Article Serotype-Specific Killing of Large Carcinoma Cells by Reovirus

Emily J. Simon, Morgan A. Howells, Johnasha D. Stuart and Karl W. Boehme * Department of Microbiology and Immunology and Center for Microbial Pathogenesis and Host Inflammatory Response, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; [email protected] (E.J.S.); [email protected] (M.A.H.); [email protected] (J.D.S.) * Correspondence: [email protected]; Tel.: +(501)-686-5189

Academic Editor: Andrew Mehle Received: 1 May 2017; Accepted: 1 June 2017; Published: 6 June 2017

Abstract: Reovirus is under development as a therapeutic for numerous types of cancer. In contrast to other oncolytic viruses, the safety and efficacy of reovirus have not been improved through genetic manipulation. Here, we tested the oncolytic capacity of recombinant strains (rs) of prototype reovirus laboratory strains T1L and T3D (rsT1L and rsT3D, respectively) in a panel of non-small cell lung cancer (NSCLC) cell lines. We found that rsT1L was markedly more cytolytic than rsT3D in the large cell carcinoma cell lines tested, whereas killing of adenocarcinoma cell lines was comparable between rsT1L and rsT3D. Importantly, non-recombinant T1L and T3D phenocopied the kinetics and magnitude of cell death induced by recombinant strains. We identified segments L2, L3, and M1 as viral determinants of -specific differences cell killing of the large cell carcinoma cell lines. Together, these results indicate that recombinant reoviruses recapitulate the cell killing properties of non-recombinant, tissue culture-passaged strains. These studies provide a baseline for the use of reverse genetics with the specific objective of engineering more effective reovirus oncolytics. This work raises the possibility that type 1 reoviruses may have the capacity to serve as more effective oncolytics than type 3 reoviruses in some tumor types.

Keywords: reovirus; oncolysis; lung cancer; reverse genetics

1. Introduction Oncolytic viruses are an emerging class of cancer therapeutics that selectively replicate in and kill transformed cells while sparing non-transformed cells [1,2]. Mammalian (reovirus) is one of a number of viruses with oncolytic potential that are in different stages of development [2–6]. Reoviruses are non-enveloped viruses with a segmented double-stranded RNA that productively infect and lyse several different tumor cell types in vitro and in experimental models [3,4,7]. In some cases, reovirus shows efficacy as a virotherapeutic agent for aggressive and refractory tumors [8]. Phases I and II clinical trials in the United States, Canada, and Europe demonstrate that pelareorep (Reolysin), a first-generation reovirus therapeutic is tolerated and safe, even in patients with advanced cancer who have undergone extensive chemotherapy [9,10]. A Phase III clinical trial to test reovirus efficacy against head and neck cancers was recently completed. Phase I and II trials indicate that the majority of oncolytic viruses under development, including reovirus, are safe for use in [11]. However, the therapeutic efficacy of many of these agents is limited [2]. The therapeutic potency of oncolytic viruses can be enhanced by genetic modifications that increase viral replication in and killing of cancer cells, re-target the to infect new tumor types, or introduce immunomodulatory factors that enhance immune-mediated killing of tumors [2]. For example, talimogene laherparepvec (T-VEC), which in 2015 became the first oncolytic virus approved by the US Food and Drug Administration (FDA) for use in humans, is a genetically-modified

Viruses 2017, 9, 140; doi:10.3390/v9060140 www.mdpi.com/journal/viruses Viruses 2017, 9, 140 2 of 19 herpes simplex virus type 1 (HSV-1) [12]. The HSV-1 strain that forms the basis for T-VEC lacks the viral ICP34.5 gene, which enhances tumor cell lysis. ICP34.5 also is required for replication in the central nervous system (CNS) and its deletion further improves the safety profile of the virus [13]. The ICP34.5 locus was replaced with the gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that functions to recruit and activate antigen presenting cells that facilitate T-cell recognition and killing of cancer cells [14]. Other prospective oncolytic viruses, including myxoma virus [15], adenovirus [16], coxsackievirus [17], measles virus [18], and vesicular stomatitis virus [19] also have been genetically altered to increase safety or oncolytic efficacy [2]. In contrast, reovirus has not undergone genetic manipulation to increase its therapeutic potency [2,20]. The reovirus in use for clinical trials (Reolysin) is a derivative of laboratory strain type 3 Dearing (T3D) [4] that was isolated from a child’s stool sample in the [21]. Although Reolysin shows tremendous efficacy in pre-clinical studies, initial clinical trials showed that the efficacy of reovirus monotherapy was low [22,23]. As a consequence, ongoing clinical trials use Reolysin in combination with chemotherapy or radiotherapy [11]. However, it may be possible to enhance the oncolytic capacity of reovirus, as large plaque variants generated by serial passage of the Reolysin parental strain T3D in L929 cells showed an increased capacity to lyse tumor cells [24]. This finding indicates that reovirus can be further adapted to more effectively kill tumors. A wealth of reovirus sequence polymorphisms that augment viral replication or host responses are known [25]. Some of these polymorphisms may have the capacity to increase reovirus oncolytic activity. The segmented reovirus genome also allows generation of reassortant viruses that contain distinct combinations of gene segments from different reovirus strains. Many reovirus strain-specific differences in properties that may enhance oncolysis, including viral replication, induction, and host cell tropism are linked to a single gene segment. Reassortant reoviruses that contain a combination of gene segments from different reovirus strains may enhance viral oncolytic efficacy. In addition, it may be possible to combine individual mutations and reassortants to increase reovirus oncolytic activity or generate reoviruses that are uniquely tailored to target specific tumor types. Cancer is a highly heterogeneous disease that can originate from any type of cell in the body [26]. The genetic or epigenetic abnormalities that lead to transformation often differ greatly between individuals that have the same type of cancer [26]. Even within a single tumor, there are often dramatic differences between the cells that comprise the mass [26]. Together, cancer heterogeneity leads to differential susceptibility to anti-cancer therapeutics, including reovirus [27]. The ability to generate reoviruses with different constellations of gene segments that enhance killing in different cell and tumor types could greatly expand the utility of reovirus as a cancer treatment. Reovirus can be genetically modified using -based reverse genetics [28–31]. Complete reverse genetics systems are available for the two most commonly studied prototype reovirus laboratory strains, T3D and type 1 Lang (T1L) [28–30]. Although recovery of recombinant reovirus is highly efficient, this technology has not been fully applied to the production or enhancement of reovirus for oncolytic purposes. In this study, we tested recombinant strains (rs) of T1L (rsT1L) and T3D (rsT3D) that were generated by plasmid-based reverse genetics for the capacity to kill a panel of non-small cell lung cancer (NSCLC) cell lines (Table1). Lung cancer is the leading cause of cancer-related deaths among both men and women in the United States [32]. The 5-year survival rate for lung cancer is approximately 17% [33] and over half of patients succumb within one year of diagnosis [34]. NSCLC causes 85% of lung cancer cases and can be divided into three subtypes NSCLC—adenocarcinoma, large cell carcinoma, and squamous cell carcinoma [35]. Our cell panel consisted of three adenocarcinoma and two large cell carcinoma cell lines. Adenocarcinomas are the most prevalent NSCLC subtype and large cell carcinomas are generally the most aggressive form of the disease [35]. We found that rsT1L and rsT3D killed cell lines derived from lung adenocarcinomas equivalently. However, rsT1L killed the large cell carcinoma cell lines tested to a substantially greater extent than rsT3D. Importantly, recombinant and non-recombinant reoviruses killed each cell line tested equivalently. Production of viral yields by rsT1L and rsT3D were comparable in the large cell Viruses 2017, 9, 140 3 of 19 carcinoma cell lines. This finding indicates that differences in viral replication do not underlie the strain-specific disparity in killing of the large cell carcinoma cell lines. Finally, we identified reovirus gene segments, L2, L3, and M1 as viral determinants of differential cell killing of large cell carcinoma cell lines. Together, our results indicate that that the oncolytic potency of reoviruses generated by reverse genetics is comparable to non-recombinant strains. Our results further suggest that a T1L-based reovirus may be more effective therapeutics than T3D-derived viruses in certain cell or tumor types.

Table 1. Non-small cell lung cancer (NSCLC) cell lines used in this study.

