Search

Reovirus is the first naturally occurring human virus reported to exploit activated Ras signaling in the host cell for infection, and is currently undergoing clinical trials as a cancer therapeutic. Recent evidence suggests that Ras transformation promotes three reoviral replication steps during the first round of infection: uncoating of the incoming virion, generation of progeny viruses with enhanced infectivity, and virus release through enhanced apoptosis. Whether oncogenic Ras also enhances reovirus spread in subsequent rounds of infection through other mechanisms has not been examined. Here, we show that compared with nontransformed cells, Ras-transformed cells are severely compromised not only in their response to IFN-β, but also in the induction of IFN-β mRNA following reovirus infection. Defects in both IFN-β production and response allow for efficient virus spread in Ras-transformed cells. We show that the MEK/ERK pathway downstream of Ras is responsible for inhibiting IFN-β expression by blocking signaling from the retinoic acid–inducible gene I (RIG-I) which recognizes viral RNAs. Overexpression of wild-type RIG-I restores INF-β expression in reovirus-infected Ras-transformed cells. In vitro–synthesized viral mRNAs also invoke robust RIG-I–mediated IFN-β production in transfected nontransformed cells, but not in Ras-transformed cells. Collectively, our data suggest that oncogenic Ras promotes virus spread by suppressing viral RNA–induced IFN-β production through negative regulation of RIG-I signaling. Cancer Res; 70(12); 4912–21. ©2010 AACR.

Purpose: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. Experimental Design: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. Results: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus’ antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. Conclusions: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

In the hopes that a better understanding of cancer biology would allow for the development of novel, more effective therapeutics, a concerted effort over the past 20 years has focussed on the molecular mechanisms of cell growth control and tumorigenesis. Recently, there has been a renewal of interest and optimism in the field, on account of a confluence of ideas from molecular oncology and virology, leading to the development of novel virus-based therapeutics for the treatment of cancer. Viruses have been used in the past as potential cancer therapeutics; a large variety of agents have been tested, including rabies, adenovirus, paramyxovirus, Newcastle disease virus and mumps virus [1]. Although ‘responses’ have been documented, little has emerged from such trials in terms of a longstanding, oncolytic virus therapeutic mainstay. Fortunately, new technologies and insight have allowed for a re-examination of oncolytic viruses; an understanding of cancer biology and signalling, for example, has permitted the characterisation of the selectivity of viruses such as reovirus for cells with an activated Ras signalling pathway [2,3]. Discovery of the ability of adenovirus E1B protein to interact with the tumor suppressor, p53, not only helped to delineate p53’s role in cell cycle control, but also provided the foundation for the development of E1B-deleted mutants as potential cancer therapeutics [4,5]. Similarly, an awareness of herpes virus biology has permitted the engineering of non-neurovirulent mutants lacking genes necessary for * Corresponding author. Tel.: +1-403-2207548; fax: +1-4032708520. E-mail address: plee@ucalgary.ca (P.W.K. Lee).

Electron microscopy and sequence analysis have suggested the presence of distinct morphological regions within the reovirus cell attachment protein sigma 1. Kinking of purified sigma 1 observed by electron microscopy and sequence-predicted flexibility profiles suggest the presence of potential flexible regions in the molecule, most notably near the N-terminus, in the neck region and near the middle of the fiber. We have mapped the trypsin and chymotrypsin cleavage sites in sigma 1 by direct amino acid sequencing of gel-purified, proteolytic fragments of purified baculovirus-expressed sigma 1. The results indicated that both proteases cleave sigma 1 several times in one, or both, of two specific regions in the molecule. Further analysis using proteases with different cleavage specificities revealed the same general digestion pattern. The two protease-sensitive regions of sigma 1 were localized to the proposed N-terminal hinge region separating the hydrophobic anchor from the coiled-coil and to the C-proximal portion of the neck separating most of the fibrous tail from the globular head. The protease susceptibility of these regions indicates an open, accessible conformation supporting the notion of flexible regions that may be important in sigma 1 function.

