ABSTRACT
RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.
Subject(s)
RNA Caps/genetics , RNA Virus Infections/genetics , Recombinant Fusion Proteins/genetics , 5' Untranslated Regions/genetics , Animals , Cattle , Cell Line , Cricetinae , Dogs , Humans , Influenza A virus/metabolism , Mice , Mutant Chimeric Proteins/genetics , Mutant Chimeric Proteins/metabolism , Open Reading Frames/genetics , RNA Caps/metabolism , RNA Virus Infections/metabolism , RNA Viruses/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Recombinant Fusion Proteins/metabolism , Transcription, Genetic/genetics , Viral Proteins/metabolism , Virus Replication/geneticsABSTRACT
PTEN is a phosphatase which metabolises PIP3, the lipid product of PI 3-Kinase, directly opposing the activation of the oncogenic PI3K/AKT/mTOR signalling network. Accordingly, loss of function of the PTEN tumour suppressor is one of the most common events observed in many types of cancer. Although the mechanisms by which PTEN function is disrupted are diverse, the most frequently observed events are deletion of a single gene copy of PTEN and gene silencing, usually observed in tumours with little or no PTEN protein detectable by immunohistochemistry. Accordingly, with the exceptions of glioblastoma and endometrial cancer, mutations of the PTEN coding sequence are uncommon (<10%) in most types of cancer. Here we review the data relating to PTEN loss in seven common tumour types and discuss mechanisms of PTEN regulation, some of which appear to contribute to reduced PTEN protein levels in cancers.
Subject(s)
Disease Susceptibility , Loss of Function Mutation , Neoplasms/etiology , Neoplasms/metabolism , PTEN Phosphohydrolase/genetics , Animals , Biomarkers, Tumor , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Mutation , Neoplasms/pathology , PTEN Phosphohydrolase/metabolism , Signal TransductionABSTRACT
The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.
Subject(s)
Influenza A virus/pathogenicity , Mutation , Reassortant Viruses/pathogenicity , Repressor Proteins/genetics , Viral Nonstructural Proteins/genetics , Animals , Chick Embryo , Chickens , Dogs , HEK293 Cells , Humans , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza A Virus, H7N1 Subtype/genetics , Influenza A Virus, H7N1 Subtype/pathogenicity , Influenza A virus/genetics , Influenza in Birds/virology , Madin Darby Canine Kidney Cells , Reassortant Viruses/geneticsABSTRACT
Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a â¼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export.IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery.
Subject(s)
Active Transport, Cell Nucleus , Influenza A Virus, H1N1 Subtype/physiology , RNA, Messenger/metabolism , RNA, Viral/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication , Cell Line , Humans , In Situ Hybridization, FluorescenceABSTRACT
The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.
Subject(s)
Influenza A virus/pathogenicity , Membrane Proteins/metabolism , Orthomyxoviridae Infections/mortality , RNA-Binding Proteins/metabolism , Alleles , Amino Acid Sequence , Animals , Cytokines/immunology , England/epidemiology , Gene Deletion , Humans , Influenza A Virus, H1N1 Subtype/classification , Influenza A Virus, H1N1 Subtype/growth & development , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H3N2 Subtype/classification , Influenza A Virus, H3N2 Subtype/growth & development , Influenza A Virus, H3N2 Subtype/pathogenicity , Influenza A virus/classification , Influenza A virus/growth & development , Influenza B virus/classification , Influenza B virus/growth & development , Influenza B virus/pathogenicity , Influenza, Human/complications , Influenza, Human/epidemiology , Influenza, Human/mortality , Influenza, Human/virology , Leukocytes/immunology , Lung/pathology , Lung/virology , Membrane Proteins/chemistry , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Orthomyxoviridae Infections/complications , Orthomyxoviridae Infections/pathology , Pneumonia, Viral/etiology , Pneumonia, Viral/pathology , Pneumonia, Viral/prevention & control , Polymorphism, Single Nucleotide/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Scotland/epidemiology , Virus ReplicationABSTRACT
UNLABELLED: Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE: Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.
