Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5.

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

Failure to activate the IFN-beta promoter by a paramyxovirus lacking an interferon antagonist

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 syste

Analysis of Paramyxovirus Transcription and Replication by High-Throughput Sequencing.

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

Sensitivity of African swine fever virus to type I interferon is linked to genes within multigene families 360 and 505.

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

Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs

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 genus Rubulavirus on 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

Classical swine fever virus Npro antagonises IRF3 to prevent IFN-independent TLR3 and RIG-I-mediated apoptosis

Classical swine fever virus (CSFV) is the causative agent of classical swine fever,a notifiable disease of economic importance that causes severe leukopenia, fever and haemorrhagic disease in domesticated pigs and wild boar across the globe. CSFV has been shown to antagonise the induction of type I IFN, partly through a function of its N-terminal protease (Npro) which binds IRF3 and targets it for proteasomal degradation. Additionally, Npro has been shown to antagonise apoptosis triggered by the dsRNA-homologpoly (I:C), however the exact mechanism by which this is achieved has not been fully elucidated. In this study we confirm the ability of Npro to inhibit dsRNA-mediated apoptosis and show that Npro is also able to antagonise Sendai virus-mediated apoptosis in PK-15 cells. Gene editedPK-15 cell lines were used to show the dsRNA-sensing pathogen recognition receptors (PRRs) TLR3 and RIG-I specifically respond to poly(I:C) and SeV respectively, subsequently triggering apoptosis through pathways that convergeon IRF3 and culminate in the cleavage of caspase-3.Importantly, this IRF3-mediated apoptosis was found to be dependent on transcription-independent functions of IRF3 and also on Bax, a pro-apoptotic Bcl-2 family protein, through a direct interaction between the two proteins. Deletion of IRF3, stable expression of Npro and infection with wild-type CSFV were found to antagonise the mitochondrial localisation of Bax, a key hallmark of the intrinsic, mitochondrial pathway of apoptosis. Together, these findings show that Npro’s putative interaction with IRF3 is involved not only in its antagonism of type I IFN, but also dsRNA-mediated mitochondrial apoptosis

The cellular localization of avian influenza virus PB1-F2 protein alters the magnitude of IFN2 promoter and NFκB-dependent promoter antagonism in chicken cells

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

LGP2 plays a critical role in sensitizing mda-5 to activation by double-stranded RNA.

The DExD/H box RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene-5 (mda-5) sense viral RNA in the cytoplasm of infected cells and activate signal transduction pathways that trigger the production of type I interferons (IFNs). Laboratory of genetics and physiology 2 (LGP2) is thought to influence IFN production by regulating the activity of RIG-I and mda-5, although its mechanism of action is not known and its function is controversial. Here we show that expression of LGP2 potentiates IFN induction by polyinosinic-polycytidylic acid [poly(I:C)], commonly used as a synthetic mimic of viral dsRNA, and that this is particularly significant at limited levels of the inducer. The observed enhancement is mediated through co-operation with mda-5, which depends upon LGP2 for maximal activation in response to poly(I:C). This co-operation is dependent upon dsRNA binding by LGP2, and the presence of helicase domain IV, both of which are required for LGP2 to interact with mda-5. In contrast, although RIG-I can also be activated by poly(I:C), LGP2 does not have the ability to enhance IFN induction by RIG-I, and instead acts as an inhibitor of RIG-I-dependent poly(I:C) signaling. Thus the level of LGP2 expression is a critical factor in determining the cellular sensitivity to induction by dsRNA, and this may be important for rapid activation of the IFN response at early times post-infection when the levels of inducer are low

Human IFIT1 inhibits mRNA translation of rubulaviruses but not other members of the Paramyxoviridae family

This work was supported by The Welcome Trust (101788/Z/13/Z, 101792/Z/13/Z) and Medical research council grant (G1100110/1, MR/K024213/1).We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus (PIV) type 5, selectively inhibiting the translation of PIV5 mRNAs. Here we report that whilst PIV2, PIV5 and mumps virus (MuV) are sensitive to IFIT1, non-rubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV) and canine distemper virus (CDV) are resistant. The IFIT1-sensitivity of PIV5 was not rescued by co-infection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. Whilst the translation of PIV2, PIV5 and MuV mRNAs were directly inhibited by IFIT1, the translation of PIV3, SeV and CDV mRNAs were not. Using purified human mRNA capping enzymes we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5’ guanosine nucleoside cap (which need not be N7-methylated) and that this sensitivity is partly abrogated by 2’ O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2’ O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2’ -O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed. Importance Paramyxoviruses cause a wide variety of diseases and yet most of their genes encode for structural proteins and proteins involved in their replication cycle. Thus the amount of genetic information that determines the type of disease paramyxoviruses cause is relatively small. One factor that will influence disease outcomes is how they interact with innate host cell defences, including the interferon (IFN) system. Here we show that different paramyxoviruses interact in distinct ways with cells in a pre-existing IFN-induced antiviral state. Strikingly, all the rubulaviruses tested were sensitive to the antiviral action of ISG56/IFIT1, whilst all the other paramyxoviruses tested were resistant. We developed novel in vitro biochemical assays to investigate the mechanism of action of IFIT1, demonstrating that the mRNAs of rubulaviruses can be directly inhibited by IFIT1 and that this is at least partially because their mRNAs are not correctly methylated.Publisher PDFPeer reviewe