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.

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...