Subject(s)
Host Specificity/genetics , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/pathogenicity , Mammals/virology , Virulence/genetics , A549 Cells , Alleles , Animals , Birds/virology , Cell Line , Cell Line, Tumor , Dogs , HEK293 Cells , Humans , Influenza in Birds/virology , Influenza, Human/virology , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/virology , Phylogeny , Reassortant Viruses/genetics , Viral Proteins/genetics , Virus Replication/geneticsABSTRACT
Loss of function of the PTEN tumour suppressor, resulting in dysregulated activation of the phosphoinositide 3-kinase (PI3K) signalling network, is recognized as one of the most common driving events in prostate cancer development. The observed mechanisms of PTEN loss are diverse, but both homozygous and heterozygous genomic deletions including PTEN are frequent, and often accompanied by loss of detectable protein as assessed by immunohistochemistry (IHC). The occurrence of PTEN loss is highest in aggressive metastatic disease and this has driven the development of PTEN as a prognostic biomarker, either alone or in combination with other factors, to distinguish indolent tumours from those likely to progress. Here, we discuss these factors and the consequences of PTEN loss, in the context of its role as a lipid phosphatase, as well as current efforts to use available inhibitors of specific components of the PI3K/PTEN/TOR signalling network in prostate cancer treatment.
Subject(s)
Carcinoma/etiology , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/metabolism , Prostatic Neoplasms/etiology , Animals , Carcinoma/diagnosis , Carcinoma/metabolism , Humans , Male , Molecular Targeted Therapy , Mutation , PTEN Phosphohydrolase/metabolism , Prognosis , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/therapy , Signal TransductionABSTRACT
The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphatase dephosphorylates PIP3, the lipid product of the class I PI 3-kinases, and suppresses the growth and proliferation of many cell types. It has been heavily studied, in large part due to its status as a tumour suppressor, the loss of function of which is observed through diverse mechanisms in many tumour types. Here we present a concise review of our understanding of the PTEN protein and highlight recent advances, particularly in our understanding of its localization and regulation by ubiquitination and SUMOylation.
Subject(s)
Cells/enzymology , PTEN Phosphohydrolase/metabolism , Protein Processing, Post-Translational , Humans , Protein Transport , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin/metabolismABSTRACT
Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.
Subject(s)
Alternative Splicing , Influenza A Virus, H5N2 Subtype/metabolism , RNA, Messenger/biosynthesis , RNA, Viral/biosynthesis , Viral Matrix Proteins/biosynthesis , Animals , Birds , Cell Line, Tumor , Disease Outbreaks , Dogs , Humans , Influenza A Virus, H5N2 Subtype/genetics , Influenza in Birds/epidemiology , Influenza in Birds/genetics , Influenza in Birds/metabolism , Influenza, Human/epidemiology , Influenza, Human/genetics , Influenza, Human/metabolism , Mice , Mice, Inbred BALB C , North America/epidemiology , RNA, Messenger/genetics , RNA, Viral/genetics , Viral Matrix Proteins/geneticsABSTRACT
PA-X is a fusion protein of influenza A virus encoded in part from a +1 frameshifted X open reading frame (X-ORF) in segment 3. We show that the X-ORFs of diverse influenza A viruses can be divided into two groups that differ in selection pressure and likely function, reflected in the presence of an internal stop codon and a change in synonymous diversity. Notably, truncated forms of PA-X evolved convergently in swine and dogs, suggesting a strong species-specific effect.
Subject(s)
Influenza A virus/genetics , Repressor Proteins/genetics , Viral Nonstructural Proteins/genetics , Animals , Codon , Codon, Terminator , Conserved Sequence , Databases, Genetic , Dogs , Evolution, Molecular , Genome, Viral , Models, Genetic , Open Reading Frames , Phylogeny , RNA, Viral/metabolism , Repressor Proteins/chemistry , Swine , Viral Nonstructural Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.
