Bacterial clones containing human tissue-type plasminogen activator (t-PA) cDNA sequences were identified in a cDNA library prepared using gel-fractionated mRNA from human melanoma cells. A plasmid containing the Escherichia coli trp promoter and the cDNA sequence coding f or the 527-amino acid mature t-PA protein was constructed for expression in E. coli. A polypeptide was produced having the fibrinolytic properties characteristic of authentic human t-PA.

Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli

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.

Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles.

Reovirus mRNA synthesized in vitro by the virus-associated RNA polymerase in the presence of S-adenosylmethionine contains blocked, methylated 5'-termini with the structure, m-7G(5')ppp(5')G-MpCp. The functional significance and possible mechanism of formation of this novel 5'-5' terminal nucleotide linkage are discussed.

https://www.pnas.org/content/pnas/72/1/362.full.pdf

Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-

A genetic approach has been used to define the molecular basis for the different patterns of virulence and central nervous system cell tropism exhibited by reovirus types 1 and 3. Intracerebral inoculation of reovirus type 3 into newborn mice causes a necrotizing encephalitis (without ependymal damage) that is uniformly fatal. Animal inoculated with reovirus type 1 generally survive and may develop epedymal cell damage (without neuronal necrosis) and hydrocephalus. Using recombinant clones derived from crosses between reovirus types 1 and 3, we have been able to determine that the S1 genome segment is responsible for the differing cell tropism of reovirus serotypes and is the major determinant of neurovirulence. The type 1 S1 genome segment is responsible for ependymal damage with subsequent hydrocephalus; the type 3 S1 genome segment is responsible for neuronal necrosis and neurovirulence. We postulate that these differences are due to the specific interaction of the σ1 outer capsid polypeptide (the protein coded for by the S1 genome segment) with receptors on the surface of either ependymal cells or neuronal cells.

Molecular basis of reovirus virulence: Role of the S1 gene

https://www.cell.com/cell/pdf/S0092-8674(02)00612-8.pdf

Structure of the Reovirus Membrane-Penetration Protein, μ1, in a Complex with Its Protector Protein, σ3

Digestion of purified reovirus with chymotrypsin in the presence of 0.15 M NaCl converts virions to infectious subviral particles (SVP(i)). The SVP(i) have an active ribonucleic acid (RNA) polymerase and are similar in composition to the partially uncoated virions which have been isolated from infected L cells. SVP(i) have a buoyant density of 1.40 g/ml in CsCl and sediment at 420S as compared to 1.37 g/ml and 630S for virions. They consist of 30% less protein and include the polypeptides of the inner structural layer, lambda(1), lambda(2), and sigma(3), and a polypeptide derived by cleavage of mu(2), a constituent of the outer shell. The genome RNA is retained within SVP(i), but more than 60% of the "adenine-rich," single-stranded RNA is released by the proteolytic treatment. Infection of L cells with SVP(i) or virions results in the transcription of all 10 genome segments. In cycloheximide-treated SVP(i)-infected cells, transcription occurs predominantly from one medium and two small genome segments, the same pattern of early messenger RNA (mRNA) observed in virion-infected cells. In contrast, SVP(i) incubated in vitro synthesize mRNA corresponding to all genome segments.

Transcription by Infectious Subviral Particles of Reovirus

Polysomes from reovirus-infected L cells were analyzed. Although all 10 transcription products of the viral genome were represented in polysomes and presumably can be translated, fewer than 10 reovirus-specific polypeptides were detected in infected cells. The 5'-terminal sequence of all species of reovirus ribonucleic acid (RNA) from polysomes was ppGpYp, as previously found for the genome double-stranded RNA and the in vitro single-stranded products of the virion-associated RNA polymerase. RNA isolated from both heavy (>30 ribosomes) and light (5 to 8 ribosomes) polysomes includes similar amounts of large, medium, and small classes of reovirus single-stranded RNA, suggesting that linkage of the genome segments observed in virions may occur at the single-strand stage of RNA replication.

A. J. LaFiandra

Papers

Transcription by Infectious Subviral Particles of Reovirus

Reovirus-Specific Ribonucleic Acid from Polysomes of Infected L Cells