Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex.

The Respiratory Syncytial Virus (RSV) fusogenic glycoprotein F(1) was characterized using biochemical and biophysical techniques. Two heptad-repeat (HR) regions within F(1) were shown to interact. Proteinase-K digestion experiments highlight the HR1 region (located proximal to the fusion peptide sequence) of the F(1) protein to which an HR2-derived (located proximal to the membrane-spanning domain) peptide binds, thus protecting both the protein and peptide from digestion. Solution-phase analysis of HR1-derived peptides shows that these peptides adopt helical secondary structure as measured by circular dichroism. Sedimentation equilibrium studies indicate that these HR1 peptides self-associate in a monomer/trimer equilibrium with an association constant of 5.2 x 10(8) M(-2). In contrast, HR2-derived peptides form random monomers in solution. CD analysis of mixtures containing peptides from the two regions demonstrate their propensity to interact and form a very stable (T(m) = 87 degrees C), helical (86% helicity) complex comprised of three HR1 and three HR2 members.

A 43-nucleotide RNA cis-acting element governs the site-specific formation of the 3' end of a poxvirus late mRNA.

The 3' ends of late mRNAs of the ati gene, encoding the major component of the A-type inclusions, are generated by endoribonucleolytic cleavage at a specific site in the primary transcript [Antczak et al., (1992), Proc. Natl. Acad. Sci. USA 89, 12033-12037]. In this study, sequence analysis of cDNAs of the 3' ends of ati mRNAs showed these mRNAs are 3' polyadenylated at the RNA cleavage site. This suggests that ati mRNA 3' end formation involves cleavage of a late transcript, with subsequent 3' polyadenylation of the 5' cleavage product. The RNA cis-acting element, the AX element, directing orientation-dependent formation of these mRNA 3' ends, was mapped to a 345-bp AluI-XbaI fragment. Deletion analyses of this fragment showed that the boundaries of the AX element are within -5 and +38 of the RNA cleavage site. Scanning mutagenesis showed that the AX element contains at least two subelements: subelement I, 5'-UUUAU downward arrowCCGAUAAUUC-3', containing the cleavage site ( downward arrow), separated from the downstream subelement II, 5'-AAUUUCGGAUUUGAAUGC-3', by a 10-nucleotide region, whose composition may be altered without effect on RNA 3' end formation. These features, which differ from those of other elements controlling RNA processing, suggest that the AX element is a component of a novel mechanism of RNA 3' end formation.

Reovirus exists in the form of 13 particle species that differ in their content of protein sigma 1.

When electrophoresed in 0.7% agarose gels, populations of reovirus particles can be resolved into 13 well-defined bands that possess from 0 to 12 projection/spike-associated trimers of protein sigma 1. This state of affairs is not an artifact of purification, of the techniques used to demonstrate it, of aggregation, or of virus particle instability. Complexes of monoclonal antibody against protein sigma 1 with virus particles that possess only 1, or, to a lesser extent, 2 sigma 1 trimers are less stable (that is, more readily dissociated by sonication) than complexes of antibody and virus particles that possess 3 or more sigma 1 trimers. The specific infectivity of virus particles that possess 3 or more sigma 1 trimers is essentially the same; virus particles that possess only 2 sigma 1 trimers are about two-thirds as infectious; and particles that possess only 1 sigma 1 trimer still possess very significant infectivity (about one-third of maximum). Reovirus particles that possess no sigma 1 trimers (about 1 in 30) are essentially noninfectious. The reason reovirus particles do not possess a full complement of sigma 1 trimers is presumably the fact that only very small amounts of protein sigma 1 are synthesized in infected cells; and since possession of 3 such trimers is sufficient for maximal infectivity, and since the average number of sigma 1 trimers per reovirus particle is 7.1, there is presumably no selection for variants that synthesize larger amounts of sigma 1. On the contrary, such variants may well be at a selective disadvantage.

Site-specific RNA cleavage generates the 3' end of a poxvirus late mRNA.

The cowpox virus late mRNAs encoding the major protein of the A-type inclusions have 3' ends corresponding to a single site in the DNA template. The DNA sequence of the Alu I-Xba I fragment at this position encodes an RNA cis-acting signal, designated the AX element, which directs this RNA 3' end formation. In cells infected with vaccinia virus the AX element functions independently of either the nature of the promoter element or the RNA polymerase responsible for generating the primary RNA. At late times during virus replication, vaccinia virus induces or activates a site-specific endoribonuclease that cleaves primary RNAs within the AX element. The 3' end produced by RNA cleavage is then polyadenylylated to form the 3' end of the mature mRNA. Therefore, the poxviruses employ at least two mechanisms of RNA 3' end formation during the viral replication cycle. One mechanism, which is operative at early times in viral replication, involves the termination of transcription [Rohrmann, G., Yuen, L. & Moss, B. (1986) Cell 46, 1029-1035]. A second mechanism, which is operative at late times during viral replication, involves the site-specific cleavage of primary RNAs.

Reovirus genome segment assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins.

Using a panel of monoclonal antibodies (MABs) against reovirus proteins, we have identified proteins that associate with reovirus messenger RNA molecules prior to the generation of progeny double-stranded (ds) genome segments and proteins that are components of the structures within which progeny ds genome segments are generated. The following conclusions can be drawn from the results obtained. (1) Three proteins rapidly become associated with mRNA molecules to form single-stranded RNA-containing complexes (ssRCCs): the nonstructural protein microNS, the nonstructural protein sigma NS, and protein sigma 3. (2) Analysis of populations of ssRCCs in density gradients and by sequential exposure to various MABs indicates that some ssRCCs contain only microNS, others microNS and sigma NS or sigma 3, and others all three proteins. Each ssRCC contains one RNA molecule and, depending on the size of the RNA, 10-30 protein molecules. (3) The relative proportions of the individual RNA species in the ssRCC populations reflect the composition of the total mRNA population present in infected cells (which differs substantially from equimolarity). (4) RCCs that contain dsRNA, which become detectable as early as 4 hr after infection, contain not only microNS, sigma NS, and sigma 3, but also lambda 2. (5) The relative proportions of the 10 genome segments in dsRCCs are equimolar. This suggests that genome segment assortment into progeny genomes is linked to the transcription of plus strands into minus strands.

Studies on the mechanism of the antiviral activity of ribavirin against reovirus.

We have examined the mechanism by which ribavirin inhibits the multiplication of reovirus. At a concentration of 12.5 microM (3 micrograms/ml) ribavirin inhibits viral multiplication, ssRNA formation, dsRNA formation, and protein synthesis by about 90%; when much higher concentrations are used for brief periods of time, the primary target of ribavirin is seen to be viral ssRNA synthesis. When the effect of ribavirin triphosphate (RTP) was tested on the in vitro transcription by cores of the dsRNA genome segments into plus-stranded RNA, elongation, that is, the formation of intact mRNA molecules, was found to be inhibited to the greatest extent; initiation was at least 2.5 times less sensitive, and cap formation and methylation were almost unaffected. The inhibition of elongation and initiation was not competitive with respect to any of the four nucleoside triphosphates. Remarkably, the transcription of plus strands into minus strands by immature reovirus particles (the replicase reaction) was insensitive to RTP. A model is proposed that envisages RTP binding to a site close to the catalytic site of the transcriptase. This binding is postulated to inhibit the helicase function of the transcriptase and lower its affinity for template RNA so that the likelihood of premature termination is greatly increased. The helicase activity is not, of course, necessary for the transcription of plus strands into minus strands, which would account for the differential sensitivity of the transcriptase and the replicase to RTP.