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Mucosal surfaces are protected from infection by both structural and sentinel cells, such as mast cells. The mast cell's role in antiviral responses is poorly understood; however, they selectively recruit natural killer (NK) cells following infection. Here, the ability of virus-infected mast cells to enhance NK cell functions was examined. Cord blood-derived human mast cells infected with reovirus (Reo-CBMC) and subsequent mast cell products were used for the stimulation of human NK cells. NK cells upregulated the CD69 molecule and cytotoxicity-related genes, and demonstrated increased cytotoxic activity in response to Reo-CBMC soluble products. NK cell interferon (IFN)-γ production was also promoted in the presence of interleukin (IL)-18. In vivo, SCID mice injected with Reo-CBMC in a subcutaneous Matrigel model, could recruit and activate murine NK cells, a property not shared by normal human fibroblasts. Soluble products of Reo-CBMC included IL-10, TNF, type I and type III IFNs. Blockade of the type I IFN receptor abrogated NK cell activation. Furthermore, reovirus-infected mast cells expressed multiple IFN-α subtypes not observed in reovirus-infected fibroblasts or epithelial cells. Our data define an important mast cell IFN response, not shared by structural cells, and a subsequent novel mast cell-NK cell immune axis in human antiviral host defense.

Annexins are a structurally related family of calcium and phospholipid-binding proteins that are involved in the regulation of a wide range of molecular and cellular processes. Annexin A2 is unique among the annexins in that it possesses redox sensitive cysteine(s). The ubiquitous and abundant expression of ANXA2 in cells and its reactivity with hydrogen peroxide led us to hypothesize that this protein could play a role in cellular redox regulation. Here we show that ANXA2 protein levels are induced by hydrogen peroxide. Furthermore, depletion of ANXA2 resulted in the elevation of cellular reactive oxygen species (ROS) upon oxidative stress, increased activation of the ROS-induced pro-apoptotic kinases, JNK, p38 and Akt and elevated sensitivity to ROS-mediated cellular damage/death. ANXA2-null mice showed significantly elevated protein oxidation in the liver and lung tissues compared to WT mice. ANXA2 depleted cancer cells showed enhanced cellular protein oxidationconcomitant with decreased tumor growth compared to control cancer cells andboth the oxidation of cellular proteins and tumor growth deficit werereversed by the antioxidant N-acetyl cysteine, indicating that ANXA2 plays akey role in the regulation of cellular redox during tumorigenesis. Ex-vivo human cancer studies showed that up-regulation of the reduced form of ANXA2 is associated with protection of the tumor proteins from oxidation. In summary, our results indicate that ANXA2 plays an important role incellular redox regulation by protecting cells from oxidative stress, aneffect that is particularly important during tumorigenesis.

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.

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus–treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor. Cancer Res; 70(6); 2435–44

The role of the checkpoint kinase 2 (Chk2) as an upstream activator of p53 following DNA damage has been controversial. We have recently shown that Chk2 and the DNA-dependent protein kinase (DNA-PK) are both involved in DNA damage-induced apoptosis but not G1 arrest in mouse embryo fibroblasts. Here we demonstrate that Chk2 is required to activate p53 in vitro as measured by its ability to bind its consensus DNA target sequence following DNA damage and is in fact the previously unidentified factor working synergistically with DNA-PK to activate p53. The gene mutated in ataxia telangiectasia is not involved in this p53 activation. Using wortmannin, serine 15 mutants of p53, DNA-PK null cells and Chk2 null cells, we demonstrate that DNA-PK and Chk2 act independently and sequentially on p53. Furthermore, the p53 target of these two kinases represents a latent (preexisting) population of p53. Taken together, the results from these studies are consistent with a model in which DNA damage causes an immediate and sequential modification of latent p53 by DNA-PK and Chk2, which under appropriate conditions can lead to apoptosis.

There are several well-documented cases in medical literature of the remission of leukemias and malignant lymphomas following natural human viral infections. In the hope of being able to reproduce these spontaneous tumor regressions, investigators have studied various viruses with distinct oncolytic properties. The first attempts to treat patients with oncolytic viruses took place > 80 years ago; however, it achieved little success. With modern technologies and current knowledge of viruses and cancer, there is an expectation for the discovery of efficient oncolytic viral therapies. This article will review the current knowledge of oncolytic viruses in relation to the treatment of lymphoma.

Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.

In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA damage or induce apoptosis to eliminate damaged cells. The checkpoint kinase, Chk2, has been implicated in both of these responses and is believed to function in an ataxia telangiectasia (Atm)-dependent manner. We show here that Chk2−/− mouse embryo fibroblasts (MEFs), unlike Atm−/− or p53−/− MEFs, behaved like normal MEFs in manifesting p21 induction and G1arrest upon exposure to γ-irradiation. Therefore, Chk2 is not involved in p53-mediated G1arrest. To examine the role of Chk2 in p53-dependent apoptotic response, we used adenovirus E1A-expressing MEFs. We show that Chk2−/− cells, like p53−/− cells, did not undergo DNA damage-induced apoptosis, whereas Atm−/− cells behaved like normal cells in invoking an apoptotic response. Furthermore, this apoptosis could occur in the absence of protein synthesis, suggesting that it is preexisting, or “latent,” p53 that mediates this response. We conclude that Chk2 is not involved in Atm- and p53-dependent G1arrest, but is involved in the activation of latent p53, independently of Atm, in triggering DNA damage-induced apoptosis.

The importance of herpes simplex viruses (HSV) as human pathogens and the emerging prospect of using mutant derivatives of HSV-1 as potential anti-cancer therapeutics have necessitated a thorough investigation into the molecular basis of host-cell permissiveness to HSV. Here we show that NIH-3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. Inhibitors of the Ras signalling pathway, such as farnesyl transferase inhibitor 1 and PD98059, effectively suppressed HSV-1 infection of ras-transformed cells. Enhanced permissiveness of the transformed cells was linked to the inhibition of virus-induced activation (phosphorylation) of the double-stranded RNA-activated protein kinase (PKR), thereby allowing viral transcripts to be translated in these cells. An HSV-1-derived oncolytic mutant, R3616, was also found to infect preferentially both transformed cells and PKR-/- (but not PKR+/+) mouse embryo fibroblasts. These observations suggest that HSV-1 specifically targets cells with an activated Ras signalling pathway, and have important ramifications in the use of engineered HSV in cancer therapy, the development of strategies against HSV infections, and the controversial role of HSV in human cancers.

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).

The p53 tumor suppressor protein is a dimer of dimers that binds its consensus DNA sequence (containing two half-sites) as a pair of clamps. We show here that after one wild-type dimer of a tetramer binds to a half-site on the DNA, the other (unbound) dimer can be in either the wild-type or the mutant conformation. An equilibrium state between these two conformations exists and can be modulated by two types of regulators. One type modifies p53 biochemically and determines the intrinsic balance of the equilibrium. The other type of regulator binds directly to one or both dimers in a p53 tetramer, trapping each dimer in one or the other conformation. In the wild-type conformation, the second dimer can bind to the second DNA half-site, resulting in drastically enhanced stability of the p53-DNA complex. Importantly, a genotypically mutant p53 can also be in equilibrium with the wild-type conformation, and when trapped in this conformation can bind DNA.

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

The C-terminal globular head of the lollipop-shaped final sigma1 protein of reovirus is responsible for interaction with the host cell receptor. Like the N-terminal fibrous tail, it has its own trimerization domain. Whereas N-terminal trimerization (formation of a triple alpha-helical coiled coil) occurs at the level of polysomes (i.e. cotranslationally) and is ATP-independent, C-terminal trimerization is a posttranslational event that requires ATP. Coprecipitation experiments using anti-Hsp70 antibodies and truncated final sigma1 proteins synthesized in vitro revealed that only regions downstream of the N-terminal alpha-helical coiled coil were associated with Hsp70. Hsp70 was also found to be associated with nascent final sigma1 chains on polysomes as well as with immature postribosomal final sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini). These latter structures were true intermediates in the final sigma1 biogenetic pathway since they could be chased into mature final sigma1 trimers with the release of Hsp70. Thus, unlike N-terminal trimerization, C-terminal trimerization is Hsp70- and ATP-dependent. The involvement of two mechanistically distinct oligomerization events for the same molecule, one cotranslational and one posttranslational, may represent a common approach to the generation of oligomeric proteins in the cytosol.

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.

Viruses function in close harmony with the signaling machinery of their host. Upon exposure to the cell, a battery of viral products become engaged in boosting friendly signaling elements of the host or suppressing harmful ones. The efficiency of viral replication is indeed the biological outcome of this interaction between cellular and host signaling molecules. Oncolytic viruses, natural or man-made, follow the same set of rules of engagement. Pro-oncogenic cell signaling machinery, therefore, is undoubtedly the most important area influencing the development of the next generation of effective, specific and rationally designed oncolytic viruses. Ras signaling, with its central role in what is known today as molecular oncology, is an attractive topic for studying the behavior of viruses versus cancer cells and to develop strategies to target cancer cells on the basis of such platform. This work reviews the development of oncolytic herpes viruses capable of targeting Ras signaling pathway along with a few other examples of viruses which are developed to contain specificity for certain pro-oncogenic characteristics of their host cells.

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.

Purpose: To our knowledge this is the first report of intravesical oncolytic reovirus for therapy of superficial bladder cancer in an orthotopic bladder tumor model. Superficial bladder carcinomas are often multifocal and have high recurrences after surgical resection. In 20% of cases intravesical immunotherapy fails to prevent recurrence and complications from bacillus Calmette-Guerin (BCG) are common. Human reovirus is an oncolytic virus that selectively destroys cancer cells with an activated Ras pathway. We examined the ability of this virus to kill bladder cancer cells in vitro and inhibit tumor growth in vivo. Materials and Methods: Following cytotoxicity assays in vitro dose escalation of intravesical reovirus was tested for tumor control and toxicity in a rat model. Treatments were done twice weekly for 3 weeks. In parallel intravesical BCG was studied. Animals were monitored on a daily basis for health status and by routine urine cytology. Animals underwent necropsy at study end point and appropriate tissues were taken for histology. Results: Side effects in reovirus groups were minor compared with BCG complications. Tumor response (animal survival) was 90% 100 days after tumor implantation in reovirus treated animals, whereas the highest survival in BCG treated groups was 50%. Animals treated with reovirus had significantly higher tumor-free survival than those treated with immunotherapy or normal saline (log rank test p 0.0002 and 0.04, respectively). Conclusions: Intravesical reovirus is safe and effective in this animal model and it may have clinical applications for bladder cancer treatment.

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.

The key role of Ras in diverse cellular functions qualifies it as a central divergence point for many signal transduction pathways, the most extensively studied of which include the ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38-kinase pathways[1,2]. Stimulation of these pathways results in the activation of different nuclear transcription factors (including SRF, ELK-1, JUN, NFκB, and ATF-2), which in turn leads to a variety of biological functions regulating cell growth, differentiation, apoptosis, migration, and survival[3,4]. In this study, a new role for Ras signaling pathway as key determinant of host cell permissiveness to viral infection was investigated. Herpes simplex virus-1 (HSV-1) was chosen because of its importance as a human pathogen and its potential use in cancer therapy[5,6,7,8,9]. The authors initially compared the level of viral infection in untransformed NIH-3T3 cells with that in cells transformed with oncogenes that activate the Ras pathway (e.g., v-erbB, sos, and ras). Only transformed cells demonstrate significant morphological changes as a result of infection (such as cell rounding and clumping), increased viral protein synthesis, and a high titer of viral progeny. Interestingly, this was specific to the cells with an overactivated Ras-pathway and was not a general feature of the transformed phenotype, as cells transformed with c-myc did not have an increase in viral protein synthesis. The demonstration that inhibitors of Ras (farnesyl transferase inhibitors or FTIs)[10] and a blocker of MEK1/2 (a downstream molecule in ERK pathway) named PD98059[11], prevented this increase in HSV-1 infection further proved that an activated Ras pathway is important for infection. As Ras is a key regulator of several signalling pathways, mutant versions of Ras that preferentially activate only one of several known pathways downstream of Ras (RAF/ERK, PI3K, or RAL/GDS) were used to find out which pathway was involved in permissiveness to infection. The authors showed that the RAF/ERK pathway was the only one whose activation significantly increased susceptibility to HSV-1. To further elucidate the role(s) of the Ras pathway in HSV-1 infection, it was important to identify the step at which HSV-1 infection is blocked in the nonpermissive NIH-3T3 cells. Since virus binding and internalization are comparable between permissive and nonpermissive cells, transcription of viral genes were considered to be a logical target for investigation. Herpes genes are transcribed in a sequential manner with α genes being the first to be expressed and required for the synthesis of subsequent polypeptide groups β and γ [5]. Accordingly, the relative amounts of HSV-1 transcripts (including the α gene α27, β genes UL29 and UL30, γ1 gene γ134.5, and γ2 gene UL44) generated in NIH-3T3 cells and their Ras-transformed version (named H-ras cells) were compared after amplification of these transcripts by quantitative RT-PCR. The results show

Mouse embryo fibroblasts (MEFs) expressing the adenovirus E1A protein undergo apoptosis upon exposure to ionizing radiation. We show here that immediately following gamma-irradiation, latent p53 formed a complex with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK(CS)). The complex formation was DNase sensitive, suggesting that the proteins came together on the DNA, conceivably at strand breaks. This association was accompanied by phosphorylation of pre-existing, latent p53 at Ser18 (corresponding to Ser15 in human p53), which was not found in DNA-PK(CS)(-/-) cells. Most significantly, DNA damage-induced apoptosis was abolished in both DNA-PK(CS)(-/-) and p53(-/-) cells. In addition, blocking synthesis of inducible p53 by cycloheximide did not abrogate apoptosis, suggesting that the latent population of p53 is sufficient for executing the apoptotic program. Finally, E1A-expressing MEFs from a p53 "knock-in" mouse where Ser18 was mutated to an alanine had an attenuated apoptotic response, indicating that phosphorylation of this site by DNA-PK is a contributing factor for apoptosis.