Cell Line Histology Tumor Source NCI-H661 Large cell carcinoma Lymph node metastasis NCI-H1299 Large cell carcinoma Lymph node metastasis NCI-H1437 Adenocarcinoma Pleural effusion metastasis NCI-H1563 Adenocarcinoma Primary NCI-H1975 Adenocarcinoma Primary

2. Results

2.1. Recombinant Reovirus Strains rsT1L and rsT3D Differ in the Capacity to Kill Large Cell Carcinoma Cell Lines To assess the oncolytic capacity of recombinant reoviruses, we infected a panel of NSCLC cell lines (Table1) with recombinant (rsT1L or rsT3D) or non-recombinant (T1L or T3D) reoviruses. The panel included two large cell carcinoma lines (H661 and H1299) and three adenocarcinoma lines (H1437, H1563, and H1975) (Table1). Each cell line was mock infected or infected with rsT1L, rsT3D, T1L, or T3D at multiplicities of infection (MOIs) of 10, 100, or 1000 plaque forming units (PFU)/cell and ATP content was measured at 24, 48, and 72 h post-infection as an indicator of cell viability (Figure1). We observed a dramatic difference in cell viability between rsT1L and rsT3D in the two large cell carcinoma cell lines (Figure1A,B). In H661 cells, each dose of rsT1L induced more cell death than rsT3D at 48 and 72 h. The peak loss of cell viability following rsT3D infection was approximately 40% of control, whereas rsT1L reduced cell viability by greater than 95%. In H1299 cells, viability of rsT3D-infected cells never dropped below 60% of control, whereas rsT1L caused almost complete cell death at MOIs of 100 and 1000 PFU/cell by 72 h. In contrast to the large cell carcinoma cell lines, rsT1L and rsT3D induced comparable dose- and time-dependent cell killing in the adenocarcinoma cell lines (Figure1C–E). Importantly, cell death induced in each NSCLC line by non-recombinant T1L and T3D phenocopied the cell killing induced by rsT1L and rsT3D, including enhanced killing of the two large cell carcinoma cell lines (H661 and H1299) by T1L compared to T3D (Figure1A,B). These results indicate that the cell killing capacity of reovirus generated by plasmid-based reverse genetics does not differ from that of native, non-recombinant strains. These data also revealed that the two large cell carcinoma lines were more resistant to killing by rsT3D than rsT1L. These findings suggest that T1 reoviruses may be more effective oncolytics than T3 reoviruses in certain tumor types, including large cell carcinomas. Viruses 2017, 9, 140 4 of 19 Viruses 2017, 9, 140 4 of 19

Figure 1. Lung cancer cells are susceptible to killing by recombinant reoviruses. (A) H661; (B) H1299; Figure 1. Lung cancer cells are susceptible to killing by recombinant reoviruses. (A) H661; (B) H1299; (C)( H1437;C) H1437; (D ()D H1563;) H1563; and and ( E(E)) H1975H1975 cells cells were were infected infected with with recombinant recombinant strains strains (rs)T1L, (rs)T1L, rsT3D, rsT3D, T1L T1Lor or T3D T3D at atmultiplicity multiplicity of infection of infection (MOIs) (MOIs) of 10, of 100, 10, or 100, 1000 or plaque 1000 plaqueforming forming units (PFU)/cell. units (PFU)/cell. Cellular CellularATP content ATP content was measured was measured at 24 h at (left 24 hpanel), (left panel), 48 h (middle 48 h (middle panel),panel), and 72 andh (right 72 h panel) (right panel)post- post-infection.infection. Cell Cell viability viability is isshown shown as as a apercentage percentage of of ATP ATP content content in in mock-infected mock-infected cells. cells. Error Error bars bars indicateindicate standard standard deviation deviation (SD). (S.D.). * p <* p 0.05; < 0.05; ** p**< p 0.01;< 0.01; *** ***p p< < 0.001 0.001 asas determineddetermined for rsT1L rsT1L vs. vs. rsT3D rsT3D by Student’sby Student’st-test. t-test. @, p@,< p 0.05; < 0.05; @@, @@,p

2.2.2.2. Reovirus Reovirus Infectivity Infectivity in in NSCLC NSCLC Cell Cell Lines Lines

WeWe performed performed a fluorescent a fluorescent focus focus assay assay to to assess assess reovirus reovirus infectivityinfectivity in the the NSCLC NSCLC cell cell panel panel (Figure(Figure2). Each 2). Each cell cell line line was was infected infected with with rsT1L rsT1L or or rsT3D rsT3D at at an an MOI MOI of of 100 100 PFU/cell, PFU/cell, which was was the the medianmedian viral viral dose dose tested tested in in the the cell cell killing killing experiments. experiments. AtAt 1818 hh post-infection,post-infection, the the cells cells were were fixed fixed and stained using reovirus-specific polyclonal antiserum and DAPI. A higher percentage of H661, Viruses 2017, 9, 140 5 of 19 Viruses 2017, 9, 140 5 of 19 and stained using reovirus-specific polyclonal antiserum and DAPI. A higher percentage of H661, H1473, H1563, and H1975 cells were infected by rsT3D compared to rsT1L. H1299 cells were the only H1473, H1563, and H1975 cells were infected by rsT3D compared to rsT1L. H1299 cells were the only cell line that rsT1L infected to a greater degree than rsT3D (~80% vs. ~60%). The largest difference cell line that rsT1L infected to a greater degree than rsT3D (~80% vs. ~60%). The largest difference was observed in H1437 cells, where rsT3D infected an approximately 2-fold greater number of cells was observed in H1437 cells, where rsT3D infected an approximately 2-fold greater number of cells than rsT1L. In H661, H1299, and H1563 lines, the majority of cells were infected by both reovirus than rsT1L. In H661, H1299, and H1563 lines, the majority of cells were infected by both reovirus strains (>60%). In contrast, a low percentage (<40%) of H1437 and H1975 cells were infected by rsT1L strains (>60%). In contrast, a low percentage (<40%) of H1437 and H1975 cells were infected by rsT1L or rsT3D. These findings indicate that each lung lines tested is susceptible to infection by or rsT3D. These findings indicate that each lung cancer cell lines tested is susceptible to infection by recombinant reoviruses. It is of note that although a higher percentage of H661 cells were infected recombinant reoviruses. It is of note that although a higher percentage of H661 cells were infected with rsT3D than rsT1L, rsT1L induced markedly more cell death in H661 cells than rsT3D. Thus, it is with rsT3D than rsT1L, rsT1L induced markedly more cell death in H661 cells than rsT3D. Thus, it unlikely that the strain-specific difference in killing of H661 cells is due to differential infectivity is unlikely that the strain-specific difference in killing of H661 cells is due to differential infectivity between the two viruses. between the two viruses.

FigureFigure 2. ReovirusesReovirusesinfect infect NSCLC NSCLC cell cell lines. lines. H661, H661 H1299,, H1299, H1437, H1437, H1563 H1563 and H1975 and H1975 cells were cells infected were infectedwith rsT1L with or rsT1L rsT3D or at rsT3D an MOI atof an 100 MOI PFU/cell. of 100 PF AtU/cell. 24 h post-inoculation, At 24 h post-inoculation, reovirus-infected reovirus-infected cells were cellsdetected were by detected indirect by immunofluorescence indirect using reovirus-specific using reovirus-specific polyclonal antiserum.polyclonal Nucleiantiserum. were Nucleistained were with DAPI.stained Results with DAPI. are expressed Results asare the expresse mean percentaged as the mean of infected percentage cells for of duplicateinfected cells samples. for duplicateError bars samples. indicate SD.Error * p

AlthoughAlthough rsT1L rsT1L and and rsT3D rsT3D infectivity infectivity did did not not differ differ in in H661 H661 cells, cells, rsT1L rsT1L infected infected H1299 H1299 cells cells moremore efficiently efficiently than than rsT3D. rsT3D. A A greater greater level level of of infection infection by by rsT1L rsT1L could could result result in in more more killing killing of of H1299 H1299 cellscells than rsT3D.rsT3D. ToTo test test whether whether the the disparity disparity in infectivityin infectivity caused caused strain-specific strain-specific differences differences in killing in killingof H1299 of H1299 cells, we cells, performed we performed cell viability cell viability assays assays in which in which the infectious the infectious dose of dose rsT1L of andrsT1L rsT3D and rsT3Dwas normalized was normalized based based on fluorescent on fluorescent focus focus unit unit (FFU) (FFU) titers titers that that were were determined determined on H1299 cells. cells. WeWe infected H1299 cells with 10,000, 1000, or 100 FFU/cell and and measured measured ATP ATP at at 72 72 h h post-infection post-infection asas an an indicator indicator of of cell cell viability viability (Figure (Figure 3).3). For reovirus, the FFU:PFU ratio is approximately 10:1 10:1 [36]. [ 36]. Thus,Thus, the the FFU FFU doses doses tested tested approximate approximate the the MOI MOI ra rangenge used used in in experiments experiments initiated initiated using using PFUs. PFUs. WeWe found that that when when infection infection was was normalized normalized by by infe infectivity,ctivity, rsT1L rsT1L retained retained th thee capacity capacity to to kill kill H1299 H1299 cellscells to to a a greater greater degree degree than than rsT3D rsT3D (Figure (Figure 33A).A). H1563 cells, which were equallyequally susceptible to rsT1L andand rsT3D rsT3D infection, infection, were were killed killed to to comparable comparable levels levels by by rsT1L rsT1L and and T3D T3D when when infection infection was was initiated initiated basedbased on on normalize normalize FFU FFU titers titers that that were were determined determined on on H1563 H1563 cells cells (Figure (Figure 33B).B). This result indicates thatthat differences differences in in virus virus infectivity infectivity do do not not lead lead to to strain-specific strain-specific differences in killing of H1299 cells.cells.

Viruses 2017, 9, 140 6 of 19 Viruses 2017, 9, 140 6 of 19

Viruses 2017, 9, 140 6 of 19

FigureFigure 3. 3.Differences Differences in in infectivity infectivity do not do underlie not underlie reovirus reovirus strain-specific strain-specific killing of H1299 killing cells. of (A H1299) H1299 or (B) H1563 cells were infected with rsT1L or rsT3D at MOIs of 100, 1000, or 10,000 fluorescent cells. (A) H1299 or (B) H1563 cells were infected with rsT1L or rsT3D at MOIs of 100, 1000, or focus units (FFU)/cell. FFU titers determined on H1299 or H1563 cells were used to calculate the MOIs 10,000Figure fluorescent 3. Differences focus unitsin infectivity (FFU)/cell. do not FFU underlie titers reovirus determined strain-specific on H1299 killing or H1563 of H1299 cells cells. were (A used) to for infection. Cellular ATP content was measured at 72 h post-infection. Cell viability is shown as a calculateH1299 the or MOIs (B) H1563 for infection. cells were Cellularinfected with ATP rsT1L content or rsT3 wasD measured at MOIs of at 100, 72 h1000, post-infection. or 10,000 fluorescent Cell viability percentage of ATP content in mock-infected cells. Error bars indicate S.D. ** p < 0.001 as determined focus units (FFU)/cell. FFU titers determined on H1299 or H1563 cells were used to calculate the MOIs is shownby Student’s as a percentage t-test. The of data ATP presented content is in compile mock-infectedd from two cells. independent Error bars experiments. indicate SD. ** p < 0.001 as for infection. Cellular ATP content was measured at 72 h post-infection. Cell viability is shown as a determined by Student’s t-test. The data presented is compiled from two independent experiments. percentage of ATP content in mock-infected cells. Error bars indicate S.D. ** p < 0.001 as determined 2.3. rsT1L and rsT3D Gene Expression and Replication in NSCLC Cell Lines by Student’s t-test. The data presented is compiled from two independent experiments. 2.3. rsT1L and rsT3D Gene Expression and Replication in NSCLC Cell Lines To assess viral in the NSCLC cell panel, each cell line was mock infected or 2.3. rsT1L and rsT3D Gene Expression and Replication in NSCLC Cell Lines Toinfected assess with viral rsT1L protein or rsT3D production at an MOI inof 10 the PFU/cell NSCLC (Figure cell panel,4). Whole each cell cell lysate lines prepared was mock at 6, infected12, 18, or infectedor 24 with Toh post-infection assess rsT1L viral or protein rsT3Dwere separated atproduction an MOI by SDS-PAGE in of the 10 NSCLC PFU/cell and immunoblottedcell (Figure panel, each4). Wholewith cell reovline cell irus-specificwas lysates mock infected preparedpolyclonal or at 6, 12, 18,infectedantiserum. or 24 hwith post-infection Reovirus rsT1L or proteins rsT3D were at were an separated MOI detected of 10 byinPFU/cell each SDS-PAGE cell (Figure line and by4). Whole18 immunoblotted h. In cell H1299 lysate ands prepared withH1975 reovirus-specific cellsat 6, 12,rsT1L 18, polyclonalorproduced 24 h post-infection antiserum. modestly Reovirushigher were separated viral proteins prot byein SDS-PAGE levels were than detected and rsT3D. immunoblotted in In each H1299 cell ce linells,with rsT1L byreov 18 irus-specificproduced h. In H1299 3.8- polyclonal and and 3.6- H1975 cells rsT1Lantiserum.fold more produced μ Reovirus1 than modestlyrsT3D proteins at 18 higherwereh and detected 24 viral h, respec protein in eachtively. levelscell Approximately line than by 18 rsT3D. h. In 22-fold H1299 In H1299 more and σcells,H19753 was rsT1L detectedcells rsT1L produced in 3.8- andproducedrsT1L-infected 3.6-fold modestlymore cells µrelative higher1 than viralto rsT3D rsT3D prot atatein 18 levels h, h but and than the 24 d h,rsT3D.ifference respectively. In decreasedH1299 Approximatelycells, to 3.7-forsT1Lld produced at 24 22-foldh. In 3.8- H1975 moreand cells, 3.6-σ3 was rsT1L produced 3- and 3.8-fold more μ1 at 18 h and 24 h, respectively. The differences in σ3 were 3.7-fold detectedfold inmore rsT1L-infected μ1 than rsT3D cells at 18 relative h and 24 to h, rsT3D respectively. at 18 h,Approximately but the difference 22-fold decreased more σ3 was to detected 3.7-fold in at 24 h. at 18 h and 2-fold at 24 h. For H1299, the difference could result from an increased number of cells In H1975rsT1L-infected cells, rsT1L cells produced relative to 3-rsT3D and at 3.8-fold 18 h, but more the differenceµ1 at 18 decreased h and 24 to h, 3.7-fo respectively.ld at 24 h. In The H1975 differences cells, in rsT1Lbecoming produced infected 3- byand rsT1L 3.8-fold compared more μ 1to at rsT3D. 18 h and However, 24 h, respecti no differencevely. The in differences reovirus gene in σ expression3 were 3.7-fold was σ3 were 3.7-fold at 18 h and 2-fold at 24 h. For H1299, the difference could result from an increased atobserved 18 h and between 2-fold rsT1Lat 24 andh. For rsT3D H1299, in H661, the difference H1437, or couldH1563 result cells. Together,from an increased these results number demonstrate of cells number of cells becoming infected by rsT1L compared to rsT3D. However, no difference in reovirus becomingthat recombinant infected reoviruses by rsT1L comparedefficiently toex rsT3D.press viral However, proteins no in difference NSCLC cell in reovirus lines. gene expression was geneobserved expression between was observed rsT1L and between rsT3D in rsT1LH661, H1437, and rsT3D or H1563 in H661, cells. H1437,Together, or th H1563ese results cells. demonstrate Together, these resultsthat demonstrate recombinant thatreoviruses recombinant efficiently reoviruses express viral efficiently proteins in express NSCLC viralcell lines. proteins in NSCLC cell lines.

Figure 4. Reovirus protein synthesis in NSCLC cell lines. (A) H661; (B) H1299; (C) H1437; (D) H1563; and (E) H1975 cells were mock infected or infected with rsT1L or rsT3D at an MOI of 10 PFU/cell. Whole cell lysates prepared at 6, 12, 18, or 24 h post-infection were analyzed by SDS-PAGE and immunoblotted Figure 4. Reovirus protein synthesis in NSCLC cell lines. (A) H661; (B) H1299; (C) H1437; (D) H1563; Figureusing 4. Reovirus reovirus-specific protein antiserum synthesis (top) in NSCLC or β- cell (bottom). lines. (A )Reovirus H661; (B proteins) H1299; are (C indicated) H1437; to (D the) H1563; right and and (E) H1975 cells were mock infected or infected with rsT1L or rsT3D at an MOI of 10 PFU/cell. Whole (E) H1975of the cellstop panel. were The mock images infected are representative or infected with of the rsT1L results or from rsT3D two at independent an MOI of 10 experiments. PFU/cell. Whole cell cell lysates prepared at 6, 12, 18, or 24 h post-infection were analyzed by SDS-PAGE and immunoblotted lysates prepared at 6, 12, 18, or 24 h post-infection were analyzed by SDS-PAGE and immunoblotted using reovirus-specific antiserum (top) or β-actin (bottom). Reovirus proteins are indicated to the right β usingof reovirus-specific the top panel. The antiserum images are (top)representative or -actin of (bottom).the results from Reovirus two independent proteins are experiments. indicated to the right of the top panel. The images are representative of the results from two independent experiments.

