A previous study has shown that rotavirus cores have an associated replicase activity which can direct the synthesis of double-stranded RNA from viral mRNA in a cell-free system (. 68:7030-7039, 1994). To define the cis-acting signals in rotavirus mRNA that are important for RNA replication, gene 8 transcripts which contained internal and terminal deletions and chimeric transcripts which linked gene 8-specific 3-terminal sequences to the ends of nonviral sequences were generated. Analysis of these RNAs in the cell-free system led to the identification of a cis-acting signal in the gene 8 mRNA which is essential for RNA replication and two cis-acting signals which, while not essential for replication, serve to enhance the process. The sequence of the essential replication signal is located at the extreme 3 end of the gene 8 mRNA and, because of its highly conserved nature, is probably a common feature of all 11 viral mRNAs. By site-specific mutagenesis of the gene 8 mRNA, residues at positions ؊1, ؊2, ؊5, ؊6, and ؊7 of the 3 essential signal were found to be particularly important for promoting RNA replication. One of the cis-acting signals shown to enhance the replication in the cell-free system was located near the 5 end of the 3 untranslated region (UTR) of the gene 8 mRNA, while remarkably the other was located in the 5 UTR of the message. The existence of an enhancement signal in the 5 UTR raises the possibility that the 5 and 3 ends of the rotavirus mRNA may interact with each other and/or with the viral replicase during genome replication.
A template-dependent, in vitro rotavirus RNA replication system was established. The system initiated and synthesized full-length double-stranded RNAs on rotavirus positive-sense template RNAs. Native rotavirus mRNAs or in vitro transcripts, with bona fide 3' and 5' termini, derived from rotavirus cDNAs functioned as templates. Replicase activity was associated with a subviral particle containing VP1, VP2, and VP3 and was derived from native virions or baculovirus coexpression of rotavirus genes. A cis-acting signal involved in replication was localized within the 26 3'-terminal nucleotides of a reporter template RNA. Various biochemical and biophysical parameters affecting the efficiency of replication were examined to optimize the replication system. A replication system capable of in vitro initiation has not been previously described for Reoviridae.
Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negativestain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VP1/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VP1, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 singlelayered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.
S U M M A R Y This study examines the relationship between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the control of cervical ripening and parturition under normal (normal term pregnancy) and abnormal (preterm labor and prolongation of pregnancy) conditions by (a) measuring changes in the collagen both visually and quantitatively, (b) localizing and characterizing iNOS and COX-2 under normal conditions, and (c) characterizing the changes in iNOS and COX-2 under abnormal conditions. Cervices are obtained from estrus and timed pregnant Sprague-Dawley rats (n54-10 per group). Preterm labor is induced with Onapristone (3 mg/rat; progesterone antagonist) and the prolongation of pregnancy with progesterone (2.5 mg, twice daily). Collagen changes are measured and visualized with the picrosirius polarization method. RT-PCR is used to characterize the mRNA expression (p,0.05), and immunohistochemistry is used to localize the protein expression for iNOS and COX-2. The organization and birefringence of the collagen during pregnancy decreased and is supported by changes in the luminosity (p,0.001). The iNOS and COX-2 enzymes were localized in cervical smooth muscle, vascular smooth muscle, and epithelium. Under normal conditions, iNOS mRNA levels decreased as COX-2 mRNA levels increased demonstrating an inverse correlation (Spearman r 5 20.497; p50.00295). Onapristone stimulated preterm labor, increasing the iNOS and COX-2 mRNA (p,0.05). The increase demonstrated a positive correlation (Spearman r 5 0.456; p50.03). Progesterone prolonged pregnancy, decreasing the iNOS and COX-2 mRNA (p50.036). In conclusion, there may be an interaction between the nitric oxide and prostaglandin pathways in cervical ripening and parturition. (J Histochem Cytochem 54:623 -639, 2006)
Background: Patients who have suffered obstetric brachial plexus injury (OBPI) have a high incidence of musculoskeletal complications stemming from the initial nerve injury. The presence of muscle imbalances and contractures leads to typical bony changes affecting the shoulder, including the SHEAR (Scapular Hypoplasia, Elevation and Rotation) deformity. The SHEAR deformity commonly occurs in conjunction with Medial Rotation Contracture (MRC) of the arm. OBPI also causes muscle imbalances at the level of the forearm, that lead to a fixed supination deformity (SD) in a small number of patients. Both MRC and SD will cause severe functional limitations without surgical intervention.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.