Equine ANP32 proteins support influenza A virus RNA polymerase activity
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ABSTRACT:
SUBMITTER: Zhang Y
PROVIDER: S-EPMC10786659 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:The influenza B virus (IBV) causes seasonal influenza and has accounted for an increasing proportion of influenza outbreaks. IBV mainly causes human infections and has not been found to spread in poultry. The replication mechanism and the determinants of interspecies transmission of IBV are largely unknown. In this study, we found that the host ANP32 proteins are required for the function of the IBV polymerase. Human ANP32A/B strongly supports IBV replication, while ANP32E has a limited role. Unlike human ANP32A/B, chicken ANP32A has low support activity to IBV polymerase because of a unique 33-amino-acid insert, which, in contrast, exhibits species specific support to avian influenza A virus (IAV) replication. Chicken ANP32B and ANP32E have even lower activity compared with human ANP32B/E due to specific amino acid substitutions at sites 129-130. We further revealed that the sites 129-130 affect the binding ability of ANP32B/E to IBV polymerase, while the 33-amino-acid insert of chicken ANP32A reduces its binding stability and affinity. Taken together, the features of avian ANP32 proteins limited their abilities to support IBV polymerase, which could prevent efficient replication of IBV in chicken cells. Our results illustrate roles of ANP32 proteins in supporting IBV replication and may help to understand the ineffective replication of IBV in birds.
| S-EPMC7580981 | biostudies-literature
Project description:ANP32 proteins have been implicated in supporting influenza virus replication, but most of the work to date has focused on the ability of avian Anp32 proteins to overcome restriction of avian influenza polymerases in human cells. Using a CRISPR approach, we show that the human acidic nuclear phosphoproteins (ANPs) ANP32A and ANP32B are functionally redundant but essential host factors for mammalian-adapted influenza A virus (IAV) and influenza B virus (IBV) replication in human cells. When both proteins are absent from human cells, influenza polymerases are unable to replicate the viral genome, and infectious virus cannot propagate. Provision of exogenous ANP32A or ANP32B recovers polymerase activity and virus growth. We demonstrate that this redundancy is absent in the murine Anp32 orthologues; murine Anp32A is incapable of recovering IAV polymerase activity, while murine Anp32B can do so. Intriguingly, IBV polymerase is able to use murine Anp32A. We show, using a domain swap and point mutations, that the leucine-rich repeat (LRR) 5 region comprises an important functional domain for mammalian ANP32 proteins. Our approach has identified a pair of essential host factors for influenza virus replication and can be harnessed to inform future interventions.IMPORTANCE Influenza virus is the etiological agent behind some of the most devastating infectious disease pandemics to date, and influenza outbreaks still pose a major threat to public health. Influenza virus polymerase, the molecule that copies the viral RNA genome, hijacks cellular proteins to support its replication. Current anti-influenza drugs are aimed against viral proteins, including the polymerase, but RNA viruses like influenza tend to become resistant to such drugs very rapidly. An alternative strategy is to design therapeutics that target the host proteins that are necessary for virus propagation. Here, we show that the human proteins ANP32A and ANP32B are essential for influenza A and B virus replication, such that in their absence cells become impervious to the virus. We map the proviral activity of ANP32 proteins to one region in particular, which could inform future intervention.
| S-EPMC6694824 | biostudies-literature
Project description:Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens.
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Project description:ObjectivesInterferons (IFNs) are one of the most important components of innate immunity against viruses, especially those carrying the RNA genomes such as influenza viruses. Upon viral infection, the IFNs are rapidly secreted, inducing the expression of several genes in the target cells and establishing an antiviral state. In this study, the effects of proteins encoded by some IFN-related genes on influenza A virus RNA-dependent RNA polymerase enzyme were investigated. We evaluated the importance of these proteins in the pathogenesis of different influenza A virus types.Materials and methodsThe IFN-related genes were amplified by polymerase chain reaction from the HEK293 cDNA library and cloned into pCHA expression vector. The expression of genes and subcellular localizations of the proteins were determined by Western blotting and immunofluorescence staining, respectively. The effects of IFNs-related proteins on virus RdRP enzyme were determined by influenza A virus mini-replicons.ResultsThe study revealed that the influenza A virus infections significantly altered the transcript level of the IFN-related CCL5, IFIT1, IFIT3, IFITM3, and OAS1 genes in HEK293 cells. It was determined that the alteration of the gene expression was also related to the virus type. The mini-replicon assays showed that the transient expression of CCL5, IFI27, OAS1, IFITM3, IFIT1, and IFIT3 have inhibitory effects on WSN and/or DkPen type virus RdRP enzymes. We observed that the proteins except OAS1 inhibited WSN type RdRP enzyme at a higher level than that of DkPen enzyme.ConclusionIt was concluded that influenza A virus infection significantly alters the IFN-related gene expression in the cells. Most of the proteins encoded from these genes showed an inhibitory effect on the virus RdRP enzymes in the HEK293 cells. The inhibition of the influenza virus RdRP with IFN-related proteins may be the result of direct or indirect interactions between the host proteins and the viral enzyme subunits.
