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Two hammerhead motif containing ribozymes (Rzs) were constructed through recombinant techniques that were directed to cleave at the conserved sites of the reovirus S1 gene segment which encodes the cell attachment protein sigma1. The two mono-ribozymes 553 and 984 cleaved the target RNA in a sequence specific manner, and Rz-553 being the more efficient. When the mono-Rzs were combined in direct tandem to make it a multitarget-Rz, very efficient cleavage of the S1 RNA was achieved that retained the specificity of the two mono-ribozymes. This cleavage was, as expected, Mg(++)-dependent but protein-independent. Almost complete cleavage of the S1 RNA was observed with multitarget ribozyme alone. Although S1-Rz-984 cleaved the short S1 synthetic RNA, it failed to cleave the full length S1 RNA (1.4 kb). On the contrary, Rz-553 cleaved the short synthetic RNA as well as the full length S1 RNA with equal efficiency. Full length S1 RNA was, however, cleaved efficiently by the multitarget-ribozyme-S1-Rz-984-553 that cleaved at both the target sites. Thus, hybridization of one ribozyme (Rz-553) to a full length S1 RNA potentially opened up the 984-Rz target site that was otherwise inaccessible to the mono-Rz-984. Multitarget ribozyme expressing mammalian cells showed reduced amounts of S1 RNA that correlated well with the levels of reovirus sigma1 protein. Potential uses of such multitarget-Rzs are discussed.

Kinetic analyses of infectivity loss during thermal inactivation of reovirus particles revealed substantial differences between virions and infectious subvirion particles (ISVPs), as well as between the ISVPs of reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The difference in thermal inactivation of T1L and T3D ISVPs was attributed to the major surface protein mu1 by genetic analyses with reassortant viruses and recoated cores. Irreversible conformational changes in ISVP-bound mu1 were shown to accompany thermal inactivation. The thermal inactivation of ISVPs approximated first-order kinetics over a range of temperatures, permitting the use of Arrhenius plots to estimate activation enthalpies and entropies that account for the different behaviors of T1L and T3D. An effect similar to enthalpy-entropy compensation was additionally noted for the ISVPs of these two isolates. Kinetic analyses with other ISVP-like particles, including ISVPs of a previously reported thermostable mutant, provided further insights into the role of mu1 as a determinant of thermostability. Intact virions, which contain final sigma3 bound to mu1 as their major surface proteins, exhibited greater thermostability than ISVPs and underwent thermal inactivation with kinetics that deviated from first order, suggesting a role for final sigma3 in both these properties. The distinct inactivation behaviors of ISVPs are consistent with their role as an essential intermediate in reovirus entry.

Reoviruses are a common class of enteric viruses capable of infecting a broad range of mammalian species, typically with low pathogenicity. Previous studies have shown that reoviruses are common in raw water sources and are often found along with other animal viruses. This suggests that in addition to the commonly monitored enteroviruses, reoviruses might serve as an informative target for monitoring fecal contamination of drinking water sources. Mammalian reoviruses were detected and identified by a combined cell culture-reverse transcription-PCR (RT-PCR) assay with novel primers targeting the L3 gene that encodes the lambda3 major core protein. Five of 26 (19.2%) cytopathic effect-positive cell culture lysates inoculated with surface water were positive for reoviruses by RT-PCR. DNA sequence analysis of RT-PCR products revealed significant sequence diversity among isolates, which is consistent with the sequence diversity among previously characterized mammalian reoviruses. Sequence analysis revealed persistence of a reovirus genotype at a single sampling site, while a sample from another site contained two different reovirus genotypes.

Reovirus mRNAs are efficiently translated within host cells despite the absence of 3' polyadenylated tails. The 3' nontranslated regions (3'NTRs) of reovirus mRNAs contain sequences that exhibit a high degree of gene-segment-specific conservation. To determine whether the 3'NTRs of reovirus mRNAs serve to facilitate efficient translation of viral transcripts, we used T7 RNA polymerase to express constructs engineered with full-length S4 gene cDNA or truncation mutants lacking sequences in the 3'NTR. Full-length and truncated s4 mRNAs were translated using rabbit reticulocyte lysates, and translation product sigma3 was quantitated by phosphorimager analysis. In comparison to full-length s4 mRNA, translation of the s4 mRNA lacking the 3'NTR resulted in a 20 to 50% decrease in sigma3 produced. Addition to translation reactions of an RNA oligonucleotide corresponding to the S4 3'NTR significantly enhanced translation of full-length s4 mRNA but had no effect on s4 mRNA lacking 3'NTR sequences. Translation of s4 mRNAs with smaller deletions within the 3'NTR identified a discrete region capable of translational enhancement and a second region capable of translational repression. Differences in translational efficiency of full-length and deletion-mutant mRNAs were independent of RNA stability. Protein complexes in reticulocyte lysates that specifically interact with the S4 3'NTR were identified by RNA mobility shift assays. RNA oligonucleotides lacking either enhancer or repressor sequences did not efficiently compete the binding of these complexes to full-length 3'NTR. These results indicate that the reovirus S4 gene 3'NTR contains a translational operator sequence that serves to regulate translational efficiency of the s4 mRNA. Moreover, these findings suggest that cellular proteins interact with reovirus 3'NTR sequences to regulate translation of the nonpolyadenylated reovirus mRNAs.

To complement evidence for nucleoside triphosphate phosphohydrolase (NTPase), RNA helicase, RNA 5' triphosphate phosphohydrolase, and nucleic acid-binding activities by the core shell protein lambda1 of mammalian orthoreoviruses (reoviruses), we determined nucleotide sequences of the lambda1-encoding L3 gene segments from three isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). The T1L and T3D L3 gene sequences and deduced lambda1 protein sequences shared high levels of identity (97.7% and 99.3%, respectively). The lambda1 sequences differed at only 9 of 1275 amino acids. Two single-nucleotide insertions relative to a previously published sequence for T3D L3 extended the lambda1 open reading frame to within 60 nucleotides of the plus-strand 3' end for T3D and the other isolates sequenced, in keeping with the short 3' nontranslated regions of the other nine gene segments. Seven of the nine amino acid differences between T1L and T3D lambda1 were located within the amino-terminal 500 residues of lambda1, a region with putative sequence similarities to NTPases and RNA helicases. The T2J L3 and lambda1 sequences were found to be more divergent, especially within the amino-terminal 180 residues of lambda1, preceding the putative CCHH zinc finger motif. The T2J L3 sequence, along with partial sequences for L3 genes from three other reovirus isolates, suggested that one or more of the polymorphisms at amino acids 71, 215, 500, 1011, and/or 1100 in lambda1 contribute to the L3-determined differences in ATPase activities by T1L and T3D cores. The findings contribute to our ongoing efforts to elucidate sequence-structure-function relationships for the lambda1 core protein., (Copyright 1999 Academic Press.)

The reovirus sigma1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis. Although the function of sigma1s is not known, the sigma1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express sigma1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the sigma1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain sigma1s. To determine whether sigma1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells. After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether sigma1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein sigma1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.

Reovirus nonstructural protein sigma NS exhibits a ssRNA-binding activity thought to be involved in assembling the reovirus mRNAs for genome replication and virion morphogenesis. To extend analysis of this activity, recombinant sigma NS (r sigma NS) was expressed in insect cells using a recombinant baculovirus. In infected-cell extracts, r sigma NS was found in large complexes (> or = 30 S) that were disassembled into smaller, 13-19 S complexes upon treatment with RNase A. R sigma NS also bound to poly(A)-Sepharose beads both before and after purification. Treatment with high salt during purification caused r sigma NS to sediment in even smaller, 7-9 S complexes, consistent with more complete loss of RNA. To localize the RNA-binding site, limited proteolysis was used to fragment the r sigma NS protein. Upon mild treatment with thermolysin, 11 amino acids were removed from the amino terminus of r sigma NS, and the resulting protein no longer bound to poly(A). In addition, when r sigma NS in cell extracts was treated with thermolysin to generate the amino-terminally truncated from, it sedimented at 7-9 S, also consistent with the loss of RNA-binding capacity. To confirm these findings, a deletion mutant lacking amino acids 2-11 was constructed and expressed in insect cells from a recombinant baculovirus. The mutant protein in cell extracts showed greatly reduced poly(A)-binding activity and sedimented as 7-9 S complexes. These data suggest that the first 11 amino acids of sigma NS, which are predicted to form an amphipathic alpha-helix, are important for both ssRNA binding and formation of complexes larger than 7-9 S.

Mutations selected in reoviruses isolated from persistently infected cultures (PI viruses) affect viral entry into cells. Unlike wild-type (wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those present in the endocytic compartment, blocks growth of wt reovirus by inhibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yields of wt and PI viruses in cells treated with E64. Prototype PI viruses L/C, PI 2A1, and PI 3-1 produced substantially greater yields than wt viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated cells. To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from independent crosses of T1L and each of the prototype PI viruses for growth in cells treated with E64. Growth of reassortant viruses in the presence of E64 segregated exclusively with the S4 gene, which encodes viral outer-capsid protein sigma3. These results suggest that mutations in sigma3 protein selected during persistent infection alter its susceptibility to cleavage during viral disassembly. To determine the temporal relationship of acid-dependent and protease-dependent steps in reovirus disassembly, cells were infected with wt strain T1L or T3D, and medium containing either ammonium chloride or E64d, a membrane-permeable form of E64, was added at various times after adsorption. Susceptibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after adsorption. These findings indicate that acid-dependent and protease-dependent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the same compartment of the endocytic pathway.

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.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.

The mammalian reoviruses are capable of inhibiting cellular DNA synthesis and inducing apoptosis. Reovirus strains type 3 Abney (T3A) and type 3 Dearing (T3D) inhibit cellular DNA synthesis and induce apoptosis to a substantially greater extent than strain type 1 Lang (T1L). We used T1L x T3A and T1L x T3D reassortant viruses to identify viral genes associated with differences in the capacities of reovirus strains to elicit these cellular responses to viral infection. We found that the S1 and M2 genome segments determine differences in the capacities of both T1L x T3A and T1L x T3D reassortant viruses to inhibit cellular DNA synthesis and to induce apoptosis. These genes encode viral outer-capsid proteins that play important roles in viral attachment and disassembly. To extend these findings, we used field isolate strains of reovirus to determine whether the strain-specific differences in inhibition of cellular DNA synthesis and induction of apoptosis are also associated with viral serotype, a property determined by the S1 gene. In these experiments, type 3 field isolate strains were found to inhibit cellular DNA synthesis and to induce apoptosis to a greater extent than type 1 field isolate strains. Statistical analysis of these data indicate a significant correlation between the capacity of T1L x T3A and T1L x T3D reassortant viruses and field isolate strains to inhibit cellular DNA synthesis and to induce apoptosis. These findings suggest that reovirus-induced inhibition of cellular DNA synthesis and induction of apoptosis are linked and that both phenomena are induced by early steps in the viral replication cycle.

The cytotoxic T-lymphocyte (CTL) response in reovirus-infected C3H mice was investigated by using reovirus-vaccinia virus recombinants. Results of cytotoxicity assays indicated that the nonstructural protein sigma1NS elicited a significant CTL response. Experiments with sigma1NS-specific CTL lines showed that both strain-specific and cross-reactive epitopes exist in the sigma1NS protein.

