Mutational analysis of a mammalian reovirus mRNA capping enzyme.

The amino-terminal 42-kDa region of the 144-kDa mammalian reovirus lambda 2 protein is a guanylyltransferase. It catalyzes the transfer of GMP from GTP to the 5' end of 5' -diphosphorylated mRNA via a phosphoamide with Lys-190. This amino acid is located at the base of a deep cleft. Based on sequence comparisons, the Kx[V/L/I]S motif is present in all known and proposed guanylyltransferases of the family Reoviridae. The requirement for this conserved sequence and other regions of the enzyme was analyzed by site-directed mutagenesis. Based on the enzymatic activity of the mutants, Lys-190 and Asp-191 are the only amino acids of the (190)KDLS sequence that are necessary for enzymatic activity. Since Asp-191 has its side chain oriented away from the cleft, most likely it plays an indirect role in forming a functional guanylyltransferase.

Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein lambda1.

A previously identified nucleoside triphosphatase activity in mammalian reovirus cores was further characterized by comparing two reovirus strains whose cores differ in their efficiencies of ATP hydrolysis. In assays using a panel of reassortant viruses derived from these strains, the difference in ATPase activity at standard conditions was genetically associated with viral genome segment L3, encoding protein lambda1, a major constituent of the core shell that possesses sequence motifs characteristic of other ATPases. The ATPase activity of cores was affected by several other reaction components, including temperature, pH, nature and concentration of monovalent and divalent cations, and nature and concentration of anions. A strain difference in the response of core ATPase activity to monovalent acetate salts was also mapped to L3/lambda1 by using reassortant viruses. Experiments with different nucleoside triphosphates demonstrated that ATP is the preferred ribonucleotide substrate for cores of both strains. Other experiments suggested that the ATPase is latent in reovirus virions and infectious subviral particles but undergoes activation during production of cores in close association with the protease-mediated degradation of outer-capsid protein mu1 and its cleavage products, suggesting that mu1 may play a role in regulating the ATPase.

The M1 gene is associated with differences in the temperature optimum of the transcriptase activity in reovirus core particles.

The reovirus core is a multienzyme complex that contains five different structural proteins and 10 segments of double-stranded RNA. The core is responsible for transcribing mRNA from the enclosed double-stranded RNA. The reovirus transcriptase has an unusual temperature profile, with optimum transcription occurring at approximately 50 degrees C and little activity occurring below 30 or above 60 degrees C. Purified reovirus serotype 1 Lang (T1L) cores transcribed most efficiently at 48 degrees C. The transcriptase temperature optimum of purified reovirus serotype 3 Dearing (T3D) cores was 52 degrees C. In addition, T1L cores produced more mRNA per particle than did T3D cores at their respective temperature optima. Core particles were purified from T1L x T3D reassortants and were used to map these differences. The M1 gene, which encodes minor core protein mu 2, was uniquely associated with the difference in temperature optimum of transcription (P = 0.0003). The L1 gene, which encodes minor core protein lambda 3 (previously implicated as the RNA polymerase), and the M1 gene were associated with the difference in absolute amounts of transcript produced (P = 0.01 and P = 0.0002, respectively). These data suggest that minor core protein mu 2 also plays a role in reovirus transcription.

Characterization and structural localization of the reovirus lambda 3 protein.

The putative reovirus RNA polymerase, protein lambda 3, was characterized using antiserum prepared against a TrpE-lambda 3 fusion protein synthesized in Escherichia coli. Immunofluorescence microscopy showed that lambda 3 accumulated in perinuclear inclusion bodies in reovirus-infected cells. Analysis of lambda 3 accumulation in infected cells indicates that, once synthesized, lambda 3 is quite stable throughout the course of infection. Anti-lambda 3 serum did not immunoprecipitate virions, core particles or iodinated surface proteins of either virions or cores. These results indicate that lambda 3 is located in the inner part of the core. Experiments involving urea denaturation of purified reovirus cores indicate that lambda 3 cannot be selectively removed from the core without total denaturation of the core structure. When the dsRNA genome was eliminated from the core, lambda 3 remained associated with the other viral proteins in the core. Thus, lambda 3 appears to be a stable, structural component of the reovirus core, not bound to genomic dsRNA or free in soluble form inside the core.