The reovirus cell attachment protein sigma 1 is a lollipop-shaped structure with the fibrous tail anchored to the virion. Since it interacts with the cell receptor, sigma 1 is a major determinant of reovirus infectivity and tissue tropism. Studies on its structure-function relationships have been facilitated by the fact that protein sigma 1 produced in any expression system is capable of binding to cell receptors. The use of site-specific and deletion mutants has led to the identification and characterization of its virion anchorage and receptor binding domains. Studies on the oligomeric status of sigma 1 have revealed that sigma 1 is a homotrimer and that two independent trimerization events at different loci (the N- and C-terminal halves, respectively) of the protein, are involved in its generation. This also accounts for a clearly demonstrable dominant negative effect by a mutant subunit in a wild-type/mutant sigma 1 heterotrimer. Current efforts are focused on the involvement of chaperones in the generation of sigma 1 and on events that take place upon sigma 1 binding to the cell receptor. Protein sigma 1 has therefore become an excellent model system for the study of both virus attachment and protein oligomerization and folding mechanisms.

Background Respiratory enteric orphan (reo)virus is a promising oncolytic viral candidate. Reoviral anticancer therapy is currently undergoing multiple clinical trials targeting various human cancers; however, there is no effective reoviral inhibitor that can be used to block unwanted reovirus replication during reoviral anticancer therapy. Methods Studies were conducted with transformed or normal cells in vitro and in vivo to characterize viral replication in the presence or absence of chemical inhibitors. Results We have identified a protease inhibitor that is very effective in the inhibition of viral replication. The dipeptide benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-FA-FMK) effectively inhibited reovirus replication in a susceptible host and cured cells of a persistent infection with reovirus in vitro. Electron microscopic analysis of Z-FA-FMK-treated cells revealed that internalized reovirus virions, retained in a perinuclear localization, no longer undergo further processing into viral factories following Z-FA-FMK treatment, suggesting that Z-FA-FMK specifically affects a reovirus virion maturation step. Animal studies showed that reovirus infection of Ras oncogenic tumours and host heart tissues is completely blocked by Z-FA-FMK treatment in severe combined immunodeficiency mice. Conclusions Z-FA-FMK is a very effective viral inhibitor that can prevent reovirus replication in vitro and reovirus-mediated myocarditis, as well as reovirus-mediated oncolysis, in vivo. A potential application of this drug for inhibition of reovirus infection is suggested.

Oligonucleotide site-directed mutagenesis was used to modify the type 3 (T3) reovirus cell attachment protein sigma 1 at residues located in the three regions (designated C, D, and E in the C-terminal one-third of sigma 1) that are highly conserved between the three reovirus serotypes. Of the eight residues targeted for mutagenesis, five (one in region C, and two each in regions D, and E) are conserved among all three proteins. Wild-type (wt) and mutant sigma 1 forms were synthesized in an vitro transcription/translation system and subjected to structural and functional analysis. None of the mutations affected the ability of sigma 1 to form trimers. However, mutation (all representing drastic changes) in any of the five triply conserved residues (Tyr326, Asn369, Phe370, Tyr450, and Pro451) caused a complete or partial abrogation of sigma 1 cell binding function, whereas mutation in any of the other three residues (Ser325, Ser327, and Asp365) had no adverse effect. The structural integrity of the mutant proteins was then probed using trypsin, chymotrypsin, and a neutralizing monoclonal anti-sigma 1 antibody. In all cases, the loss of cell binding function was associated with a drastic conformational change in the C-terminal globular head of sigma 1. These results suggest that conserved residues in the three highly conserved regions in the C-terminal portion of sigma 1 play important structural and functional roles and are involved in proper head folding and generation of a conformation-dependent receptor binding domain.

We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.

