• C protein
• TlmPV
• paramyxovirus
• viral RNA synthesis

• Paramyxovirinae/genetics
• Paramyxovirinae/physiology
• RNA, Viral/genetics
• Viral Proteins/genetics
• Virus Replication
• Animals
• Cricetinae
• Cell Line

• 2021B1111090002 R & D projects in key areas of Guangdong province

• IFN antagonism
• NF-kappa B (NF-κB)
• live-attenuated vaccine
• next-generation vaccines
• type I interferons (IFNs)
• veterinary vaccine
• veterinary virology
• viral immune evasion

Paramyxoviridae is a viral family within the order of Mononegavirales; they are negative single-strand RNA viruses that can cause significant diseases in both humans and animals. In order to replicate, paramyxoviruses–as any other viruses–have to bypass an important protective mechanism developed by the host’s cells: the defensive line driven by interferon. Once the viruses are recognized, the cells start the production of type I and type III interferons, which leads to the activation of hundreds of genes, many of which encode proteins with the specific function to reduce viral replication. Type II interferon is produced by active immune cells through a different signaling pathway, and activates a diverse range of genes with the same objective to block viral replication. As a result of this selective pressure, viruses have evolved different strategies to avoid the defensive function of interferons. The strategies employed by the different viral species to fight the interferon system include a number of sophisticated mechanisms. Here we analyzed the current status of the various strategies used by paramyxoviruses to subvert type I, II, and III interferon responses.

• JAK-STAT
• KPNA1
• interferon signaling
• karyopherin α1
• nonstructural protein 7
• nsp7
• porcine epidemic diarrhea virus

• Animals
• Cell Line
• Interferons/metabolism
• Janus Kinase 1/metabolism
• Porcine epidemic diarrhea virus/genetics
• STAT1 Transcription Factor/metabolism
• Signal Transduction
• Swine
• Viral Nonstructural Proteins/genetics
• Viral Nonstructural Proteins/metabolism
• alpha Karyopherins/metabolism

• Ministry of Science and Technology of the People's Republic of China (MOST)
• National Natural Science Foundation of China (NSFC)

SARS-CoV-2 is the causative agent of COVID-19. Overproduction and release of proinflammatory cytokines are the underlying cause of severe COVID-19. Treatment of this condition with JAK inhibitors is a double-edged sword, which might result in the suppression of proinflammatory cytokine storm and the concurrent enhancement of viral infection, since JAK signaling is essential for host antiviral response. Improving the current JAK inhibitor therapy requires a detailed molecular analysis on how SARS-CoV-2 modulates interferon (IFN)-induced activation of JAK-STAT signaling.

In this study, we focused on the molecular mechanism by which SARS-CoV-2 NSP13 helicase suppresses IFN signaling. Expression of SARS-CoV-2 NSP13 alleviated transcriptional activity driven by type I and type II IFN-responsive enhancer elements. It also prevented nuclear translocation of STAT1 and STAT2. The suppression of NSP13 on IFN signaling occurred at the step of STAT1 phosphorylation. Nucleic acid binding-defective mutant K345A K347A and NTPase-deficient mutant E375A of NSP13 were found to have largely lost the ability to suppress IFN-β-induced STAT1 phosphorylation and transcriptional activation, indicating the requirement of the helicase activity for NSP13-mediated inhibition of STAT1 phosphorylation. NSP13 did not interact with JAK1 nor prevent STAT1-JAK1 complex formation. Mechanistically, NSP13 interacted with STAT1 to prevent JAK1 kinase from phosphorylating STAT1.

SARS-CoV-2 NSP13 helicase broadly suppresses IFN signaling by targeting JAK1 phosphorylation of STAT1.

