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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

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.

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.

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).

A conformational change was detected in reovirus upon its attachment to mouse L fibroblasts. Specifically, the capsid proteins of cell-bound virions became more resistant to pepsin digestion. Similar observations were made using glutaraldehyde-fixed cells or plasma membranes instead of live cells, indicating that virus internalization was not necessary for this effect. This conformational change was totally reversible, since bound virions reverted back to the pepsin-sensitive state upon release from the cell surface. Not unexpectedly, a conformational change was also detected in the reovirus cell attachment protein sigma 1 when it alone bound to cells. The alteration was mapped, by deletion mutagenesis, to a region proximal to the N-terminal (virion-anchoring) end of the protein and was also found to be reversible. Structural changes in sigma 1 were also detectable following its interaction with sialic acid (conjugated to bovine serum albumin) shown previously to the minimal receptor determinant recognized by reovirus. These results suggest that upon cell attachment, a signal is transmitted from the C-terminal receptor-binding region of sigma 1 to the N terminus in a ripple-like progression that eventually leads to conformational changes in the other reovirus capsid proteins. An altered conformational state may be necessary for subsequent viral entry and programmed disassembly of viral capsids inside susceptible cells.

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.

As the incidence of cancer continues to escalate, the demand for alternative anticancer therapeutics increases. The use of viruses in cancer treatment is well documented (1); however, as knowledge in the fields of virology, cell biology, and oncology expands, refined techniques and novel viral-based therapeutics emerge. The oncolytic potential of a wide spectrum of viruses has been explored, such as adenovirus (2,3), herpes simplex virus (HSV) (4,5), vesicular stomatitis virus (6), vaccinia virus (7), poliovirus (8), and mammalian reovirus (9).

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.

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 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 reovirus cell attachment protein (protein σl) is a minor outer capsid protein (49K molecular weight) located at the 12 vertices of the viral icosahedron.1,2 This protein possesses intrinsic cell binding function,1 and plays a pivotal role in viral infectivity and tissue tropism.3 Considerable effort has been made to probe the structure-function relationships of this protein. The S1 gene segment, which encodes σ1, has been sequenced for all three reovirus serotypes,4–8 and the deduced amino acid sequence has been analyzed to predict various structural motifs. Based on sequence analysis, Bassel-Duby et al.6 first suggested that the N-terminal portion of σl possesses oligomerization potential. First of all, the N-terminal one-third of σl was found to be highly a-helical. The additional presence, in the same region, of an extended heptad repeat (a-b-c-d-e-f-g)n, where a and d are characterically apolar residues, further indicates the propensity of this region to adopt a coiled-coiled rope-like structure. The C-terminal portion of σl, on the other hand, does not possess any distinct pattern and is therefore predicted to assume a complex globular structure. It was therefore postulated that the N- and C-terminal portions represent the proximal and the distal ends, respectively, of protein σl, and that the C-terminal portion interacts with the cellular receptor. This theoretically-deduced morphology of σl was subsequently confirmed by electron microscopic studies which showed purified al as rod-like structures (fibers) topped with knobs.2,9,10 Similar lollipop-shaped structures have also been found on the surface of the virion with the globular head region most distal from the virion.2 Thus, the reovirus σl protein morphologically resembles the adenovirus fiber protein which is also responsible for the attachment of the virus to cellular receptors.11,12

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.

Oncolytic virus therapy is a novel and expanding approach for treatment of cancer. However, its success is dependent on clarification of signaling mechanisms involved in determination of cell permissiveness to such viral agents. Such data can lead to the development of strategies for increasing efficiency and specificity of oncolytic viruses. We have shown that transformation with oncogenes in Ras signaling pathway plays an essential role in the determination of cell permissiveness to herpes virus and some of its oncolytic mutants. In this study we provide evidence that transcription factors down-stream of Ras can be used as a marker to predict permissiveness of human lymphoma cell lines to R3616, an oncolytic version of herpes simplex virus-1 with deletions in both copies of the viral g134.5 gene. Different lymphoma cell lines including Radji, Daudi. Jurkat, CA46, ST486, NC37, U937, Ramas and Molt4 were infected with R3616 at MOI~0.5 and their permissiveness to this virus was evaluated by different methods such as western blotting for viral proteins and plaque titration for progeny virus.

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.

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.

"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.

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.