Viruses 2017, 9, 140 7 of 19 Viruses 2017, 9, 140 7 of 19

ToTo assess assess viral viral replication replication of of the the NSCLC NSCLC cells, cells, we we infected infected each each cell cell line line with with rsT1L rsT1L or rsT3Dor rsT3D at an at MOIan MOI of 1 of PFU/cell 1 PFU/cell and and quantified quantified viral viral titers titers at 0, 24,at 0, and 24, 48and h post-infection48 h post-infection (Figure (Figure5). We 5). used We a used lower a multiplicitylower multiplicity infection infection for assessing for assessing viral replication viral replic thanation cell killingthan cell (1 PFU/cellkilling (1 versus PFU/cell 10–1000 versus PFU/cell) 10–1000 toPFU/cell) provide to a largerprovide dynamic a larger range dynamic in which range to in measure which to viral measure yields. viral We foundyields. that We rsT1Lfound and that rsT3D rsT1L replicatedand rsT3D in replicated each lung in cancer each celllung line, cancer with cell progeny line, with yields progeny ranging yields from ranging 100- to 10,000-foldfrom 100- to increase 10,000- overfold input.increase We over found input. that We rsT1L found and that rsT3D rsT1L produced and rsT3D comparable produced yieldscomparable in H661 yields and in H1299 H661 cells. and ThisH1299 result cells. indicates This result that indicates differences that viral differences in replication viral do in notreplication account do for not differential account killingfor differential of H661 andkilling H1299 of H661 cells byand rsT1L H1299 and cells rsT3D. by rsT1L H1437 and cells rsT3D. produced H1437 the cells lowest produced viral yields the oflowest the cell viral lines yields tested. of Yieldsthe cell of lines rsT1L tested. and rsT3DYields wereof rsT1L equivalent and rsT3D in H1437were equivalent cells at 24 in h. H1437 However, cells rsT3Dat 24 h. produced However, higher rsT3D progenyproduced yields higher than progeny rsT1L atyields 48 h. than Although rsT1L rsT1Lat 48 h. and Although rsT3D replicated rsT1L and in rsT3D H1563 replicated and H1975, in H1563 rsT1L producedand H1975, higher rsT1L progeny produced yields higher than rsT3Dprogeny in these yields cell than lines. rsT3D These in results these indicate cell lines. that These recombinant results reovirusesindicate that replicate recombinant efficiently reoviruses in all of replicate the NSCLC efficiently cell lines in tested. all of the NSCLC cell lines tested.

FigureFigure 5.5.T1 T1 and and T3 T3 reoviruses reoviruses replicate replicate in NSCLC in NSCLC cell lines.cell lines. (A) H661; (A) H661; (B) H1299; (B) H1299; (C) H1437; (C) H1437; (D) H1563; (D) andH1563; (E) H1975and (E) cells H1975 were cells infected were infected with rsT1L with or rsT1L rsT3D or at rs anT3D MOI at an of MOI 1 PFU/cell. of 1 PFU/cell. Viral titersViral weretiters determinedwere determined in cell in lysates cell lysates at 0, at 24, 0, and24, and 48 h 48 post-infection h post-infection by by plaque plaque assay assay on on L929 L929 cells. cells. Results Results areare expressedexpressed as as the the mean mean viral viral yield yield at 24 at and 24 and48 h 48forh triplicate for triplicate samples. samples. Error bars Error indicate bars indicateS.D. * p < SD.0.05 *differencep < 0.05 difference between betweenrsT1L and rsT1L rsT3D and rsT3Das determined as determined by Student’s by Student’s t-test.t -test.The Thedata data presented presented are arerepresentative representative of the of results the results from from two independent two independent experiments. experiments.

2.4.2.4. RecombinantRecombinant ReovirusesReoviruses KillKill NSCLCNSCLC CellCell LinesLines byby aa Caspase-IndependentCaspase-Independent MechanismMechanism

InIn manymany cellcell types,types, includingincluding L929L929 andand HeLaHeLa cells,cells, T3T3 reovirusesreoviruses induceinduce higherhigher levelslevels ofof apoptosisapoptosis thanthan T1T1 reovirusesreoviruses [[25].25]. ToTo determinedetermine whetherwhether differencesdifferences inin apoptosisapoptosis underlieunderlie differentialdifferential cellcell killingkilling byby rsT1LrsT1L andand rsT3D,rsT3D, wewe assessed assessed caspase-3/7 caspase-3/7 activityactivity (Figure(Figure6 )6) and and poly-ADP poly-ADP ribose ribose polymerasepolymerase (PARP)(PARP) cleavage cleavage (Figure (Figure7 )7) in in each each NSCLC NSCLC cell cell line line following following rsT1L rsT1L and and rsT3D rsT3D infection. infection. TumorTumor necrosisnecrosis factor- factor-αα/cycloheximide/cycloheximide (TNF-(TNF-αα/CHX)/CHX) induced caspase-3/7 and and PARP cleavagecleavage inin eacheach NSCLCNSCLC cellcell line,line, indicatingindicating thatthat apoptoticapoptotic responsesresponses areare functionalfunctional inin thethe cellcell panel. panel. InfectionInfection withwith rsT1LrsT1L oror rsT3DrsT3D diddid notnot elicitelicit caspase-3/7caspase-3/7 activity (Figure6 6A,B)A,B) oror PARPPARP cleavage cleavage (Figure (Figure7 A,B)7A,B) inin thethe twotwo large large cell cell carcinoma carcinoma lines lines (H661 (H661 and and H1299). H1299). We We detected detected modest modest caspase-3/7 caspase-3/7 activityactivity inin H1437H1437 cellscells followingfollowing infectioninfection withwith thethe highesthighest multiplicitymultiplicity ofof rsT3DrsT3D (Figure(Figure6 6C).C). However, However, minimal minimal PARPPARP cleavage cleavage waswas observedobserved inin thisthis cellcell lineline followingfollowing reovirusreovirus infectioninfection (Figure(Figure7 7C).C). In In H1563 H1563 cells, cells, rsT1LrsT1L andand rsT3DrsT3D induced induced caspase-3/7 caspase-3/7 activityactivity inin aa dose-dependentdose-dependent mannermanner atat 2424 andand 4848 hh (Figure(Figure6 6d).D). However,However, rsT1LrsT1L andand rsT3D rsT3D did did not not induce induce PARP PARP cleavage cleavage in in H1563 H1563 cellscells (Figure(Figure7 D).7D). In In H1975 H1975 cells, cells, rsT1LrsT1L andand rsT3DrsT3D elicitedelicited caspase-3/7caspase-3/7 activity at 48 h (Figure6 6E),E), butbutminimal minimalPARP PARP cleavage cleavage at at all all of of thethe timetime pointspoints testedtested (Figure(Figure7 7E).E).

Viruses 2017, 9, 140 8 of 19 Viruses 2017, 9, 140 8 of 19

FigureFigure 6. Caspase-3/7 6. Caspase-3/7 activation activationin in NSCLCNSCLC cell lines lines during during reovirus reovirus infection. infection. (A)( AH661;) H661; (B) H1299; (B) H1299; (C)( H1437;C) H1437; (D ()D H1563;) H1563; andand ( E)) H1975 H1975 cells cells were were mock mock infected infected or infected or infected with rsT1L with or rsT1L rsT3D orat an rsT3D MOI of 100 PFU/cell. Caspase-3/7 activity was quantified at 24 h (left panel) or 48 h (right panel) post- at an MOI of 100 PFU/cell. Caspase-3/7 activity was quantified at 24 h (left panel) or 48 h (right infection. As a control, each cell line was treated with tumor necrosis factor-α (TNFα), 5 ng/mL) and panel) post-infection. As a control, each cell line was treated with tumor necrosis factor-α (TNFα), 5 cycloheximide (CHX, 25 μg/mL) 12 h prior to each time point. Results are presented as the mean ng/mL) and cycloheximide (CHX, 25 µg/mL) 12 h prior to each time point. Results are presented as caspase-3/7 activity relative to mock infection. Error bars indicate S.D. * p < 0.05 relative to mock the meaninfection caspase-3/7 as determined activity by Student’s relative t-test. to mock The data infection. presented Error is repr barsesentative indicate of SD. three * pindependent< 0.05 relative to mockexperiments. infection as determined by Student’s t-test. The data presented is representative of three independent experiments. To directly test whether caspases are required for reovirus killing of NSCLC cells, we assessed theTo effect directly of the test pan-caspase whether caspases inhibitor are Z-VAD-FMK required for (Z-VAD) reovirus on killing reovirus of NSCLCcell killing cells, (Figure we assessed8). Each the effectlung of thecancer pan-caspase cell line was inhibitor infected Z-VAD-FMK with rsT1L or (Z-VAD) rsT3D at on an reovirus MOI of 100 cell PFU/cell killing (Figure in the absence8). Each or lung presence of 25 μM Z-VAD and ATP content was measured at 48 h post-infection as an indicator of cancer cell line was infected with rsT1L or rsT3D at an MOI of 100 PFU/cell in the absence or presence cell viability. TNF/CHX-induced caspase-3/7 activity was completely inhibited in each cell line by of 25 µM Z-VAD and ATP content was measured at 48 h post-infection as an indicator of cell viability. both Z-VAD concentrations, indicating that the dose of Z-VAD tested was functional. As before, TNF/CHX-inducedrsT1L killed H661 caspase-3/7and H1299 cells activity to a greater was completelyextent than rsT3D inhibited (Figure in each8A,B). cell Similarly, line by rsT1L both and Z-VAD concentrations,rsT3D induced indicating comparable that levels the dose of cell of death Z-VAD in testedH1437, wasH1563, functional. and H1975As cells before, (Figure rsT1L 8C–E). killed We H661 and H1299 cells to a greater extent than rsT3D (Figure8A,B). Similarly, rsT1L and rsT3D induced comparable levels of cell death in H1437, H1563, and H1975 cells (Figure8C–E). We found that Z-VAD VirusesViruses2017 2017, 9,, 1409, 140 9 of9 19 of 19 Viruses 2017, 9, 140 9 of 19 found that Z-VAD had little effect on killing by rsT1L or rsT3D in any of the NSCLC cell lines. hadTogether,found little that effect these Z-VAD on results killing had indicate bylittle rsT1L effect that or caspase on rsT3D killing activity in by any rsT1L is of dispensable the or NSCLC rsT3D for in cell reovirus-inducedany lines. of the Together, NSCLC cell these cell death lines. results in indicatetheTogether, NSCLC that these caspase cell linesresults activity tested. indicate isThese dispensable that findings caspase also foractivity reovirus-induced sugg isest dispensable that strain-specific cellfor reovirus-induced death differences in the NSCLC in cell apoptosis death cell lines in tested.inductionthe NSCLC These are findings cell not lines likely also tested. the suggest underlyingThese that findings strain-specific reason also for sugg the differencesest disparity that strain-specific inin apoptosiscell killing differences induction between in arersT1L apoptosis not and likely thersT3Dinduction underlying in H661 are reason notand likelyH1299 for the the cells. disparity underlying in cell reason killing for between the disparity rsT1L in and cell rsT3D killing in H661between and rsT1L H1299 and cells. rsT3D in H661 and H1299 cells.

FigureFigure 7. 7.Reovirus-induced Reovirus-induced poly-ADP poly-ADP ribose ribose polymerase polymerase (PARP) (PARP) cleavage cleavage in NSCLC in NSCLC cell lines. cell ( lines.A) Figure 7. Reovirus-induced poly-ADP ribose polymerase (PARP) cleavage in NSCLC cell lines. (A) (A)H661; H661; (B (B) H1299;) H1299; (C (C) H1437;) H1437; (D (D) H1563;) H1563; and and (E ()E H1975) H1975 cells cells were were mock mock infe infectedcted (M) (M) or orinfected infected with with H661; (B) H1299; (C) H1437; (D) H1563; and (E) H1975 cells were mock infected (M) or infected with rsT1LrsT1L (T1) (T1) or or rsT3D rsT3D (T3) (T3) at at an an MOI MOI of of 100 100 PFU/cell. PFU/cell. Each cell cell line line was was treated treated with with TNF TNFα α(5(5 ng/mL) ng/mL) andrsT1L CHX (T1) (25 or μrsT3Dg/mL) (T3) (T/C) at 6an h MOIprior of to 100 the PFU/cell.24 h time Each point. cell Whole line was cell treatedlysates prwithepared TNF αat (524, ng/mL) 48, or and CHX (25 µg/mL) (T/C) 6 h prior to the 24 h time point. Whole cell lysates prepared at 24, 48, or 72and h CHX post-infection (25 μg/mL) were (T/C) analyzed6 h prior toby the SDS-PA 24 h timeGE point.and immunoblotted Whole cell lysates using prepared a PARP-specific at 24, 48, or 72 h post-infection were analyzed by SDS-PAGE and immunoblotted using a PARP-specific monoclonal monoclonal72 h post-infection antibody (top)were andanalyzed β-actin by(bottom). SDS-PA Full-lengthGE and andimmunoblotted cleaved PARP using proteins a PARP-specific are indicated antibody (top) and β-actin (bottom). Full-length and cleaved PARP proteins are indicated to the right tomonoclonal the right of antibody the upper (top) panel. and Theβ-actin images (bottom). are representative Full-length and of twocleaved independent PARP proteins experiments. are indicated of theto the upper right panel. of the Theupper images panel. are The representative images are representative of two independent of two independent experiments. experiments.

Figure 8. Reovirus-induced killing in NSCLC cell lines is caspase-independent. (A) H661; (B) H1299; Figure(FigureC) H1437; 8. Reovirus-induced8. Reovirus-induced (D) H1563; and killing( Ekilling) H1975 in inNSCLC cellsNSCLC were cellcell mock lineslines infected is caspase-independent. or infected with rsT1L(A (A) H661;) H661;or rsT3D (B ()B H1299;) at H1299; an (C)MOI( H1437;C) H1437; of 100 (D PFU/cell()D H1563;) H1563; in and theand (absenceE ()E H1975) H1975 or cellspresence cells were were of mock mock25 μM infectedinfected Z-VAD. or Cellular infected infected ATP with with content rsT1L rsT1L was or or rsT3D quantified rsT3D at atan an MOIatMOI 48 of 100hof post-infection.100 PFU/cell PFU/cell in in theCell the absence absenceviability oror is presencepresence shown as of a 25 percentage μµMM Z-VAD. Z-VAD. of CellularATP Cellular content ATP ATP contentin content mock-infected was was quantified quantified cells at 48(left).at h48 post-infection. Eachh post-infection. cell line was Cell Cell treated viability viability with is shown 25is shownμM asZ-VAD aas percentage a percentagefor 1 h prior of ATPof to ATP mock content content treatment in mock-infected in mock-infected or treatment cells withcells (left). EachTNF(left). cellα Each(5 line ng/mL) wascell line treated and was CHX withtreated (25 25 μwithµg/mL).M Z-VAD25 μ Caspase-3/7M Z-VAD for 1 h for prior activity 1 h toprior mockwas to qu mock treatmentantified treatment at or 24 treatment hor and treatment results with with TNFare α (5 ng/mL)presentedTNFα (5 and ng/mL) as CHXthe mean and (25 µCHXcaspase-3/7g/mL). (25 Caspase-3/7μg/mL). activity Caspase-3/7 relative activity to activitymock was quantified infection was qu antified(right). at 24 h Error andat 24 results barsh and indicate are results presented S.D. are as* thepresented p mean< 0.05 caspase-3/7asrelative the mean to caspase-3/7mock activity infection relative activity as to determinedrelative mock infection to mock by Student’s (right).infection Error (right).t-test. bars TheError indicate data bars presentedindicate SD. * p S.D.< is 0.05 relativerepresentative* p < to0.05 mock relative of infection two to independent mock as determined infection experiments. as by determined Student’s t -test.by Student’s The data t presented-test. The data is representative presented is of tworepresentative independent of experiments. two independent experiments.