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Project description:An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells.
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Project description:The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.
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Project description:The influenza A RNA polymerase possesses endonuclease activity to digest the host mRNA. Thus this endonuclease domain can be a target of anti-influenza A virus drug. Here we report that green tea catechins inhibit this viral endonuclease activity and that their galloyl group is important for their function. Docking simulations revealed that catechins with galloyl group fit well into the active pocket of the endonuclease domain to enable stable binding. Our results provide useful data that make it possible to refine and optimize catechin-based drug design more readily for stability.
| S-EPMC2762814 | biostudies-literature
Project description:Emerging influenza virus poses a health threat to humans and animals. Domestic cats have recently been identified as a potential source of zoonotic influenza virus. The influenza virus minigenome replication system based on the ribonucleic acid (RNA) polymerase I (PolI) promoter is the most widely used tool for investigating polymerase activity. It could help determine host factors or viral proteins influencing influenza virus polymerase activity in vitro. However, influenza virus polymerase activity has never been studied in feline cells thus far. In the present study, the feline RNA PolI promoter was identified in the intergenic spacer regions between adjacent upstream 28S and downstream 18S rRNA genes in the cat (Felis catus) genome using bioinformatics strategies. The transcription initiation site of the feline RNA PolI promoter was predicted. The feline RNA PolI promoter was cloned from CRFK cells, and a promoter size of 250 bp contained a sequence with sufficient PolI promoter activity by a dual-luciferase reporter assay. The influenza virus minigenome replication system based on the feline RNA PolI promoter was then established. Using this system, the feline RNA PolI promoter was determined to have significantly higher transcriptional activity than the human and chicken RNA PolI promoters in feline cells, and equine (H3N8) influenza virus presented higher polymerase activity than human (H1N1) and canine (H3N2) influenza viruses. In addition, feline myxovirus resistance protein 1 (Mx1) and baloxavir were observed to inhibit influenza virus polymerase activity in vitro in a dose-dependent manner. Our study will help further investigations on the molecular mechanism of host adaptation and cross-species transmission of influenza virus in cats.
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Project description:UNLABELLED:Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections. IMPORTANCE:Highly pathogenic avian influenza A (HPAI) viruses have recently emerged from wild and domestic birds to cause fatal human disease. In human patients, it is thought that adaptation of the viral polymerase, a complex of viral proteins responsible for viral gene expression and RNA genome replication, to interactions with mammalian rather than avian host proteins contributes to disease severity. In this study, we used computational analysis and RNA interference (RNAi) experiments to identify a biological network of human proteins that regulates an H5N1 HPAI virus polymerase, in comparison to a mammalian H1N1 virus. Of 31 proteins tested, 18 (58%) were required for polymerase function in both HPAI and H1N1 viruses. Remarkably, we also found proteins such as DDX17 that governed the HPAI virus polymerase's adaptation to human cells. These virus-host interactions may thus control pathogenicity of HPAI virus in humans and are promising therapeutic targets for antiviral drugs in severe influenza infections.
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Project description:The influenza A virus causes a highly contagious respiratory disease that significantly impacts our economy and health. Its replication and transcription is catalyzed by the viral RNA polymerase. This enzyme is also crucial for the virus, because it is involved in the adaptation of zoonotic strains. It is thus of major interest for the development of antiviral therapies and is being intensively studied. In this article, we will discuss recent advances that have improved our knowledge of the structure of the RNA polymerase and how mutations in the polymerase help the virus to spread effectively among new hosts.
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