Members of the Paramyxovirinae subfamily include viruses such as measles, mumps, parainfluenza viruses (PIV) of humans, Newcastle Disease virus of birds, Sendai virus (SeV) of rodents, and simian virus 5 (SV5), which has been isolated from monkeys, dogs, pigs, and humans. Paramyxoviruses also have zoonotic potential, as has been observed with the newly emergent Hendra (HeV) and Nipah viruses, which naturally infect fruit bats but can cause serious, often fatal infections when transmitted to farm and domestic animals and to humans (reviewed in ref.2). Like all viruses, upon infection of cells, paramyxoviruses are subjected to a variety of intracellular antiviral responses, including the IFN response (reviewed in refs. 3-5). Over the last few years, it has become clear that protein products of the P͞V͞C gene of viruses within the Paramyxovirinae subfamily (for review of the molecular biology of paramyxoviruses, see ref. 1) specifically reduce the effectiveness of the IFN response. For example, the V protein of SV5 targets signal transducer and activator of transcription 1 (STAT1) for degradation, thereby blocking both IFN-␣͞␤ and IFN-␥ signaling within infected cells (6), whereas the C proteins of SeV block IFN signaling by interfering with STAT phosphorylation or stability (reviewed in refs. 7-9). As well as blocking IFN signaling, these viruses also specifically limit the production of IFN by virus-infected cells (10-12). The block on IFN-␤ production is at the level of transcription, because very little IFN-␤ mRNA is induced in cells infected with SV5. In contrast, large amounts of IFN-␤ mRNA (and thus IFN-␤) are produced by cells infected with a recombinant of SV5 (SV5V⌬C) that produces a truncated V protein lacking the cysteine-rich C terminus (which is dispensable for virus replication), suggesting that the V protein is responsible for the block on IFN production. This conclusion is supported by the observation that in gene reporter assays, the V proteins of SV5, PIV2, and SeV inhibit the activation of the IFN-␤ promoter in response to intracellular dsRNA (11).Initial transcription from the IFN-␤ promoter requires the activation of a number of cellular transcription factors, including IFN regulatory factor (IRF)-3 and NF-B, leading to the formation of an enhanceosome complex that associates with the basal transcriptional machinery to recruit RNA polymerase II to the IFN-␤ promoter (reviewed in refs. 3 and 13). The molecular details of how the V proteins of paramyxoviruses block IFN production are not known, but the block affects the signal transduction pathway that activates both NF-B and IRF-3 in response to dsRNA. Thus, these transcription factors are not activated in cells infected with wild-type SV5 but are activated in cells infected with SV5V⌬C. Furthermore, ectopic expression of SV5 V inhibits the activation of IRF-3 and NF-B by both dsRNA and infection with SV5V⌬C (10, 11). Unlike the targeted degradation of signal transducer and activator of transcription 1 (STAT1), which requires both the N-and C-t...

The induction of IFN-beta by the paramyxovirus PIV5 (formerly known as SV5) is limited by the action of the viral V protein that targets the cellular RNA helicase mda-5. Here we show that 12 other paramyxoviruses also target mda-5 by a direct interaction between the conserved cysteine-rich C-terminus of their V proteins and the helicase domain of mda-5. The inhibition of IFN-beta induction is not species-restricted, being observed in a range of mammalian cells as well as in avian cells, and we show that the inhibition of mda-5 function is also not restricted to mammalian cells. In contrast, the V proteins do not bind to the related RNA helicase RIG-I and do not inhibit its activity. The relative contributions of mda-5 and RIG-I to IFN-beta induction are discussed.

We have identified two novel proteins that interact specifically with the C-terminal repression domain of Interferon Regulatory Factor-2 (IRF-2). These proteins, which we term IRF-2 binding proteins 1 and 2 (IRF-2BP1 and IRF-2BP2, the latter having two splicing isoforms, A and B), are nuclear proteins, and have the properties of IRF-2-dependent transcriptional co-repressors that can inhibit both enhancer-activated and basal transcription in a manner that is not dependent upon histone deacetylation. IRF-2BP1 and IRF-2BP2A/B contain an N-terminal zinc finger and a C-terminal RING finger domain of the C3HC4 subclass, but show no homology to other known transcriptional regulators; they therefore define a new family of co- repressor proteins. An alternatively spliced form of IRF-2 that lacks two amino acids (valines 177 and 178) in the central portion of the protein (IRF-2[S]) cannot bind to these co-repressors and cannot mediate repression despite having the same C- terminal repression domain as IRF-2, suggesting that the relative conformation of the DNA binding domain and the C-terminal region of IRF-2 is crucial for transcriptional repression.