Reovirus is a benign human virus that was recently found to have oncolytic properties and is currently in clinical trials as a potential cancer therapy. We have previously demonstrated that activation of Ras signaling, a common event in cancer, renders cells susceptible to reovirus oncolysis. In this study, we investigate which elements downstream of Ras are important in reovirus infection. By using a panel of NIH 3T3 cells transformed with activated Ras mutated in the effector-binding domain, we found that only the RasV12G37 mutant, which was unable to signal to Raf or phosphatidylinositol 3-kinase but retained signaling capability to guanine nucleotide-exchange factors (GEFs) for the small G protein, Ral (known as RalGEFs), was permissive to reovirus. Expression of the activated mutant of the RalGEF, Rlf, also allowed reovirus replication. Specific inhibition of the Ral pathway by using dominant-negative RalA rendered normally permissive H-Ras cells (cells expressing activated Ras) resistant to reovirus. To further identify elements downstream of RalGEF that promote reovirus infection, we used chemical inhibitors of the downstream signaling elements p38 and JNK. We found that reovirus infection was blocked in the presence of the p38 inhibitor but not the JNK inhibitor. Together, these results implicate a Ras/RalGEF/p38 pathway in the regulation of reovirus replication and oncolysis.

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.

Up to 50% of the transitional cell carcinomas (TCC) express an activated EGF pathway involving MAP/MEK and RAF kinase thus providing a novel means to selectively eliminate transformed cells expressing such proteins. This EGF pathway expression phenotype was also confirmed in our MGH-U3 and room temperature-112 human TCC cell lines, which makes them a suitable model target for the reovirus oncolysis. We report here on an in vitro assay of co-culture spheroids using either human or rat TCC cells with their corresponding fibroblasts to examine the potential of viral selective lysis for TCC. Reovirus, a respiratory enteric orphan virus, which mammals are exposed to early in life, was used in this study. Selective killing of transformed versus normal cells was assayed by time-lapse photography, vital dye staining, immunohistochemistry, and MTT assay. In this in vitro bladder cancer model, reovirus selectively destroyed the transformed cells by lysis or induction of apoptosis. Based on these findings we have initiated an in vivo pre-clinical study on intravesical administration of reovirus in an animal model to further explore the effect of reovirus-mediated oncolysis of TCC.

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.

Reovirus is a double-stranded RNA-containing virus that possesses the distinctive ability to replicate in transformed cells while sparing normal cells, both in vitro and in vivo, in rodent models of cancer. The discovery of this property only arose through years of basic research on the biology of reovirus infection. Upon elucidation of the intracellular factors that govern cellular susceptibility, it became clear that reovirus Type 3 Dearing was capable of replicating in cells with an activated Ras signaling pathway, whereas normal, untransformed cells were unable to support reovirus infection (1). Because normal cells are resistant to reovirus, it is not surprising that reovirus infection in humans is usually subclinical (2, 3). Altogether, the potential impact of such findings is impressive when one considers that activating mutations in the ras genes alone contribute to more than 30% of all human cancers and that many other mutations in elements of the Ras pathway can also contribute to oncogenesis (4, 5). Recently, research using murine cancer models has revealed that this genetically unmodified laboratory strain of reovirus can indeed selectively destroy tumor cells, with no manifestations of animal morbidity or mortality (6). This has led the way to investigation into its therapeutic potential in human cancer.

NIH-3T3 cells, which are resistant to reovirus infection, became susceptible when transformed with activated Sos or Ras. Restriction of reovirus proliferation in untransformed NIH-3T3 cells was not at the level of viral gene transcription, but rather at the level of viral protein synthesis. An analysis of cell lysates revealed that a 65 kDa protein was phosphorylated in untransformed NIH-3T3 cells, but only after infection with reovirus. This protein was not phosphorylated in infected or uninfected transformed cells. The 65 kDa protein was determined to be the double-stranded RNA-activated protein kinase (PKR), whose phosphorylation leads to translation inhibition. Inhibition of PKR phosphorylation by 2-aminopurine, or deletion of the Pkr gene, led to drastic enhancement of reovirus protein synthesis in untransformed cells. The emerging picture is one in which early viral transcripts trigger PKR phosphorylation in untransformed cells, which in turn leads to inhibition of translation of viral genes; this phosphorylation event is blocked by an element(s) in the Ras pathway in the transformed cells, allowing viral protein synthesis to ensue. The usurpation of the Ras signaling pathway therefore constitutes the basis of reovirus oncolysis.

The reovirus cell attachment protein, sigma1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple alpha-helical coiled-coil) is located at the N-terminal one-third of sigma1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of sigma1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) sigma1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated sigma1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of sigma1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL sigma1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent sigma1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.

The demonstration that sialic acid is the minimal determinant recognized by human reovirus is compatible with the finding that this virus binds to multiple sialoglycoproteins on the host cell surface. However, the identities of these proteins have remained unknown, By applying detergent-solubilized plasma membranes from the human epidermoid carcinoma A431 cell line to immobilized reovirions, we have identified the 150- to 170-kDa epidermal growth factor (EGF) receptor as one of the cell surface proteins recognized by reovirus. Direct interaction between the N-terminal extracellular domain of the EGF receptor and reovirus was confirmed by the demonstration that of a number of proteins secreted by A431 cells, the 105-kDa N-terminal cell surface domain of the EGF receptor was the major protein recognized by the virus. However, as expected, reovirus and EGF did not compete for the same binding site on the EGF receptor of intact A431 cells.

Reovirus binds to multiple sialoglycoproteins on the host cell surface. In an attempt to probe additional specific determinants that dictate host cell susceptibility to reovirus infection, we found that two mouse cell lines (NR6 and B82) previously shown to express no endogenous epidermal growth factor (EGF) receptors were relatively resistant to reovirus infection, whereas the same cell lines transfected with the gene encoding the EGF receptor manifested significantly higher susceptibility as determined by induction of cytopathic effects, viral protein synthesis, and plaque titration. This enhancement of infection efficiency requires a functional EGF receptor since it was not observed in cells expressing a mutated (kinase-inactive) EGF receptor. The observed difference in infection efficiency is not due to differences in virus binding or internalization. These studies suggest that the reovirus infection process is closely coupled to the EGF receptor-mediated cell signal transduction pathway.

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.

The N-terminal portion of the reovirus cell attachment protein sigma 1 has recently been shown to possess intrinsic oligomerization and virion-anchoring functions. Sequence analysis of the sigma 1 proteins of the three reovirus serotypes has revealed the presence of distinct structural domains within this region: a terminal hydrophobic tail, a hinge, and an extended coiled-coil. To probe the inter-relationship between the virion-anchoring function and the oligomerization function, we constructed two serotype 3 (T3) sigma 1 deletion mutants in SV40 expression vectors, one lacking the hydrophobic tail and the hinge, and the other lacking an adjacent region which constituted part of the coiled-coil. These mutants were (i) expressed in uninfected COS-1 cells and assayed for their ability to form oligomers, and (ii) expressed in type 1 (T1) reovirus-infected COS-1 cells and assayed for their incorporability into progeny T1 virions. It was found that, whereas both truncated sigma 1 proteins were capable of forming stable oligomers, neither could be incorporated into virions. These observations, coupled with structural characteristics deduced from sequence analysis, are compatible with a model in which the hydrophobic tail is the bona fide sigma 1 anchorage domain and whose precise association with the virion spikes is dictated by an adjacent heptad repeat region linked to the former structure via a flexible hinge region.

Plasma membranes from mouse L fibroblasts were isolated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were electroblotted to nitrocellulose paper and probed with 125I-labeled type 3 (T3) reovirus. Multiple protein bands with molecular weights ranging from 26 to 200 kDa were consistently recognized by the virus. Such binding was specific since it was blocked in the presence of unlabeled virus. That these proteins were exposed on the cell surface was confirmed by their susceptibility to sulfo-NHS-LC-biotin labeling of intact cells prior to membrane purification. Blots probed with wheat germ agglutinin (WGA)-gold showed a similar pattern of protein bands. These findings are consistent with the ability of WGA to block reovirus binding to L cells, and with our recent demonstration that the alpha-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus (R. W. Paul, A. H. C. Choi, and P. W. K. Lee, Virology 172, 382-385, 1989). Both type 1 and type 3 reoviruses were found to recognize the same set of multiple proteins on the blot, which is again consistent with the previous observation that the two serotypes compete with each other for binding to intact L cells.