Interference of wild-type reovirus growth by some temperature-sensitive (ts) mutant viruses under non-permissive conditions or by other wild-type isolates has been demonstrated; however, the stage of the virus replication cycle at which interference occurs has not been defined. Examination of the time-course of the yields of T1 Lang (T1L) dsRNA in the progeny of mixed infections of T1L with T3 Dearing (T3D) or with a panel of T3D ts mutants at a non-permissive temperature revealed that interference takes place by 8-10 h post-infection and occurs prior to or at the same time as accumulation of reovirus dsRNA. Taken together with our previous results, these data indicate that interference occurs during a window between virus uncoating and synthesis of dsRNA in the reovirus replication cycle, probably at the stage of assembly of primary reovirus 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.

Reovirus mu 2 protein can be expressed via the mouse phosphoglycerate kinase promoter to low levels in stably transfected L cells. To increase mu 2 expression, the terminal regions of the M1 gene cDNA constructs were modified and the effect on mu 2 expression was analyzed. The M1 gene has a single large open reading frame beginning at nucleotide 14 with another, in frame, AUG codon at nucleotide 161 reported to be used for translation initiation. Unexpectedly, deletions of the M1 5' terminal sequence upstream of the reported translation initiation codon, AUG161, resulted in loss of detection of mu 2 expression. When expression was driven by the stronger T7 promoter in the presence of recombinant vaccinia virus expressing the T7 RNA polymerase, constructs with the M1 5'-terminal deletion produced a smaller protein product of approximately 68 kDa, compared to approximately 73 kDa for the protein produced from the full-length M1-containing constructs consistent with the loss of 49 amino acids. The amount of shorter mu 2 product was increased by producing an improved 'Kozak' consensus sequence around the AUG codon at nucleotide 161 or by introducing an internal ribosome entry site at this location. Full-length M1 gene constructs produced a protein of the same size as the authentic mu 2 protein from virus-infected cells. It was further shown that the approximately 73 kDa product was expressed when the M1 gene was in different plasmid backgrounds and even when the M1 gene transcript was preceded by a 1 kb gene. This study demonstrated that translation of the reovirus M1 gene initiates from the first AUG codon in both infected and transfected cells.

The reovirus M2 gene is associated with the capacity of type 3 strain Abney (T3A) intermediate subviral particles (ISVPs) to permeabilize cell membranes as measured by chromium (51Cr) release (P. Lucia-Jandris, J. W. Hooper, and B. N. Fields, J. Virol. 67:5339-5345, 1993). In addition, reovirus mutants with lesions in the M2 gene can be selected by heating virus at 37 degrees C for 20 min in 33% ethanol (D. R. Wessner and B. N. Fields, J. Virol. 67:2442-2447, 1993). In this report we investigated the mechanism by which the reovirus M2 gene product (the mu 1 protein) influences the capacity of reovirus ISVPs to permeabilize membranes, using ethanol-selected T3A mutants. Each of three T3A ethanol-resistant mutants isolated (JH2, JH3, and JH4) exhibited a decreased capacity to cause 51Cr release relative to that of wild-type T3A. Sequence analysis of the M2 genes of wild-type T3A and the T3A mutants indicated that each mutant possesses a single amino acid substitution in a central region of the 708-amino-acid mu 1 protein: JH2 (residue 466, Tyr to Cys), JH3 (residue 459, Lys to Glu), and JH4 (residue 497 Pro to Ser). Assays performed with reovirus natural isolates, reassortants, and a set of previously characterized type 3 strain Dearing (T3D) ethanol-resistant mutants revealed a strong correlation between ethanol sensitivity and the capacity to cause 51Cr release. We found that ISVPs generated from the T3A and T3D mutants were stable when heated to 50 degrees C, whereas wild-type T3A ISVPs are inactivated under these conditions. Together, these data suggest that amino acid substitutions in a central region of the mu 1 protein affect the capacity of the ISVP to permeabilize L-cell membranes by altering the stability of the virus particle.

In cells simultaneously infected with any two of the three reovirus serotypes ST1, ST2, and ST3, up to 15% of the yields are intertypic reassortants that contain all possible combinations of parental genome segments. We have now found that not all genome segments in reassortants are wild type. In reassortants that possess more ST1 than ST3 genome segments, all ST1 genome segments appear to be wild type, but the incoming ST3 genome segments possess mutations that make them more similar to the ST1 genome segments that they replace. In reassortants resulting from crosses of the more distantly related ST3 and ST2 viruses that possess a majority of ST3 genome segments, all incoming ST2 genome segments are wild type, but the ST3 S4 genome segment possesses two mutations, G74 to A and G624 to A, that function as acceptance signals. Recognition of these signals has far-reaching implications for the construction of reoviruses with novel properties and functions.

The 2G10 B-cell hybridoma was found to be persistently infected with reovirus serotype 3 (RV3). The persistently infected 2G10 culture produced approximately 1 x 10(8) plaque-forming units of virus per milliliter of culture lysate, and a majority of cells in the culture were infected, as determined by infectious center assay and immunocytochemistry. Cure of the persistent infection was achieved by passaging 2G10 cells for 33 days (12 passages) in medium containing polyclonal anti-RV3 antiserum and a monoclonal antibody specific for the RV3 attachment protein. After several passages in antibody-free medium, cured 2G10 cells had (1) nondetectable levels of RV3 in cell-culture lysates, (2) no infectious centers per 3 x 10(5) cells, (3) no immunocytochemically detectable RV3 antigen, and (4) no detectable reovirus-specific RNA by reverse transcription-polymerase chain reaction amplification. Additionally, mice inoculated with cured 2G10 cell lysates did not generate antibodies directed against RV3. These observations demonstrate that persistent reovirus infection of a B-cell hybridoma can be cured by passage in medium containing anti-reovirus antibodies and suggest that the maintenance of this persistent infection is dependent on horizontal cell-to-cell transmission of virus in the culture.

Reovirus type 3 has been implicated in the origin and pathogenesis of extrahepatic biliary atresia and idiopathic neonatal hepatitis, but routine detection of this virus in hepatobiliary tissues from affected infants by culture and histological techniques has been unsuccessful. In this study, oligonucleotide primers specific to the M3 genome segment of reovirus 3 (Dearing) were used in a reverse transcriptase-mediated polymerase chain reaction technique to develop a sensitive and specific assay for the detection of reovirus 3 RNA in formalin-fixed, paraffin-embedded patient samples. Optimal reaction conditions were determined by testing infected murine tissues and preserved human liver tissue supplemented with reovirus 3. Archival specimens from 50 infants, including 14 with extrahepatic biliary atresia, 20 with idiopathic neonatal hepatitis, and 16 age-matched controls, were evaluated. Successful amplification of human albumin complementary DNA from the preserved tissues confirmed the presence of intact RNA in every patient specimen tested. Analysis of the amplification reactions by agarose gel electrophoresis and Southern blot hybridization detected the presence of reoviral RNA only once in a single patient sample. These results do not support a strong role for reovirus 3 in the development of neonatal cholestatic liver disease. The recent association of other RNA viruses of the Reoviridae family with murine liver disease and human extrahepatic biliary atresia indicates that continued investigation into a viral cause for idiopathic neonatal hepatobiliary disease is warranted.

Temperature-sensitive mutants provide an ideal means for dissecting viral assembly pathways. The morphological variants produced by and biological characteristics of tsA279, a previously uncharacterized mutant from the Fields' panel of temperature-sensitive mutants of reovirus, were determined under restrictive growth conditions. The mutant showed a distinctive pattern of increased temperature sensitivity as the temperature was raised from 39 degrees to 40 degrees. Wild-type reovirus type 1 Lang and the mutant were crossed to generate reassortants. Efficiency of plating analyses of the reassortants showed that tsA279 has temperature-sensitive lesions in two genes, a mildly temperature-sensitive one in L2, which encodes core spike protein lambda 2, and a stronger, dominant lesion in M2, which encodes major outer capsid protein mu 1. Electron microscopic examination of thin-sectioned tsA279-infected cells showed three ways in which the mutant phenotypes were expressed. The mutant appeared to be blocked in transmembrane transport of virions, a phenotype that mapped to the M2 gene; the mutant produced significantly reduced amounts of identifiable particles; and those particles that were produced appeared to be morphological variants. Immunofluorescent microscopy and immunoprecipitations of tsA279- and various T1L x tsA279 reassortant-infected cells suggested that the reduction in observed progeny was caused by a decreased production of viral proteins at the nonpermissive temperature. This phenotype also mapped to the mutant M2 gene.

A panel of serotype 3 (T3) reovirus strains was screened to determine their relative capacities to cause lethal infection and hepatobiliary disease following peroral inoculation in newborn mice. A wide range of 50% lethal doses (LD50s) was apparent after peroral inoculation of the different virus strains. Two of the strains, T3 Abney and T3 clone 31, caused mice to develop the oily fur syndrome associated with biliary atresia. The capacity to cause biliary atresia was not related to the capacity to cause lethal infection, however, because the LD50s of T3 Abney and T3 clone 31 were grossly disparate. Examination of liver and bile duct tissues revealed histopathologic evidence of biliary atresia and hepatic necrosis in T3 Abney-infected mice but not in mice inoculated with a T3 strain of similar virulence or with the hepatotropic T1 Lang strain. The consistency with which T3 Abney-infected mice developed biliary atresia-associated oily fur syndrome permitted us to determine the viral genetic basis of reovirus-induced biliary atresia. Analysis of reassortant viruses isolated from an in vitro coinfection with T3 Abney and T1 Lang indicated a strong association of the hepatobiliary disease-producing phenotype with the T3 Abney S1 gene, which encodes the viral cell attachment protein, sigma 1. Amino acid residues within the sigma 1 protein that were unique to disease-producing T3 strains were identified by comparative sequence analysis. Specific changes exist within two regions of the protein, one of which is thought to be involved in binding to host cell receptors. We hypothesize that changes within this region of the protein are important in determining the tropism of this virus for bile-ductular epithelium.

The S4 gene of reovirus encodes a double-stranded RNA-binding protein, sigma 3, that can inhibit activation of the interferon-induced dsRNA-dependent protein kinase, PKR. In this study, we attempted to localize the region of sigma 3 involved in dsRNA-binding by constructing deletion and point mutations, expressing the mutated proteins in COS cells, and testing the ability of the native mutated proteins to bind dsRNA-agarose. Transfection of S4 into COS cells resulted in expression of two forms of sigma 3, a full-length protein, and a protein containing a small truncation at the amino-terminal end. The truncation is likely due to a proteolytic event. Deletions of as few as 10 amino acids from the amino-terminal end of the protein or 10 amino acids from the carboxyl-terminal end of the protein resulted in loss of dsRNA-binding activity. A putative dsRNA-binding domain has previously been localized to an 85 amino acid region located between amino acids 234 and 297 (Miller, J. E., and Samuel, C. E., J. Virol. 66, 5347-5356 1992). Mutagenesis of basic residues located within two distinct motifs of this region showed that some basic residues are absolutely required for binding to dsRNA while others can be changed with little effect.

The reovirus group C temperature-sensitive mutant tsC447, whose defect maps to the S2 gene, which encodes the major core protein sigma 2, fails to assemble core particles at the nonpermissive temperature. To identify other proteins that may interact with sigma 2 during assembly, we generated and examined 10 independent revertants of the mutant. To determine which gene(s) carried a compensatory suppressor mutation(s), we generated intertypic reassortants between wild-type reovirus serotype 1 Lang and each revertant and determined the temperature sensitivities of the reassortants by efficiency-of-plating assays. Results of the efficiency-of-plating analyses indicated that reversion of the tsC447 defect was an intragenic process in all revertants. To identify the region(s) of sigma 2 that had reverted, we determined the nucleotide sequences of the S2 genes. In all revertant sequences examined, the G at nucleotide position 1166 in tsC447 had reverted to the A present in the wild-type sequence. This reversion leads to the restoration of a wild-type asparagine (in place of a mutant aspartic acid) at amino acid 383 in the sigma 2 sequence. These results collectively indicate that the functional lesion in tsC447 is Asp-383 and that this lesion cannot be corrected by alterations in other core proteins. These observations suggest that this region of sigma 2, which may be important in mediating assembly of the core particle, does not interact significantly with other reovirus proteins.

When mouse L fibroblasts are infected with various combinations of recombinant vaccinia viruses possessing thymidine kinase (TK) genes with inserted reovirus genes that encode core components, particles are formed that closely resemble reovirus cores. In cells infected with vaccinia viruses that express reovirus proteins lambda 1 and sigma 2, particles are formed that are very similar to reovirus core shells; if, in addition, the cells are also infected with vaccinia virus that expresses protein lambda 2, particles are formed that also possess the characteristic icosahedrally located projections/spikes that are present on reovirus cores. If, in either case, the cells are also infected with vaccinia virus that expresses the reovirus RNA polymerase, protein lambda 3, the resultant particles are morphologically identical with those formed in its absence, but also contain protein lambda 3.

The m1 species of reovirus RNA, which encodes the minor protein component mu 2, possesses two initiation codons, one "strong" according to Kozak rules and preceded by 13 residues (IC1), the other "weak" and located 49 codons downstream of the first (IC2). In reovirus-infected cells only IC2 is used, but initiation from IC1 can be activated, and efficiency of initiation from either initiation codon modulated over a wide range, by coupling unrelated sequences to either or both ends of m1 RNA. For example, when the M1 genome segment is cloned into the thymidine kinase gene of vaccinia virus in such a way that various "irrelevant" stretches of nucleotides comprising restriction endonuclease cleavage sites or promoter remnants are coupled to the 5' end of m1 RNA, translation of the resultant transcripts is also initiated at IC2, with frequencies controlled by the nature of the attached sequences. However, in rabbit reticulocyte lysates these same transcripts are translated from IC1 as well as from IC2, and transcripts in which m1 RNA is preceded by long sequences of encephalomyocarditis virus RNA (from the T7 polymerase-controlled pTM1 vector) are translated exclusively from IC1. By contrast, m1 RNA itself is translated only from IC2. It appears that the most important factor that controls the extent to which translation is initiated from IC1 and IC2 is their "availability," which is likely to be a function of the extent to which the regions on either side of them interact with each other (and also, to a lesser extent, with the 3' untranslated region) either directly or via interaction with host cell proteins. The effects described here are of considerable potential significance when genetic material is rearranged as a result of translocations, insertions, deletions, and amplifications--that is, when sequences that are normally separated are brought into apposition.

In this study, we investigated the interaction of reovirus particles with cell membranes by using a 51Cr release assay. We confirmed prior observations (J. Borsa, B. D. Morash, M. D. Sargent, T. P. Copps, P. A. Lievaart, and J. G. Szekely, J. Gen. Virol. 45:161-170, 1979) that intermediate subviral particles (ISVPs) of reovirus type 3 strain Abney (T3A) induced the release of 51Cr from preloaded L cells and showed that the intact virion and core forms did not. Reovirus type 1 strain Lang (T1L) ISVPs were found to be less efficient at 51Cr release than T3A ISVPs. Reassortants between these strains indicated that the 51Cr release phenotype segregates with the M2 gene segment. Biochemical studies indicated that the ISVPs' acquisition of the capacity to induce 51Cr release followed the cleavage of the viral M2 gene product mu 1/mu 1C to fragments delta and phi during virion conversion to ISVP but did not directly correlate with this cleavage. These studies suggest that the reovirus M2 gene product (in its cleaved form) plays a role in interacting with cell membranes.

The stimulation of reporter gene expression following co-transfection with the S4 gene of mammalian reoviruses was analysed. The sigma 3 protein of type 3 reovirus gave a five- to eightfold increase in expression of chloramphenicol acetyltransferase and beta-galactosidase but this was found to be dependent upon the nature of the promoter being used to drive reporter gene expression. The sigma 3 protein of reovirus type 1 failed to stimulate reporter gene expression under any of the conditions used. Hybrid constructs between the S4 genes of reoviruses type 1 and 3 were used to map the stimulation characteristic to the carboxy-terminal third of the gene. Analysis of the level of sigma 3 protein accumulation in transfected cells showed that the reovirus type 3 protein accumulated to a much higher level than that of reovirus type 1. Using the hybrid gene constructs this higher level of protein accumulation was shown to co-segregate with the ability to stimulate reporter gene expression.