• COVID-19
• Helicase
• JAK1
• NSP13
• SARS-CoV-2
• STAT1

For efficient replication, viruses have developed multiple strategies to evade host antiviral innate immunity. Paramyxoviruses are a large family of enveloped RNA viruses that comprises diverse human and animal pathogens which jeopardize global public health and the economy. The accessory proteins expressed from the P gene by RNA editing or overlapping open reading frames (ORFs) are major viral immune evasion factors antagonizing type I interferon (IFN-I) production and other antiviral innate immune responses. However, the antagonistic mechanisms against antiviral innate immunity by accessory proteins differ among viruses. Here, we summarize the current understandings of immune evasion mechanisms by paramyxovirus accessory proteins, specifically how accessory proteins directly or indirectly target the adaptors in the antiviral innate immune signaling pathway to facilitate virus replication. Additionally, some cellular responses, which are also involved in viral replication, will be briefly summarized.

• China Postdoctoral Science Foundation

Particles of many paramyxoviruses include small amounts of proteins with a molecular weight of about 20 kDa. These proteins, termed “C”, are basic, have low amino acid homology and some secondary structure conservation. C proteins are encoded in alternative reading frames of the phosphoprotein gene. Some viruses express nested sets of C proteins that exert their functions in different locations: In the nucleus, they interfere with cellular transcription factors that elicit innate immune responses; in the cytoplasm, they associate with viral ribonucleocapsids and control polymerase processivity and orderly replication, thereby minimizing the activation of innate immunity. In addition, certain C proteins can directly bind to, and interfere with the function of, several cytoplasmic proteins required for interferon induction, interferon signaling and inflammation. Some C proteins are also required for efficient virus particle assembly and budding. C-deficient viruses can be grown in certain transformed cell lines but are not pathogenic in natural hosts. C proteins affect the same host functions as other phosphoprotein gene-encoded proteins named V but use different strategies for this purpose. Multiple independent systems to counteract host defenses may ensure efficient immune evasion and facilitate virus adaptation to new hosts and tissue environments.

• ESCRT
• Orthoparamyxovirinae
• Paramyxoviridae
• budding
• defective-interfering RNA
• immune evasion
• inflammasome
• nucleocapsid
• processivity
• replication

• Animals
• Defective Interfering Viruses
• Genome, Viral
• Humans
• Immune Evasion
• Immunity, Innate/immunology
• Inflammasomes
• Open Reading Frames
• Paramyxoviridae Infections/immunology
• Paramyxovirinae/genetics
• Paramyxovirinae/physiology
• Phosphoproteins/chemistry
• Phosphoproteins/genetics
• Phosphoproteins/immunology
• Phosphoproteins/metabolism
• Phylogeny
• Viral Proteins/chemistry
• Viral Proteins/genetics
• Viral Proteins/immunology
• Viral Proteins/metabolism
• Virus Assembly
• Virus Replication/physiology

• R21 AI159230 NIAID NIH HHS
• R01 AI143791 NIAID NIH HHS

• Accessory protein
• Caprine parainfluenza virus 3
• Interferon antagonist
• JAK/STAT signaling
• STAT1

Signal transducer and activator of transcription (STAT2) is a member of the STAT family that plays an essential role in immune responses to extracellular and intracellular stimuli, including inflammatory reactions, invasion of foreign materials, and cancer initiation. Although the majority of STAT2 studies in the last few decades have focused on interferon (IFN)-α/β (IFNα/β) signaling pathway-mediated host defense against viral infections, recent studies have revealed that STAT2 also plays an important role in human cancer development. Notably, strategic research on STAT2 function has provided evidence that transient regulatory activity by homo- or heterodimerization induces its nuclear localization where it to forms a ternary IFN-stimulated gene factor 3 (ISGF3) complex, which is composed of STAT1 and/or STAT2 and IFN regulatory factor 9 (IEF9). The molecular mechanisms of ISGF3-mediated ISG gene expression provide the basic foundation for the regulation of STAT2 protein activity but not protein quality control. Recently, previously unknown molecular mechanisms of STAT2-mediated cell proliferation via STAT2 protein quality control were elucidated. In this review, we briefly summarize the role of STAT2 in immune responses and carcinogenesis with respect to the molecular mechanisms of STAT2 stability regulation via the proteasomal degradation pathway.