Subject(s)
Gene Expression Regulation, Viral , Influenza A virus/genetics , Peptide Chain Initiation, Translational , RNA, Viral/chemistry , Regulatory Sequences, Ribonucleic Acid , Viral Proteins/biosynthesis , Virus Assembly , Amino Acid Sequence , Base Sequence , Codon, Initiator , Codon, Terminator , HEK293 Cells , Humans , Influenza A virus/metabolism , Influenza A virus/physiology , Molecular Sequence Data , Mutation , Open Reading Frames , Peptide Biosynthesis , Peptides/genetics , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Viral/metabolism , Viral Proteins/genetics , Virion/physiologyABSTRACT
Influenza A virus is one of the world's major uncontrolled pathogens, causing seasonal epidemics as well as global pandemics. This was evidenced by the recent emergence and now prevalence of the 2009 swine origin pandemic H1N1 influenza A virus. In this study, quantitative proteomics using stable isotope labelling with amino acids in cell culture was used to investigate the changes in the host cell proteome in cells infected with pandemic H1N1 influenza A virus. The study was conducted in A549 cells that retain properties similar to alveolar cells. Several global pathways were affected, including cell cycle regulation and lipid metabolism, and these could be correlated with recent microarray analyses of cells infected with influenza A virus. Taken together, both quantitative proteomics and transcriptomic approaches can be used to identify potential cellular proteins whose functions in the virus life cycle could be targeted for chemotherapeutic intervention.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza, Human/metabolism , Lung/metabolism , Lung/virology , Proteome/analysis , Blotting, Western , Cell Line, Tumor , Epithelial Cells/metabolism , Epithelial Cells/virology , Host-Pathogen Interactions , Humans , Influenza, Human/pathology , Influenza, Human/virology , Isotope Labeling , Lung/pathology , Proteome/chemistry , Proteomics/methods , Reproducibility of ResultsABSTRACT
The cellular protein tetherin is thought to act as a 'leash' that anchors many enveloped viruses to the plasma membrane and prevents their release. We found that replication of multiple strains of influenza A virus was generally insensitive to alteration of tetherin levels, as assessed by output titre or scanning electron microscopy of cell-associated virions. This included human, swine, avian and equine isolates, strains that form filamentous or spherical particles and viruses that lack the M2 or NS1 proteins. Levels of cell-surface tetherin were not reduced by influenza infection, but tetherin and the viral haemagglutinin co-localized on the plasma membrane. However, tetherin could not be detected in filamentous virions, suggesting that influenza may possess a mechanism to exclude it from virions. Overall, if influenza does encode a specific antagonist of tetherin, it is not M2 or NS1 and we find no evidence for a role in host range specificity.
Subject(s)
Antigens, CD/metabolism , Host-Pathogen Interactions , Influenza A virus/physiology , Virus Release , Animals , Cell Line , Cell Membrane/chemistry , GPI-Linked Proteins/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Influenza A virus/growth & development , Microscopy, Confocal , Microscopy, Electron, Scanning , Viral LoadABSTRACT
Loss PTEN function is one of the most common events driving aggressive prostate cancers and biochemically, PTEN is a lipid phosphatase which opposes the activation of the oncogenic PI3K-AKT signalling network. However, PTEN also has additional potential mechanisms of action, including protein phosphatase activity. Using a mutant enzyme, PTEN Y138L, which selectively lacks protein phosphatase activity, we characterised genetically modified mice lacking either the full function of PTEN in the prostate gland or only lacking protein phosphatase activity. The phenotypes of mice carrying a single allele of either wild-type Pten or PtenY138L in the prostate were similar, with common prostatic intraepithelial neoplasia (PIN) and similar gene expression profiles. However, the latter group, lacking PTEN protein phosphatase activity additionally showed lymphocyte infiltration around PIN and an increased immune cell gene expression signature. Prostate adenocarcinoma, elevated proliferation and AKT activation were only frequently observed when PTEN was fully deleted. We also identify a common gene expression signature of PTEN loss conserved in other studies (including Nkx3.1, Tnf and Cd44). We provide further insight into tumour development in the prostate driven by loss of PTEN function and show that PTEN protein phosphatase activity is not required for tumour suppression.
Subject(s)
PTEN Phosphohydrolase , Prostatic Neoplasms , Animals , Male , Mice , Lipids , Phosphatidylinositol 3-Kinases/metabolism , Phosphoprotein Phosphatases , Prostate/metabolism , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolismABSTRACT
Avian influenza A viruses often do not propagate efficiently in mammalian cells. The viral polymerase protein PB2 is important for this host restriction, with amino-acid polymorphisms at residue 627 and other positions acting as 'signatures' of avian- or human-adapted viruses. Restriction is hypothesized to result from differential interactions (either positive or inhibitory) with unidentified cellular factors. We applied fluorescence recovery after photobleaching (FRAP) to investigate the mobility of the viral polymerase in the cell nucleus using A/PR/8/34 and A/Turkey/England/50-92/91 as model strains. As expected, transcriptional activity of a polymerase with the avian PB2 protein was strongly dependent on the identity of residue 627 in human but not avian cells, and this correlated with significantly slower diffusion of the inactive polymerase in human but not avian nuclei. In contrast, the activity and mobility of the PR8 polymerase was affected much less by residue 627. Sequence comparison followed by mutagenic analyses identified residues at known host-range-specific positions 271, 588 and 701 as well as a novel determinant at position 636 as contributors to host-specific activity of both PR8 and Turkey PB2 proteins. Furthermore, the correlation between poor transcriptional activity and slow diffusional mobility was maintained. However, activity did not obligatorily correlate with predicted surface charge of the 627 domain. Overall, our data support the hypothesis of a host nuclear factor that interacts with the viral polymerase and modulates its activity. While we cannot distinguish between positive and inhibitory effects, the data have implications for how such factors might operate.
Subject(s)
Host Specificity , Influenza A virus/enzymology , Influenza in Birds/virology , Influenza, Human/virology , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Birds , Cell Line , Humans , Influenza A Virus, H1N1 Subtype , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza A virus/physiology , Molecular Sequence Data , Protein Transport , Quail , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.
Subject(s)
Influenza A virus/physiology , RNA-Binding Proteins/physiology , Viral Core Proteins/physiology , Virus Assembly , Amino Acid Substitution/genetics , Animals , Cell Line , Chick Embryo , Chickens , Dogs , Hot Temperature , Humans , Mutant Proteins/physiology , Mutation, Missense , Nucleocapsid Proteins , RNA, Viral/biosynthesis , Viral Matrix Proteins/metabolism , Viral Plaque Assay , Viral Proteins/biosynthesis , Virion/ultrastructure , Virus ReplicationABSTRACT
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.
Subject(s)
Influenza A virus/physiology , Peptide Fragments/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Viral/genetics , Viral Proteins/genetics , Humans , Influenza A virus/genetics , Peptide Fragments/metabolism , Peptides/genetics , Peptides/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Viral Proteins/metabolism , Virus ReplicationABSTRACT
The sensing of viral nucleic acids by the innate immune system activates a potent antiviral response in the infected cell, a key component of which is the expression of genes encoding type I interferons (IFNs). Many viruses counteract this response by blocking the activation of host nucleic acid sensors. The evolutionarily conserved influenza A virus (IAV) protein PA-X has been implicated in suppressing the host response to infection, including the expression of type I IFNs. Here, we characterise this further using a PA-X-deficient virus of the mouse-adapted PR8 strain to study activation of the innate immune response in a mouse model of the early response to viral infection. We show that levels of Ifna4 and Ifnb1 mRNAs in the lungs of infected mice were elevated in the absence of PA-X and that this was completely dependent on MAVS. This therefore suggests a role for PA-X in preventing the accumulation of early type I IFN mRNAs in the lung during IAV infection.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Influenza A virus/physiology , Interferon Type I/metabolism , Repressor Proteins/genetics , Viral Nonstructural Proteins/genetics , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/metabolism , Animals , Immunity, Innate , Influenza A virus/metabolism , Interferon Type I/genetics , Lung/metabolism , Lung/virology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/virology , RNA, Messenger/metabolism , Repressor Proteins/deficiency , Repressor Proteins/metabolism , Signal Transduction , Toll-Like Receptor 7/deficiency , Toll-Like Receptor 7/genetics , Viral Nonstructural Proteins/deficiency , Viral Nonstructural Proteins/metabolismABSTRACT
OBJECTIVE: The multifunctional NS1 protein of influenza A virus has roles in antagonising cellular innate immune responses and promoting viral gene expression. To better understand the interplay between these functions, we tested the effects of NS1 effector domain mutations known to affect homo-dimerisation or interactions with cellular PI3 kinase or Trim25 on NS1 ability to promote nuclear export of viral mRNAs. RESULTS: The NS1 dimerisation mutant W187R retained the functions of binding cellular NXF1 as well as stabilising NXF1 interaction with viral segment 7 mRNAs and promoting their nuclear export. Two PI3K-binding mutants, NS1 Y89F and Y89A still bound NXF1 but no longer promoted NXF1 interactions with segment 7 mRNA or its nuclear export. The Trim25-binding mutant NS1 E96A/E97A bound NXF1 and supported NXF1 interactions with segment 7 mRNA but no longer supported mRNA nuclear export. Analysis of WT and mutant NS1 interaction partners identified hsp70 as specifically binding to NS1 E96A/E97A. Whilst these data suggest the possibility of functional links between NS1's effects on intracellular signalling and its role in viral mRNA nuclear export, they also indicate potential pleiotropic effects of the NS1 mutations; in the case of E96A/E97A possibly via disrupted protein folding leading to chaperone recruitment.
Subject(s)
Influenza A virus/genetics , Mutation , Viral Nonstructural Proteins/genetics , Active Transport, Cell Nucleus , Influenza A virus/pathogenicity , Protein Binding , Protein Folding , RNA, Messenger/metabolismABSTRACT
Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines.