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The accessory protein, PB1-F2, of influenza A virus (IAV) functions in a chicken host to prolong infectious virus shedding and thus the transmission window. Here we show that this delay in virus clearance by PB1-F2 in chickens is accompanied by reduced transcript levels of type 1 interferon (IFN)-induced genes and NFκB-activated pro-inflammation cytokines. In vitro, two avian influenza isolate-derived PB1-F2 proteins, H9N2 UDL01 and H5N1 5092, exhibited the same antagonism of the IFN and pro-inflammation induction pathways seen in vivo, but to different extents. The two PB1-F2 proteins had different cellular localization in chicken cells, with H5N1 5092 being predominantly mitochondrial-associated and H9N2 UDL being cytoplasmic but not mitochondrial-localized. We hypothesized that PB1-F2 localization might influence the functionality of the protein during infection and that the protein sequence could alter cellular localization. We demonstrated that the sequence of the C-terminus of PB1-F2 determined cytoplasmic localization in chicken cells and this was linked with protein instability. Mitochondrial localization of PB1-F2 resulted in reduced antagonism of an NFκB-dependent promoter. In parallel, mitochondrial localization of PB1-F2 increased the potency of chicken IFN 2 induction antagonism. We suggest that mitochondrial localization of PB1-F2 restricts interaction with cytoplasmic-located IKKβ, reducing NFκB-responsive promoter antagonism, but enhances antagonism of the IFN2 promoter through interaction with the mitochondrial adaptor MAVS. Our study highlights the differential mechanisms by which IAV PB1-F2 protein can dampen the avian host innate signalling response.

Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo. They impact the outcome of natural infections, may help drive virus‐host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification., During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into “infectious” virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification. IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo. They impact the outcome of natural infections, may help drive virus‐host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

Childhood onset clinical syndromes involving intellectual disability and dysmorphic features, such as polydactyly, suggest common developmental pathways link seemingly unrelated phenotypes. We identified a consanguineous family of Saudi origin with varying complex features including intellectual disability, speech delay, facial dysmorphism and polydactyly. Combining, microarray based comparative genomic hybridisation (CGH) to identify regions of homozygosity, with exome sequencing, led to the identification of homozygous mutations in five candidate genes (RSPH6A, ANKK1, AMOTL1, ALKBH8, TRAPPC6A), all of which appear to be pathogenic as predicted by Proven, SIFT and PolyPhen2 and segregate perfectly with the disease phenotype. We therefore looked for differences in expression levels of each protein in HEK293 cells, expressing either the wild-type or mutant full-length cDNA construct. Unexpectedly, wild-type TRAPPC6A appeared to be unstable, but addition of the proteasome inhibitor MG132 stabilised its expression. Mutations have previously been reported in several members of the TRAPP complex of proteins, including TRAPPC2, TRAPPC9 and TRAPPC11, resulting in disorders involving skeletal abnormalities, intellectual disability, speech impairment and developmental delay. TRAPPC6A joins a growing list of proteins belonging to the TRAPP complex, implicated in clinical syndromes with neurodevelopmental abnormalities.

High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs., We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses. IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.

SummaryInfluenza 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 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 does not replicate in chicken cells that lack ANP32A. Amino acid differences in LRR5 domain 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 resistant genome edited chickens.

Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses., Author summary As well as causing acute infections that result in mild to serious disease, many RNA viruses can establish prolonged or persistent infections in some infected individuals, that occasionally lead to chronic or reactive disease. Little is known about the molecular mechanisms involved in the establishment of such infections. Using parainfluenza virus type 5 (PIV5) as a model, we show how lytic and persistent variants of the virus can be selected on the basis of single amino acid substitutions and propose that the selection of persistent variants as the adaptive immune response develops following an acute infection might be a mechanism these viruses have evolved to enhance their transmission rates. As well as being of fundamental interest, understanding the molecular basis by which RNA viruses establish persistent infections may improve our understanding of virus epidemiology (and hence improve the control of virus infections) and of virus:host interactions that influence the relationship between virus persistence and chronic/relapsing disease. Furthermore, the knowledge of how RNA viruses, such as PIV5, establish persistent infections may lead to improve vaccine design since vectors which can establish persistent infections may induce longer-lasting more robust immunity.

The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate the translation initiation factor 2α (eIF2α) and avoid shut-off of global protein synthesis and downstream activation of the pro-apoptotic factor CHOP. Residues V16 and F18A were critical for binding of DP71L to PP1. Mutation of this PP1 binding motif or deletion of residues between 52 and 66 reduced the ability of DP71L to cause dephosphorylation of eIF2α and inhibit CHOP induction. The residues LSAVL, between 57 and 61, were also required. PP1 was co-precipitated with wild type DP71L and the mutant lacking residues 52- 66 or the LSAVL motif, but not with the PP1 binding motif mutant. The residues in the LSAVL motif play a critical role in DP71L function but do not interfere with binding to PP1. Instead we propose these residues are important for DP71L binding to eIF2α., Highlights • The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate translation initiation factor eIF2α (eIF2α). • The residues V16, F18 of DP71L are required for binding to the α, β and γ isoforms of PP1 and for DP71L function. • The sequence LSAVL downstream from the PP1 binding site (residues 57–61) are also important for DP71L function. • DP71L mutants of the LSAVL sequence retain ability to co-precipitate with PP1 showing these sequences have a different role to PP1 binding.

This work, including the efforts of Richard E Randall, was funded by Wellcome Trust (101788/Z/13/Z). This work, including the efforts of Steve Goodbourn, was funded by Wellcome Trust (101792/Z/13/Z). We have sequenced the parainfluenza virus 5 strain that persistently infects the commonly used AGS human cell line without causing cytopathology. This virus is most closely related to human strains, indicating that it may have originated from biopsy material or from laboratory contamination during generation of the cell line. Publisher PDF

It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-β promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-β promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system.

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of pigs. There are conflicting reports on the role of interferon in ASFV infection. We therefore analysed the interaction of ASFV with porcine interferon, in vivo and in vitro. Virulent ASFV induced biologically active IFN in the circulation of pigs from day 3-post infection, whereas low virulent OUR T88/3, which lacks genes from multigene family (MGF) 360 and MGF505, did not. Infection of porcine leucocytes enriched for dendritic cells, with ASFV, in vitro, induced high levels of interferon, suggesting a potential source of interferon in animals undergoing acute ASF. Replication of OUR T88/3, but not virulent viruses, was reduced in interferon pretreated macrophages and a recombinant virus lacking similar genes to those absent in OUR T88/3 was also inhibited. These findings suggest that as well as inhibiting the induction of interferon, MGF360 and MGF505 genes also enable ASFV to overcome the antiviral state., Highlights • Virulent strains of African swine fever virus induces interferon during infection. • Virulent, but not attenuated strains of ASFV are resistant to the antiviral effects of interferon in porcine macrophages. • Sensitivity to interferon is linked to genes within multigene family 360 and multigene family 530. • Dendritic cells are a potential source of the interferon detected in vivo.

Many viruses, including human influenza A virus, have developed strategies for counteracting the host type I interferon (IFN) response. We have explored whether avian influenza viruses were less capable of combating the type I IFN response in mammalian cells, as this might be a determinant of host range restriction. A panel of avian influenza viruses isolated between 1927 and 1997 was assembled. The selected viruses showed variation in their ability to activate the expression of a reporter gene under the control of the IFN-β promoter and in the levels of IFN induced in mammalian cells. Surprisingly, the avian NS1 proteins expressed alone or in the genetic background of a human influenza virus controlled IFN-β induction in a manner similar to the NS1 protein of human strains. There was no direct correlation between the IFN-β induction and replication of avian influenza viruses in human A549 cells. Nevertheless, human cells deficient in the type I IFN system showed enhanced replication of the avian viruses studied, implying that the human type I IFN response limits avian influenza viruses and can contribute to host range restriction.

Mapuera virus(MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genusRubulaviruson the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.

The V protein of the paramyxovirus Nipah virus (NiV) has been shown to antagonize the interferon (IFN) response in human cells via sequestration of STAT1 and STAT2. This study describes a mutant of the NiV V protein, referred to as V(AAHL), that is unable to antagonize IFN signalling and demonstrates that a single amino acid substitution is responsible for its inactivity. The molecular basis for this was identified as a failure to interact with STAT1 and STAT2. It was also shown that NiV V, but not V(AAHL), was functional as an IFN antagonist in human, monkey, rabbit, dog, horse, pig and bat cells, which suggests that the ability of NiV to block IFN signalling is not a major constraint that prevents this virus from crossing species barriers.

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression from both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity.

The immunomodulatory drug thalidomide has been shown to be clinically useful in a number of situations due to its ability to inhibit TNF-α synthesis. However, its use is restricted by potentially serious side effects, including teratogenicity and neuorotoxicity; furthermore, insolubility may present problems in terms of systemic bioavailability. Recently, structural modifications of thalidomide have been designed enabling greatly enhanced anti-TNF-α activity in LPS-treated mice. In contrast to thalidomide (LPS-induced TNF-α IC50 ∼200 μM in DMSO) and other analogs tested, one of these compounds, CC-3052 (IC50 ∼1 μM in water), is water soluble. Furthermore, this analog exhibits increased stability in human plasma (t1/2 ∼17.5 vs 1.5 h for thalidomide) and appears to be nontoxic, nonmutagenic, and nonteratogenic. At pharmacologically active levels, cellular proliferation and LPS-induced IL-6 mRNA and IL-12p40 mRNA (as well as IL-1β and IL-6 protein levels) in whole blood cultures were not affected; apparent inhibition of NK activity by CC-3052 was reversed upon addition of exogenous rTNF-α. In addition, IL-10 mRNA and protein levels were increased. These properties are consistent with results indicating inhibition of phosphodiesterase type IV activity by CC-3052. Furthermore, CC-3052 did not increase the degradation rate of macrophage TNF-α transcripts nor inhibit LPS-induced primary macrophage NF-κB activation. Taken together, the potency of selective TNF-α inhibition, water solubility, and increased plasma stability make CC-3052 an excellent candidate for further development and clinical evaluation for the treatment of TNF-α-mediated disease.

This work was supported by the Wellcome Trust. Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2'-O-methyltransferase activity, an enzyme that methylates the 2'-hydroxyl group of ribose sugars in the 5'-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2'-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5. Publisher PDF

Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-beta promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade. Publisher PDF

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.

Chicken, which is almost universally-acceptable, will shortly become the most consumed meat in the developed and developing world. Viruses of chickens, whether enzootic (e.g. Newcastle, Mareks or infectious bursal disease viruses or infectious bronchitis virus) or zoonotic (especially avian influenza viruses such as H5N1 and H7N9), pose major threats to the supply of this globally important source of protein. Understanding the nature of the chicken innate responses will help us better understand these viruses, their evolution and their pathogenesis, and may help us develop more effective methods of controlling them. Although first discovered in chicken cells, details of the chicken interferon response are only just being elucidated, because chicken interferon was only cloned in 1994 and the chicken genome sequence was only reported in 2004. It is noteworthy that the identity of interferon-stimulated genes is far better described in mammals. Although there are increasing numbers of reports of chicken responses to virus infection, most of the regulated genes described are involved in the cell cycle, metabolic pathways and cellular structures required for virus replication. We have conducted a study aimed at describing the complement of potential anti-viral genes, identifying those induced by recombinant type I interferon in a common research and vaccine production substrate, namely primary chick embryo fibroblasts. The results are robust because 155 of the 193 genes induced were detected by at least two of the three transcriptomic technologies employed: Illumina RNA-seq, the Affymetrix 32K GeneChip Chicken Genome Array and the Chicken Gene 1.0 ST Array.

We conducted microarray analysis of the increasingly popular chicken DF-1 cell line and its primary progenitor, chicken embryo fibroblast cells (CEF), in both normal and stimulated conditions using recombinant chicken IFN- α and the CEF-adapted infectious bursal disease virus vaccine strain PBG98. We found that DF-1 cells have an attenuated innate immune response compared to CEF, and also that chicken SOCS1 (ChSOCS1), a negative regulator of cytokine signaling, is expressed at levels 13-fold higher in DF-1 cells than in CEF. We found that overexpressing ChSOCS1 in CEF reduced levels of phosphorylated STAT1 protein in response to viral infection but that its “SOCS box protein” domain (which, in mammalian SOCS1, interacts with an E3 ubiquitin ligase complex) is not essential for the inhibition of cytokine-induced JAK/STAT signaling activation in DF-1 cells. Overexpression of SOCS1 in DF-1 cells led to a significant relative decrease in expression of IFN- β , MX1, IFIT5 and MDA5 in response to PBG98 infection, and increased viral yield. Conversely, knockdown of SOCS1 increased ISG induction and reduced viral yield in IFN-stimulated DF-1 cells. Our results show that, like its mammalian counterpart, ChSOCS1 reduces induction of the IFN signaling pathway.