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.

A series of synthetic sialosides were evaluated for their ability to interact with reovirus serotype 3. It was found that sialosides with terminal N-acetylneuraminic acid (NeuNAc) linked in either an alpha 2,3 or alpha 2,6 configuration effectively blocked the binding of reovirus to mouse L fibroblasts, in contrast to a monosaccharide mixture containing the oligosaccharide constituents. Direct binding of reovirus to the sialosides was also demonstrable using sialosides conjugated to bovine serum albumin as ligands in a solid phase binding system. Of particular significance was the finding that the conjugate containing alpha-sialic acid alone (linked to bovine serum albumin) was capable of being recognized by reovirus at a level comparable to that of the other sialoside conjugates. Virus binding was abrogated by pretreating such conjugates with neuraminidase. These results suggest that the alpha-anomeric form of sialic acid is the minimal receptor determinant for reovirus recognition.

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.

Nineteen hybridoma cell lines derived from the spleen cells of a mouse immunized with reovirus serotype 3 (strain Dearing) were cloned and the IgGs secreted by them characterized. Four of the IgGs were directed against polypeptide μNS and two against polypeptide σNS, the two nonstructural reovirus-specified polypeptides. Two IgGs were directed against polypeptide λ2 and two against polypeptide σ2, both components of the reovirus inner capsid shell. Four IgGs were directed against polypeptide σ1, the most type specific of all reovirus-specified polypeptides, three against polypeptide μl/gmlC and one against polypeptide σ3, all of them components of the reovirus outer capsid shell. One IgG was directed against a complex composed of polypeptides μl/gmlC and σ3, but failed to react with either of these polypeptides alone. Thus the 19 hybridoma cell lines secreted IgGs with specificities for 7 of the 10 reovirus-specified proteins. The ability of all these IgGs to precipitate the proteins specified by the two heterologous reovirus serotypes, serotypes 1 and 2, was also measured.

A reovirus (type 3) receptor has previously been identified on the mouse thymoma R1.1 cell line and shown to be structurally similar to the mammalian beta-adrenergic receptor [M. S. Co, G. N. Gaulton, A. Tominaga, C.J. Homcy, B.N. Fields, and M.I. Greene (1985). Proc. Natl. Acad. Sci. USA 82, 5315-5318]. To determine whether beta-adrenergic receptors are universal recognition signals for reovirus binding, we studied the human epidermoid carcinoma cell line A431 which is known to possess large numbers of functional beta-adrenergic receptors and was found in the present study to be susceptible to reovirus infection. It was observed that unlike beta-adrenergic agonists, reovirus binding and internalization did not result in the triggering of cellular adenylate cyclase activity. The presence of reovirus also had no effect on cellular response to the agonist (-)-isoproterenol, nor on the binding of the hydrophilic antagonist [3H]CGP-12,177 to intact A431 cells. Furthermore, sequestration of beta-adrenergic receptors from the cell surface by (-)-isoproterenol had no effect on reovirus binding. Conversely, the binding of [3H]CGP-12,177 to cells with internalized reovirus receptors was found to be normal. These data strongly suggest that reovirus and beta-adrenergic receptors on A431 cells are distinct from each other. Similar observations were also made with the mouse L fibroblasts, with the exception that these cells appear to possess few functional beta-adrenergic receptors.

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.