Reovirus protein lambda 3 is a poly(C)-dependent poly(G) polymerase.

Reovirus protein lambda 3 has been isolated from cells infected with two recombinant vaccinia viruses into the TK gene of which the reovirus serotype3 strain Dearing L1 genome segment under the control of the bacteriophage T7 RNA polymerase promoter, or the T7 polymerase gene itself, had been cloned. Highly purified protein lambda 3 does not transcribe double-stranded reovirus RNA into single-stranded RNA, or plus-stranded reovirus RNA into minus-stranded RNA, but it does transcribe poly(C) into poly(G). It prefers Mn2+ to Mg2+. A polymer consisting of poly(C) linked linearly to poly(U) provided template activity only for its poly(C) moiety. Protein lambda 3 forms complexes with protein lambda 1, as well as with protein lambda 2, and with both lambda 1 and lambda 2, which are sufficiently stable to be precipitated by monospecific antisera. None of these complexes are capable of transcribing either ds- or ssRNA.

Isolation and enzymatic characterization of protein lambda 2, the reovirus guanylyltransferase.

Protein lambda 2 of reovirus serotype 3 has been purified to homogeneity from extracts of cells infected with hybrid vaccinia virus strain WR into whose TK gene of the reovirus L2 genome segment under the control of the CPV ATI protein gene promoter had been inserted. Protein lambda 2 is formed in large amounts (final purification factor about 180) as a monomer that shows no tendency to pentamerize into the reovirus core projections/spikes. Isolated protein lambda 2 is reversibly guanylylated by GTP (that is, it carries out the GTP-PPi exchange reaction) and can transfer the -GMP moiety to GTP to form GppppG, to GDP to form GpppG, and to 5'-pp-terminated RNA to form GpppG- caps. These studies confirm previous studies on reovirus cores that indicated that protein lambda 2 is the reovirus guanylyltransferase. Protein lambda 2 possesses neither nucleoside nor RNA triphosphatase activities, nor methyltransferase activities; thus it is the reovirus capping enzyme, but provides neither the required 5'-ppG-terminated substrate nor does it methylate the cap structure. These must be functions of lambda 2 pentamers or of other individual or complexed components of reovirus cores.

Active site localization in a viral mRNA capping enzyme.

Capping of reovirus mRNAs is catalyzed by a guanylyltransferase that corresponds to virion structural polypeptide lambda 2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. The nucleotide attachment site on lambda 2 was localized to a region between amino acids 213 and 269 by incubating virus particles with [alpha-32P]GTP followed by proteolytic cleavage and analysis of the resulting fragments using sequence-directed antibodies as probes. The 213-269 region contains as potential GMP acceptors a single lysine, 1 arginine, and 4 histidine residues, as deduced from the nucleotide sequence of the L2 gene encoding lambda 2. Digestion of 32P-labeled capping intermediate with alkali after oxidation and beta-elimination yielded phospholysine as the only phosphoamino acid, localizing the active site to a region in lambda 2 that includes the lysine at position 226.

Reovirus guanylyltransferase is L2 gene product lambda 2.

Reovirus guanylyltransferase, studied as a covalent enzyme-GMP intermediate, was used to guanylate appropriate acceptor molecules in vitro to produce authentic cap structures. Guanylyltransferase activity was associated with lambda 2, the 140-kilodalton product of the L2 gene segment of reovirus serotypes 1 and 3.

Switch-on of transcriptase function in reovirus: analysis of polypeptide changes using 2-D gels.

Two-dimensional gel electrophoresis (IEF and SDS-PAGE) was used to examine virion polypeptide changes associated with switch-on of transcriptase function in reovirus. Results reveal that switch-on is correlated with altered electrophoretic behavior of a specific minor polypeptide (delta 1) which is present in intermediate subviral particles. A second finding is that each of the molecular weight classes of viral polypeptides exists as a series of subspecies with different isoelectric points. This suggests that extensive posttranslational modification of progeny viral polypeptides occurs during particle morphogenesis. These findings have important theoretical and practical implications.

