The N-terminal heptad repeat region of reovirus cell attachment protein sigma 1 is responsible for sigma 1 oligomer stability and possesses intrinsic oligomerization function.

The oligomerization domain of the reovirus cell attachment protein (sigma 1) was probed using the type 3 reovirus sigma 1 synthesized in vitro. Trypsin cleaved the sigma 1 protein (49K molecular weight) approximately in the middle and yielded a 26K N-terminal fragment and a 23K C-terminal fragment. Under conditions which allowed for the identification of intact sigma 1 in the oligomeric form (approximately 200K) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the N-terminal 26K fragment was found to exist as stable trimers (80K) and, to a less extent, as dimers (54K), whereas the C-terminal fragment remained in the monomeric form. A polypeptide (161 amino acids) containing the N-terminal heptad repeat region synthesized in vitro was capable of forming stable dimers and trimers. Using various criteria, we demonstrated that the stability of the intact sigma 1 oligomer is conferred mainly by the N-terminal heptad repeat region. Our results are summarized in a model in which individual heptad repeats are held together in a three-stranded alpha-helical coiled-coil structure via both hydrophobic and electrostatic interactions.

A characterization of the nonstructural protein from which the virus-specified tubules in epizootic haemorrhagic disease virus-infected cells are composed.

The complete nucleotide sequence of segment 6 of epizootic haemorrhagic disease virus serotype 2 (Alberta) which encodes nonstructural protein NS1 was determined from a cDNA clone containing a full-length copy of the gene. The gene was found to be 1806 bp in length, constituted by one open reading frame of 1656 bp which is flanked by 5' and 3' noncoding regions of 32 and 118 bp, respectively. The conserved 5' and 3' terminal hexanucleotide sequences were identical to those of BTV-10. The 5' noncoding nucleotide sequences of cognate genome segments 4, 6 and 8 were found to be highly conserved in EHDV-2 and BTV-10. The 3' noncoding regions are less conserved but share common characteristics. The predicted EHDV-2 NS1 gene product, a 552 amino acid polypeptide, is predominantly hydrophobic and has a net charge of +5 at neutral pH. Comparison to its BTV-10 counterpart revealed a high degree of homology. Regions of high amino acid similarity were shown to correlate with hydrophobic domains on the proteins whilst regions of lower amino acid similarity corresponded with the hydrophilic domains. Thirteen conserved cysteine residues of which the majority occurred in hydrophobic regions with more than 80% amino acid similarity were identified.

Development and optimization of a hybridization assay for epizootic hemorrhagic disease viruses.

Hybridization assays for the double-stranded RNA (dsRNA) orbiviruses causing bluetongue and epizootic hemorrhagic disease are more labor-intensive and less sensitive than enzyme-linked immunosorbent assay (ELISA) and immunoperoxidase assay. Cell-culture EHD virus amplification, rapid extraction, and optimization of RNA blotting conditions were examined to increase sensitivity and decrease labor. Aldehyde RNA denaturations and nylon hybridization membranes resulted in stronger positive hybridization signals. Treatment of infected cells with protease did not increase the yield of viral RNA target. Because RNA extraction is a tedious process, a simple non-phenolic diethyl pyrocarbonate extraction procedure was developed. The sensitivity, versatility, and the reproducibility of hybridization assays are addressed.

Biophysical studies on the morphology of baculovirus-expressed bluetongue virus tubules.

Bluetongue virus tubules were purified from Spodoptera frugiperda cells infected with a recombinant baculovirus containing the NS1 gene from bluetongue virus serotype 10, and expressed under control of the Autographa californica nuclear polyhedrosis virus polyhedrin promoter. These tubules were subjected to a variety of chemical and physical treatments and the resulting effects on tubule morphology were examined by electron microscopy. A number of morphological similarities were noted between bluetongue virus tubules and cellular microtubules despite a lack of homology between the component proteins at the primary sequence level. A possible multistranded helical configuration is proposed for the tubule structure.

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.

Identification of conserved domains in the cell attachment proteins of the three serotypes of reovirus.

