Antiviral immunity against a pathogen is mounted upon recognition by the host of virally associated structures. One of these viral 'signatures', double-stranded (ds) RNA, is a replication product of most viruses within infected cells and is sensed by Toll-like receptor 3 (TLR3) and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I, also known as Ddx58) and Mda5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard). Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay an undefined signal resulting in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB. Here we describe Cardif, a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKKalpha, IKKbeta and IKKvarepsilon kinases by means of its C-terminal region, leading to the activation of NF-kappaB and IRF3. Overexpression of Cardif results in interferon-beta and NF-kappaB promoter activation, and knockdown of Cardif by short interfering RNA inhibits RIG-I-dependent antiviral responses. Cardif is targeted and inactivated by NS3-4A, a serine protease from hepatitis C virus known to block interferon-beta production. Cardif thus functions as an adaptor, linking the cytoplasmic dsRNA receptor RIG-I to the initiation of antiviral programmes.

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses.

We have recovered infectious Sendai virus (SeV) from full‐length cDNA (FL‐3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL‐3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5′ nontranslated region of the NP gene, creating a new, out‐of‐frame, 5′ proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL‐3 and pGEM‐NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL‐3 with that of pGEM‐NP. When a fifth plasmid containing a new genomic 3′ end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3′ end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy‐back nondefective virus was generated which interferes with wild‐type virus replication.

The Sendai virus P/C mRNA expresses the P and C proteins from alternate reading frames. The C reading frame of this mRNA, however, is responsible for three proteins, C‘, C and Y, none of which appear to be precursors to each other in vivo. Using site‐directed and deletion mutagenesis of the P/C gene cloned in SP6 and in vitro translation of the mRNAs, we show that the 5′ most proximal initiation codon of the mRNA is an ACG at position 81, responsible for C’ synthesis. The succeeding initiation codons, all ATGs, are responsible for the P protein (position 104), the C protein (position 114) and the Y protein(s) (either positions 183 or 201). Examination of the relative molar amounts of the C′, P and C proteins found in vivo suggests that an ACG in an otherwise favorable context is almost as efficient for ribosome initiation as an ATG in a less favored context, but only 10‐20% as efficient as an ATG in a more favored context. The judicious choice of increasingly more favorable initiation codons in the P/C gene allows multiple proteins to be made from a single mRNA.

The template for paramyxovirus RNA synthesis is not naked RNA but the helical nucleocapsid core of the virus, in which each nucleocapsid protein (N protein) is predicted to be associated with precisely 6 nucleotides (nt) (11). Presumably as a consequence of this association, paramyxovirus genomes are replicated efficiently only when they are a multiple of 6 nt in length, and this has been dubbed the "rule of six" (4). The structure of paramyxovirus nucleocapsids is thus central to understanding how this rule might operate.

No abstract

The P gene of Sendai virus expresses as many as eight proteins, two of which (V and W) are expressed only from edited mRNAs; only the P protein is known to be involved in RNA synthesis. To examine the functions of the other P gene proteins, we developed an in vivo system in which genome replication is driven by plasmid generated viral proteins. We found that P was essential for this process, whereas V and W were not only non‐essential, they were inhibitory. By using various P gene deletions and varying the amounts of plasmids transfected, we provide evidence that P is a modular protein. The N‐terminal domain (shared with V and W) binds the L or polymerase protein, whereas the C‐terminal domain binds the nucleoprotein NP. A model of paramyxovirus RNA synthesis is presented, and the implications of negative regulation during persistent infection are discussed.

Paramyxovirus P genes are transcribed into two mRNAs which differ from each other by either one (measles and Sendai virus) or two (SV5 and mumps virus) G insertions, and which code for either the P or V proteins. The G insertions always occur within a short run of Gs, and a stuttering mechanism for the insertions has been suggested in which the viral polymerase reiteratively copies a template C residue during mRNA synthesis. Support for this mechanism was obtained by varying the reaction conditions during Sendai virus mRNA synthesis in vitro. A stuttering model is proposed which accounts for how the ratio of inserted to uninserted mRNAs is controlled, and why some paramyxoviruses insert one G and others two Gs when insertions occur.