Mammalian reovirus L3 gene sequences and evidence for a distinct amino-terminal region of the lambda1 protein.
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To complement evidence for nucleoside triphosphate phosphohydrolase (NTPase), RNA helicase, RNA 5' triphosphate phosphohydrolase, and nucleic acid-binding activities by the core shell protein lambda1 of mammalian orthoreoviruses (reoviruses), we determined nucleotide sequences of the lambda1-encoding L3 gene segments from three isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). The T1L and T3D L3 gene sequences and deduced lambda1 protein sequences shared high levels of identity (97.7% and 99.3%, respectively). The lambda1 sequences differed at only 9 of 1275 amino acids. Two single-nucleotide insertions relative to a previously published sequence for T3D L3 extended the lambda1 open reading frame to within 60 nucleotides of the plus-strand 3' end for T3D and the other isolates sequenced, in keeping with the short 3' nontranslated regions of the other nine gene segments. Seven of the nine amino acid differences between T1L and T3D lambda1 were located within the amino-terminal 500 residues of lambda1, a region with putative sequence similarities to NTPases and RNA helicases. The T2J L3 and lambda1 sequences were found to be more divergent, especially within the amino-terminal 180 residues of lambda1, preceding the putative CCHH zinc finger motif. The T2J L3 sequence, along with partial sequences for L3 genes from three other reovirus isolates, suggested that one or more of the polymorphisms at amino acids 71, 215, 500, 1011, and/or 1100 in lambda1 contribute to the L3-determined differences in ATPase activities by T1L and T3D cores. The findings contribute to our ongoing efforts to elucidate sequence-structure-function relationships for the lambda1 core protein. (+info)
Interferon regulatory factor 3 is required for viral induction of beta interferon in primary cardiac myocyte cultures.
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Viral myocarditis affects an estimated 5 to 20% of the human population. The antiviral cytokine beta interferon (IFN-beta) is critical for protection against viral myocarditis in mice. That is, nonmyocarditic reoviruses induce myocarditis in mice that lack IFN-alpha/beta, and nonmyocarditic reoviruses both induce more IFN-beta and are more sensitive to the antiviral effects of IFN-beta than myocarditic reoviruses in primary cardiac myocyte cultures. Induction of IFN-beta in certain cell types involves viral activation of the transcription factor interferon regulatory factor 3 (IRF-3). To address whether IRF-3 can induce IFN-beta in cardiac myocytes, primary cardiac myocyte cultures and control L929 cells were transfected with a plasmid constitutively expressing IRF-3. Overexpression of IRF-3 resulted in induction of IFN-beta in the absence of viral infection in both cell types. To address whether IRF-3 is required for viral induction of IFN-beta, cell cultures were transfected with a plasmid constitutively expressing a dominant negative IRF-3 protein. The dominant negative IRF-3 reduced reovirus induction of IFN-beta in control L929 cells and completely eliminated induction in primary cardiac myocyte cultures. This provides the first identification of a cardiac cellular factor required for viral induction of IFN-beta and the first report of any cell type requiring IRF-3 for this response. (+info)
Genome-sized RNA from reovirus particles.
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Spider-like forms of purified reovirus RNA have been characterized by electron microscopy. These structures contained 11.3 +/- 0.8 mum of double-stranded RNA and up to 20 arms, each of distinct length. Length measurements of 64 "spiders" suggest that in these structures the viral genome is folded fairly specifically. (+info)
Control of transcription of the reovirus genome.
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The ten double-stranded RNA segments of the reovirus genome are not transcribed at equal frequencies until later times during the course of infection. When an inhibitor of protein synthesis such as cycloheximide was added to infected cells at the beginning of infection, only four of the ten segments were transcribed. By two hr post infection, five and possibly seven of the segments were being transcribed. By four hr post infection all ten segments were being transcribed but not yet at equal relative frequencies. The transcription pattern at intermediate (4 hr) and late (10 hr) times were verified without using cycloheximide. A repressor present in the host cell may be responsible for controlling transcription of the reovirus genome. (+info)
Synthesis of reovirus-specific polypeptides in cells pretreated with cycloheximide.
