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The ability of some Sendai virus stocks to strongly activate IFNbeta has long been known to be associated with defective-interfering (DI) genomes. We have compared SeV stocks containing various copyback and internal deletion DI genomes (and those containing only nondefective (ND) genomes) for their ability to activate reporter genes driven by the IFNbeta promoter. We found that this property was primarily due to the presence of copyback DI genomes and correlated with their ability to self-anneal and form dsRNA. The level of IFNbeta activation was found to be proportional to that of DI genome replication and to the ratio of DI to ND genomes during infection. Over-expression of the viral V and C proteins was as effective in blocking the copyback DI-induced activation of the IFNbeta promoter as it was in reducing poly-I/C-induced activation, providing evidence that these DI infections activate IFNbeta via dsRNA. Infection with an SeV stock that is highly contaminated with copyback DI genomes is thus a very particular way of potently activating IFNbeta, presumably by providing plentiful dsRNA under conditions of reduced expression of viral products which block the host antiviral response.

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.

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We examined the 5 ends of Hantaan virus (HTN) genomes and mRNAs to gain insight into the manner in which these chains were initiated. Like those of all members of the family Bunyaviridae described so far, the HTN mRNAs contained 5 terminal extensions that were heterogeneous in both length and sequence, presumably because HTN also ''cap snatches'' host mRNAs to initiate the viral mRNAs. Unexpectedly, however, almost all of the mRNAs contained a G residue at position ؊1, and a large fraction also lacked precisely one of the three UAG repeats at the termini. The genomes, on the other hand, commenced with a U residue at position ؉1, but only 5 monophosphates were found here, indicating that these chains may not have initiated with UTP at this position. Taken together, these unusual findings suggest a prime-and-realign mechanism of chain initiation in which mRNAs are initiated with a G-terminated host cell primer and genomes with GTP, not at the 3 end of the genome template but internally (opposite the template C at position ؉3), and after extension by one or a few nucleotides, the nascent chain realigns backwards by virtue of the terminal sequence repeats, before processive elongation takes place. For genome initiation, an endonuclease, perhaps that involved in cap snatching, is postulated to remove the 5 terminal extension of the genome, leaving the 5 pU at position ؉1.

The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (g) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (d) a set of overlapping peptides completely spanning the identified nudeoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous i'mmunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunizd with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.

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.