Isolation of Reovirus T3D Mutants Capable of Infecting Human Tumor Cells Independent of Junction Adhesion Molecule-A

Abstract

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.

Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

“…These proof of concept experiments suggest that it should be also possible to adapt the virus to different cell types in order to further optimize reovirus oncolytic ability. This idea has been previously suggested and, accordingly, novel viruses were found to be better adapted as oncolytic agents (Kim et al, 2011; Page 16 of 27 Rudd et al, 2005;Shmulevitz et al, 2012;van den Wollenberg et al, 2012). Viruses selected for their large-plaque phenotype using L929 cells, somewhat reminiscent of larger plaques formed by VeroAV on Vero cells in the absence of chymotrypsin, were shown to be better oncolytic viruses both in vitro and in animal models (Shmulevitz et al, 2012).…”

“…This has led to numerous clinical studies as reviewed by others (Black and Morris, 2012;Clements et al, 2014;Harrington et al, 2010;Kelly et al, 2009;Maitra et al, 2012). Despite the fact that reoviruses are naturally oncolytic without prior genetic modifications, there is still a significant research effort ongoing to obtain novel virus variants better adapted to infect, replicate in, and kill cancer cells while sparing non-transformed cells (van den Hengel et al, 2013;Kim et al, 2011;Rudd and Lemay, 2005;Shmulevitz et al, 2012;van den Wollenberg et al, 2009van den Wollenberg et al, , 2012. One possible approach is to take advantage of novel viral variants that could be selected during establishment of viral persistence in different cell types.…”

Changing Faces in Virology: The Dutch Shift from Oncogenic to Oncolytic Viruses

“…Genetically targeted viruses were generated that could use an artificial receptor on cells that lack expression of the canonical reovirus receptor JAM-A on the cell surface (van den Wollenberg et al, 2008). The bioselection approach yielded tropism-and host-range-expanded reoviruses capable of infecting cells independent of JAM-A, purportedly via interaction via sialic acids (van den Wollenberg et al, 2012). These viruses may be useful as oncolytic agents in tumors that do not express JAM-A on their cell surface.…”

8th International Conference on Oncolytic Virus Therapeutics

“…The group modified the spike protein by inserting a His-tag in the reovirus spike protein so that the modified spike can bind a scFv-His expressed on cells. The scFv-His can acts as a surrogate receptor on cells lacking the reovirus receptor protein JAM-A (van denWollenberg et al, 2008). Viruses were produced to express the His-tagged spike, but one had lost the His-tag.…”

“…This mutant virus (jin-1) showed a much wider tropism to many cell lines which lack both JAM-A and the scFv-His. This and two more jin mutants have mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the tail of the spike protein(Reiter et al, 2011;van den Wollenberg et al, 2012) and depend on sialic acids for infection. There is no nice location to insert a transgene in the reovirus genome.…”