Perturbation of the switch-on of transcriptase activity in intermediate subviral particles from reovirus.

Intermediate subviral particles (ISVP) derived from reovirus represent a simple model system for the switch-on of transcriptase function. In such particles the endogenous transcriptase is present in a switched-off form, one step removed from the switched-on state. Switch-on of transcriptase function is an active process in this system and can be triggered by K+ions. A variety of agents which affect gene expression in cells were tested for an effect on switch-on in ISVP. Marked effects on switch-on in ISVP were observed with a diverse group of test agents, including DMSO and other solvents, BUdR, TdR, caffeine, theophylline, and temperature. The correlation in response between ISVP and cells suggests that the ISVP system may be useful as a model for studying the biochemical mechanisms underlying the perturbative effects of such agents on gene expression in cells.

Excess synthesis of viral mRNA 5-terminal oligonucleotides by reovirus transcriptase.

Short oligonucleotides corresponding to the 5'-terminal sequence of reovirus mRNAs were produced in vitro by virion-associated transcriptase activity. Both capped and uncapped oligonucleotides were synthesized in molar excess relative to mRNA. Yields of uncapped oligomers including ppG-C and ppG-C-U were severalfold greater than the homologous capped structures. In partial reaction mixtures that were nonsupportive for mRNA chain elongation, capped oligomer synthesis was increased. Similarly, oligonucleotide formation was differentially resistant in viral preparations that were inactivated with respect to mRNA synthesis by modification of the genome RNA by dimethyl sulfate alkylation or psoralen photoreaction. The results suggest that reovirus mRNA synthesis involves excessive initiation by reiterative transcription of promoter sites by the reovirus polymerase. Only a small fraction of the resulting oligonucleotides are capped and extended to form full length mRNAs during a subsequent elongation step which is apparently mediated by transcriptase molecules that escape the reiterative phase of transcription.

Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity.

We previously reported that virus-specific particles with polycytidylate [poly(C)]-dependent RNA polymerase activity accumulated at 30 degrees C in reovirus-infected cells. These particles sedimented heterogeneously from 300 to 550S and traversed through a 40% glycerol cushion to the pellet in 3 h at 190,000 x g. In the present report, we found that smaller particles with poly(C)-dependent RNA polymerase activity remained in the glycerol cushion. These smaller, enzymatically active particles, when purified, sedimented at 15 to 1S. They were spherical or triangular with a diameter of 11 to 12 nm. They were comprised mostly, and likely solely, of one reovirus protein, sigma NS. No particles with poly(C)-dependent RNA polymerase activity were found in mock-infected cells. Chromatography on the cation exchanger, CM-Sephadex, ascertained that sigma NS was the poly(C)-dependent RNA polymerase and showed its existence in two forms. In one form, it was enzymatically active and eluted from the column at 0.5 M KCl. In the enzymatically inactive state, it did not bind to the column. Our results suggest that the enzymatically active form of sigma NS carries a greater net positive charge than the inactive form. They also suggest that both forms of sigma NS are associated with a particle which has poly(C)-dependent RNA polymerase activity.

mRNA capping enzymes are masked in reovirus progeny subviral particles.

We examined the enzyme activities associated with progeny subviral particles isolated from L-cells infected with reovirus at 12 h postinfection. Activities normally present in reovirus cores were also found to be present in the progeny subviral particles, with the exception of the capping enzymes. The methylase and guanyl transferase activities, which constitute the capping system, were present in a masked form that could be activated by chymotrypsin digestion. The appearance of these progeny subviral particles in infected cells coincided with the time when mRNA synthesis was maximal, suggesting that viral mRNA synthesized at later times is uncapped.

Differential dependence of reovirus-associated enzyme activities on genome RNA as determined by psoralen photosensitivity.

Irradiation with long wavelength UV light in the presence of 4'-substituted psoralens abolished the in fectivity of reovirus and reovirus subviral particles Photoreaction of intact virions, cores, or genome RNA resulted in the formation of psoralen monoadducts and cross-links, demonstrating that the viral genome RNA in situ is double-stranded. Virus-associated RNA polymerase activity was lost following psoralen photoreaction, consistent with dependence on the genome RNA as template for viral mRNA synthesis. By contrast, reovirus guanylyl transferase and methyl transferase activities that catalyze the formation of mRNA 5'-terminal cap, m GpppG, were relatively unaffected by photoreaction. Cations that inhibit psoralen binding to nucleic acids also protected against loss of RNA polymerase activity. Partially photoinactivated viral cores produced decreased amounts of full length viral mRNAs. The absence of prematurely terminated transcripts indicates that template RNA modification by psoralen adducts has an all-or-none effect on initiation of mRNA synthesis by the core-associated RNA polymerase.

Ionizing radiation perturbs the switch-on of transcriptase in a model transcription complex in vitro.

Ionizing radiation markedly alters the response of the reovirus transcriptase unblocking mechanism to stimulation by K+ ions, which normally trigger the switch-on of transcription in this system. In irradiated subviral particles the concentration of K+ ions needed to trigger switch-on is reduced in a dose-dependent way. The observed alteration of switch-on characteristics appears to correlate with alteration of the electrophoretic behaviour of a single major polypeptide species. These observations have important implications for understanding some of the effects of ionizing radiation on cells, most notably the induction of both latent virus and cell differentiation.

Reovirus-specific enzyme(s) associated with subviral particles responds in vitro to polyribocytidylate to yield double-stranded polyribocytidylate-polyriboguanylate.

In reovirus-infected cells, virus-specific particles accumulate that have associated with them a polyribocytidylate [poly(C)]-dependent polymerase. This enzyme copies in vitro poly(C) to yield the double-stranded poly(C).polyriboguanylate [poly(G)]. The particles with poly(C)-dependent polymerase were heterogeneous in size, with most sedimenting from 300S to 550S. Exponential increase in these particles began at 23 h, and maximal amounts were present by 31 h, the time of onset of exponential growth of virus at 30 degrees C. Maximal amounts of particles with active transcriptase and replicase were present at 15 and 18 h after infection. Thereafter, there was a marked decrease in particles with active transcriptase and replicase until base line levels were reached at 31 h. Thus, the increase in poly(C)-responding particles occurred coincident with the decrease in particles with active transcriptase and replicase. The requirement for poly(C) as template was specific because no RNA was synthesized in vitro in response to any other homopolymer, including 2'-O-methyl-poly(C). Synthesis was optimal in the presence of Mn(2+) as the divalent cation, and no primer was necessary for synthesis. In contrast, the dinucleotide GpG markedly stimulated synthesis in the presence of 8 mM Mg(2+). The size of the poly(C).poly(G) synthesized in vitro was dependent on the size of the poly(C) used as template. This suggested that the whole template was copied into a complementary strand of similar size. The T(m) of the product was between 100 and 130 degrees C. Hydrolysis of the product labeled in [(32)P]GMP with alkali or RNase T2 yielded GMP as the only labeled mononucleotide. This does indicate that the synthesis of the poly(G) strand in vitro did not proceed by end addition to the poly(C) template, but proceeded on a separate strand.

Circular dichroism of intermediate subviral particles of reovirus. Elucidation of the mechanism underlying the specific monovalent cation effects on uncoating.

1. Circular dichroic (CD) spectra of purified intermediate subviral particles of reovirus were determined in the presence of different monovalent cations. 2. The CD spectra reveal that reo intermediate subviral particles can exist in two conformationally different forms. The two forms are readily distinguished by comparison of their ellipticities in the wavelength regions 210 nm and 220 nm, with a Na+-induced form exhibiting a reduced negative ellipticity relative to a Cs+-induced form. 3. The transition between the Na+- and Cs+-induced forms is reversible by manipulation of the species of monovalent cation present and appears to be temperature independent. 4. Temperature variation studies on dilute suspensions of particles indicate that the Na+-induced form is stable, whereas the Cs+-induced from undergoes a second transition, temperature dependent and irreversible, to become a viral core. 5. A model is presented relating these observations to the known properties of reovirus uncoating and transcriptase activation.