The activity of STAT2, a protein stimulated by molecular signalling systems to activate selected genes in ways that can lead to cancer, is regulated by factors controlling its rate of degradation. Yong-Yeon Cho and colleagues at The Catholic University of Korea in South Korea review the role of STAT2 in links between molecular signals of the immune response and the onset of cancer. They focus on the significance of factors that regulate the stability of STAT2. One key factor appears to be the molecular mechanisms controlling the degradation of STAT2 by cellular structures called proteasomes. These structures break down proteins as part of routine cell maintenance. Deeper understanding of the stimulation, action and degradation of STAT2 will assist efforts to treat the many cancers in which STAT2 activity is involved.

• irf3
• prr
• apoptosis
• host-pathogen interactions
• innate immunity
• interferons
• mitochondria
• viral pathogenesis

• C protein
• JAK-STAT pathway
• JAK1
• Sendai virus
• TYK2
• interferon
• respirovirus

• Sendai virus
• defective interfering genome
• innate immunity
• interferons
• nucleocapsid
• paramyxovirus

• Amino Acid Substitution/genetics
• Amino Acid Substitution/immunology
• Animals
• Cell Line
• DEAD Box Protein 58
• Defective Viruses/genetics
• Defective Viruses/immunology
• Female
• Gene Expression Regulation
• Genome, Viral
• HeLa Cells
• Host-Parasite Interactions/genetics
• Host-Parasite Interactions/immunology
• Humans
• Immunity, Innate
• Interferon Regulatory Factor-3/analysis
• Interferon Type I/immunology
• Interferon-beta/metabolism
• Mice
• Mice, Inbred C57BL
• Mutation
• Nucleocapsid/metabolism
• Nucleoproteins/genetics
• Nucleoproteins/immunology
• Paramyxoviridae Infections/immunology
• Paramyxoviridae Infections/virology
• Paramyxovirinae/genetics
• Paramyxovirinae/immunology
• Pathogen-Associated Molecular Pattern Molecules/immunology
• Pathogen-Associated Molecular Pattern Molecules/metabolism
• RNA, Viral/genetics
• Receptors, Immunologic
• Sendai virus/genetics
• Sendai virus/immunology
• Virus Replication

• Japan Agency for Medical Research and Development (AMED)
• Ministry of Education, Culture, Sports, Science and Technology (MEXT)
• MEXT | Japan Society for the Promotion of Science (JSPS)

• inflammasome
• innate immunity

• Janus kinase/signal transducer and activator of transcription signaling
• Janus kinases
• interferons
• signal transducer and activator of transcriptions
• viral antagonism
• viral attenuation

• defective interfering rna
• rig-i
• sendai
• c protein

• Adjuvants, Immunologic/metabolism
• DEAD Box Protein 58/metabolism
• Gene Deletion
• Gene Expression Regulation, Viral
• Genetic Complementation Test
• HeLa Cells
• Humans
• RNA, Small Interfering/metabolism
• RNA, Viral/metabolism
• Receptors, Immunologic
• Sendai virus/genetics
• Sendai virus/immunology
• Viral Proteins/genetics