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When L cells are infected with reovirus in the presence of cycloheximide neither virus-specific polypeptides nor viral double-stranded RNA are synthesized. There is some synthesis of viral single-stranded RNA, transcribed mainly from segments L1, M3, S3, and S4 of the 10 viral genomic segments, and in previous work this has been termed the early mRNA pattern. In an attempt to determine whether these early transcripts are functional mRNA's, the transcripts were allowed to accumulate for a period of 17.5 h at 31 C in cycloheximide-treated cells. The cycloheximide was removed and the cells were exposed for various periods to radioactive amino acids to label any virus-specific polypeptides that might be synthesized. An immunoprecipitation technique was used to separate the viral polypeptides from cellular extracts and this precipitate was then analyzed on sodium dodecyl sulfate-polyacrylamide gels. Within 30 min of cycloheximide removal, four major polypeptides (lambda2, mu0, sigma2a, and sigma3) and two minor polypeptides (lambda1 and mu2) were found. In infected cells without cycloheximide eight viral polypeptides (lambda1, lambda2, mu0, mu2, sigma1, sigma2, sigma2a, sigma3) were found at 17.5 h after infection and the same pattern was found between 3 to 4 h after removal of cycloheximide which had been present for 17.5 h after infection. The latter result shows that the cycloheximide inhibition is reversible and that the cells readily recovered and synthesized the normal complement of viral polypeptides. In one set of experiments cordycepin was added to infected cells immediately after the removal of cycloheximide at 17.5 h to inhibit the synthesis of new viral transcripts. During the succeeding 4 h in the presence of cordycepin, the pattern of protein synthesis was the same as that obtained during the 30 min after cycloheximide removal. It is concluded that the polypeptides formed right after removal of cycloheximide are the translation products of transcripts accumulated during cycloheximide treatment and, therefore, that these transcripts are functional viral mRNA's. (+info)
Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses.
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The reovirus attachment protein, sigma1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The sigma1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of sigma1 that binds cell surface carbohydrate. Chimeric and truncated sigma1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-sigma1 antibodies, and oligomerization indicates that the chimeric and truncated sigma1 proteins are properly folded. To assess carbohydrate binding, recombinant sigma1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated sigma1 proteins, the sialic acid-binding domain of type 3 sigma1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted beta-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of sigma1 protein purified from virions. In contrast, the homologous region of T1L sigma1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 sigma1 tail. Furthermore, our findings indicate that T1L and T3D sigma1 proteins contain different arrangements of receptor-binding domains. (+info)
The reovirus S4 gene 3' nontranslated region contains a translational operator sequence.
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Reovirus mRNAs are efficiently translated within host cells despite the absence of 3' polyadenylated tails. The 3' nontranslated regions (3'NTRs) of reovirus mRNAs contain sequences that exhibit a high degree of gene-segment-specific conservation. To determine whether the 3'NTRs of reovirus mRNAs serve to facilitate efficient translation of viral transcripts, we used T7 RNA polymerase to express constructs engineered with full-length S4 gene cDNA or truncation mutants lacking sequences in the 3'NTR. Full-length and truncated s4 mRNAs were translated using rabbit reticulocyte lysates, and translation product sigma3 was quantitated by phosphorimager analysis. In comparison to full-length s4 mRNA, translation of the s4 mRNA lacking the 3'NTR resulted in a 20 to 50% decrease in sigma3 produced. Addition to translation reactions of an RNA oligonucleotide corresponding to the S4 3'NTR significantly enhanced translation of full-length s4 mRNA but had no effect on s4 mRNA lacking 3'NTR sequences. Translation of s4 mRNAs with smaller deletions within the 3'NTR identified a discrete region capable of translational enhancement and a second region capable of translational repression. Differences in translational efficiency of full-length and deletion-mutant mRNAs were independent of RNA stability. Protein complexes in reticulocyte lysates that specifically interact with the S4 3'NTR were identified by RNA mobility shift assays. RNA oligonucleotides lacking either enhancer or repressor sequences did not efficiently compete the binding of these complexes to full-length 3'NTR. These results indicate that the reovirus S4 gene 3'NTR contains a translational operator sequence that serves to regulate translational efficiency of the s4 mRNA. Moreover, these findings suggest that cellular proteins interact with reovirus 3'NTR sequences to regulate translation of the nonpolyadenylated reovirus mRNAs. (+info)
Reovirus as an oncolytic agent against experimental human malignant gliomas.
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BACKGROUND: Reovirus is a naturally occurring oncolytic virus that usurps activated Ras-signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases. The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment for malignant gliomas. METHODS: We investigated whether reovirus would infect and lyse human glioma cell lines in vitro. We also tested the effect of injecting live reovirus in vivo on human gliomas grown subcutaneously or orthotopically (i.e., intracerebrally) in mice. Finally, reovirus was tested ex vivo against low-passage cell lines derived from human glioma specimens. All P values were two-sided. RESULTS: Reovirus killed 20 (83%) of 24 established malignant glioma cell lines tested. It caused a dramatic and often complete tumor regression in vivo in two subcutaneous (P =.0002 for both U251N and U87) and in two intracerebral (P =.0004 for U251N and P =.0009 for U87) human malignant glioma mouse models. As expected, serious toxic effects were found in these severely immunocompromised hosts. In a less immunocompromised mouse model, a single intratumoral inoculation of live reovirus led to a dramatic prolongation of survival (compared with control mice treated with dead virus; log-rank test, P<.0001 for both U251N and U87 cell lines). The animals treated with live virus also appeared to be healthier and gained body weight (P =.0001). We then tested the ability of reovirus to infect and kill primary cultures of brain tumors removed from patients and found that it killed nine (100%) of nine glioma specimens but none of the cultured meningiomas. CONCLUSIONS: Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers. (+info)