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.

Human reovirus requires an activated Ras signaling pathway for infection of cultured cells. To investigate whether this property can be exploited for cancer therapy, severe combined immune deficient mice bearing tumors established from v- erbB –transformed murine NIH 3T3 cells or human U87 glioblastoma cells were treated with the virus. A single intratumoral injection of virus resulted in regression of tumors in 65 to 80 percent of the mice. Treatment of immune-competent C3H mice bearing tumors established from ras -transformed C3H-10T1/2 cells also resulted in tumor regression, although a series of injections were required. These results suggest that, with further work, reovirus may have applicability in the treatment of cancer.

The tumour suppressor p53 becomes activated as a transcription factor in response to DNA damage, but the mechanism for this activation is unclear. A good candidate for an upstream activator of p53 is the DNA-dependent protein kinase (DNA-PK) that depends on the presence of DNA breaks for its activity. Here we investigate the link between DNA damage and the activation of DNA-PK and of p53. To determine whether DNA-PK is an upstream mediator of the p53 DNA-damage response, we analysed a severe combined-immunodeficiency (SCID) mouse cell line, SCGR11, and the human glioma cell line M059J . Both cell lines lack any detectable DNA-PK activity. We find that p53 is incapable of binding to DNA in the absence of DNA-PK, that DNA-PK is necessary but not sufficient for activation of p53 sequence-specific DNA binding, and that this activation occurs in response to DNA damage. Our results establish DNA-PK as a link between DNA damage and p53 activation, and reveal the existence of a mammalian DNA-damage-response pathway.

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.

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 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.

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.

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.

Bovine rotavirus-infected MA-104 cells maintained in the presence and absence of CaCl 2 displayed cytopathic effects (cpe) distinct from each other. Lysates of calcium-free cultures were unable to induce cpe in subsequent passages in MA-104 cells, an observation reflected by the demonstration that virus titers of such lysates were drastically reduced. The minimum concentration of CaCl 2 in the growth medium required to maintain maximum virus yield was determined to be approximately 0.17 m M . The period of calcium-dependency for infectious virus formation was between 6 and 12 hr postinfection at 37°, a time corresponding to the entire log phase of virus growth. Viruses produced in the absence of calcium were found to be exclusively incomplete single-shelled particles (D particles), as determined by cesium chloride density gradient analysis, electron microscopy, and SDS-polyacrylamide gel electrophoresis. Subsequent examination of virus-specified proteins in infected cells revealed that there was a reduction in the level of the major outer capsid protein (42K) in the absence of calcium. Thus, total inhibition of mature virus production under this condition could be due to the combined effect of reduced production of the 42K protein and incomplete assembly of the virus.

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.

Previously we reported that calcium plays an important role in the maturation of bovine rotavirus ( M. S. Shahrabadi and P. W. K. Lee, 1986 . Virology 152, 298–307). We now demonstrate that the formation of mature double-shelled (L) particles was strictly dependent on the concentration of calcium present in the growth medium. The formation of single-shelled (D) particles did not appear to be a calcium-mediated process. Subsequent labeling studies using 45Ca revealed that calcium was incorporated into the L particles but not the D particles. The previously noted decreased level of the outer capsid protein VP7 (42K) in calcium-deprived cultures was now found to be due to the preferential degradation, and not to the impaired synthesis, of this protein in the absence of calcium. It was further demonstrated that calcium had a stabilizing effect on VP7 and that VP7 synthesized in the presence of calcium was not degraded upon subsequent calcium deprivation. Protein degradation during calcium deprivation was apparently limited to the mature form of VP7 since the unglycosylated precursor (pVP7), formed in the presence of tunicamycin, was found to be stable under this condition. Electron microscopic examination of infected cells revealed that in the presence of calcium, virus maturation took place by the budding of viral cores through the endoplasmic reticulum (ER). No such budding was observed in calcium-deprived cells. In these cells mature virions were absent and membrane fragments could be found associated with viral cores or single-shelled particles.

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

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.

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.

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.