• U19 AI083025 NIAID NIH HHS

The non-structural protein, NS1, is a virulence factor encoded by influenza A viruses (IAVs). In this report, we provide evidence that the conserved residue, tyrosine (Y) 84, in a conserved putative SH2-binding domain in A/Duck/Hubei/2004/L-1 [H5N1] NS1 is critical for limiting an interferon (IFN) response to infection. A phenylalanine (F) substitution of this Y84 residue abolishes NS1-mediated downregulation of IFN-inducible STAT phosphorylation, and surface IFNAR1 expression. Recombinant IAV (rIAV) [H1N1] expressing A/Grey Heron/Hong Kong/837/2004 [H5N1] NS1-Y84F (rWSN-GH-NS1-Y84F) replicates to lower titers in human lung epithelial cells and is more susceptible to the antiviral effects of IFN-β treatment compared with rIAV expressing the intact H5N1 NS1 (rWSN-GH-NS1-wt). Cells infected with rWSN-GH-NS1-Y84F express higher levels of IFN stimulated genes (ISGs) associated with an antiviral response compared with cells infected with rWSN-GH-NS1-wt. In mice, intranasal infection with rWSN-GH-NS1-Y84F resulted in a delay in onset of weight loss, reduced lung pathology, lower lung viral titers and higher ISG expression, compared with mice infected with rWSN-GH-NS1-wt. IFN-β treatment of mice infected with rWSN-GH-NS1-Y84F reduced lung viral titers and increased lung ISG expression, but did not alter viral titers and ISG expression in mice infected with rWSN-GH-NS1-wt. Viewed altogether, these data suggest that the virulence associated with this conserved Y84 residue in NS1 is, in part, due to its role in regulating the host IFN response.

• influenza A viruses
• interferon signaling
• interferon-stimulated genes
• interferon-β
• non-structural protein 1

• Anti-viral
• IFN-stimulated gene (ISG)
• Influenza virus
• Interferon-alpha (IFN-α)
• Janus kinase/signal transducer of activator of transcription (JAK/STAT)
• STAT3
• Vaccinia virus

• 2',5'-Oligoadenylate Synthetase/metabolism
• Animals
• Cell Line
• Gene Knockdown Techniques
• Humans
• Influenza A virus/physiology
• Interferon-alpha/metabolism
• Mice
• Myxovirus Resistance Proteins
• STAT3 Transcription Factor/metabolism
• Signal Transduction
• Vaccinia virus/physiology
• eIF-2 Kinase/metabolism

• Health Research Board
• Science Foundation Ireland

• Foot-and-mouth disease virus
• IFN-γ
• janus kinase 1
• signaling pathway
• structural protein VP3

• Innate immunity
• Interferon gamma
• Macrophage
• NKLAM
• STAT1
• Ubiquitination

• Animals
• Bone Marrow Cells/cytology
• DNA/metabolism
• HEK293 Cells
• Humans
• Interferon-gamma/pharmacology
• Janus Kinase 1/metabolism
• Lysine/metabolism
• Macrophages/drug effects
• Macrophages/metabolism
• Membrane Proteins/metabolism
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Models, Biological
• Phosphorylation
• Protein Binding/drug effects
• RAW 264.7 Cells
• Receptors, Interferon/metabolism
• STAT1 Transcription Factor/metabolism
• Transcription, Genetic/drug effects
• Ubiquitination/drug effects
• Interferon gamma Receptor

• I01 BX000705 BLRD VA
• R56 AI089758 NIAID NIH HHS

• JAK/STAT pathway
• immune evasion
• innate immunity
• interferon
• vaccines
• viruses

• Alternative interferon response pathway
• Cytokines/interferon
• Host-pathogen interactions
• ISGF3
• STAT2 transcription factor
• STAT2/IRF9
• Signal transduction

Short linear motifs (SLiM) are short peptides that facilitate protein function and protein-protein interactions. Viruses utilize these motifs to enter into the host, interact with cellular proteins, or egress from host cells. Studying functional motifs may help to predict protein characteristics, interactions, or the putative cellular role of a protein. In virology, it may reveal aspects of the virus tropism and help find antiviral therapeutics. This review highlights the recent understanding of functional motifs utilized by viruses. Special attention was paid to the function of proteins harboring these motifs, and viruses encoding these proteins. The review highlights motifs involved in (i) immune response and post-translational modifications (e.g., ubiquitylation, SUMOylation or ISGylation); (ii) virus-host cell interactions, including virus attachment, entry, fusion, egress and nuclear trafficking; (iii) virulence and antiviral activities; (iv) virion structure; and (v) low-complexity regions (LCRs) or motifs enriched with residues (Xaa-rich motifs).

• APOBEC
• PDZ domain
• agnoprotein
• clathrin endocytosis
• inhibitor of apoptosis (IAP)
• low-complexity repeats
• pentraxin
• retinoblastoma
• transposition
• ubiquitylation

Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents.