Viruses 2017, 9, 140 10 of 19 Viruses 2017, 9, 140 10 of 19 2.5. Reovirus Gene Segments L2, L3, and M1 Correlate with Strain-Specific Differences in Cell Killing 2.5. Reovirus Gene Segments L2, L3, and M1 Correlate with Strain-Specific Differences in Cell Killing To identify reovirus gene segments responsible for differential killing of the two large cell carcinomaTo identify lines by reovirus rsT1L geneand segmentsrsT3D, we responsible used reverse for genetics differential to killingengineer of thereciprocal two large panels cell carcinoma of T1L × T3Dlines single-gene by rsT1L and reassortant rsT3D, we used viruses. reverse We genetics generated to engineer a complete reciprocal set panelsof 20 ofsingle-geneT1L × T3D reassortantsingle-gene virusesreassortant in which viruses. all We ten generated gene segments a complete were set individually of 20 single-gene replaced reassortant in rsT1L viruses or inrsT3D which genetic all ten backgrounds.gene segments We were infected individually H661 and replaced H1299 in rsT1Lcells with or rsT3D rsT1L, genetic rsT3D, backgrounds. or the reassortant We infected viruses H661 at andan MOIH1299 of cells100 PFU/cell with rsT1L, and rsT3D, measured or the ATP reassortant content virusesat 72 h post-infection at an MOI of 100 as PFU/cellan indicator and of measured cell viability ATP (Figurecontent 9). at 72In hboth post-infection cell lines, asthe an parental indicator viruses of cell retained viability (Figurethe serotype-specific9). In both cell differences lines, the parental in cell killingviruses described retained above the serotype-specific (Figure 1). No single differences gene fr inom cell T3D killing reduced described the cell above killing (Figure capacity1). No of rsT1L single togene levels from observed T3D reduced for rsT3D the in cell either killing cell capacity line. In H661 of rsT1L cells, to replacement levels observed of T1L for L2, rsT3D L3, inM1, either M3, S3, cell orline. S4 gene In H661 with cells, the T3D replacement allele decreased of T1L killing L2, L3, by M1, viruses M3, S3, with or S4an geneotherwise with T1L the T3Dgenetic allele background decreased (Figurekilling by9A, viruses upper withpanel). an In otherwise H1299 cells, T1L geneticall of the background individual(Figure T3D gene9A, upperreplacements panel). except In H1299 for cells, M2 decreasedall of the individualthe cell killing T3D capacity gene replacements of the virus (Figure except for9B, M2upper decreased panel). In the both cell H661 killing and capacity H1299 ofcells, the T1Lvirus (Figure L2,9 L3,B, upperand M1 panel). enhanced In both cell H661killing and of an H1299 otherwise cells, T1LT3D genes virus, L2, although L3, and not M1 to enhanced the level ofcell rsT1L killing (Figure of an 9A,B, otherwise lower T3D panels). virus, These although findings not toindicate the level that of the rsT1L L2, (FigureL3, and9 M1A,B, gene lower segments panels). haveThese the findings highest indicatecorrelation that with the differences L2, L3, and between M1 gene rsT1L- segments and rsT3D-mediated have the highest cell correlation killing in H661 with anddifferences H1299 cells. between rsT1L- and rsT3D-mediated cell killing in H661 and H1299 cells.

FigureFigure 9. 9. ReovirusReovirus gene gene segments segments L2, L2, L3, L3, and and M1 M1 mediate mediate serotype-specific serotype-specific differences differences in killing in killing of largeof large cell cellcarcinoma carcinoma cell lines. cell lines. (A) H661 (A) H661or (B) orH1299 (B) H1299 cells were cells mock were infected mock infected or infected or infected with rsT1L, with rsT3D,rsT1L, rsT3D,T3D single-gene T3D single-gene reassortant reassortant viruses viruses (upper (upper panel), panel), or T1L or T1L single-gene single-gene reassortant reassortant viruses viruses (lower(lower panel) panel) at at an an MOI MOI of of 100 100 PFU/cell. PFU/cell. Cellular Cellular ATP ATP content content was was quantified quantified at at 48 48 hh post-infection. post-infection. CellCell viability viability is isshown shown as asa percentage a percentage of ATP of ATP content content in mock-infected in mock-infected cells. cells. Error Error bars indicate bars indicate S.D. * SD.p <* 0.05p < 0.05 relativerelative to tomock mock infection infection as asdetermined determined by by Student’s Student’s t-test.t-test. The The data data presented presented is is representativerepresentative of of two two independent independent experiments. experiments.

WeWe next next assessed assessed replication replication of of L2, L2, L3, L3, and and M1 M1 single-gene single-gene reassortment reassortment viruses viruses in in H661 H661 and and H1299H1299 cells cells (Figure (Figure 10). 10). We We focused focused on on L2, L2, L3, L3, an andd M1 M1 because because those those genes genes exhibited exhibited the the highest highest correlationcorrelation with with differences differences in in cell cell killing killing in in the the reassortant reassortant panel. panel. In In H661 cells, rsT1L/T3D-L3 and and rsT1L/T3D-M1rsT1L/T3D-M1 produced produced lower lower yields yields at at 24 24 h h rela relativetive to to rsT1L. rsT1L. However, However, only only rsT1L/T3D-M1 was was reduced significantly at 48 h. In H1299 cells, only replication of rsT1L/T3D-L3 was substantially lower

Viruses 2017, 9, 140 11 of 19

Viruses 2017, 9, 140 11 of 19 reduced significantly at 48 h. In H1299 cells, only replication of rsT1L/T3D-L3 was substantially lowerthan than rsT1L rsT1L at at24 24h. h.However, However, yields yields for for all threethree T3DT3D single single gene gene replacements replacements (rsT1L/T3D-L2, (rsT1L/T3D-L2, rsT1L/T3D-L3,rsT1L/T3D-L3, and and rsT1L/T3D-M1) rsT1L/T3D-M1) were were at leastat least 10-fold 10-fold reduced reduced compared compared to wild to wild type type rsT1L rsT1L at 48 at h. 48 Inh. the In T3D the T3D genetic genetic background, background, only on thely virusthe virus with with the the T1L T1L L2 geneL2 gene replicated replicated to higherto higher levels levels than than rsT3DrsT3D at 48at 48 h inh bothin both H661 H661 and and H1299 H1299 cells. cells. These These findings findings indicate indicate that that reovirus reovirus replication replication in thein the largelarge cell cell carcinoma carcinoma cell cell lines lines is influencedis influenced by by the the genetic genetic complement complement of theof the virus. virus.

FigureFigure 10. 10.Replication Replication of of single-gene single-gene reassortant reassortant viruses viruses in in large large cell cell carcinoma carcinoma cell celllines. lines. ((AA)) H661H661 or or (B) H1299 cells were were infected infected with with rsT1L, rsT1L, rsT3D, rsT3D, or or the the indicated indicated reassortant reassortant viruses viruses at at an an MOI MOI of 1 of 1PFU/cell. PFU/cell. Viral Viral titers titers were were determined determined in incell cell lysates lysates at at0, 0,24, 24, and and 48 48 hours h post-infection post-infection by by plaque plaque assayassay on on L929 L929 cells. cells. Results Results are are expressed expressed as theas the mean mean viral viral yield yield for for triplicate triplicate samples samples at 24at 24 h (right)h (right) andand 48 h48 (left) h (left) post-infection. post-infection. Error Error bars bars indicate indicate SD. S.D. * p < * 0.05 p < difference0.05 difference between between rsT1L rsT1L and rsT3D and rsT3D as determinedas determined by Student’s by Student’st-test. The t-test. data presentedThe data ispresented representative is re ofpresentative two independent of two experiments. independent experiments. 3. Discussion 3. Discussion Our results indicate that recombinant reoviruses effectively kill NSCLC cell lines (Table2). Importantly,Our results cell killing indicate by non-recombinant that recombinant reoviruses reovirus wases effectively indistinguishable kill NSCLC from cellcell death lines induced(Table 2). byImportantly, recombinant cell strains, killing indicating by non-recombinant that there are notreov significantiruses was differences indistinguishable in cell death from induction cell death betweeninduced recombinant by recombinant and native strains, reoviruses. indicatingThese that results there suggest are not that significant use of recombinant differences reoviruses in cell death to treatinduction cancer can between be as effective recombinant as the nativeand native strains. reoviruses. We also found These that results rsT1L suggest killed the that large use cell of recombinant carcinoma cellreoviruses lines tested to totreat a greater cancer extent can be than as effective rsT3D, suggesting as the native that strains. rsT1L orWe T1-based also found vectors that rsT1L may be killed more the effectivelarge cell oncolytics carcinoma than cell T3-based lines tested viruses to a in greater certain ex tumortent than types. rsT3D, A similar suggestin observationg that rsT1L was madeor T1-based in a mousevectors mammary may be tumor more line,effective where oncolytics T1L induced than more T3-based cell death viruses than in T3D, certain indicating tumor that types. differential A similar susceptibilityobservation of was cancer made cells in to a reovirus mouse strainsmammary is not tumor limited line, to thewhere cells T1L tested induced in this studymore [cell37]. death than T3D,It remains indicating to bethat determined differential why susceptibility the large cellof ca carcinomancer cells to lines reovirus (H661 strains and H1299) is not limited differed to in the susceptibilitycells tested in to this reovirus study killing[37]. (Figure1). One possibility is that H661 and H1229 cells harbor mutationsIt remains in cancer-related to be determined genes or why pathways the large that cell differ carcinoma from those lines in (H661 the adenocarcinoma and H1299) differed lines in (H1437,susceptibility H1563, andto reovirus H1975). killing H1299 (Figure cells contain 1). One a mutation possibility in theis that NRAS H661 gene. and H661H1229 cells cells harbor harbor mutationsmutations in in the cancer-related genes for CDKN2A, genes or SMARCA4, pathways that and differ TP53. from All threethose adenocarcinomain the adenocarcinoma cell lines lines contain(H1437, mutations H1563, inand the H1975). CDKN2A H1299 gene, cells H1437 contain and H1975a mutation cells containin the NRAS mutations gene. in H661 the TP53 cells gene,harbor andmutations H1975 cells in havethe genes mutations for CDKN2A, in the EGFR SMARCA4, and PIK3CA and genes. TP53. It All is possible three adenocarcinoma that mutations in cell other lines cancer-relatedcontain mutations genes associatein the CDKN2A with strain-specific gene, H1437 and differences H1975 cells in cell contain killing. mutations However, in based the TP53 on thegene, currentand H1975 information cells have available, mutations no commonin the EGFR genetic and PIK3CA marker correlatesgenes. It is withpossible differences that mutations in cell deathin other inductioncancer-related between genes rsT1L associate and rsT3D with in strain-specific H661 and H1299 differences cells. A second in cell possibilitykilling. However, is whether based the on cell the linescurrent derive information from primary available, or metastatic no common tumors (Tablegenetic1). marker Large cell correlates carcinoma with cell differences lines H661 andin cell H1299 death wereinduction isolated between from metastatic rsT1L and growths, rsT3D in whereas H661 and adenocarcinoma H1299 cells. A cellsecond lines possibility H1563 and is H1975whether derive the cell fromlines a primary derive from tumor primary mass. However, or metastatic H1437 tumors cells were(Table generated 1). Large from cell acarcinoma lung metastasis. cell lines Moreover, H661 and H1299 were isolated from metastatic growths, whereas adenocarcinoma cell lines H1563 and H1975 derive from a primary tumor mass. However, H1437 cells were generated from a lung metastasis. Moreover, data from mouse models and clinical trials indicate that T3D (Reolysin) can target and kill

Viruses 2017, 9, 140 12 of 19 data from mouse models and clinical trials indicate that T3D (Reolysin) can target and kill metastases in mouse models and humans [22,38,39]. Thus, whether cell lines derive from primary versus metastatic tumors is unlikely to underlie strain-specific differences in cell killing. Third, differential susceptibility to reovirus-induced cell death could reflect properties of the cell type of origin. Large cell carcinomas are undifferentiated tumors that originate from lung transformed epithelial cells [40], whereas adenocarcinomas derive from epithelial glands or ducts [41]. Although we found differential susceptibility to rsT3D between large cell carcinoma and adenocarcinoma cell lines, a previous study found that different adenocarcinoma and large cell carcinoma cell lines were equivalently susceptible to killing by Reolysin [42]. However, the same study identified bronchiolar carcinoma and lung squamous cell carcinoma cell lines that were Reolysin resistant [42]. Variable resistance to reovirus is observed in other cancer cell lines, including head and neck squamous cell carcinoma cell lines and pancreatic cancer cell lines [43,44]. It is common for human cancers to vary in numerous properties, such as growth rate, drug sensitivity, and invasiveness [45]. The same type of tumor can differ greatly between patients, and individual tumors are comprised of strikingly heterogeneous cell populations [45]. Tumor heterogeneity is a primary source of resistance to conventional chemotherapeutics [45]. Presumably, the mixed cell populations that comprise tumors will contain at least some cells that are resistant to reovirus. In clinical trials, some tumors respond well to reovirus therapy, whereas others were refractory to treatment [23]. Our results indicate that cancer cell lines resistant to one strain of reovirus may be susceptible to a different strain. In addition, serial passage in cancer cells can be used to select reovirus variants with enhanced cancer cell killing capacity [24]. Thus, many opportunities exist to use reverse genetics to expand the oncolytic range or potency of reovirus.

Table 2. Summary of results. Cell killing, infectivity, replication, and apoptosis induction by recombinan strains (rs)T1L and rsT3D in each cell line was ranked on a graded scale. Cell killing was scored based on the percent loss of cell viability at 72 h at a multiplicity of infection (MOI) of 1000 plaque forming units (PFU)/cell (Figure1) (+, less than 50%; ++, 50–80%; +++, 80–100%). Infectivity was scored based on the percentage of cells infected at 24 h (Figure2) (+, less than 30%; ++, 30–80%; +++, 80–100%). Replication was scored based on viral progeny yields generated at 48h (Figure5) (+, 0–2 log10; ++, 2–4 log10; +++, greater than 4 log10). Apoptosis induction was scored based on fold caspase-3/7 activation at 48 h at an MOI of 1000 PFU/cell (Figure6)( −, no apoptosis induction; +, 1–2-fold; ++, 2–4-fold; +++, greater than 4-fold).

Cell Killing Infectivity Replication Apoptosis Induction Cell Line rsT1L rsT3D rsT1L rsT3D rsT1L rsT3D rsT1L rsT3D H661 +++ ++ +++ +++ +++ +++ − − H1299 +++ + +++ ++ ++ ++ − − H1437 ++ ++ + ++ + ++ − + H1563 +++ +++ ++ ++ +++ ++ +++ +++ H1975 ++ ++ + + ++ ++ + +

The difference in cell killing between rsT1L and rsT3D in H661 and H1299 cells also could result from distinct cell death mechanisms induced by T1 and T3 reoviruses. In cultured cells and in vivo, T3 reoviruses induce apoptosis, whereas T1 reoviruses induce minimal apoptosis and kills cells by an undefined mechanism [46–49]. Reolysin induces apoptosis in many different primary cancer cells and cancer cell lines, tumor xenografts in mice, and patients [44,50–52]. It is possible that H661 and H1299 cells have impaired apoptotic responses, which is a common feature of transformed cells. The inability to undergo apoptosis could prevent killing by rsT3D, but not rsT1L. However, we found that TNF-α/CHX induced apoptotic markers (caspase-3/7 activation (Figure6) and PARP cleavage (Figure7)) in H661 and H1299 cells, indicating that apoptotic responses remain intact in both cell lines. Moreover, minimal caspase-3/7 activity (Figure6) and PARP cleavage (Figure7) were observed following infection with either rsT1L or rsT3D in any of the cell lines tested. Furthermore, treatment Viruses 2017, 9, 140 13 of 19 with the broad-spectrum caspase inhibitor Z-VAD-FMK did not prevent killing by either reovirus strain in any of the cell lines tested (Figure8). Taken together, these findings indicate that rsT1L and rsT3D can induce caspase-independent cell death in each NSCLC cell line used in this study. Caspase activity also is dispensable for killing of head and neck cancer cell lines [44]. In cancer patients, the mechanism of reovirus oncolysis is not well understood. In addition to apoptosis, reovirus also can induce autophagy and programmed necrosis [53,54]. It is possible that reovirus elicits multiple cell death pathways and that the cellular environment dictates the mechanism of cell killing. We found that the L2, L3, and M1 genes associate with differences in killing between rsT1L and rsT3D in H661 and H1299 cells. The L2, L3, and M1 genes encode viral proteins λ2, λ1, and µ2, respectively [25]. Protein λ2 is an outer capsid component that forms pentamers at the 5-fold vertices of the reovirus virion [55,56]. A channel in the center of the λ2 pentamer serves as the insertion site for the σ1 protein [57]. Once σ1 dissociates during viral entry into target cells, the channel functions as the exit site for newly synthesized viral RNAs [58]. The λ2 protein also has guanylyltransferase [59] and methyltransferase [57] activity that is required for addition of 50 cap to nascent reovirus transcripts. The λ1 protein is an inner capsid constituent that is present in low amounts in the virion (~120 copies) [60]. Protein λ1 has RNA helicase activity that may function in unwinding of genomic dsRNAs to initiate [61–63]. The λ1 protein also has RTPase activity [63,64], which in combination with its helicase activity suggests that λ1 contributes to capping of reovirus mRNAs. The µ2 protein also is a low abundance inner capsid component (~24 copies) [60]. Protein µ2 is hypothesized to function as a polymerase cofactor that enhances reovirus replication in a number of cell lines by enhancing RNA synthesis [57]. However, the mechanism by which µ2 promotes reovirus RNA production is undefined. The µ2 protein also is implicated in serotype-specific differences in type 1-interferon induction and sensitivity [65–67]. It is not known whether λ1, λ2, and µ2 function independently or in concert to enhance rsT1L-mediated killing of H661 and H1299 cells. Although rsT1L and rsT3D replication in H661 and H1299 cells did not differ (Figure5), replication of several of the single-gene reassortant viruses differed from the parental strains. We observed a general trend that insertion of T3D genes into a T1L genetic background decreased viral replication, whereas insertion of T1L genes into the T3D genetic background increased viral progeny production (Figure 10). Although differences in viral replication do not underlie differential cell killing by the parental viruses, variation in cell killing by single gene reassortant viruses may simply reflect differences in viral replication. It is important to note that although rsT3D and Reolysin originally derive from laboratory strain T3D, each has a unique passage history and genetic differences between the two strains that could impact oncolytic efficacy. It remains to be determined whether H661 and H1299 cells are more resistant to Reolysin than rsT1L. Next generation sequencing of Reolysin stocks revealed 32 nucleotide changes between Reolysin and T3D reference sequences in Genbank, 18 of which resulted in protein coding changes [68]. A subsequent comparison of the Reolysin sequence and sequences of plasmid-encoded rsT3D gene segments identified 20 amino acid differences between the viruses [69]. These findings indicate that Reolysin and rsT3D are not highly divergent. Construction of the Reolysin polymorphisms in the rsT3D background will be important for mechanistic studies of reovirus oncolysis and an important step in moving recombinant reovirus to the clinic. In this study, we assessed the oncolytic potential of recombinant reoviruses generated by plasmid-based reverse genetics. To our knowledge, this is the first characterization of cancer cell killing by recombinant reoviruses generated using plasmid-based reverse genetics [20]. Our findings suggest that in certain types of tumors, T1 reovirus-based oncolytics may be more effective than T3-based vectors. Importantly, the cell killing profiles of recombinant reoviruses mirrors that of the native strains, suggesting that recombinant viruses will be as effective as traditional strains with respect to their oncolytic capacity. This work will inform future efforts to improve the efficacy and safety of reovirus oncolytics. Viruses 2017, 9, 140 14 of 19

