Comparison of the Virion Polymerase of Reovirus with the Enzyme Purified from Reovirus-Infected Cells

Abstract

Reovirus has in its protein coat an enzyme which catalyzes the net synthesis of the three size classes of virus-specific, single-stranded ribonucleic acid (RNA). For synthesis of 24, 19, and 14S single-stranded RNA, Mn++ was the preferred divalent cation, and ammonium sulfate at an optimal concentration of 4.2% of saturation was an absolute requirement. During synthesis, the parental double-stranded RNA was conserved in the viral core and the newly synthesized completed RNA chains were released as free RNA. The viral cores synthesizing RNA had properties consistent with the presence of nascent RNA on their outer surface. The enzymetemplate complex from the infected cells described in an earlier paper was comprised of viral cores already active in the in vivo synthesis of single-stranded RNA. This pool of viral cores was newly made during infection, and exponential increase in the number of particles in this pool, as detected by the increase in enzymatic activity, occurred 2 hr earlier than that in mature virus. MATERIALS AND METHODS Cell culture. L cells, strain 929, a continuous cell line derived from mouse fibroblasts, were grown in suspension in Eagle's spinner medium supplemented with 5% fetal bovine serum (3). Virus. Dearing strain of reovirus type 3 cloned was used as stock virus after two passages (6). Infectivity was determined by plaque assay (8). The procedures for infection of cells, virus growth, harvest of infected cells or of medium containing released virus, and subsequent purification of released or cell-associated virus have been described previously (9). For the preparation of 3H-labeled cell-associated reovirus, 3H-labeled uridine and actinomycin D were added to final concentrations, respectively, of 1 pCi/ml and 0.15 ug/ml in the viral growth medium. The specific activity of the virus labeled with radioactivity was 100 to 900 counts per min per 108 plaque-forming units (PFU).

Unilateral Synthesis of Reovirus Double-Stranded Ribonucleic Acid by a Cell-Free Replicase System

“…2, inset). This result is consistent with the conclusion of others that transcriptase is associated with the subviral particles of reovirus (1,4,5,10,11). In contrast, replicase activity was not associated with the subviral particles but was deposited variously over the gradient.…”

“…In the present paper, this polymerase will be tentatively designated "replicase," to distinguish it from the reovirus-bound "transcriptase." Transcriptase is found in the subviral particles of reovirus (virus core) and synthesizes single-stranded RNA (ssRNA) by transcribing only one strand of reovirus dsRNA (1,4,5,10,11). For our purposes, the ssRNA synthesized by transcriptase will be referred to as plus-RNA, and the RNA complementary to it, as minus-RNA.…”

Isolation and Properties of an RNA Polymerase from Influenza Virus-Infected Cells

“…The pellets were resuspended in 0.1 M Tris-hydrochloride, 0.001 M MgCl2 containing 1% NP-40, and centrifuged at 2,000 X g for 10 min. The supernatant fluids (200 jsliters) were layered over 4.8-ml, 15 to 30% sucrose gradients in RSB anid centrifuged at 35,000 rpm for 75 min in a Spinco SW50 rotor. Fractions of -0.25 ml were collected, and 25-,gliter portions of the gradient from labeled cells were used for radioactivity determination in 0.2 ml of NCS solubilizer (Amersham/Searle Corp., Des Plaines, Ill.) and 10 ml of toluene-Liquifluor scintillationfluid.…”

“…These observations support the view that RNA synthesis in influenza virus-infected cells is carried out in structures which each contain single RNA molecules, rather than by a subviral particle containing all of the genome RNAs. In contrast, in reovirus-infected cells the templates for synthesis of both single-stranded messenger RNA and progeny viral RNA appear to reside in subviral particles containing all the RNA species (1,15,32).…”

Reovirus-Specific Enzyme(s) Associated with Subviral Particles Responds In Vitro to Polyribocytidylate to Yield Double-Stranded Polyribocytidylate·Polyriboguanylate

“…There were also no particles in uninfected cells that synthesized RNA in vitro in response to the addition of poly(C). Purified complete reovirus cores derived after treatment of purified virus with chymotrypsin in low salt (3,13,17,31) or subviral particles obtained after chymotrypsin treatment in phosphate-buffered saline (8, 22) did not synthesize RNA in response to poly(C) in the presence of [a-32P]GTP. Moreover, subviral particles derived from inoculum virus in cells infected in the presence of cycloheximide (7,20,35) were active in the synthesis of reovirus single-stranded RNAs in the presence of the four nucleoside triphosphates, but they were inactive in response to poly(C) in the presence of only GTP.…”