• antiviral agent
• broad-spectrum
• cellular defense
• replication
• viral entry

• C protein
• Interferon-β
• Lipopolysaccharide
• Nitric oxide
• Sendai virus

The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share a common N-terminus (PNT), and C, which overlaps PNT. Overlapping genes are of particular interest because they encode proteins originated de novo, some of which have unknown structural folds, challenging the notion that nature utilizes only a limited, well-mapped area of fold space. The C proteins cluster in three groups, comprising measles, Nipah, and Sendai virus. We predicted that all C proteins have a similar organization: a variable, disordered N-terminus and a conserved, α-helical C-terminus. We confirmed this predicted organization by biophysically characterizing recombinant C proteins from Tupaia paramyxovirus (measles group) and human parainfluenza virus 1 (Sendai group). We also found that the C of the measles and Nipah groups have statistically significant sequence similarity, indicating a common origin. Although the C of the Sendai group lack sequence similarity with them, we speculate that they also have a common origin, given their similar genomic location and structural organization. Since C is dispensable for viral replication, unlike PNT, we hypothesize that C may have originated de novo by overprinting PNT in the ancestor of Paramyxovirinae. Intriguingly, in measles virus and Nipah virus, PNT encodes STAT1-binding sites that overlap different regions of the C-terminus of C, indicating they have probably originated independently. This arrangement, in which the same genetic region encodes simultaneously a crucial functional motif (a STAT1-binding site) and a highly constrained region (the C-terminus of C), seems paradoxical, since it should severely reduce the ability of the virus to adapt. The fact that it originated twice suggests that it must be balanced by an evolutionary advantage, perhaps from reducing the size of the genetic region vulnerable to mutations.

• Amino Acid Sequence
• Binding Sites
• Evolution, Molecular
• Molecular Sequence Data
• Paramyxovirinae/genetics
• Paramyxovirinae/metabolism
• Phosphoproteins/chemistry
• Phosphoproteins/genetics
• Phosphoproteins/metabolism
• STAT1 Transcription Factor/metabolism
• Sequence Alignment
• Sequence Analysis
• Sequence Homology, Nucleic Acid
• Species Specificity
• Viral Proteins/chemistry
• Viral Proteins/genetics
• Viral Proteins/metabolism

• Wellcome Trust
• T32 AI007610 NIAID NIH HHS
• 090005 Wellcome Trust

• DC
• ISG
• ORF
• WT
• dendritic cells
• innate immunity
• interferon
• interferon-stimulated gene
• moDC
• monocyte-derived DC
• open reading frame
• pDC
• paramyxovirus
• plasmacytoid DC
• wild type

The Sendai virus (SeV) C proteins are shown to exert multiple functions during the course of infection. Perhaps reflecting their many functions, they occur at multiple sites of the cell. In this study, we focused on the nuclear-localizing ability of the smaller C protein, Y1, and found that this translocation is mediated by Ran GTPase but not by passive diffusion, and that basic residues within the 149-157 amino acid region are critical for that. The mechanism of inhibition of interferon (IFN)-signaling seemed to differ between the C and Y1 proteins, since deletion of 12 C-terminal amino acids resulted in a loss of the function for the C but not for the Y1 protein. The ability of Y1 mutants to inhibit IFN-α-induced, ISRE-driven expression of a reporter gene almost paralleled with that to localize in the nucleus. These results suggest that nuclear localization of the Y1 protein might be important for the inhibitory effect on type-I IFN-stimulated gene expression.

