Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.
Human Orthoreovirus Type 3 Dearing is not pathogenic to humans and has been evaluated clinically as an oncolytic agent. Its transduction efficiency and the tumor cell selectivity may be enhanced by incorporating ligands for alternative receptors. However, the genetic modification of reoviruses has been difficult, and genetic targeting of reoviruses has not been reported so far. Here we describe a technique for generating genetically targeted reoviruses. The propagation of wild-type reoviruses on cells expressing a modified s1-encoding segment embedded in a conventional RNA polymerase II transcript leads to substitution of the wild-type genome segment by the modified version. This technique was used for generating reoviruses that are genetically targeted to an artificial receptor expressed on U118MG cells. These cells lack the junction adhesion molecule-1 and therefore resist infection by wild-type reoviruses. The targeted reoviruses were engineered to carry the ligand for this receptor at the C terminus of the s1 spike protein. This demonstrates that the C terminus of the s1 protein is a suitable locale for the insertion of oligopeptide ligands and that targeting of reoviruses is feasible. The genetically targeted viruses can be propagated using the modified U118MG cells as helper cells. This technique may be applicable for the improvement of human reoviruses as oncolytic agents.
Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.
Glioblastoma (GB) is a devastating disease for which new treatment modalities are needed. Efficacious therapy requires the removal of stem-cell like cells, these cells drive tumor progression because of their ability to self-renew and differentiate. In glioblastoma, the GB stem-like cells (GSC) form a small population of tumor cells and possess high resistance to chemo and radiation therapies. To assess the sensitivity of GSC to reovirus-mediated cytolysis, a panel of GSC cultures was exposed to wild-type reovirus Type 3 Dearing (T3D) and its junction adhesion molecule-A (JAM-A)-independent mutant, jin-1. Several parameters were evaluated, including the fraction of cells expressing the JAM-A reovirus receptor, the fraction of cells synthesizing reovirus proteins, the number of infectious reovirus particles required to reduce cell viability, the amount of infectious progeny reovirus produced and the capacity of the reoviruses to infect the GSC in 3-dimensional (3D) tumor cell spheroids. Our data demonstrate a marked heterogeneity in the susceptibility of the cultures to reovirus-induced cytolysis. While in monolayer cultures the jin-1 reovirus was generally more cytolytic than the wild-type reovirus T3D, in the 3D GSC spheroids, these viruses were equally effective. Despite the variation in reovirus sensitivity between the different GSC cultures, our data support the use of reovirus as an oncolytic agent. It remains to be established whether the variation in the reovirus sensitivity correlates with a patient's response to reovirus therapy. Moreover, our data show that the expression of the JAM-A receptor is not a major determinant of reovirus sensitivity in 3D GSC cultures.
The capacity to modify the reovirus genome facilitates generation of new therapeutic reoviruses. We describe a method for generating replication-competent reoviruses carrying a heterologous transgene. The strategy is based on the expanded-tropism reovirus mutant jin-3, which can infect cells independent of the reovirus receptor junction-adhesion molecule A (JAM-A). Jin-3 harbors a mutation in the S1 segment, resulting in a G196R substitution in the tail of the spike protein σ1. The use of the jin-3 tail-encoding S1 segment allows replacing the codons for the JAM-A-binding head domain by up to 522 nucleotides of foreign sequences, without exceeding the size of the wild-type S1 segment. We inserted the codons for the porcine teschovirus-1 2A element fused with those encoding the fluorescent protein iLOV. Replicating rS1His-2A-iLOV reoviruses were generated by co-transfection of expression plasmids for all reovirus segments. These reoviruses contain the S1His-2A-iLOV segment in the absence of the wild-type S1 segment. Density-gradient centrifugation confirmed the association of the σ1-tail fragment with the capsid. Both JAM-A-positive and -negative cells exposed to the rS1His-2A-iLOV reoviruses exhibited iLOV fluorescence, confirming the jin-3-derived expanded-tropism phenotype. These data demonstrated the feasibility of generating decapitated replication-competent T3D reoviruses carrying a heterologous transgene.
In the canonical pathway, infection of cells by the wild-type mammalian orthoreovirus Type 3 Dearing (T3D) is dependent on the interaction of the viral spike protein σ1 with the high-affinity cellular receptor junction adhesion molecule-A (JAM-A). We previously demonstrated that the human glioblastoma cell line U-118 MG does not express JAM-A and resists reovirus T3D infection in standard cell culture conditions (SCCC). Heterologous JAM-A expression sensitises U-118 MG cells to reovirus T3D. Here we studied reovirus infection in U-118 MG cells grown in spheroid cultures with the premise that cells in such cultures resemble cells in tumours more than those grown under standard adherent cell culture conditions on a plastic surface. Although the U-118 MG cells in spheroids do not express JAM-A, they are susceptible to reovirus T3D infection. We show that this can be attributed to factors secreted by cells in the spheroids. The concentration of active extracellular proteases cathepsin B and L in the medium of spheroid cultures was increased 19- and 24-fold, respectively, as compared with SCCC. These enzymes can convert the reovirus particles into a form that can infect the U-118 MG cells independent of JAM-A. Taken together, these data demonstrate that infection of tumour cells by wild-type reovirus T3D is not strictly dependent on the expression of JAM-A on the cell surface.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.