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acidinducible 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.Mammalian cells contain a variety of pattern recognition receptors that recognize foreign macromolecules termed pathogenassociated molecular patterns (PAMPs). Viral PAMPs generated in the cytosol during replication are recognized by the DExD/H-box RNA helicases coded for by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) (reviewed in reference 30) and stimulate the production of type I interferon (IFN), which constitutes a major component of the innate immune response to virus infection (reviewed in reference 21). It is becoming clear that viruses generate a variety of different PAMPs and that, rather than being redundant, mda-5 and RIG-I show ligand specificity and are therefore differentially sensitive to activation by different viruses. For example, RIG-I seems to be more important for IFN induction in response to hepatitis C virus (HCV) (6, 24) and influenza A virus (12, 19), while mda-5 is necessary for responses to picornaviruses (7,12). Both mda-5 and RIG-I can be activated by the synthetic double-stranded RNA (dsRNA) poly(I-C), but a recent study suggests that the length of the dsRNA influences whether IFN induction is dependent on mda-5 or RIG-I, with mda-5 being more important for induction by long dsRNA and RIG-I more important for induction by short dsRNA (11). In addition to length, other structural features of viral RNAs can also determine receptor activation. For example, single-stranded RNA (ssRNA) and dsRNA molecules bearing a 5Ј triphosphate induce IFN via RIG-I and not mda-5 (10, 19). This motif is recognized as nonself, since most cellular RNAs are either capped or have a 5Ј monophosphate. IFN induction by RNA purified from influenza A virus, vesicular stomatitis virus, and rabies virus requires RIG-I and is dependent on the presence of a 5Ј triphosphate, underlining the importance of this motif as a genuine viral PAMP (8,10,19).mda-5 and RIG-I share a common domain structure with two tandem C...

b RIG-I and mda-5 are activated by viral RNA and stimulate type I interferon production. Laboratory of genetics and physiology 2 (LGP2) shares homology with RIG-I and mda-5 but lacks the CARD domains required for signaling. The V proteins of paramyxoviruses limit interferon induction by binding mda-5 and preventing its activation; however, they do not bind RIG-I and have not been considered inhibitors of RIG-I signaling. Here we uncover a novel mechanism of RIG-I inhibition in which the V protein of parainfluenzavirus type 5 (PIV5; formerly known as simian virus type 5 [SV5]) interacts with LGP2 and cooperatively inhibits induction by RIG-I ligands. A complex between RIG-I and LGP2 is observed in the presence of PIV5-V, and we propose that this complex is refractory to activation by RIG-I ligands. The V proteins from other paramyxoviruses also bind LGP2 and demonstrate LGP2-dependent inhibition of RIG-I signaling. This is significant, because it demonstrates a general mechanism for the targeting of the RIG-I pathway by paramyxoviruses. Virus infection stimulates innate immune responses in the host, among which the production of type I interferon (IFN) plays a critical role in restricting virus replication, via the upregulation of a large number of IFN-stimulated genes, and through the modulation of subsequent adaptive immune responses (reviewed in reference 37). IFN induction is triggered by the recognition of pathogen-associated molecular patterns (PAMPs), principally those associated with viral nucleic acids, which are seen as foreign by cellular pattern recognition receptors (PRRs; reviewed in reference 21). The RNA helicases RIG-I and mda-5 are the two bestcharacterized cytoplasmic PRRs; RIG-I preferentially recognizes RNA molecules with an uncapped 5=-triphosphate in a short region of blunt double-stranded RNA (dsRNA), while mda-5 recognizes longer molecules of dsRNA which need not be 5=-triphosphorylated (13,16,32,33,42,44; reviewed in reference 43). Viral RNA binds to the C-terminal regulatory domain (RD) of RIG-I or mda-5, and it is this region that confers specificity of PAMP recognition, although the central RNA helicase domain may also be involved in binding longer RNA molecules (22,24,28,49,52). RNA binding causes a conformational change which promotes oligomerization and allows interaction of the N-terminal CARD domains of RIG-I or mda-5 with the mitochondrial adapter protein IPS-1 (also known as VISA, MAVS, and CARDIF). This initiates a signaling cascade leading to activation and nuclear translocation of the transcription factors IRF-3 and NF-B, which are needed to turn on transcription of the IFN-␤ gene.While the interactions between synthetic PAMPs and RIG-I or mda-5 have been well characterized, the types of PAMPs recognized by RIG-I and mda-5 during viral infections are less clear. Mice lacking mda-5 showed no IFN induction in response to the picornavirus encephalomyocarditis virus (EMCV) (10, 17), consistent with the generation of a dsRNA replicative intermediate for this virus; RIG-I 0/0 mice...

