**3. Tumour Specificity and Replication**

The reovirus life-cycle is shown in Figure 1. Viral entry occurs over multiple steps, the first being a low-affinity "tethering" of the reovirus σ1 protein to cell surface sialic acid [29,30]. Subsequently, σ1 engages junctional adhesion molecule A (JAM-A), the canonical reovirus receptor [31–33], which is ubiquitously expressed throughout the body and has several important roles in normal cellular processes including tight junction formation, leukocyte migration, and angiogenesis [34]. Fortuitously, JAM-A is also overexpressed in several cancers, including both haematological and solid malignancies [35–41]. Following reovirus engagement with JAM-A and receptor-mediated endocytosis, the viral particle undergoes acid-dependent cathepsin-mediated proteolysis within the endosome [42,43] to form an intermediate subviral particle (ISVP) characterised by the loss of σ3 and cleavage of µ1 [44]. The proteolytic uncoating, principally by cathepsins L and B, is critical for penetration of the endosome membrane by µ1; ISVPs undergo a conformational change causing autocleavage of µ1 into µ1N which triggers pore formation in the endocytic membrane [45] and delivers transcriptionally active reovirus into the cytosol [46,47] for replication. Capped, positive-sense single stranded (ss) RNA serves as

mRNA for protein translation and provides a template for replication of nascent dsRNA genomes [48]. Transcription and translation occur in cytoplasmic "viral factories" [49,50], with packaging of the segmented genome into virions occurring concomitantly with RNA synthesis [51,52]. Viral egress can be non-cytolytic in the absence of transformation; however, the release of progeny virus is typically lytic in permissive, transformed cells [53,54]. delivers transcriptionally active reovirus into the cytosol [46,47] for replication. Capped, positivesense single stranded (ss) RNA serves as mRNA for protein translation and provides a template for replication of nascent dsRNA genomes [48]. Transcription and translation occur in cytoplasmic "viral factories" [49,50], with packaging of the segmented genome into virions occurring concomitantly with RNA synthesis [51,52]. Viral egress can be non-cytolytic in the absence of transformation; however, the release of progeny virus is typically lytic in permissive, transformed cells [53,54].

*Cancers* **2020**, *12*, x FOR PEER REVIEW 3 of 26

**Figure 1.** Reovirus replication: **1**. Reovirus is first tethered via a weak interaction between σ1 and cell surface sialic acid; σ1 then binds with high affinity to junctional adhesion molecule A (JAM-A) resulting in internalization of the virus via receptor-mediated endocytosis. **2.** Once internalized, the virus is transported to early and late endosomes where it undergoes proteolytic digestion to remove the outer capsid protein σ3 resulting in the formation of infectious subvirion particles (ISVPs). **3.** Alternatively, ISVPs may be formed by extracellular proteases within the tumour environment allowing direct entry into cells via membrane penetration. **4.** After further proteolytic degradation a transcriptionally active viral core is released into the cytoplasm. Transcription and translation occur ultimately leading to the assembly of new viral progeny, host cell death and progeny release. Figure created using Biorender (https://biorender.com/). **Figure 1.** Reovirus replication: **1**. Reovirus is first tethered via a weak interaction between σ1 and cell surface sialic acid; σ1 then binds with high affinity to junctional adhesion molecule A (JAM-A) resulting in internalization of the virus via receptor-mediated endocytosis. **2.** Once internalized, the virus is transported to early and late endosomes where it undergoes proteolytic digestion to remove the outer capsid protein σ3 resulting in the formation of infectious subvirion particles (ISVPs). **3.** Alternatively, ISVPs may be formed by extracellular proteases within the tumour environment allowing direct entry into cells via membrane penetration. **4.** After further proteolytic degradation a transcriptionally active viral core is released into the cytoplasm. Transcription and translation occur ultimately leading to the assembly of new viral progeny, host cell death and progeny release. Figure created using Biorender (https://biorender.com/).

The molecular features associated with the oncolytic capacity of reovirus have been the subject of decades of research. Initially, an association between reovirus permissiveness and epidermal growth factor receptor (EGFR) status was revealed [55,56], along with evidence that activation of downstream signalling pathways, induced after transfection with the oncogene *v-erb*, are important [57]. Subsequent transfection of cells with constitutively active elements of the RAS pathway, a group of small GTP-binding proteins that regulate cell fate and growth, identified a role for RAS in reovirus The molecular features associated with the oncolytic capacity of reovirus have been the subject of decades of research. Initially, an association between reovirus permissiveness and epidermal growth factor receptor (EGFR) status was revealed [55,56], along with evidence that activation of downstream signalling pathways, induced after transfection with the oncogene *v-erb*, are important [57]. Subsequent transfection of cells with constitutively active elements of the RAS pathway, a group of small GTP-binding proteins that regulate cell fate and growth, identified a role for RAS in reovirus

permissiveness [58]. Therefore, although JAM-A is important for host cell entry, gain-of-function mutations activating RAS signalling [59] could promote reovirus replication and the release of virus progeny [60]. *RAS* mutations are prevalent in cancer [61], supporting the use of reovirus as a potential therapeutic agent [58,62]. The link between reovirus and cellular RAS status was further strengthened by observations that tumour cell susceptibility could be influenced by modulating RAS and/or its downstream effectors using short-hairpin RNA or small-molecule inhibitors [63,64]. Mechanistically, modulation of RAS signalling may promote susceptibility via inhibition of PKR [58]. In healthy cells, binding of dsRNA by PKR results in its dimerization, autophosphorylation and activation. Activated PKR subsequently phosphorylates the translation initiation factor, eIF2, rendering it inactive, which prevents the translation of viral transcripts [65]; however, in *RAS*-transformed cells PKR remains inactive and viral replication can occur [58,66,67]. Currently, the mechanism that coordinates *RAS*-transformation and PKR inactivation remains unclear [68].

Although the RAS–PKR axis provides a plausible explanation for the susceptibility of cancer cells to reovirus, the true molecular mediator has been the subject of debate, with doubt being cast by the survival of some infected *RAS*-transformed cells [69,70]. Moreover, the absence of a correlation between total or phospho-PKR with RAS expression or cell death contradicts previous studies [71], as does the lack of association between oncolysis and EGFR signalling [72]. It has become increasingly apparent that viral replication and cell death are not inextricably linked. Indeed, it is possible that RAS activation does not underlie viral replication but rather sensitivity to apoptosis which can occur independently of replication [53,64]. Sensitivity to reovirus oncolysis is likely to be dependent on multiple cellular and molecular determinants, many of which may yet be undiscovered.