The paramyxoviruses are a family of > 30 viruses that variously infect humans, other mammals and fish to cause diverse outcomes, ranging from asymptomatic to lethal disease, with the zoonotic paramyxoviruses Nipah and Hendra showing up to 70% case-fatality rate in humans. The capacity to evade host immunity is central to viral infection, and paramyxoviruses have evolved multiple strategies to overcome the host interferon (IFN)-mediated innate immune response through the activity of their IFN-antagonist proteins. Although paramyxovirus IFN antagonists generally target common factors of the IFN system, including melanoma differentiation associated factor 5, retinoic acid-inducible gene-I, signal transducers and activators of transcription (STAT)1 and STAT2, and IFN regulatory factor 3, the mechanisms of antagonism show remarkable diversity between different genera and even individual members of the same genus; the reasons for this diversity, however, are not currently understood. Here, we review the IFN antagonism strategies of paramyxoviruses, highlighting mechanistic differences observed between individual species and genera. We also discuss potential sources of this diversity, including biological differences in the host and/or tissue specificity of different paramyxoviruses, and potential effects of experimental approaches that have largely relied on in vitro systems. Importantly, recent studies using recombinant virus systems and animal infection models are beginning to clarify the importance of certain mechanisms of IFN antagonism to in vivo infections, providing important indications not only of their critical importance to virulence, but also of their potential targeting for new therapeutic/vaccine approaches.

• Paramyxoviridae
• Innate immunity
• Signal transducers and activators of transcription 1
• Signal transducers and activators of transcription 2
• Melanoma differentiation associated factor 5
• Retinoic acid-inducible gene-I

The P gene of paramyxoviruses is unique in producing not only P but also “accessory” C and/or V proteins. Successful generation of C- or V-deficient recombinant viruses using a reverse genetics technique has been revealing their importance in viral pathogenesis as well as replication. As for Sendai virus (SeV), the C proteins, a nested set of four polypeptides C’, C, Y1, and Y2, have been shown to exert multiple functions in escaping from the host innate immunity, inhibiting virus-induced apoptosis, promoting virus assembly and budding, and regulating viral RNA synthesis. In this study, we subjected the 4C(-) recombinant lacking expression of all four C proteins to serial passages through eggs, and found the rapid emergence of a C-recovered revertant virus. Unlike the SeV strains or the recombinants reported previously or tested in this study, this was caused by an exceptionally quick accumulation of U-to-C transitions in a limited region of the 4C(-) genome causing recovery of the C protein expression. These results suggest that a lack of C proteins could lead unexpectedly to strong selective pressures, and that the C proteins might play more critical roles in SeV replication than ever reported.

• Animals
• Cell Line
• Chick Embryo
• Cytosine/chemistry
• Genome, Viral
• Macaca mulatta
• RNA, Viral/biosynthesis
• RNA, Viral/chemistry
• RNA, Viral/genetics
• Recombination, Genetic
• Sendai virus/genetics
• Sendai virus/growth & development
• Sendai virus/pathogenicity
• Uracil/chemistry
• Viral Proteins/genetics
• Viral Proteins/metabolism
• Virus Replication

• ubiquitin
• interferon
• e3 ligase
• virus

• Amino Acid Sequence
• Animals
• Gene Expression Regulation
• Humans
• Interferons/immunology
• Interferons/metabolism
• Molecular Sequence Data
• Signal Transduction/physiology
• Ubiquitin/metabolism
• Ubiquitin-Protein Ligases/metabolism
• Ubiquitination
• Viruses/immunology
• Viruses/metabolism

• R01 CA092900 NCI NIH HHS
• R01 CA142425 NCI NIH HHS
• CA142425 NCI NIH HHS
• CA092900 NCI NIH HHS

We have previously shown that hepatitis B virus (HBV) protein X (HBX), a regulatory protein of HBV, activates Stat1, leading to type I interferon (IFN) production. Type I IFN secreted from HBX-expressing hepatic cells enforces antiviral signals through its binding to the cognate type I IFN receptor. We therefore investigated how cells handle this detrimental situation. Interestingly, compared to Chang cells stably expressing an empty vector (Chang-Vec), Chang cells stably expressing HBX (Chang-HBX) showed lower levels of IFN-α receptor 1 (IFNAR1) protein, a subunit of type I IFN receptor. The levels of IFNAR1 transcripts detected in Chang-HBX cells were lower than the levels in Chang-Vec cells, indicating that HBX regulates IFNAR1 at the transcriptional level. Moreover, we observed that HBX induced the translocation of IFNAR1 to the cytoplasm. Consistent with these observations, HBX also downregulated Tyk2, which is required for the stable expression of IFNAR1 on the cell surface. Eventually, Chang-HBX cells consistently maintained a lower level of IFNAR1 expression and displayed no proper response to IFN-α, while Chang-Vec cells exhibited a proper response to IFN-α treatment. Taken together, we propose that HBX downregulates IFNAR1, leading to the avoidance of extracellular IFN-α signal transduction.