Sequence analysis of reovirus serotype 1 (ST1) and 2 (ST2) S1 genome segment cDNAs identified several differences from previously reported versions of their sequences. The sequences reported here comprise 1463 and 1440 base pairs, respectively; for comparison, the ST3 S1 genome segment is 1416 nucleotides long. The serotype 1 and 2 sigma 1 proteins are predicted to contain 470 and 462 amino acids, respectively; the ST3 sigma 1 protein is 455 amino acids long. As previously observed, the ST1 and ST2 sigma 1 proteins are much more closely related to each other than to that of ST3 (about 48 and 25% similarity, respectively, using a computer program that finds about 14% similarity among unrelated proteins). The sequences of the three S1 genome segments have diverged very extensively in all three codon positions, in some cases almost to the extent of randomness. Despite this, not only function but also shape and configuration have been retained (since the three sigma 1 proteins can be incorporated efficiently into completely heterologous capsids). Seventy-nine amino acid residues are conserved among all three serotypes, many of them clustered into five regions in which one-third or more of the residues are triply conserved. These regions may represent functionally conserved domains involved in oligomerization, cell attachment, and hemagglutination.

[Polyacrylamide gel electrophoresis (PAGE) as a method for detecting enteric adenovirus (EAd)].

This paper presents a method which could provide a simple, rapid, economical, and reliable means of detecting or identifying adenoviruses (Advs), rotaviruses (RVs) and reoviruses (ReoVs) in stool suspensions or tissue cultures. The method is based on polyacrylamide gel electrophoresis (PAGE) of the virus nucleic acids, but sample preparation does not need the use of radioactive label, specific DNA probe or antisera. Comparison of the results of PAGE of Adv, RV and ReoV with those of electron microscopy (EM) and/or enzyme-linked immunoabsorbent assay (ELISA) was made. The method is of comparable sensitivity to electron microscopy, and is not limited by amount of specimens obtained, and is thus suitable for application as a batch testing method.

In vitro and in vivo characterization of avian reoviruses. I. Pathogenicity and antigenic relatedness of several avian reovirus isolates.

Pathogenicity, pathogenesis, and antigenic relatedness of four avian reovirus isolates obtained from commercially reared broilers were investigated. Chickens of various ages were inoculated both orally and intratracheally with reovirus. Based on disease signs, mortality, weight depression, tissue lesions, invasiveness, and viral persistence in chickens inoculated at 1 day of age, the isolates were classified as being of low, intermediate, or high pathogenicity. The low-pathogenicity isolate (2177) did not cause mortality, weight depression, or clinical disease. The isolate of intermediate pathogenicity (2035) produced low mortality rates (8%), some weight reduction by 7 weeks postinoculation, and microscopic lesions in the intestine and gastrocnemius tendons. The pathogenic isolates, 2408 and 1733, caused severe clinical disease characterized by stunting, feathering abnormalities, mortality as high as 84%, and microscopic lesions in the liver, intestine, pancreas, and/or gastrocnemius tendon. Highly pathogenic isolates also persisted longer in tissues of infected birds and elicited a more prompt and prolonged antibody response. Birds inoculated at 1 day or 1 week of age were more susceptible to reovirus-induced disease than birds inoculated at 2 weeks, suggesting an age-associated resistance. All isolates produced mortality with equal frequency in embryos. The isolates characterized were found to be antigenically similar based on cross-neutralization and cross-protection studies.

Virological studies of water from the Cape Flats reclamation plant.

The virological quality of sewage effluent treated by a pilot reclamation plant on the Cape Flats was evaluated. The potable water produced by the treatment was repeatedly shown to be free of virus contamination.

The cell attachment proteins of type 1 and type 3 reovirus are differentially susceptible to trypsin and chymotrypsin.

Purified native sigma 1 proteins from [35S]methionine-labeled reovirions [serotypes 1 (T1) and 3 (T3)] were subjected to limited trypsin and chymotrypsin digestion. It was found that T1 sigma 1 was resistant to both trypsin and chymotrypsin, whereas T3 sigma 1 (49K molecular weight) was cleaved by trypsin to yield a 24K and a 25K fragment, and by chymotrypsin to yield a 42K fragment. The 24K tryptic fragment, but not the 25K tryptic fragment, was shown to possess L-cell binding capacity, and represents the carboxy-terminal half of T3 sigma 1 since it contains the single cysteine residue (amino acid 351) as revealed by tryptic analysis of [35S]cysteine-labeled sigma 1. Neither tryptic fragment was able to bind to glycophorin, the reovirus receptor on human erythrocytes. Thus, the mechanism of reovirus host cell attachment is distinct from that of reovirus hemagglutination. The two tryptic fragments were recognized by different neutralizing monoclonal anti-sigma 1 antibodies, indicating that neutralizing and cell attachment sites are not necessarily equivalent. The 42K chymotryptic fragment of T3 sigma 1 was shown to be generated by a cleavage proximal to the carboxy-terminus. Like intact T3 sigma 1, the 42K protein retained its capacity to bind to both L cells and glycophorin, and was recognized by all the neutralizing monoclonal anti-sigma 1 antibodies tested. Thus, the host cell receptor binding site on T3 sigma 1 is located between the trypsin-sensitive and the chymotrypsin-sensitive sites.

Dissociation of double-stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay.

A new technique has been applied to the study of the RNA secondary structure unwinding activity of the eukaryotic initiation factors (eIFs) 4F, 4A, and 4B. Secondary structures were generated at the 5' ends of reovirus and globin mRNA molecules by hybridization with 32P-labeled cDNA molecules 15 nucleotide residues long. The dissociation of the labeled cDNAs from the mRNAs was assayed by a gel filtration chromatography procedure which separates the free cDNAs from mRNAs and mRNA/cDNA hybrids. When the three factors were tested alone, only eIF-4F stimulated dissociation of hybrids. The combination of eIF-4A plus eIF-4B also exhibited a strong hybrid dissociating activity, which was markedly temperature dependent. Under optimum conditions, up to 90% of the hybrid structures are disrupted in 60 min. These results demonstrate for the first time that stable double-stranded regions can be melted and dissociated by eIFs. They also characterize more precisely the first step in the structure unwinding reaction.

Comparison of proteins and nucleic acids of Echinochloa ragged stunt and rice ragged stunt viruses.

Echinochloa ragged stunt virus (ERSV) and rice ragged stunt virus (RRSV; Reoviridae) were purified from their respective host plants and disrupted in SDS. Evidence for the double-strandedness of ERSV RNA was obtained. Electrophoresis in 10% polyacrylamide gels resolved the RNAs of each virus into 10 segments, ranging for ERSV from 0.61 x 10(6) to 2.8 x 10(6) daltons, with a sum representing the total genome of 17.89 x 10(6) daltons, and for RRSV from 0.6 x 10(6) to 2.98 x 10(6) daltons, with a sum of 18.15 x 10(6) daltons. Although the overall RNA pattern of the two viruses was similar, there were distinctive features. Using 7.5-15% polyacrylamide gels, electrophoresis of SDS-dissociated RRSV particles yielded five major proteins of 129 x 10(3), 123 x 10(3), 63 x 10(3) and 35 x 10(3) daltons, and two minor proteins of 113 x 10(3) and 88 x 10(3) daltons, respectively, while comparable samples of ERSV yielded four major proteins of 127 x 10(3), 123 x 10(3), 63 x 10(3) and 34 x 10(3) daltons, and three minor proteins of 103 x 10(3), 50 x 10(3) and 49 x 10(3) daltons, respectively. We conclude from this and other evidence that ERSV and RRSV are closely related viruses and should be classified in the same subgroup, despite reported differences in the morphology of their outer capsids.

Characterization of a nonstructural phosphoprotein of two orbiviruses.

A phosphorylated nonstructural protein, NS2, was detected in bluetongue and African horsesickness virus (BTV and AHSV) infected-radiolabeled-cell lysates by electrophoresis on SDS-polyacrylamide gels (SDS-PAGE). The NS2 proteins of both viruses have similar migration on one-dimensional (1D) 10% SDS-PAGE. Examination of infected cell lysates on two-dimensional (2D) gels (isoelectric focusing followed by SDS-PAGE) separated two phosphorylated isoelectric forms of BTV NS2 and four phosphorylated forms of AHSV NS2. The isoelectric points of both species of BTV NS2 were acidic relative to all forms of AHSV NS2. Nonphosphorylated NS2 polypeptides were not detected by 2D gels. A nonphosphorylated host protein, which comigrated with NS2 on 1D gels, could be distinguished from viral proteins by isoelectric focusing on 2D gels. High performance liquid chromatography (HPLC) elution profiles of NS2 tryptic peptides from the two orbiviruses were compared. Three 32P-labeled tryptic peptides were generated from both AHSV and BTV NS2 proteins, which had been isolated and eluted from SDS-polyacrylamide gels. The elution profile from reverse phase HPLC was very similar for the tryptic phosphopeptides; in contrast, 35S-labeled tryptic peptides displayed considerable differences in elution profiles for the two NS2 proteins. Phosphoamino acid analysis revealed only phosphoserine in hydrolysates of BTV and AHSV NS2.

Isolation and characterization of epizootic hemorrhagic disease virus from sheep and cattle in Colorado.

Epizootic hemorrhagic disease virus was isolated from cattle and sheep in northeastern Colorado during July and August 1984. The isolates were identified as serotype 2 by plaque-inhibition serotyping, genome electropherotyping, and protein analysis.

Localization of the nonstructural protein NS1 in bluetongue virus-infected cells and its presence in virus particles.

Seven monoclonal antibodies to the nonstructural protein NS1 of an Australian isolate of bluetongue virus (BTV) have been used in immunofluoresence and immunogold procedures to locate NS1 in virus-infected cells and cytoskeletons. The antibodies fall into three groups indicating that NS1 contains at least three antigenic sites. One group consists of four antibodies which react solely with cytoskeleton-associated virus-specific tubules. A second group contains one antibody which reacts with cytoskeleton-associated virus particles, released viruses, and purified virus and core particles. Two antibodies constituting a third group react with both tubules and cytoskeleton-associated and released virus particles. NS1 was found in [35S]methionine-labeled, purified virus and core particles. Immunofluorescence tests reveal that those antibodies which react with virus particles also bind to cytoskeleton-associated virus inclusion bodies (VIB). The nature of this association was examined by probing cytoskeletons of BTV-infected cells with antibodies to NS1 and protein A-gold. VIB observed in thin sections were not uniformly labeled. Gold was associated with fibrillar arrays found around virus particles either leaving or in close proximity to the VIB. Fibrillar material was not found in association with all virus particles elsewhere in the cell and this suggests that fibril-virus complexes may be intermediate in virus morphogenesis.

Electron microscopic and physicochemical analysis of a reo-like virus of the planthopper Peregrinus maidis.

A virus, determined to be a member of the Reoviridae, was found naturally infecting colonies of Peregrinus maidis, the maize planthopper. The virus, referred to as peregrinus maidis-virus (PgMV), was found by electron microscopy throughout all P. maidis tissues examined, including salivary glands, testes, ovaries, and intestines. The virus particles were associated with fibrillar viroplasmic inclusions in the cytoplasm, of a type characteristic of the Reoviridae. The particles, purified from P. maidis, had morphologies typical of the Reoviridae, with a mean diameter of 68.7 nm. The purified virus contained 12 double-stranded (ds)RNAs, while dsRNA extracts of PgMV-infected P. maidis showed these, plus one additional high-molecular weight dsRNA. No similar dsRNAs were present in extracts of another planthopper. Perkinsiella saccharicida, the corn leafhopper (Dalbulus maidis), or from Zea mays upon which the P. maidis had been reared. Six major and four minor proteins were identified in purified PgMV preparations, but we were unable to determine which of these were definitely of viral or host origin. PgMV was found to be serologically unrelated to three other members of the Reoviridae. The similarities of PgMV to other members of the Reoviridae are discussed.

Association of reovirus proteins with the structural matrix of infected cells.

The interaction of reovirus with the cytoskeleton was investigated. The soluble components of infected cells were extracted with the nonionic detergent NP-40 in a physiological buffer, and a cytoskeletal extract was prepared from the detergent-insoluble fraction. We observed a selective association of viral-specified products with the cytoskeleton that was temporally controlled. Viral dsRNA appeared first on the framework but after several hours was found also in the soluble phase, encapsidated in mature virions. The initial viral translation products were associated exclusively with the soluble fraction, but concomitant with the appearance of dsRNA, viral proteins microNS and sigma 3 were detected on the cytoskeleton. Several hours later, all viral proteins were detected on the framework. Viral polypeptide microNS exhibited unique spatial distribution patterns that correlated with viral assembly: Before dsRNA replication, it appeared as diffusely distributed protein; a few hours later, it was detected in punctate foci interconnected by tiny filaments; several hours later, it appeared as an extensive fiber network that traversed the foci. The other viral proteins were detected only within viral foci. MicroNS remained bound to the matrix fraction after treatment with DNase, Mg2+, and high salt, treatments that released other viral proteins. This distribution pattern was virus-directed because passage of virus at high multiplicity of infection induced mutations that prevented assembly of the microNS-coated filament organization. A small fraction of the viral-specified products that included polypeptide microNS, but not viral dsRNA, was coprecipitated from cytoskeletal extracts with proteins of mol wt approximately 55K by monoclonal antibodies that recognized tubulin and vimentin. Disruption of this interaction by long exposure to colchicine did not prevent association of viral proteins or RNA with the matrix, indicating that viral products were not transported through these interactions. The results indicate that reovirus morphogenesis includes temporal and spatial controls not described previously.