Previously a reovirus (serotype 3) S1 gene cDNA was inserted into the lac cloning site of pUC13 and expressed in Escherichia coli to yield a sigma 1 fusion protein (F-sigma 1) capable of binding to mouse L fibroblasts and of agglutinating human red blood cells (S.A. Masri, L. Nagata, D. C. W. Mah, and P. W. K. Lee, 1986, Virology 149, 83-90). To probe the functional domains on the sigma 1 protein, restriction enzymes which divide the S1 gene into four segments (5'-I-II-III-IV-3') of similar size were used to generate five in-frame deletion mutants (D1-D5). Corresponding mutant forms of sigma 1 were expressed in E. coli and were assayed for (i) host cell (mouse L fibroblasts) binding activity; (ii) glycophorin (reovirus erythrocyte receptor) binding activity (R. W. Paul and P. W. K. Lee, 1987. Virology 159, 94-101 and (iii) recognizability by a library of neutralizing monoclonal anti-sigma 1 antibodies. It was found that mutant sigma 1 forms with segment III or segment IV deleted did not exhibit any detectable L-cell binding activity, whereas mutants with these two segments intact (but lacking segment II or segments I and II) were capable of attaching to L-cell receptors, albeit with reduced efficiencies. On the other hand, only F-sigma 1, but none of the mutants, could bind immobilized glycophorin. These data clearly suggest that the host cell binding domain of sigma 1 is distinct from its hemagglutination domain. Also, the five neutralizing anti-sigma 1 monoclonal antibodies tested were all found to recognize epitopes on either the middle segments or the carboxy-terminal half of sigma 1.

"Saturation binding experiments" were carried out to characterize the attachment of reovirus to mouse L fibroblasts. Scatchard analysis of data obtained from such experiments suggests that one homogeneous set of noncooperative, high affinity binding sites are involved in reovirus attachment. It is estimated that L cells possess between 3 and 5 X 10(5) reovirus binding sites per cell and that the equilibrium dissociation constant (KD) is approximately 3 X 10(-9) M. Scatchard analysis of data from similar experiments, carried out in the presence of anti-sigma 1 and anti-sigma 3 antibodies, revealed that although both antibodies prevent viral attachment, they exhibit distinct binding inhibition characteristics: anti-sigma 1 effectively abolishes high-affinity, specific binding, whereas anti-sigma 3 apparently blocks low-affinity, nonspecific interactions. The nature of the L-cell receptor was then probed using various enzymes and reagents, and compared with that of the reovirus receptor on human type O erythrocytes. It was found that whereas reovirus hemagglutination (HA) is inhibited by pretreatment of erythrocytes with various proteases or neuraminidase, virus binding to L cells is unaffected by such treatments. Neither HA nor cell binding is inhibited by the various sugars tested, including N-acetyl-D-glucosamine, which was previously reported to inhibit reovirus HA (L. D. Gelb and A. M. Lerner, 1965, Science 147, 404-405). Both L cells and erythrocyte reovirus receptors are nevertheless highly sensitive to periodate treatment, which presumably destroys the high-affinity reovirus binding sites since protein sigma 1, which is capable of attaching to L cells by itself, does not bind to cells pretreated with periodate. It is therefore concluded that sugar residues on the receptor may be involved in this specific interaction. The possibility that gangliosides may serve as reovirus receptors was also probed. It was found that bovine brain gangliosides, but not cerebrosides, readily aggregate reovirus, inhibit HA, and block viral attachment to L cells. However, binding of protein sigma 1 to L cells is unaffected by gangliosides. Inhibition of reovirus HA and L-cell binding by these gangliosides is therefore most likely due to a steric hindrance effect brought about by interactions between the gangliosides and other components of the outer viral capsid.

The complete sequence of the reovirus (serotype 3) S1 gene was obtained by using cloned cDNA derived from the RNA segment. This gene is 1416 nucleotides in length and contains two open reading frames. The first reading frame has a coding capacity of 455 amino acids, sufficient to account for the known S1 product, protein sigma 1 (42,000 MW). It possesses a signal peptide as well as three possible glycosylation sites. No homology could be detected when this gene sequence and the deduced amino acid sequence were compared to published sequences of the corresponding gene of a human rotavirus. The second reading frame (not in phase with the first) starts at the second ATG recently shown to be a functional initiation site. It has a coding capacity of 120 amino acids. Its outstanding feature is the highly basic amino-terminal region, a characteristic apparently shared by a number of DNA binding proteins. It is speculated that this protein, hitherto undetected, may play a role in mediating viral and/or host nucleic acid transcription.

