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Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)

The receptor-recognition interaction that initiates reovirus infection is mediated by the sigma 1 protein, located at the vertices of the icosahedral virion. We have applied computer-based image-averaging techniques to electron micrographs of negatively stained preparations of sigma 1 purified from virions (serotype 2 Jones). Combining these results with inferences based on the amino acid sequence has led to a molecular model in which the overall folding of the chains is described; its conformation embodies motifs, coiled-coil alpha-helices and nodular multichain elements rich in beta-sheets, previously detected in the corresponding proteins of other viruses, but with some novel variations. Sigma 1 is a filamentous lollipop-shaped molecule with an overall length of approximately 48 nm; it has a flexible "tail," approximately 40 nm long by 4 to 6 nm wide, terminating at its distal end in a globular "head," approximately 9.5 nm in diameter. The purified protein is a tetramer (4 by 50 kilodaltons) consisting of two similarly oriented dimers bonded side by side and in register. For each chain, a cluster of hydrophobic residues at its amino terminus resides at the proximal end of the tail; next, an alpha-helical domain (residues 25 to 172) participates in a two-chained coiled coil, 22 nm long, with two such coiled coils pairing laterally to form the proximal half of the tail. The remainder of the tail (residues 173 to approximately 316) is less uniform in width and is expected to be rich in beta-sheet; the interdimer bonding is evidently sustained through this portion of the molecule. Finally, the globular head consists of the carboxy-terminal domains (which contain the receptor-binding sites) folded into compact globular conformations; in appropriate side views, the head is resolved into two subunits, presumably contributed by the respective dimers. This model for how the four sigma 1 polypeptide chains are threaded in parallel through the fiber is supported by the observed match between an empirical curvature profile, which identifies the locations of relatively flexible sites along the tail, and the flexibility profile predicted on the basis of the model. Appraisal of the interactions that stabilize the coiled coils suggests that (i) the alpha-helices are individually only marginally stable, a property that may be of significance with regard to the retracted conformation in which sigma 1 is accommodated in the intact virion, and (ii) the predominant interactions between the two coiled coils are likely to involve hydrogen bonding between patches of uncharged residues.

We were interested in screening a series of isolates of the protozoan Leishmania for the presence of viruses. The experimental procedure we used was based on an enzymatic assay originally developed for viral RNA-dependent RNA polymerases. Simultaneously, total promastigote nucleic acid preparations were analyzed for the presence of viral genome and/or transcripts. Two isolates, both classified as L. braziliensis guyanensis, were found to be positive for RNA polymerase activity and to carry a large (6 kilobases) RNA species. The polymerase reaction products hybridized to the 6-kilobase RNA, believed to be the viral genome. In conjunction with electron microscopical observations these results indicate the presence of an RNA virus in these Leishmania isolates.

The transposon TnphoA was used to generate fusions between phoA, the gene for alkaline phosphatase (PhoA), and genes encoding proteins that are secreted by Vibrio cholerae. One of the PhoA+ mutants isolated showed a dramatic reduction in its ability to colonize the intestines of suckling mice. This mutant no longer produced a 20.5-kDa protein (TcpA) that we show is the major subunit of a V. cholerae pilus. Amino-terminal sequence analysis of the TcpA pilus subunit showed that it shares amino acid homology with the pilins produced by several other pathogenic bacteria. The TcpA pilus was coordinately expressed with cholera toxin under various culture conditions, and this effect appeared to be dependent on the transcriptional activator encoded by the toxR gene. We conclude that the toxR gene plays a central role in the transcriptional regulation of multiple virulence genes of V. cholerae.

Electron microscopy revealed structures consisting of long fibers topped with knobs extending from the surfaces of virions of mammalian reoviruses. The morphology of these structures was reminiscent of the fiber protein of adenovirus. Fibers were also seen extending from the reovirus top component and intermediate subviral particles but not from cores, suggesting that the fibers consist of either the mu 1C or sigma 1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma 1 contains an extended fiber domain (R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizell, Jr., and B. N. Fields, Nature [London] 315:421-423, 1985). When sigma 1 protein was released from viral particles with mild heat and subsequently obtained in isolation, it was found to have a morphology identical to that of the fiber structures seen extending from the viral particles. The identification of an extended form of sigma 1 has important implications for its function in cell attachment. Other evidence suggests that sigma 1 protein may occur in virions in both an extended and an unextended state.