One of the accessory proteins of Sendai virus (SeV), C, translated from an alternate reading frame of P/V mRNA has been shown to function at multiple stages of infection in cell cultures as well as in mice. C protein has been reported to counteract signal transduction by interferon (IFN), inhibit apoptosis induced by the infection, enhance the efficiency of budding of viral particles, and regulate the polarity of viral genome-length RNA synthesis to maximize production of infectious particles. In this study, we have generated a series of SeV recombinants containing substitutions of highly conserved, charged residues within the C protein, and characterized them together with previously-reported C′/C(−), 4C(−), and F170S recombinant viruses in infected cell cultures in terms of viral replication, cytopathogenicity, and antagonizing effects on host innate immunity. Unexpectedly, the amino acid substitutions had no or minimal effect on viral growth and viral RNA synthesis. However, all the substitutions of charged amino acids resulted in the loss of a counteracting effect against the establishment of an IFN-α-mediated anti-viral state. Infection by the virus (Cm2′) containing mutations at K77 and D80 induced significant IFN-β production, severe cytopathic effects, and detectable amounts of viral dsRNA production. In addition to the Cm2′ virus, the virus containing mutations at E114 and E115 did not inhibit the poly(I:C)-triggered translocation of cellular IRF-3 to the nucleus. These results suggest that the C protein play important roles in viral escape from induction of IFN-β and cell death triggered by infection by means of counteracting the pathway leading to activation of IRF-3 as well as of minimizing viral dsRNA production.

• Amino Acids/metabolism
• Cell Nucleus/drug effects
• Cell Nucleus/metabolism
• Conserved Sequence
• Cytopathogenic Effect, Viral/drug effects
• Genome, Viral/genetics
• HeLa Cells
• Humans
• Immune Evasion/drug effects
• Immune Evasion/immunology
• Immunity, Innate/drug effects
• Immunity, Innate/immunology
• Interferon Regulatory Factor-3/metabolism
• Interferon-beta/biosynthesis
• Kinetics
• Mutation/genetics
• Poly I-C/pharmacology
• Protein Transport/drug effects
• RNA, Double-Stranded/biosynthesis
• Recombination, Genetic/drug effects
• Recombination, Genetic/genetics
• Respirovirus Infections/immunology
• Respirovirus Infections/virology
• Sendai virus/drug effects
• Sendai virus/genetics
• Sendai virus/metabolism
• Signal Transduction/drug effects
• Structure-Activity Relationship
• Viral Proteins/chemistry
• Viral Proteins/metabolism
• Virion/genetics

STAT1 belongs to the STAT family of transcription factors, which comprises seven factors: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. STAT1 is a 91 kDa protein originally identified as the mediator of the cellular response to interferon (IFN) α, and thereafter found to be a major component of the cellular response to IFNγ. STAT1 is, in fact, involved in the response to several cytokines and to growth factors. It is activated by cytokine receptors via kinases of the JAK family. STAT1 becomes phosphorylated and forms a dimer which enters the nucleus and triggers the transcription of its targets. Although not lethal at birth, selective gene deletion of STAT1 in mice leads to rapid death from severe infections, demonstrating its major role in the response to pathogens. Similarly, in humans who do not express STAT1, there is a lack of resistance to pathogens leading to premature death. This indicates a key, non-redundant function of STAT1 in the defence against pathogens. Thus, to successfully infect organisms, bacterial, viral or parasitic pathogens must overcome the activity of STAT1, and almost all the steps of this pathway can be blocked or inhibited by proteins produced in infected cells. Interestingly, some pathogens, like the oncogenic Epstein–Barr virus, have evolved a strategy which uses STAT1 activation.

A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.

Respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children, elderly and immuno-compromised individuals. Their RNA genome encodes for structural proteins that compose viral particles, but also for virulence factors that alter cell biology to enhance virus replication and spreading. Among them, the C protein plays a critical role by blocking cellular response to type I interferons, the main antiviral cytokines secreted during virus infections. To provide molecular basis to this activity, we found that the C protein of human parainfluenza virus type 3 (hPIV3-C), the most frequent human Respirovirus, interacts with STAT1, a key component of type I interferon receptor complex. But hPIV3-C was also found to interact with GRB2, an adaptor molecule involved in cellular response to Epidermal Growth Factor (EGF), and to enhance cell response to this cytokine. This pathway increases protein translation, promotes cell survival and contributes to airway inflammation and mucus secretion. Thus, our findings show that hPIV3-C not only inhibits the antiviral response but also stimulates cellular response to EGF, which benefits virus replication and induces an excessive inflammation of airways during infection.

• Amino Acids/genetics
• Amino Acids/metabolism
• Animals
• Cell Line
• Chlorocebus aethiops
• Dengue Virus/genetics
• Dengue Virus/metabolism
• Gene Expression Regulation
• Humans
• Proteasome Endopeptidase Complex/metabolism
• Protein Binding
• STAT2 Transcription Factor/genetics
• STAT2 Transcription Factor/metabolism
• Ubiquitin/metabolism
• Viral Nonstructural Proteins/genetics
• Viral Nonstructural Proteins/metabolism

• U54 AI057158 NIAID NIH HHS
• U54 AI57158 NIAID NIH HHS

An overview is given on the development of technologies to allow reverse genetics of RNA viruses, i.e., the rescue of viruses from cDNA, with emphasis on nonsegmented negative-strand RNA viruses ( Mononegavirales ), as exemplified for measles virus (MV). Primarily, these technologies allowed site-directed mutagenesis, enabling important insights into a variety of aspects of the biology of these viruses. Concomitantly, foreign coding sequences were inserted to (a) allow localization of virus replication in vivo through marker gene expression, (b) develop candidate multivalent vaccines against measles and other pathogens, and (c) create candidate oncolytic viruses. The vector use of these viruses was experimentally encouraged by the pronounced genetic stability of the recombinants unexpected for RNA viruses, and by the high load of insertable genetic material, in excess of 6 kb. The known assets, such as the small genome size of the vector in comparison to DNA viruses proposed as vectors, the extensive clinical experience of attenuated MV as vaccine with a proven record of high safety and efficacy, and the low production cost per vaccination dose are thus favorably complemented.

• Animals
• Cell Line, Tumor
• Chlorocebus aethiops
• Humans
• Interferon Type I/immunology
• Mice
• Rinderpest/immunology
• Rinderpest/virology
• Rinderpest virus/physiology
• Transfection
• Vero Cells
• Viral Nonstructural Proteins/immunology
• Virus Replication/physiology

• BBS/E/I/00001014 Biotechnology and Biological Sciences Research Council
• BBSEI00001014 Biotechnology and Biological Sciences Research Council

• Bronchi/metabolism
• Bronchi/virology
• Cilia/virology
• Humans
• Interferon-alpha/metabolism
• Interferon-beta/metabolism
• Interferons/metabolism
• Microscopy, Confocal
• Models, Biological
• Mutation
• Parainfluenza Virus 1, Human/genetics
• Parainfluenza Virus 1, Human/metabolism
• Phenotype
• Protein Structure, Tertiary
• Reverse Transcriptase Polymerase Chain Reaction
• Temperature
• Trachea/metabolism
• Trachea/virology

• R01 HL077844 NHLBI NIH HHS
• Intramural NIH HHS
• R01 HL77844-1 NHLBI NIH HHS