We have reported previously that reovirus, when used in combination with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) chemotherapy, mediates the rejection of murine ascites tumors. Surviving animals reject a challenge with the same, but not a different, tumor, which suggests that tumor-specific immunity is induced by the treatment regimen. The present study was designed to characterize the interaction between reovirus and murine peritoneal macrophages, both in vitro and in vivo, to determine whether such a relationship may play a role in immune modulation resulting in tumor rejection. The results demonstrated that reovirus can efficiently infect peritoneal macrophages in vitro and stimulate the secretion of tumor necrosis factor-alpha (TNF-alpha). In vivo administration of reovirus, however, did not produce high levels of infection in peritoneal exudate cells, even though the cells were stimulated to express detectable levels of membrane TNF-alpha. These results suggested that infection is not necessary for TNF-alpha expression and this hypothesis was supported by the observation that this expression was also stimulated in vitro by UV-inactivated reovirus. These findings suggest that one mechanism for immune stimulation by reovirus may be through the induction of TNF-alpha.

The aims of this experiment were: (1) to establish a reovirus type 3-induced murine model of biliary atresia/neonatal hepatitis that as far as possible corresponds to the human disease; (2) to demonstrate that the disease is histologically similar to the human disease, and to investigate the natural history of reovirus type 3 infection in this model; (3) to study the host-virus interrelationships at a molecular level; and (4) to develop sensitive assays that could be translated to the human disease. In this study we were unable to produce an exact model for extrahepatic biliary atresia (EHBA) in the laboratory mouse following a perinatal reovirus type 3 infection. However, the ability of reovirus type 3 to persist in the murine liver and the effects produced in the offspring of infected pregnant mice indicate that this preparation may provide the basis for the eventual development of the experimental model of EHBA.

The mammalian reoviruses possess a genome composed of 10 double stranded RNA segments. The serotype 1 strain Lang M1 segment was sequenced and compared to the published type 3 sequence. Both segments were 2304 base-pairs long coding for the mu 2 protein predicted to be 736 amino acids long. The sequences were highly conserved with 97.2% conservation of nucleotide sequence and 98.6% conservation of amino acid sequence. The M1 segments of serotypes 1 and 3 have recently diverged as indicated by the distribution of variation with respect to codon positions. The conservation of amino acid sequence indicated that the mu 2 protein has a relatively high functional density.

Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope.

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.

The oligomerization state of the reovirus cell attachment protein sigma 1 (49K monomeric molecular weight) was determined by biochemical and biophysical means. Full-length (protein product designated A) and C-terminal truncated (protein product designated B) serotype 3 reovirus S1 mRNA transcripts synthesized in vitro were cotranslated in a rabbit reticulocyte lysate, and the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under conditions which allowed for the identification of oligomeric forms of sigma 1. A total of four oligomeric protein bands (corresponding to A3, A2B1, A1B2, and B3, respectively) was consistently observed, which suggests that the protein is made up of three monomeric subunits. Biophysical characterization of purified sigma 1 using column filtration and sucrose gradient sedimentation analysis confirmed the highly asymmetric shape of sigma 1 and allowed us to determine the molecular weight of the native protein to be approximately 132K (a trimer). Similar biophysical analysis on the two tryptic fragments of the sigma 1 [N-terminal fibrous tail (26K monomeric molecular weight) and the C-terminal globular head (23K monomeric molecular weight)] yielded molecular weights of 77K and 64K, respectively, both again corresponding to trimers. We therefore conclude that protein sigma 1 is a homotrimer and provide, with supportive experimental evidence, a rationale for the anomalous behavior of the oligomeric protein in SDS-polyacrylamide gels, which, coupled with chemical cross-linking studies, has in part led to the previous suggestion that sigma 1 might be a higher order oligomer.

We studied the mRNA-binding properties of eukaryotic initiation factor (eIF) 2. This Met-tRNA-binding factor interacts with the cap structure of reoviral mRNA in an ATP-independent manner. Both the beta- and gamma-subunit of eIF-2 are involved in the UV-induced cross-linking of eIF-2 to the cap. The interaction of eIF-2 with a messenger is sensitive to the cap analogue 7-methyl-guanosine 5'-triphosphate as measured by cross-linking and by mRNA retention on nitrocellulose filters. The cap-binding property of eIF-2 does not conflict with the current mRNA-binding model of initiation factors eIF-4A, -4B, and -4F: cross-linking of eIF-4E and of eIF-4B is stimulated by eIF-2. The eIF-2-mediated increase of eIF-4E interaction results in a decrease of the cross-linking of the beta- and gamma-subunits of eIF-2. The presence of GTP in the cross-linking assay interferes with the interaction of eIF-2 with the cap structure but does not inhibit the eIF-2 stimulated eIF-4E and -4B cross-linking. These observations indicate a role for eIF-2 in the mRNA recognition.

Viral pesticidal agents must be evaluated for potential health hazards prior to utilization. Assessment of the likelihood of replication in humans has included in vitro exposure of human cells to the potential pesticidal agent. Previous in vitro evaluation strategies have lacked positive controls. Thus, negative results, interpreted as no effect of the virus on human cells, could reflect basic deficiencies in the testing protocols. We designed a testing scheme for viral pesticides and used it to test the nuclear polyhedrosis virus of Autographa californica. Tests were aimed at evaluating potential replication or gene expression in primate cells. Parallel tests were run utilizing identical protocols with primate viruses known to produce the biological effect being evaluated. Thus protocols described were tested with positive viral controls.

Conditions under which reovirus RNA is infectious have been worked out. In brief, single-stranded (plus-stranded, ss) and/or double-stranded (ds) RNA of reovirus serotype 3 (ST3 virus) is lipofected into L929 mouse fibroblasts together with a rabbit reticulocyte lysate in which ss or melted dsRNA has been translated. After 8 hr the cells are then infected with a helper virus, ST2 reovirus. Virus yields are harvested 24 or 48 hr later. Under these conditions virus that forms plaques by 5 days is produced, all of which is ST3 virus; ST2 virus forms plaques only after 12 days. No reassortants are present among the progeny. The virus yields are about 0.2 PFU/cell; immunofluorescence assays show that this progeny is derived from about 4% of the cells. Double-stranded RNA is 20 times as infectious as ssRNA; ds and ssRNA together yield 10 times as much infectious virus as dsRNA alone, the reason being that dsRNA greatly increases the infectiousness of ssRNA. All species of both ss and dsRNA are required for the operation of this additive effect. The primed rabbit reticulocyte lysate is not essential, but increases virus yields by 100-fold. Its activity is proportional to the time for which translation has proceeded; however, this activity is not due solely to newly synthesized proteins because destruction of the RNA following translation abolishes activity which cannot be restored by simple addition of more RNA. Translation of all species of RNA is essential. Whereas no reassortants are formed when ss and dsRNA of different genotypes are lipofected together, mixtures of dsRNAs of different genotypes do yield reassortants. The same is true for such mixtures of ssRNA. These findings will permit the introduction of new or altered genome segments into the reovirus genome. They open the way to the identification of encapsidation and assortment signals on reovirus genome segments, the characterization of functional domains on reovirus proteins, and the development of reovirus as an expression vector.

Previously the receptor recognition domain of the reovirus serotype 3 (T3) cell attachment protein (sigma 1) was mapped to the C-terminal half of the protein using deletion mutagenesis of the reovirus S1 gene. A similar approach has been adopted in the present study to map the domain on T3 sigma 1 that is responsible for incorporation into the virion (i.e., the anchoring domain). Restriction enzymes which divide the T3 S1 cDNA into four segments (5'-I-II-III-IV-3') of similar size were used to generate four mutants, each with a particular segment deleted. The mutants were cloned into SV40 expression vectors and used to transfect COS-1 cells which were subsequently with reovirus serotype 1. Progeny viral particles with truncated T3 sigma 1 proteins incorporated were then identified by radioimmunoprecipitation with a serotype-specific anti-T3 sigma 1 serum. It was found that the mutant lacking I (mutant dl) was totally incapable of being incorporated into the virion, whereas the mutant lacking domain II (mutant dII) was incorporated efficiently. Due to altered antigenicities of the mutants lacking domain III (mutant dIII) or domain IV (mutant dIV), incorporation of these two proteins into virions was less detectable using the above assay. Evidence that domain I (the N-terminal 121 amino acids) alone dictates the incorporation of sigma 1 into the virion came from the subsequent demonstration that a chimeric protein containing domain I fused to chloramphenicol acetyltransferase (CAT) was incorporated into the virion (detectable with an anti-CAT serum) as efficiently as the full-length sigma 1 protein.

The S1 gene nucleotide sequences of 10 type 3 (T3) reovirus strains were determined and compared with the T3 prototype Dearing strain in order to study sequence diversity in strains of a single reovirus serotype and to learn more about structure-function relationships of the two S1 translation products, sigma 1 and sigma 1s. Analysis of phylogenetic trees constructed from variation in the sigma 1-encoding S1 nucleotide sequences indicated that there is no pattern of S1 gene relatedness in these strains based on host species, geographic site, or date of isolation. This suggests that reovirus strains are transmitted rapidly between host species and that T3 strains with markedly different S1 sequences circulate simultaneously. Comparison of the deduced sigma 1 amino acid sequences of the 11 T3 strains was notable for the identification of conserved and variable regions of sequence that correlate with the proposed domain organization of sigma 1 (M.L. Nibert, T.S. Dermody, and B. N. Fields, J. Virol. 64:2976-2989, 1990). Repeat patterns of apolar residues thought to be important for sigma 1 structure were conserved in all strains examined. The deduced sigma 1s amino acid sequences of the strains were more heterogeneous than the sigma 1 sequences; however, a cluster of basic residues near the amino terminus of sigma 1s was conserved. This analysis has allowed us to investigate molecular epidemiology of T3 reovirus strains and to identify conserved and variable sequence motifs in the S1 translation products, sigma 1 or sigma 1s.

Core particles of reovirus type 3 Dearing (T3D) crystallized in the face-centered cubic space group F432 with dimensions of 1270 A along each edge of the unit cell. Core particles of reovirus type 1 Lang (T1L) did not crystallize. Experiments with core particles derived from 27 different T1L x T3D reassortant viruses indicated that the L2 genome segment determined the capacity of cores to crystallize. This finding indicates important differences in the surface topography of the L2-translation product, the lambda 2 protein, of these two isolates, and suggests that important crystal contacts are mediated by this protein. These data are used to generate a model of the packing of reovirus core particles within the unit cell.

The S1 species of reovirus mRNA contains two overlapping open reading frames. Both are utilized in either virus-infected or S1 DNA transfected mammalian cells, resulting in two different polypeptides from a single mRNA. Consistent with ribosome scanning, expression of the downstream reading frame was increased by sequence changes that diminished the consensus around the upstream initiator site. However, the upstream product was not decreased by the same changes, suggesting that its synthesis is rate-limited at elongation. The results suggest a model for regulating bicistronic mRNA translation in which the ribosomes in one reading frame interfere with the movement of ribosomes in the other frame due to different rates of elongation.

A full-length cDNA copy of reovirus double-stranded RNA genome segment S4 which codes for a major virion structural polypeptide, sigma 3, has been completely sequenced. The 1,196-nucleotide cDNA contains a single long open reading frame in the plus strand extending 1,095 nucleotides from the 5'-proximal A-T-G to a single stop codon. This corresponds to translation of 92% of the S4 gene. The inferred sigma 3 polypeptide is hydrophilic and consists of 365 amino acids, totalling 41,164 daltons.

Mutant L cells, designated LR cells, were isolated after "curing" a persistently infected cell line (L/C) with antireovirus serum. The LR cells were shown to be virus-free; no reovirus was detectable by infectious center assays, plaque assays, presence of viral proteins, presence of viral dsRNA and immunofluorescence studies. Persistent infections were readily established n LR cells following infection with either cloned, low passage wild-type reovirus or cloned, low passage reovirus isolated from carrier cultures. Reovirus isolated from carrier cultures, however, grew much better than wild-type reovirus in LR cells and showed complete dominance over wild-type reovirus in coinfection experiments. Infection of LR cells with wild-type reovirus resulted in a low-level persistent infection with inefficient viral replication; these mutant L cells were partially resistant to infection with wild-type reovirus. In contrast, infection of the mutant L cells with virus isolated from the persistently infected cells resulted in a persistent infection accompanied with efficient viral replication. Infection of the original L cells with either wild-type reovirus or reovirus isolated from the persistently infected cells resulted in a lytic infection with no surviving cells. Thus the host cell plays a crucial role in the maintenance of persistent reovirus infection. Our results show that there is a coevolution of both mutant L cells and mutant reovirus during persistent infection.

S1 nuclease analysis and 3' terminal sequencing of the reovirus genome double-stranded RNAs and in vitro produced viral mRNAs has been used to establish the viral mRNA is a full length copy of the genome template. Sequence determination at the 3' end of the genome minus strand has by transposition allowed determination of the 5' terminal sequences of the viral mRNAs. In all but one case an AUG codon which could function in initiation of protein synthesis has been found within the determined sequence. The 3' ends of the plus strands from all genome segments were found to have a common sequence. The implications of these results on the mechanism of virus replication are discussed.

The sigma 3 protein of reovirus is believed to play a role in the control of protein synthesis in reovirus-infected cells. In this paper we describe the establishment of a line of L-cells expressing the sigma 3 protein from the cloned S4 gene of reovirus serotype 3, under the control of the SV40 early promoter. The protein was enriched in the crude initiation factor fraction prepared by a high-salt wash of ribosomes. There was no apparent detrimental effect on the cell line.

Previous studies have demonstrated the importance of the hemagglutinin (HA) of reovirus in determining virus-specific immune responses. In this study, we have used anti-HA monoclonal antibodies and reoviruses with antigenically altered HA proteins to further analyze the CTL response against the HA. We showed that reovirus-specific CTL primarily recognize one distinct antigenic domain on the HA. These results emphasize the fine specificity of the T cell receptor that recognizes viral determinants. The CTL response is directed against the region of the HA that is also responsible for neurotropism and binding to neutralizing antibody.

The reovirus S1 gene codes for two polypeptides: sigma 1 and sigma s. In order to characterize the structure and function of the sigma 1 polypeptide, we have expressed the sigma 1 protein in Escherichia coli. The S1 gene from mammalian reovirus type 3 (Dearing strain) and the variant K strain were subcloned into an expression vector containing the tac (trp-lac) promoter designed to express foreign gene products in E. coli efficiently. The hybrid plasmids, upon induction with isopropyl-beta-D-thiogalactopyranoside, expressed two polypeptides that were detected by [35S]methionine labelling. One of the induced proteins had a relative molecular mass (Mr) of approx. 46,000 and corresponded to sigma 1, as shown by immunoprecipitation with goat anti-reovirus antibody and a monoclonal antibody against sigma 1. The second induced protein had a Mr of approx. 12,000 and was very similar to sigma s as judged by comparative tryptic peptide map analysis. Protein sigma 1 produced in E. coli was shown to be functional as judged from its ability to bind to mouse L fibroblasts.

Genome double-stranded RNAs isolated from purified human reovirus (serotype 3) and rotavirus (Wa strain) were modified at the 3' termini by addition of oligo(C) approximately 15 with T4 RNA ligase. These RNAs were transcribed into cDNA by oligo(dG)-primed reverse transcriptase and cloned after insertion into pBR322 at the Pst I site. Hybridization of plasmid-transformed Escherichia coli RR1 colonies with 32P-labeled viral genome RNAs demonstrated the presence of DNA clones representative of each of the 10 reovirus RNAs and 10 of the 11 constituent segments of the rotavirus genome. Analyses of the size and terminal nucleotide sequences of insert DNAs indicated that some clones contained a full-length copy of the virus genome segment. The complete nucleotide sequence of rotavirus genome segment 11 double-stranded RNA was obtained by using this procedure. It provides a general method for cloning double-stranded RNAs and also nonpolyadenylylated single-stranded RNAs.

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.

A xenogeneic antiserum raised to antireovirus immunoglobulin was used to define an idiotypic determinant present on antibodies to reovirus type 3 hemagglutinin. The same idiotype was identified on nonimmune lymphoid cells and on neuronal cells that specifically bind the hemagglutinin of type 3 reovirus. This idiotypic determinant, called Id3, is shared by (a) a monoclonal antibody to the neutralization site of hemagglutinin from type 3 reovirus; (b) BALB/c serum antibodies to the hemagglutinin of reovirus type 3; (c) R1.1, a murine thymoma cell line that binds reovirus type 3; (d) primary cultures of murine neuronal cells. The presence of an idiotype shared by antihemagglutinin antibodies and by structures on nonlymphoid cells suggests a general relationship between disparate receptors that recognize a common determinant. Furthermore, this suggests a novel approach for the study of viral receptor interactions and for analysis of mechanisms of autoimmune responses.