Most paramyxoviruses circumvent the IFN response by blocking IFN signaling and limiting the production of IFN by virus-infected cells. Here we report that the highly conserved cysteine-rich C-terminal domain of the V proteins of a wide variety of paramyxoviruses binds melanoma differentiation-associated gene 5 (mda-5) product. mda-5 is an IFN-inducible host cell DExD/H box helicase that contains a caspase recruitment domain at its N terminus. Overexpression of mda-5 stimulated the basal activity of the IFN- β promoter in reporter gene assays and significantly enhanced the activation of the IFN- β promoter by intracellular dsRNA. Both these activities were repressed by coexpression of the V proteins of simian virus 5, human parainfluenza virus 2, mumps virus, Sendai virus, and Hendra virus. Similar results to the reporter assays were obtained by measuring IFN production. Inhibition of mda-5 by RNA interference or by dominant interfering forms of mda-5 significantly inhibited the activation of the IFN- β promoter by dsRNA. It thus appears that mda-5 plays a central role in an intracellular signal transduction pathway that can lead to the activation of the IFN- β promoter, and that the V proteins of paramyxoviruses interact with mda-5 to block its activity.

Interferon-alpha (IFNα) binds to receptors on the cell surface, which initiate a cascade of signal transduction pathways that leads to transcription of selected genes. This transduction pathway involves binding of transcription factors to a common cis-acting DNA sequence called IFN-stimulated response element (ISRE). To test whether these signaling pathways are functional in hepatitis C virus (HCV)-replicating cells, we studied the regulation of ISRE-mediated transcription of firefly luciferase gene in stable replicon cell lines. A plasmid construct was prepared (pISRELuc) which contains four tandem repeats of 9-27 ISRE sequences positioned directly upstream of the herpes virus 1 thymidine kinase promoter TATA box that drives the expression of firefly luciferase. Regulation of ISRE-mediated expression of firefly luciferase by IFNα was studied by transfecting this clone into Huh-7 cells replicating HCV subgenomic HCV RNA. The significance of ISRE-mediated transcriptional activation was studied in a replicon cell line by pretreatment of cells with actinomycin D, which inhibits cellular DNA-dependent RNA transcription. IFN treatment activates ISRE-mediated expression of luciferase, indicating that this pathway is functional in Huh-7 cells. Activation of ISRE-mediated transcription of luciferase is relatively high in two Huh-7 stable cell lines replicating HCV subgenomic RNA. Inhibition of ISRE-mediated transcription of luciferase by actinomycin D also makes HCV replication totally resistant to IFNα. These in vitro studies suggest that activation of IFN-inducible genes is important in mounting a successful antiviral response against HCV.

The African Swine Fever Virus (ASFV) is an acute virus with a tropism for the pig macrophage and the ability to persist. In spite of these characteristics, no individual ASFV gene has been described which interferes with the impact of interferon (IFN) response. Moreover, bioinformatic searching failed to detect any ASFV genes with any homology to the IFN system. Therefore, we cloned and screened a total of 16 early expressed, non-homologous ASFV genes in luciferase reporter assays for their effect on the induction and impact of IFN responses. Five of these viral genes were demonstrated to inhibit the impact of IFN. One, the ASFV open reading frame (ORF) DP148R coding for protein MGF360-18R, inhibited luciferase reporter assays monitoring activation of the host cell response to both type I and type II IFN’s. Cells expressing this viral protein exhibited a clear reduction in the levels of STAT1, a critical transcription factor of the Jak-STAT pathway. Inhibition of the ubiquitin–proteasome pathway using the 26S proteasome inhibitor MG132 reversed the MGF360-18R-mediated degradation of STAT1. We conclude that ASFV has evolved multiple mechanisms for the manipulation of IFN responses and that MGF360-18R, in particular, manipulates the polyubiquitination system to induce degradation of STAT1, a critical signalling intermediate of the Jak-STAT pathway. Finally, the hypothesis that early expressed, non-homologous ASFV genes might include mechanisms for the inhibition of IFN responses, and indeed for host cell manipulation in general, has been confirmed.