Search

Reovirus mRNAs synthesized in vitro by the virionassociated enzyme have a 5' 'cap 1' structure (m7G(5')ppp(5')GmpCp...). However, about one third to one half of the reovirus mRNAs formed in mouse L929 cells have a 5' 'cap 2' structure (m7G(5')ppp(5')GmpCmp...) and the rest have a 5' 'cap 1' structure. The finding that virus mRNA 'cap' methylation is impaired in extracts of interferon-treated cells prompted us to study the effect of interferon on virus mRNA 'cap' methylation in vivo. Using labeling with [3H]-guanosine and dual labeling with [3H]methionine and [14C]uridine we compared the 5' structures of reovirus mRNAs accumulating between 5 and 11 h after infection in: L929 cells treated with 390 to 2600 U/ml of a partially purified mouse interferon preparation and untreated L929 cells. The treatment resulted in a 70 to 98% decrease in the 24 h virus yield and in a 50 to 55% decrease in the label accumulated in virus mRNAs. The 'capping' of virus mRNAs and the methylation of their 5' terminal and adjacent G residues were not diminished in interferon-treated cells. However, the percent of 'cap 2' termini was 36 to 47% lower in virus mRNAs from interferon-treated cells than in virus mRNAs from control cells. The interferon treatment did not result in the appearance of additional methylated nucleotides in the virus mRNAs.

In this report, we study the reovirus serotype 3 (strain Dearing) sigma 1 protein obtained from various sources: from Escherichia coli expressing sigma 1 protein, from reovirus-infected mouse L cells, and from purified reovirions. We demonstrate that the sigma 1 protein is a multimer in its undisrupted form and present biochemical evidence suggesting that the multimer is made up of four sigma 1 subunits.

Reovirus-associated methylases, located in the viral core, function independently of RNA polymerase and guanylyl transferase to modify the 5′-termini of single-stranded oligonucleotides present in the virion. A previously undiscovered class of oligomers, 5′-terminated by GpppG, serve as substrate and are converted to m 7 GpppGp(Xp)n and m 7 GpppG m p(Xp)n by addition of methyl groups from S-adenosyl methionine. This reaction occurs under conditions distinct from those stimulating mRNA methylation.

Human fibrin foam as a matrix for three-dimensional organ cultures was successfully employed for the cultivation of newborn rat and hamster colon tissue. Colonic tissue was maintained under different oxygen atmospheres and with various synthetic media. Explants of rat and hamster colon, maintained in Leibovitz's L-15 medium buffered with HEPES in the presence of 5% CO2 and air, retained their normal columnar epithelial architecture for 24 days in culture. Outgrowth of epithelial cells from colonic explants similar to those of the mother fragments grew into the foam. Viability of the explants was indicated by radioactive thymidine incorporation, and by the ability of the fragments to support viral replication.

The biochemical mechanism of the interferon-mediated inhibition of reovirus messenger RNA translation in cell-free extracts prepared from mouse ascites tumor cells was investigated. The following results were obtained: (1) Cell-free extracts from interferon-treated cells catalyze the formation of methionyl-X initiation dipeptides in response to reovirus mRNA at a rate and extent only slightly less than that of extracts from untreated cells. Under conditions where the overall translation in vitro of reovirus mRNA is inhibited by 75% or more, the formation of methionyl-X initiation dipeptides is inhibited much less, about 30%. (2) Neither methionyl-X initiation dipeptide formation nor overall translation of reovirus mRNA is significantly affected by the addition of either S -adenosyl- l -methionine or S -adenosyl- l -homocysteine to cell-free systems prepared from either interferon-treated or untreated cells. (3) Reovirus mRNA synthesized in vitro in the presence of S -adenosyl- l -methionine is translated slightly more efficiently than either reovirus mRNA synthesized in the presence of S -adenosyl- l -homocysteine or reovirus mRNA synthesized in the absence of both S -adenosyl- l -methionine and S -adenosyl- l -homocysteine by cell-free systems prepared from untreated ascites cells; by contrast, all of the reovirus mRNA preparations were translated very poorly by cell-free systems prepared from interferon-treated cells. (4) Exogenously added mouse ascites cell transfer RNA partially reverses the inhibition of viral mRNA translation in vitro catalyzed by cell-free systems prepared from interferon-treated cells in response to reovirus mRNA synthesized in the absence or in the presence of either S -adenosyl- l -methionine or S -adenosyl- l -homocysteine. Yeast and rat liver tRNA as well as tRNA from two prokaryotes, Escherichia coli and Streptococcus faecalis , do not significantly stimulate viral mRNA translation in the interferon-treated cell-free system. Translation catalyzed by untreated control extracts is not dependent upon the addition of tRNA. (5) Reovirus [ 32 P]mRNA recovered from interferon-treated cell-free protein synthesis systems after 15-min incubation possesses a profile similar to that of mRNA recovered from untreated control systems when analyzed by polyacrylamide-agarose-urea gel electrophoresis. These results indicate that the interferon-mediated inhibition of viral mRNA translation in vitro is exerted at a step of polypeptide chain biosynthesis which is subsequent to formation of the first peptide bond and which either directly or indirectly involves the participation of transfer RNA.

The mechanism by which viruses bind to and infect specific tissues to cause disease has only recently begun to be understood. The mammalian reoviruses provide an especially attractive model for studying the details of cell surface recognition. The cell and tissue tropism of reovirus is determined by a portion of the viral hemagglutinin termed the neutralization domain. We have reported previously on the generation of both monoclonal and polyclonal anti-idiotypic antibodies that mimic the viral hemagglutinin in the specificity of binding to the reovirus receptor. By using these anti-idiotypic antibodies as specific probes, we have successfully isolated the mammalian reovirus receptor from neuronal and lymphoid cells. In the present study, we report that the reovirus receptor is structurally similar to the mammalian beta-adrenergic receptor. This conclusion is based on the following observations: (i) purified beta-adrenergic receptor is immunoprecipitable by anti-reovirus receptor antibody; (ii) purified reovirus receptor obtained from murine thymoma cells and beta-adrenergic receptor obtained from calf lung exhibit identical molecular masses and isoelectric points; (iii) trypsin digests of purified reovirus and beta-adrenergic receptors display indistinguishable fragment patterns; (iv) purified reovirus receptor binds the beta-antagonist [125I]iodohydroxybenzylpindolol and this binding is blocked by the beta-agonist isoproterenol.

We have examined the effects of heat and several chemical inactivating agents on the buoyant density, particle-associated polypeptides and ultrastructure of reovirus particles. Treatment at pH 11 removed polypeptide sigma 1 from the outer capsid of reovirus type 2 but not from type 1; resultant particles were unchanged in their buoyant density and morphology. Treatment of reovirus types 2 and 3 with 2.5 M-guanidine-HCl produced particles with unchanged polypeptide content but an increased buoyant density, and caused aggregation of type 3 but not type 2. Treatment with 1% SDS removed polypeptide sigma 3 from both types 1 and 2 and increased the buoyant density of the virus particles. The outer capsid of SDS-treated virions was greatly altered and often indistinct. Treatment of type 3 with either 1% phenol or 33% ethanol produced particles that had a full complement of polypeptides, were unaltered in buoyant density, but were greatly aggregated. Thus, these inactivating agents affect reovirus particles in specific and distinct ways. The differential effects of such treatments can thus be used to study the structure and function of the reovirus capsid components.

The relationship between reovirus type 3 and persistent infantile cholestasis was studied by measuring antibody to the virus in the sera of affected and control babies younger than 1 year of age. One hundred sixty-seven infants were divided into four groups: those with extrahepatic biliary atresia, idiopathic neonatal hepatitis, or other cholestatic disorders, and controls. When available, maternal sera obtained simultaneously with infant sera were also studied. The results indicate that 62% of babies with extrahepatic biliary atresia and 52% of infants with idiopathic neonatal hepatitis have reovirus 3 antibodies. In contrast, less than 12% of either normal infants or babies with other cholestatic disorders have antibodies. These observations suggest that perinatal infection with reovirus type 3 may serve as an initiating event in the genesis of two closely related forms of infantile obstructive cholangiopathy: extrahepatic biliary atresia and idiopathic neonatal hepatitis.

The electrokinetics of vaccinia virus and reovirus type 3 were studied in dilute electrolyte solutions by whole-particle microelectrophoresis with dark-field laser illumination. The electrophoretic mobilities were dependent upon pH and electrolyte composition. The isoelectric point of vaccinia virus in dilute NaCl was 4.8 +/- 0.1; that of reovirus was 3.8 +/- 0.1. The mobility of reovirus was -2.65 +/- 0.30 micrometer . S-1 . V-1 . cm-1. Treatments known to cause irreversible changes in the structure of vaccinia, such as heating to 90 degrees C or exposure to pH 10, altered the virus's mobility under the reference conditions. The practical range of whole-particle microelectrophoresis thus extends to microorganisms as small as reovirus, allowing their electrokinetic and related surface properties to be studied.

The 3′-terminal sequence of the plus-stranded RNA species transcribed in vitro from the S2 gene of reovirus serotype 3 (Dearing strain) was found to be identical with the 3′-terminal sequence of the plus strand of the S2 gene. The 5′-terminal sequence of the former RNA species was determined by M. Kozak (Nature ( London ) 269 , 390–394, 1977); this sequence has now been found to be exactly complementary to the 3′-terminal sequence of the minus strand of the S2 gene. Thus the transcript of the S2 gene, which is most probably the in vivo messenger RNA, is identical in base sequence (barring base modifications) to the plus strand of this gene. In all likelihood this situation holds for all reovirus messenger RNA species. Certain secondary structure features of s2 plus strands are described: they possess complementary sequences near their 5′- and 3′-termini which may permit them to assume a circular configuration, and they possess a sequence near their 5′-terminus that is complementary to a 3′-proximal sequence of 18 S ribosomal RNA.

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.

We used [ methyl - 3 H] dimethyl sulfate to probe the genome structures of several RNA and DNA viruses. We compared sites of modification in nucleic acids that were methylated chemically before and after extraction from purified virions. With both single-stranded and double-stranded substrates alkylation occurred mainly at the N7 position of guanine. However, adenine N1 atoms were differentially accessible in single-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirus mRNA and deproteinized genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae methylated in situ yielded RNAs with ratios of 0.04 to 0.08, indicating that the intravirion genomes were double stranded. We obtained ratios of 0.26 and 0.35 for the RNAs of dimethyl sulfate-treated brome mosaic and avian sarcoma virions, respectively, which was consistent with partial protection of adenine N1 sites by structural proteins or genome conformation or both. The ratios of 1-methyladenosine to 7-methylguanosine for vaccinia virus DNAs methylated in situ (0.10) and after phenol extraction (0.14) were less than the ratios for φX174 and M13 DNAs (0.39 to 0.64) but considerably greater than the ratio observed with adenovirus DNA (0.002 to 0.02). The presence of a single-stranded region(s) in the vaccinia virus genome was confirmed by S1 nuclease digestion of [ methyl - 3 H] DNA; the released radiolabeled fraction had a ratio of 0.41, compared with 0.025 for the residual duplex DNA. In addition to the structure-dependent accessibility of adenine N1, methylation of adenine N3 was severalfold lower in the intravirion genomes of vaccinia virus, φX174, and adenovirus than in the corresponding extracted DNAs. Chemical methylation of virions and subviral particles should be useful for in situ analyses of specific regions of RNA and DNA genomes, such as the sites of protein binding during virus maturation.

Compounds of general structure N(5')pn(5')N were used by the reovirus-associated RNA polymerase as primers for template-directed synthesis of virus-specific oligonucleotides and RNA. Structural requirements for activity included a guanosine residue and at least four phosphates--i.e., G(5')pppp(5')N. Gp4G incubated with viral cores in the presence of CTP yielded Gp4GpC and CpGp4GpC. In a complete transcription mixture, Gp4G was also incorporated into the 5' termini of full-length transcripts in the unmethylated forms Gp4GpC and CpGp4GpC, in contrast to viral mRNAs that contain 5'-terminal m7GpppGmpC formed de novo. Gp5G, Gp6G, and Gp4A but not Gp2G, Gp3G, and Ap4A also primed reovirus transcription and inhibited RNA methylation.

There were marked differences in the abilities of eight different soil materials to remove and retain viruses from settled sewage, but for each soil material the behavior of two different viruses, poliovirus type 1 and reovirus type 3, was often similar. Virus adsorption to soil materials was rapid, the majority occurring within 15 min. Clayey materials efficiently adsorbed both viruses from wastewater over a range of pH and total dissolved solids levels. Sands and organic soil materials were comparatively poor adsorbents, but in some cases their ability to adsorb viruses increased at low pH and with the addition of total dissolved solids or divalent cations. Viruses in suspensions of soil material in settled sewage survived for considerable time periods, despite microbial activity. In some cases virus survival was prolonged in suspensions of soil materials compared to soil-free controls. Although sandy and organic soil materials were poor virus adsorbents when suspended in wastewater, they gave ≥95% virus removal from intermittently applied wastewater as unsaturated, 10-cm-deep columns. However, considerable quantities of the retained viruses were washed from the columns by simulated rainfall. Under the same conditions, clayey soil material removed ≥99.9995% of the viruses from applied wastewater, and none were washed from the columns by simulated rainfall.

A cytoplasmic polyhedrosis virus (CPV) from Heliothis armigera contains 10 double-stranded RNA genome segments which are all well resolved on polyacrylamide gel electrophoresis. The relationship between log molecular weight and electrophoretic mobility in polyacrylamide gels has been studied using 32 P-labeled CPV RNA, and has been shown to deviate markedly from linearity at the higher molecular weight values. A new relationship between molecular weight and electrophoretic mobility for doubles-tranded RNA has therefore been defined and used to determine the absolute molecular weight of the CPV RNA by comparison with reovirus type 3 RNA.

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.

Subviral particles which sedimented at 400 and 600 S were identified in reovirus-infected cells. The 600 S particles (immature virions) contained 10 segments of double-stranded RNA, the major capsid polypeptides λ1, λ2, μ2, σ2, σ3, and a small amount of the nonstructural polypeptide μ0. They lacked A-rich oligonucleotides. Kinetic experiments demonstrated that the acquisition of these oligonucleotides by virions is a very late event in viral morphogenesis. The 400 S particles, which contained 10 segments of double-stranded RNA, consisted of two kinds of particles: (1) short-lived intermediates which contained the core polypeptides λ1, λ2 (in reduced amounts), σ2, and polypeptide μ0; and (2) particles that accumulated in infected cells and that contained the core polypeptides only. When observed in the electron microscope, the latter particles ranged in size from 25 to 54 nm. They lacked the spikes present on the surface of viral cores, but possessed surface detail similar to that on viral cores.

Human reovirus s1 mRNA, which codes for the viral hemagglutinin, also directs the synthesis of a previously unrecognized polypeptide of molecular mass 14 kDa in reticulocyte and wheat germ extracts. Hybrid-arrest of translation by selected restriction fragments of cloned S1 DNA indicated that synthesis of the 14-kDa polypeptide initiates at the second AUG. This was confirmed by NH2-terminal sequence analyses. The coding sequence for the 14-kDa polypeptide thus lies entirely within the hemagglutinin gene but in a different reading frame. Although not found in virions, the 14-kDa polypeptide apparently is formed in virus-infected mouse L cells, as demonstrated by comparison of [35S]methionine-labeled polypeptides in cell extracts with the corresponding in vitro products.

Four enteric viruses, poliovirus type 1, echovirus type 1, reovirus type 3, and simian adenovirus SV-11, were concentrated from seeded 1.3-liter volumes of raw, finished, and granular activated carbon-treated waters by adsorption to 47-mm-diameter (17 cm2), electropositive ( Virosorb 1MDS ) filters at pH 7.5 or electronegative ( Filterite ) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Removal of particulates from raw and finished waters by 0.2-micron prefiltration before virus addition and pH adjustment had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared with granular activated carbon-treated water, but the extent of interference varied with virus type and adsorption conditions. For electropositive 1MDS filters, organic interference was similar with all virus types. For Filterite filters, organic interference was evident with poliovirus and echovirus, but could be overcome by adding MgCl2. Reovirus and SV-11 were not adversely affected by organics during adsorption to Filterite filters. Elution of reovirus and adenovirus was inefficient compared with that of poliovirus and echovirus. None of the three adsorption schemes ( 1MDS at pH 7.5 and Filterite with and without 5 mM MgCl2 at pH 3.5) could be judged superior for all viruses and water types tested.

The reovirus oligoadenylates exist in two states within the virion: free and bound to viral proteins. The latter class of oligonucleotides, after digestion with Penicillium (P1) nuclease, yields adenylic acid and an adenosine-containing compound that is positively charged at pH 1.7, 3.5, or 6.5. In a mixture of [35S]methionine- and [3H]adenosine-labeled reovirus disrupted by sodium dodecyl sulfate/urea, approximately 4% of the radioactivity in [35S]methionine-labeled proteins coelutes with [3H]adenosine-labeled material at a net charge of -1.5 when analyzed by ion-exchange chromatography on DEAE-cellulose. This material migrates in sodium dodecyl sulfate/polyacrylamide gels with mu polypeptides and with a small protein, viii. Radioactivity is not released when the complex is boiled in buffer containing sodium dodecyl sulfate and urea or boiled in 80% dimethyl sulfoxide or when viral RNA is extracted with phenol. Digestion with Pronase converts the [3H]adenosine-labeled compound to oligomers of net charge -8 to -12 which contain nuclease P1- and alkaline phosphatase-sensitive adenylic acid residues as well as adenosine in a P1- and phosphatase-resistant linkage. These data indicate that reovirus contains structural proteins that are covalently bound to an oligoadenylate moiety.

Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

Summary The regulation of translation in reovirus-infected cells has been investigated by double-infection experiments. Different cell lines were able to translate uncapped encephalomyocarditis (EMC) virus mRNA and capped vesicular stomatitis virus (VSV) mRNAs both early and late during reovirus infection. These results are not fully in agreement with a previously suggested model in which, in the early phase of reovirus infection, only capped mRNAs are translated, whereas in the late phase the cells are modified to translate exclusively uncapped mRNAs. The observations that EMC virus and VSV shut down host protein synthesis more efficiently than reovirus translation in the late phase in double-infections indicate that competition for a message-discriminatory factor may not be involved in the shut-off of host protein synthesis in these animal virus-infected cells.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.

In standard pairwise crosses there was no detectable recombination between defective reovirus lacking the largest genomic segment and prototypes of the seven known classes of ts mutants. However, in such crosses between R2A (201) and the various prototypes frequencies of ts+ recombinants between 2.6 and 6.1% were observed, as others have found (Fields, 1971; Fields and Joklik, 1969). An infectious center assay was devised to measure recombination in this system, and it was found that all mixedly infected cells gave rise to ts+ recombinants in crosses between prototype ts mutants, but no recombination was detectable when the defective virus was crossed with three different ts mutants. The ts mutation of mutant R2A (201) was efficiently rescued when crossed with UV-inactivated wild-type virus but not when crossed with UV-inactivated defective virus. It is concluded from these various experiments that if there is any recombination between these defective reovirions and any known class of ts mutants it is too low to be measured by methods presently available. The kinetics of recombination were measured in cells mixedly infected with R2A (201) and R2B (352) mutants. At the earliest time progeny virus could be found in the cells the frequency of ts+ recombinants was 4.5%, and this frequency remained unchanged despite a subsequent 1,000-fold increase in progeny virus.

A number of field isolates of reovirus 3 were examined to determine their relative neurovirulence after intracerebral inoculation. One isolate was found that had decreased neurovirulence. This "avirulent" strain showed the typical type 3 neural tropism but grew significantly less well in brain tissue than T3 (Dearing) and the other type 3 reoviruses. The avirulent virus was not temperature-sensitive, and its yield in mouse L cells in vitro was similar to that of the laboratory strains. To determine the reason that this clone was avirulent, we isolated a series of reassortant progeny clones from crosses between the avirulent strain and T1 (Lang) and T3 (Dearing). Using these reassortants, we showed that avirulence was a property of the M 2 gene segment. The M2 segment was also responsible for conferring greater sensitivity to chymotryptic digestion on the avirulent strain, compared to more virulent strains. Prior studies have determined that the localization of virus in different cell types in the brain (tropism) is a property of the viral hemagglutinin, the product of the S1 RNA genome segment. Our studies thus indicate that the basis for relative neurovirulence does not reside in the viral hemagglutinin and clearly illustrate the multigenic nature of neurovirulence.

The aggregation of poliovirus and reovirus was followed in buffers at various pH values by means of a single particle analysis (SPA) test. The SPA test used here was modified from the original test reported earlier to prevent disaggregation of virus clumps from invalidating the results. The modified SPA test demonstrated that the efficiency of aggregation, which is a measure of the percentage of collisions which are effective in producing an aggregate, may vary widely depending on the conditions in which the virus is placed. The modified SPA test was also used to demonstrate that the kinetic features of viral aggregation follow the classical laws of colloid particle aggregation, which in turn are solely dependent upon diffusion of the particles as caused by brownian motion.

This report describes immunocytochemical and ultrastructural methods which led to the identification of Reovirus type 3 (Reo-3) in the porta hepatis of a patient with extrahepatic biliary atresia. The study indicates that Reo-3 antigenic sites are demonstrable by the avidin-biotinylated complex peroxidase method following formalin fixation and paraffin embedding, but are destroyed by freezing and thawing prior to fixation. Deparaffinization of the block and subsequent rembedding in epon-araldite did not alter immunoperoxidase staining. This procedure offered the advantage of higher light microscopic resolution of semithin (1 micron) sections and assisted in the selection of specific areas for ultrastructural studies. Localization of Reo-3 in extrahepatic biliary atresia was confined to a biliary remnant in which there were acutely inflammed, partially necrotic microscopic ducts. Electron microscopic examination of the immunoreactive sites revealed virus-like particles similar in appearance to Reo-3 particles in infected tissue culture cells. The observations presented here support previously reported serologic data which have shown an association between Reo-3 infection and extrahepatic biliary atresia.

Infection with reovirus 3 (Reo-3) has been suggested as the cause of extrahepatic biliary atresia and idiopathic neonatal hepatitis, but confirmation has been lacking. Therefore we have searched for a specific anti-Reo-3 antibody response in the sera of patients with biliary atresia or neonatal hepatitis and for Reo-3 antigens in their hepatobiliary tissues. Sera from 23 infants with extrahepatic biliary atresia, 12 with neonatal hepatitis, 30 age-matched control patients with other liver diseases, and 55 control patients without liver disease were tested by an enzyme-linked immunosorbent assay for total (IgA, IgG, and IgM) anti-Reo-3 antibodies; sera of infants younger than 6 months of age were tested also for IgM anti-Reo-3 antibodies alone. There was no difference between either total or IgM anti-Reo-3 antibody levels in infants with extrahepatic biliary atresia or neonatal hepatitis and levels in control infants. Reo-3 antigens were not detected in the hepatobiliary tissues of 19 infants (18 with biliary atresia, one with neonatal hepatitis) by an immunoperoxidase method that readily demonstrated Reo-3 in control infected HEp-G2 cells. Our data do not support a relationship between neonatal liver diseases and infection with Reo-3.

The synthesis of adenine-rich RNA has been examined in cells infected with temperature-sensitive mutants of Reovirus type 3. Prototypes of all mutant groups produce adenine-rich RNA at the permissive temperature. However, cells similarly infected at the nonpermissive temperature fail to produce measurable amounts of adenine-rich RNA under these same conditions.

The electrophoretic analysis of reovirus-specific polypeptides in infected cells using a discontinuous gel system has allowed the resolution of additional viral-specific polypeptides, including one large-sized gamma3 and two (or possibly three) medium-sized (mu3, mu4, mu5(?)) species. The proteins designated mu0, sigma1, and sigma2 based on electrophoretic mobility in gel systems containing phosphate-urea correspond to mu4, sigma2, and sigma1, respectively, when analyzed in systems containing Tris-glycine. It is likely that protein modifications (phosphorylation and glycosylation) are responsible for at least some of these differences.

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.

Helix-coil transitions of double-stranded RNA from reovirus and infectious bursal disease virus were measured optically in aqueous medium of different ionic strengths. In RNA from reovirus four transitions and in RNA from infectious bursal disease virus two transitions were resolved and evaluated quantitatively. The ionic strength dependence of their midpoint temperatures dTm/dlog[Na+] were 13.5 +/- 0.3 degrees C for reovirus RNA and 14.9 +/-0.7 degrees C for infectious bursal disease virus RNA. The midpoint temperatures extrapolated to 1 M ionic strength were 102.9, 104.3, 105.6, and 108.8 +/- 0.3 degrees C for reovirus RNA, and 108.8 +/- 1.8 and 109.6 +/- 1.0 degrees C for infectious bursal disease virus RNA. The G + C content of the regions in reovirus RNA melting in the different transitions were determined from the spectrum of the hypochromicity. The quantitative interpretation of the data is carried out on the basis of the ion condensation theory. It is estimated for double-stranded RNA of 100% G + C, that dTm/dlog[na+] = 8.4 degrees C. The two-dimensional dependence Tm = Tm (ionic strength, G + C content) is given. The ionic strength dependence in different double-stranded RNAs is correlated to the spacing of the phosphate backbone, secondary structure, and tertiary structure.

The aim of this study was to investigate methods to eliminate pathogenic viral agents while preserving the 'normalizing' properties of the gut microflora of mice. Mouse hepatitis virus A59 (MHV), Reo 3 virus and Theiler GD VII virus were added to the caecal contents of 'normal' mice and following dilution, with or without subsequent culturing, given to germ-free mice. Four weeks later antibody titres against these and other viruses were determined. MHV and Theiler CD VII virus survived dilution but were eliminated during culturing. Reo-virus survived the 10-1 dilution-culture step. All dilutions and dilutioncultures of caecal contents resulted in 'normalization' in germ-free recipients of the relative caecal weight, percentage faecal fusiform-shaped bacteria, faecal bile acids and colonization of small intestine by segmented filamentous bacteria.

In this paper we provide a step by step comparison of the pathogenesis of murine infection caused by reovirus type 3 (Dearing) and an antigenic variant (K) selected by its resistance to neutralization with a monoclonal antibody (G5) directed against the T3 hemagglutinin. To show that specific changes in the biologic properties of variant K were due to mutation in the S1 double-stranded RNA segment (gene), which encodes the viral hemagglutinin, we generated a reassortant virus ("1 HA K") containing the variant K S1 gene and compared its properties to variant K and to a reassortant ("1 HA 3") containing the T3 (Dearing) S1 gene. These studies, in conjunction with our previous nucleotide sequence analysis of the S1 genes of variant K and T3 (Dearing) [R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizel, Jr., and B. N. Fields, Nature (London) 315:421-423, 1985; R. Bassel-Duby, D. R. Spriggs, K. L. Tyler, and B. N. Fields, submitted for publication], indicate that a single amino acid change in the T3 hemagglutinin can alter viral growth and tropism within the central nervous system without affecting either its primary replication in the intestine or its pattern of spread to or within the central nervous system.

An anti-idiotypic antibody has been characterized that forms a functional internal image of the cell-attachment site of reovirus type 3. This antibody shares sequence similarity with the reovirus type 3 cell attachment protein. Studies utilizing synthetic peptides have shown that the region of sequence similarity corresponds to the cell-attachment site of the virus and the antibody. Studies are described demonstrating that these peptides possess biological activity similar to that of the virus and the antibody. Three-dimensional models of the cell-attachment site of the virus are presented and compared to a similar model for the antibody. These studies indicate that anti-idiotypic antibodies can be utilized to understand the three-dimensional basis of ligand - receptor interactions, and can lead to the development of novel biologically active substances.

We describe a method which uses protein blots of whole animal sections to map the distribution of exogenous and endogenous antigens to specific nuclei and tracts within the nervous system (NS). We propose this method as a new approach to analysis of the expression of NS gene products during development and to charting the course and molecular pathogenesis of infectious and immune disorders of the NS.

We report the sequence of the L1 genome segment of reovirus serotype 3 strain Dearing, which encodes the minor core component protein lambda 3. It is 3854 bp long, with a long open reading frame starting at position 19 that is 1267 codons long. Protein lambda 3 is not detectably related to any other protein, nor does it appear to possess motifs indicative of recognized specialized functions. We have also sequenced the L1 genome segments of reovirus serotypes 1 and 2. The serotype 1 and 3 L1 genome segments are extremely closely related; there are only 154 mismatches (4.1%), 80% of which are in third base codon positions, so that these two lambda 3 proteins are 98.3% related (only 22 mismatches out of 1267). The serotype 2 L1 genome segment is only 75% related to the serotype 1 and 3 genome segments, and the serotype 2 lambda 3 protein is 92% related to the serotype 1 and 3 lambda 3 proteins. We have also analyzed the divergence patterns by which the various reovirus genome segments evolved into the three serotype forms. It appears that serotype 2 separated from the serotype 1/3 precursor long before serotypes 1 and 3 themselves diverged. In all cases the third base codon positions in the various genome segments have diverged about 80% toward randomness. The first and second base codon positions have diverged much less and to varying degree, depending, presumably, on each protein's ability to accept changes without significant loss of function. For the separation into the serotype 1 and 3 forms, the extent of divergence of the various genome varies over a very wide range. The S1 genome segments have again diverged most extensively, the extent of divergence in the first, second, and third base codon positions being about 50, 35 and 75%, respectively. For seven other genome segments that we examined the extent of third base codon position divergence is 56, 53, 48, 29, 22, 13, and 6%, whereas first and second base codon position divergence ranges from no more than 6 to 2 and 3 to less than 1%, respectively. The most likely explanation of these patterns is that the separation of the various genome segments into the present-day serotype 1 and 3 associated forms occurred at different times during evolution, from progenitors that were genome segment reassortants with survival rates as high as or higher than those of homologous genome segment sets.

When pyridoxal phosphate (PLP) binds reversibly to a select population of reovirus λ protein molecules, the transcriptase activity in virus cores is inhibited [Morgan, E. M., and Kingsbury, D. W. (1980), Biochemistry 19, 484–489]. We now report that each of the enzymes involved in post-transcriptional modifications of virus mRNA molecules (nucleotide phosphohydrolase, guanylyltransferase, and both methyltransferases) is also inhibited reversibly by PLP. This supports the view that reovirus mRNA transcription and modification are accomplished by a topographically related group of enzyme molecules and suggests that these enzymatic activities reside in one or more of the λ protein species. PLP did not interact with the RNA binding sites of the methyltransferases or guanylyltransferase, with the GTP binding site of guanylyltransferase, or with the nucleotide binding site of the phosphohydrolase, as shown by the inability of these substrates to compete with PLP in kinetic assays or to block PLP-directed reductive alkylation of the λ proteins. However, kinetic data suggested that PLP interacts with the AdoMet binding sites of the reovirus methyltransferases.

The binding and subsequent infectivity of reovirus to target cells are mediated by interaction with specific cell surface viral receptors. To gain a more detailed understanding of the biochemistry of the reovirus receptor and the cellular consequences of viral attachment, we have studied the binding of type 3 reovirus (Dearing strain) in a quantitative manner utilizing an antiidiotypic antibody probe. A syngeneic monoclonal antiidiotypic antibody (87.92.6) was prepared by immunization with hybridoma cells which secrete an antireovirus hemagglutinin-specific antibody. This antiidiotypic antibody was previously shown to specifically recognize the cell surface receptor for reovirus type 3. In this report, we demonstrate that antiidiotype mimicked reovirus tropism in binding to murine thymomas; antiidiotype inhibited the binding of reovirus to specific targets, but not the binding of anti-H-2; and cross linking of receptor-bound antiidiotype by antiimmunoglobulin induced patching, but not capping of reovirus receptors. Utilizing radiolabeled antiidiotype, we next quantitate the number of reovirus receptors on R1.1 and YAC thymoma cells and, finally, report on the preliminary identification of the reovirus receptor as a 67,000-Da membrane glycoprotein.

The virus-specific double-stranded genome RNA and polypeptides present in virions and cells infected with the three mammalian reovirus serotypes have been examined by co-electrophoresis in several different polyacrylamide gel systems. The double-stranded RNA and polypeptide species previously described for type 3 Dearing were found to have corresponding species in the other serotypes examined. In each serotype several RNA and polypeptide species were found to have different electrophoretic mobilities from the corresponding RNA or polypeptide species of type 3 Dearing. The combination of electrophoretic variants among the RNAs and polypeptides of the reovirus serotypes gave electrophoretic markers in all 10 of the reovirus genes. The usefulness of these electrophoretic markers in "mapping" the reovirus genome is discussed.

The nucleotide sequence of the M2 gene segment of the mammalian reovirus prototype strain, type 3 Dearing, was determined from a cloned full-length cDNA copy of the viral double-stranded RNA segment. The gene comprises 2203 nucleotides and has a single long open reading frame that spans bases 30 through 2154 and encodes the 708 amino acid outer capsid protein mu 1. Aminoterminal sequence analysis of mu 1C, the proteolytically cleaved form of mu 1 that is found in purified reovirions, has identified the site of mu 1 to mu 1C cleavage between residues 42 and 43 in the mu 1 sequence. Aminoterminal sequence analysis of delta, the proteolytically cleaved product of mu 1C that is found in chymotrypsin-generated intermediate subviral particles, has indicated that the mu 1C to delta cleavage occurs near the carboxyterminus of mu 1C. Lastly, stoichiometric determinations using new sequence information have suggested that approximately equimolar amounts of mu 1C and the other major outer capsid component sigma 3 are present in virions. The data presented in this study should be useful for understanding the molecular basis of the functions of the mu 1 protein in reovirus entry into cells and in pathogenesis in the host animal.

It has previously been shown that of all the soluble reovirus-specified proteins present in the infected cell lysate, protein sigma 1 alone possesses the capacity to bind to host cells (P.W.K. Lee, E.C. Hayes, and W.K. Joklik, 1981, Virology 108, 156-163). We found that sigma 1 from urea-disrupted reovirus particles was also capable of such specific binding. Reovirions were therefore used as a source of functional sigma 1. Accordingly, a simple procedure has been developed to purify sigma 1 by subjecting urea-disrupted reovirions to DEAE ion-exchange chromatography. Protein sigma 1 thus isolated was electrophoretically homogeneous and the recovery was estimated to be 50 to 60% of the theoretical yield. The purified protein presumably maintained its native conformation since it was recognized by a panel of monoclonal anti-sigma 1 antibodies previously isolated, and was capable of specifically binding to host cell receptors, agglutinating human erythrocytes and inducing neutralization and hemagglutination-inhibition antibodies. Subsequent chemical crosslinking studies revealed the presence of oligomeric (mostly dimeric) sigma 1 forms in the preparation. The amino acid composition of the purified sigma 1 was found to closely match that inferred from the S1 gene sequence. However, attempts to determine its amino-terminal sequence have not been successful. The p/ of the purified protein was determined to be 6.8. Circular dichroic measurements of the purified sigma 1 indicated that 54 and 19% of its residues were arranged in alpha-helical and beta-sheet secondary structures, respectively.

A series of 94 isolates of reovirus from humans, cattle, and mice, showed extensive variability in the patterns of migration of the ten double-stranded RNA genome segments. This variation was found in all three serotypes, and involved all ten genome segments, including the segment responsible for serological specificity. Although a single pattern was present among several samples isolated from individuals and collected at a single time and place, there were often multiple genetic variants of a single serotype present in a population. Samples isolated from widely different geographic origins or different mammalian hosts showed different patterns; samples from a single species from the same area over a period of time showed more limited variations. Among most isolates, the migration of the slowest S segment, the segment that encodes the hemagglutinin and is responsible for serological specificity in laboratory strains, was similar to reference strains for type 1 and type 3 isolates. However, the type 2 isolates showed considerable variation in this segment.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.

Cytolytic T lymphocytes (Tc) specific for cells infected with reovirus type 3 were shown to lyse an uninfected B-cell hybridoma line (designated 87.92.6). This hybridoma expresses and secretes an anti-idiotypic antibody that reacts with a monoclonal antibody (termed G-5). G-5 recognizes a domain on the hemagglutinin of the reovirus that is relevant to virus tropism. The Tc cell response was H-2 restricted and could be inhibited by G-5. As shown by limiting dilution analysis, identical clones lysed reovirus-infected and anti-idiotype-bearing target cells. Tc cells induced by a variant of wild-type reovirus type 3 (immunologically selected by resistance to neutralization by G-5) were unable to recognize the anti-idiotype-bearing cells although they lysed reovirus type 3-infected tumor cells. We conclude that Tc cells must bear determinants that bind anti-idiotype molecules and, furthermore, that B cells and cytolytic T cells can share these idiotypic determinants.

In our laboratory, virus adsorbed to talc–Celite layers is eluted with 100 mL of 10% fetal calf serum (FCS) in normal saline (pH 9.0). A further 10-fold reduction in the volume of the eluate was necessary before its inoculation into cell cultures.A 100-mL volume of an experimentally contaminated sample was placed in a dialysis sac and hydroextracted overnight (4 °C) with polyethylene glycol (PEG) 6000. The viscous material remaining in the sac was resuspended in 10 mL of Earle's balanced salt solution. After membrane filtration (0.2 μm), the concentrate was plaque assayed in BS-C-1 cells. Using this technique, recoveries of five laboratory-adapted enteric viruses (polio 1, echo 6, coxsackie B5, coxsackie A9, and reo 3) and four freshly isolated enteric virus strains (polio 1, echo 1, coxsackie B3, and reo) ranged from 87 to 97%. In comparative tests, PEG hydroextraction was simpler and superior to organic flocculation.

The virological requirements for the recognition of infected target cells by cytolytic T lymphocytes (CTL), using reovirus, a nonenveloped, icosahedral virus has been investigated. Using mouse L cells infected at the nonpermissive temperature with is (temperature-sensitive) mutants of reovirus in complementation groups C and G, it has been shown that the production of complete viral particles is not necessary for efficient lysis of infected cells by CTL. In addition, adsorption of purified viral particles and viral top component (TC), empty capsids lacking genome ds-RNA, to L cells just prior to use in cytolytic T cell assays is sufficient to produce target cells capable of being lysed, though target production is less efficient than with L cells infected with reovirus. Membrane fluorescence analysis of cells infected with reovirus is mutants at the nonpermissive temperature and with adsorbed viral particles revealed the presence of the viral σ 1 protein on the cell surface. For adsorbed particles, the degree of membrane fluorescence paralleled the capacity of CTL to lyse target cells. It is concluded that cells infected with icosahedral, nonenveloped viruses, like cells infected with enveloped viruses, express viral antigens on the cell surface even in the absence of the production of complete viral particles; adsorbed viral particles can be incorporated into the cell membrane in a manner sufficient for recognition and lysis by CTL, in the absence of actual infection of the cells.

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.

We have examined the effect of reovirus infection on the CV-1 cytoskeleton. Reovirus infection produces a major disruption of vimentin filaments without producing a discernable disorganization of microtubules or microfilament bundles in CV-1 cells. In addition to disrupting the organization of vimentin filaments, reovirus infection appears to cause a reorganization of vimentin filaments. Viral inclusions contain vimentin filamentous structures. Viral infection also alters the cytoplasmic distribution of mitochondria, consistent with the proposed role of vimentin filaments in determining the distribution of mitochondria.

Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wehat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation. S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypepetide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.