The genes of the Dearing strain of reovirus serotype 3, which consist of double-stranded RNA, have been cloned into pBR322 by tailing both strands of each gene with poly(A), transcribing them with reverse transcriptase, self-hybridizing the cognate plus and minus cDNA strands, incubating them with Escherichia coli DNA polymerase I to ensure that they are complete, and cloning the double-stranded cDNA molecules by standard procedures. The sequence of the cloned S2 gene has been determined. The sequence at the termini are exactly the same as those at the ends of the native double-stranded RNA gene. The gene is 1,329 nucleotides long and possesses a single long open reading frame that starts at the first initiation codon (residue 19) and extends for 331 codons, sufficient to encode a protein of the same size as the known S2 gene product, protein sigma 2, a major reovirus core component (Mr, 38,000). A second open reading frame of 85 codons, in a different phase, starts close to where the first ends. The protein translated from this reading frame is unknown.

We have recently described the isolation of 19 hybridoma cell lines that secrete IgGs directed against reovirus-coded proteins, and characterized their specificities as judged by their ability to precipitate virus-coded proteins from lysates of infected cells. We describe in this paper the characterization of these hybridoma-secreted IgGs by their ability to react with reovirus particles. IgGs directed against three reovirus proteins were able to neutralize infectivity, inhibit hemagglutination, and precipitate/aggregate reovirus particles: those directed against polypeptides σ1 and σ3, both components of the outer reovirus capsid shell, and those directed against polypeptide λ2, a component of reovirus cores. In addition, some, but not all, of the IgGs directed against polypeptides μ1/μ1C and μNS also precipitate/aggregate reovirus particles. The fact that IgGs directed against λ2 possess strong neutralizing, hemagglutination inhibiting, and virus precipitating activities suggests that the projections or spikes that are present on reovirus cores and that are composed primarily or exclusively of λ2 project through the outer capsid shell to the surface of reovirus particles. This conclusion was confirmed by the demonstration that polypeptide λ2 could be labeled by means of the lactoperoxidase-catalyzed radioiodination technique under conditions when only known components of the reovirus outer capsid shell became labeled.

Purified glycophorin (predominantly type A) from human erythrocytes was found to effectively inhibit reovirus hemagglutination (HA) in contrast to other glycoproteins such as fetuin or ovalbumin. Glycophorin was also a potent inhibitor of reovirus and protein σ1 binding to mouse L fibroblasts. Glycophorin pretreated with neuraminidase lost these inhibitory properties. Using a solid phase binding assay, it was demonstrated that reovirus as well as protein σ1 could specifically bind to glycophorin immobilized on polystyrene plates. This binding was inhibited by wheat germ agglutinin (WGA) but not by other lectins such as peanut agglutinin (PA), Maclura pomifera agglutinin (MPA), Bauhinia purpurea agglutinin (BPA), or concanavalin A (Con A). Binding of reovirus to glycophorin was also partially inhibited by a monoclonal antibody (10F7) (W. L. Bigbee, R. G. Langlois, M. Vanderlaan, and R. H. Jensen, 1984 , J. Immunol. 133, 3149–3155), which recognizes a determinant common to the M and N forms of glycophorin, but not by N-specific monoclonal antibodies NN4 and NN5 or an M-specific monoclonal antibody, 6A7. Taken together, these results clearly indicate that the M, N blood group antigen, glycophorin, is the erythrocyte receptor for reovirus.

A cDNA clone encompassing the complete reovirus (serotype 3) S1 gene was constructed using two partial clones containing overlapping sequences. The gene (with the first 15 bases at the 5' end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter. The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-sigma 1 antibodies. Like authentic soluble sigma 1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors. Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E. coli cells, were also found to exhibit hemagglutinating (HA) activity. Such hemagglutination was inhibited by a monoclonal anti-sigma 1 antibody previously shown to inhibit reovirus HA activity. It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact.