar to the MTS of the p13(II) protein of human T-cell leukemia virus type 1. We formally demonstrate the functional interchangeability of the two sequences for mitochondrial localization of PB1-F2. Mutation analysis of the putative amphipathic helix in the PB1-F2 reveals that replacement of five basic amino acids with Ala abolishes mitochondrial targeting, whereas mutation of two highly conserved Leu to Ala does not. These findings demonstrate that PB1-F2 possesses an MTS similar to other viral proteins and that this MTS, when fused to EGFP, is capable of independently compromising mitochondrial function and cellular viability.