Template-dependent, in vitro replication of rotavirus RNA

Abstract

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.

cis-Acting signals that promote genome replication in rotavirus mRNA

“…The cores were recovered from the sample by pelleting through 40% sucrose in TBS, resuspended in TBS, and then centrifuged to equilibrium at 4ЊC in a solution of 42.8% (wt/wt) CsCl in TBS. The cores (density of 1.44 to 1.49 g/cm 3 ) were recovered and dialyzed initially against TBS for 6 h and then against LSB (2 mM Tris-HCl [pH 7.6], 0.5 mM EDTA, 0.5 mM dithiothreitol) for 18 h to open the cores (4). The open cores were stored on ice and retained high levels of replicase activity for at least 2 months.…”

“…Cell-free replication assay and analysis of replication products. The ability of reporter RNAs to serve as templates for dsRNA synthesis was evaluated with the cell-free system developed by Chen et al (4). Unless otherwise noted, reaction mixtures contained 50 mM Tris-HCl (pH 7.1); 10 mM magnesium acetate; 1.5% polyethylene glycol; 1.5 mM dithiothreitol; 1.5 U of RNasin; 1.25 mM (each) ATP, CTP, GTP, and UTP; 15 Ci of [ 32 P]UTP; 4 to 8 g of reporter RNA; and 1.6 g of open cores, and the final volume was 20 l. Reaction mixtures were incubated for 3 to 4 h at 37ЊC.…”

“…Following cleavage with SacII, the linearized vector was used in runoff transcription reactions to produce full-length gene 8 mRNA containing authentic 5Ј and 3Ј termini. The ability of the wild-type gene 8 mRNA to serve as a template for replication was assayed in the template-dependent cell-free replication system developed for the rotaviruses by Chen et al (4). In addition to saturating levels of the input gene 8 mRNA, the system contained open core particles as a source of replicase activity and [ 32 P]UTP to label new synthesized RNA products.…”

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

“…A template-dependent in vitro replication system (8) was used to test the replicase activity of the various VLPs and VP6 complexes. The in vitro system, which contained [␣-32 P]UTP and 400 ng of double-layered VLPs or 800 ng of single-layered VLPs (determined by Bio-Rad protein assay), was programmed with a reporter mRNA transcript and incubated for 4 h at 37ЊC, and the reaction products were resolved by SDS-PAGE as described previously (8). The presence of dsRNA product in the gel was determined by autoradiography and comparison with 32 P i -labelled rotavirus strain OSU dsRNA markers.…”

“…VP3 binds GTP specifically and is assumed to be a guanylyltransferase (23,29). Core-like particles containing VP1, VP2, and VP3 derived from either virions or baculovirus coexpression of VP1/2/3/6 have recently been shown to direct the synthesis of dsRNA, synthesizing negativestrand RNA on positive-strand RNA templates (8), an enzymatic activity called the viral replicase. Temperature-sensitive mutants have been used to demonstrate a role for VP2 in viral replicase activity (24).…”