Interferon-alpha-induced endogenous superantigen. a model linking environment and autoimmunity.

We earlier proposed that a human endogenous retroviral (HERV) superantigen (SAg) IDDMK(1,2)22 may cause type I diabetes by activating autoreactive T cells. Viral infections and induction of interferon-alpha (IFN-alpha) are tightly associated with the onset of autoimmunity. Here we establish a link between viral infections and IFN-alpha-regulated SAg expression of the polymorphic and defective HERV-K18 provirus. HERV-K18 has three alleles, IDDMK(1,2)22 and two full-length envelope genes, that all encode SAgs. Expression of HERV-K18 SAgs is inducible by IFN-alpha and this is sufficient to stimulate V beta 7 T cells to levels comparable to transfectants constitutively expressing HERV-K18 SAgs. Endogenous SAgs induced via IFN-alpha by viral infections is a novel mechanism through which environmental factors may cause disease in genetically susceptible individuals.

Inhibition of Sendai virus genome replication due to promoter-increased selectivity: a possible role for the accessory C proteins.

The role of the negative-stranded virus accessory C proteins is difficult to assess because they appear sometimes as nonessential and thereby of no function. On the other hand, when a function is found, as in the case of Sendai virus, it represents an enigma, in that the C proteins inhibit replication under conditions where the infection follows an exponential course. Furthermore, this inhibitory function is exerted differentially: in contrast to the replication of internal deletion defective interfering (DI) RNAs, that of copy-back DI RNAs appears to escape inhibition, under certain experimental conditions (in vivo assay). In a reexamination of the C effect by the reverse genetics approach, it was found that copy-back RNA replication is inhibited by C in vivo as well, under conditions where the ratio of C to copy-back template is increased. This effect can be reversed by an increase in P but not L protein. The "rule of six" was differentially observed in the presence or absence of C. Finally, a difference in the ability of the replicating complex to tolerate promoter modifications in RNA synthesis initiation was shown to occur in the presence or the absence of C as well. We propose that C acts by increasing the selectivity of the replicating complex for the promoter cis-acting elements governing its activity. The inhibitory effect of C becomes the price to pay for this increased selectivity.

Partial characterization of a Sendai virus replication promoter and the rule of six.

We have used a cDNA copy of a natural, internally deleted, Sendai virus defective interfering genome to study the effect of insertions and deletions (which maintain the hexamer genome length) on the ability of viral genomes to be amplified in a transfected cell system. The insertion of 18 nt at nt72 (In the 5' untranslated region of the N gene, just downstream of the le+ region) was found to be lethal, whereas similar insertions further from the genome ends were well tolerated. Curiously, the insertion of 6 nt on either side of the le+/N junction (at nt47 and nt87) was well tolerated, but the insertion of 12 nt at either site, or of 6 nt at both sites, largely ablated genome amplification. These results suggest that an element of this replication promoter is located downstream of nt87, in the 5' untranslated region of the first gene. Remarkably, the addition of 6 nt by the insertion of 2, 3, or 4 nt at nt47 plus the insertion of 4, 3, or 2 nt, respectively, at nt87 was poorly tolerated, presumably because the hexamer phase of the intervening sequence was altered with respect to the N subunits of the template. These results suggest that the rule of six operates, at least in part, at the level of the initiation of antigenome synthesis.

Rescue of Sendai virus cDNA templates with cDNA clones expressing parainfluenza virus type 3 N, P and L proteins.

Several years ago, we reported that a Sendai virus (SeV) defective genome (DIH4UV) could be rescued in vivo with human parainfluenza virus type 1 (hPIV1) and bovine PIV3 but not by measles virus or vesicular stomatitis virus. It was concluded that the cis-acting RNA sequences were conserved within the SeV/PIV1/PIV3 group but that interactions between the polymerase complex (P-L) and the template protein N were unique for each virus. We have re-examined these conclusions using proteins expressed from cloned N, P and L genes for SeV and PIV3. The results demonstrate the specificity of the protein-protein interactions between polymerase and template, and confirm the prediction of the earlier work that PIV3 N, P and L proteins are capable of assembling and replicating SeV mini-genomes also expressed from a cDNA clone.

A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus.

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.

An acidic activation-like domain of the Sendai virus P protein is required for RNA synthesis and encapsidation.

The Sendai virus polymerase is composed of the P and L proteins and carries out both mRNA synthesis and genome replication from the same nucleocapsid template. For mRNA synthesis, P interacts with the assembled NP of the nucleocapsid, and for genome replication, P interacts as well with unassembled NP for nascent chain assembly. The V and W nonstructural proteins, which are translated from edited P gene mRNAs and contain only the N-terminal half of the P protein, were found to inhibit genome replication but not mRNA synthesis. As genome replication is thought of as RNA synthesis plus concurrent encapsidation of the nascent chain, this half of P presumably plays a specific role in RNA encapsidation. Deletion analysis of the P gene found that residues 1-77 in the N-terminal half were in fact essential for RNA encapsidation. Moreover, either residues 1-77 or 78-144 also provided a function that was essential for RNA synthesis per se. Unlike other regions of P, such as those which bind NP in the C-terminal half, the N-terminal domains are very poorly conserved even among related viruses, show signs of acting in a position-independent manner, and, at least for RNA synthesis, are functionally redundant, similar to acidic activation domains of cellular transcription factors.

The P gene of human parainfluenza virus type 1 encodes P and C proteins but not a cysteine-rich V protein.

The nucleotide sequence of the P gene of human parainfluenza virus type 1 (PIV1) was determined from cloned cDNA copies of the mRNA. By analogy with the gene organization of Sendai virus, two open reading frames in the mRNA sense of the gene were identified as coding sequences for the P protein (568 amino acids with an estimated molecular weight of 64,655) and the C protein (204 amino acids with an estimated molecular weight of 24,108). Comparison of the deduced amino acid sequences of the P and C proteins of PIV1 with those of Sendai virus showed a high degree of homology. However, a sequence for the cysteine-rich V protein, which was considered a common feature of other paramyxoviruses, was interrupted by the presence of multiple stop codons. The sequence analysis of three P-gene-specific cDNA clones generated from genomic RNA by polymerase chain reaction and one additional clone generated from mRNA confirmed that the coding sequence for the cysteine-rich region is silent in the PIV1 gene and thus is not translated into protein. Two potential editing sites with the consensus sequence 3'UUYUCCC were found in the PIV1 P gene at positions 564 to 570 and 1430 to 1436. However, examination of the PIV1 mRNA population by a primer extension method indicated that neither of these sites is utilized. These results indicate that the PIV1 P gene has a coding strategy different from those of other paramyxovirus P genes.

The P gene of bovine parainfluenza virus 3 expresses all three reading frames from a single mRNA editing site.

The P gene of bovine parainfluenza virus 3 (bPIV3) contains two downstream overlapping ORFs, called V and D. By comparison with the mRNA editing sites of other paramyxoviruses, two editing sites were predicted for bPIV3; site a to express the D protein, and site b to express the V protein. Examination of the bPIV3 mRNAs, however, indicates that site b is non-functional whereas site a operates frequently. Insertions at site a give rise to both V and D protein mRNAs, because a very broad distribution of Gs is added when insertions occur. This broad distribution is very different from the editing sites of Sendai virus or SV5, where predominantly one form of edited mRNA containing either a one or two G insertion respectively is created, to access the single overlapping ORF of these viruses. A model is proposed to explain how paramyxoviruses control the range of G insertions on that fraction of the mRNAs where insertions occur. The bPIV3 P gene is unique as far as we know, in that a sizeable portion of the gene expresses all 3 reading frames as protein. bPIV3 apparently does this from a single editing site by removing the constraints which control the number of slippage rounds which take place.