The V protein of simian virus 5 (SV5) facilitates the ubiquitination and subsequent proteasome-mediated degradation of STAT1. Here we show, by visualizing direct protein-protein interactions and by using the yeast two-hybrid system, that while the SV5 V protein fails to bind to STAT1 directly, it binds directly and independently to both DDB1 and STAT2, two cellular proteins known to be essential for SV5-mediated degradation of STAT1. We also demonstrate that STAT1 and STAT2 interact independently of SV5 V and show that SV5 V protein acts as an adaptor molecule linking DDB1 to STAT2/STAT1 heterodimers, which in the presence of additional accessory cellular proteins, including Cullin 4a, can ubiquitinate STAT1. Additionally, we show that the avidity of STAT2 for V is relatively weak but is significantly enhanced by the presence of both STAT1 and DDB1, i.e., the complex of STAT1, STAT2, DDB1, and SV5 V is more stable than a complex of STAT2 and V. From these studies we propose a dynamic model in which SV5 V acts as a bridge, bringing together a DDB1/Cullin 4a-containing ubiquitin ligase complex and STAT1/STAT2 heterodimers, which leads to the degradation of STAT1. The loss of STAT1 results in a decrease in affinity of binding of STAT2 for V such that STAT2 either dissociates from V or is displaced from V by STAT1/STAT2 complexes, thereby ensuring the cycling of the DDB1 and SV5 V containing E3 complex for continued rounds of STAT1 ubiquitination and degradation.Simian virus type 5 (SV5) is classified within the genus Rubulavirus of the subfamily Paramyxovirinae of the family Paramyxoviridae (18). It is now well established that most members of the Paramyxovirinae subfamily at least partially circumvent the interferon (IFN) response by blocking IFN signaling and reducing the production of IFN by infected cells (for reviews see (1,10,13,22,32). In human cells, SV5 blocks both IFN-␣/␤ and IFN-␥ signaling by targeting STAT1 (a transcription factor which is essential for IFN signaling) for proteasome-mediated degradation (3,7,23,25,36,37). The molecular mechanisms by which SV5 targets STAT1 for degradation have been the subject of several recent investigations, and of the virus proteins, only the V protein is required to mediate this process (3,7,26).The SV5 V protein is the 222-amino-acid product of a faithful mRNA copy of the second open reading frame (the V/P gene) of the virus genome. V has been shown to be a multifunctional protein that, apart from its involvement in STAT1 degradation, also interacts with an IFN-inducible DExD/H box helicase mda-5 to limit the production of IFN (1), binds singlestranded RNA (20) and may act as a chaperone keeping the nucleoprotein of the virus soluble (28). STAT1 degradation, mediated by SV5 V protein, is independent of IFN signaling or phosphorylation of STAT proteins (3, 24). However, there is an absolute requirement for STAT2 in STAT1 degradation and consequently, SV5 infection fails to induce the degradation of STAT1 in STAT2-deficient (U6A) cells (26) or in 2fTGH cells that ex...

The African swine fever virus (ASFV) DP71L protein is present in all isolates as either a short form of 70 to 72 amino acids or a long form of about 184 amino acids, and both of these share sequence similarity to the C-terminal domain of the herpes simplex virus ICP34.5 protein and cellular protein GADD34. In the present study we expressed DP71L in different mammalian cells and demonstrated that DP71L causes dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2␣) in resting cells and during chemical-induced endoplasmic reticulum stress and acts to enhance expression of cotransfected reporter genes. We showed that DP71L binds to all the three isoforms (␣, ␤, and ␥) of the protein phosphatase 1 catalytic subunit (PP1c) and acts by recruiting PP1c to eIF2␣. We also showed that DP71L inhibits the induction of ATF4 and its downstream target, CHOP. We investigated the eIF2␣ phosphorylation status and induction of CHOP in porcine macrophages infected by two ASFV field isolates, Malawi Lil20/1 and Benin 97/1, and two DP71L deletion mutants, Malawi⌬NL and E70⌬NL. Our results showed that deletion of the DP71L gene did not cause an increase in the level of eIF2␣ phosphorylation or induction of CHOP, indicating that DP71L is not the only factor required by the virus to control the phosphorylation level of eIF2␣ during infection. We therefore hypothesize that ASFV has other mechanisms to prevent the eIF2␣ phosphorylation and the subsequent protein synthesis inhibition.

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.