4. Materials and Methods

4.1. Cell Lines Lung cancer cell lines H661, H1299, H1437, H1563, and H1975 were obtained from the American Type Culture Collection (ATCC). All lung cancer cells were maintained at 37 ◦C in 5% CO2 in RPMI Medium 1640 (Gibco, Gaithersburg, MD, USA) supplemented to contain 10% fetal bovine serum ◦ (FBS) and 2mM L-glutamine. L929 cells were maintained in spinner culture at 37 C in Joklik’s MEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented to contain 5% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 ng/mL amphotericin B (Sigma-Aldrich).

4.2. Viruses Non-recombinant reovirus strains T1L and T3D are laboratory stocks. Recombinant reoviruses were generated using plasmid-based reverse genetics as described [28,29]. Purified viral stocks were generated using second or third passage L929 cell lysates as described previously [70]. Viral particles were Vertrel-XF (Microcare, New Britain, CT, USA)-extracted from infected cell lysates, layered onto 1.2- to 1.4-g/cm3 CsCl gradients, and centrifuged at 29,000 rpm for 4 h. Bands corresponding to virions 3 (1.36 g/cm )[71] were collected and dialyzed in virion-storage buffer (150 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl (pH 7.4)). Plaque assay using L929 cells was used to determine viral titer [72].

4.3. Cell Viability Assay Monolayers of cells in 96-well plates (1 × 104 cells/well) were mock infected or adsorbed in triplicate with reovirus at room temperature (RT) for 1 h. The MOIs were calculated on PFU titers determined on L929 cells or FFU titers determined on H1299 or 1563 cells, as indicated. Monolayers were washed twice with phosphate-buffered saline (PBS) and incubated at 37 ◦C in completed media for various intervals. CellTiter-Glo (Promega, Madison, WI, USA) was used to assess cell viability [73–75]. Briefly, cells were cooled to RT, 100 µL of CellTiter-Glo was added to each well, and the plate was placed on a multi-purpose rotator for 2 min. Plates were incubated at RT for 10 min and luminescence was measured using a FluoStar Omega plate reader (BMG LabTech, Ortenberg, Germany). Where indicated, cells were treated with 25 µM Z-VAD-FMK (Sigma-Aldrich) or DMSO vehicle control for 1 h prior to infection. Following adsorption, the cells were overlaid with media containing DMSO or Z-VAD-FMK.

4.4. Virus Replication Monolayers of cells in 24-well plates (1 × 105 cells/well) were adsorbed with reovirus at a range of multiplicities at RT for 1 h. The monolayers were washed twice with PBS and incubated in completed media at 37 ◦C. At the indicated time points, cells were frozen and thawed twice, and viral titers in lysates were determined using plaque assay on L929 cells [72]. Viral yields were calculated using the following formula:

log 10yieldtx = log 10(PFU/mL)tx − log 10(PFU/mL)t0 (1) where tx is the time post-infection.

4.5. Active Caspase-3/7 Assay Monolayers of cells (1 × 104 cells/well) in 96-well plates were mock infected or adsorbed in triplicate with reovirus at the indicated multiplicities at RT for 1 h. The monolayers were washed twice with PBS and incubated at 37 ◦C in completed media for various intervals. The cells were cooled to RT, 100 µL of Caspase-Glo 3/7 (Promega) was added to each well and the plates were incubated in the dark at RT for 1 h. Luminescence was measured using a FluoStar Omega plate reader (BMG LabTech). Viruses 2017, 9, 140 15 of 19

4.6. Immunoblot Assay Monolayers of cells (5 × 105 cells/well) in 12-well plates were mock infected or adsorbed with reovirus at an MOI of 10 PFU/cell for 1h at RT. The monolayers were washed twice with PBS and incubated at 37 ◦C in completed media for various intervals. Cells were lysed with RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 0.1% SDS, 0.1% deoxycholate) with 1% protease inhibitor cocktail (Sigma-Aldrich) and 1% phosphatase inhibitor cocktail (Sigma-Aldrich). Proteins were separated by SDS-PAGE and immunoblotted [49] using reovirus—(1:10,000 dilution), PARP—(Cell Signaling; 1:1000 dilution), or β-actin-specific (Sigma-Aldrich; 1:10,000 dilution) antibodies. Reovirus and β-actin antibodies were detected using goat-anti-rabbit Alexa-488 and goat-anti-mouse Alexa-546, respectively (1:5000 each). PARP and β-actin were detected using appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA, 1:5000) and Super Signal West Pico reagent (Thermo-Fisher Scientific, Waltham, MA, USA). All blots were scanned using a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). Imaging times were kept consistent for each cell line for replicate experiments. For viral protein quantitation, the band intensity for viral proteins was normalized to the intensity of the actin band in the corresponding land. The actin-normalized values for rsT1L and rsT3D were then compared. Viral protein quantitation was performed using Image Lab software, version 4.1 (Bio-Rad).

4.7. Indirect Immunofluorescence Monolayers of cells in 96-well dishes (1 × 104 cells/well) were adsorbed with reovirus at an MOI of 100 PFU/cell for 1h at RT. The monolayers were washed twice with PBS and incubated at 37 ◦C in completed media for 24 h. The media was removed and cells were fixed in methanol for 30 min at −20 ◦C. Fixed cells were incubated with PBS + 5% bovine serum albumin (BSA) for 15 min at RT followed by staining with reovirus-specific polyclonal antiserum (1:500 dilution in PBS + 0.5% Triton X-100) for 30 min at 37 ◦C. The cells were washed three times with PBS and incubated with Alexa-488-conjugated goat-anti-rabbit IgG ( Technologies, Thermo-Fisher Scientific, 1:1000 dilution in PBS + 0.5% Triton X-100) and DAPI (1:1000 dilution) at 30 min at 37 ◦C. Cells were washed three times with PBS and visualized with an EVOS-FL Auto imaging system (Life Technologies, Thermo-Fisher Scientific). Infected cells were quantified by counting the number of reovirus antigen-positive cells along with the number of DAPI-stained nuclei (total cell number) in three separate fields of view in two independent experiments. The percentage of infected cells was calculated by dividing the number of reovirus antigen-positive cells by the number of DAPI-positive nuclei.

4.8. Statistical Analysis Means for cell viability experiments were determined for three independent experiments, each of which contained three replicates. Differences were determined using random effects analysis of variance. Differences in cell killing between recombinant and non-recombinant viruses, cell infectivity, viral replication, and caspase-3/7 activity were determined using an unpaired Student’s t-test. Statistical analysis was performed using Prism software (GraphPad Software Inc., La Jolla, CA, USA). p values <0.05 were considered to be statistically significant.

Acknowledgments: We thank Pranav Danthi, Joseph Koon, and Matthew Phillips for critical review of the manuscript. This work was supported by Public Health Service awards K22 AI94079 (K.W.B.), P20 GM103625 (K.W.B.), and the University of Arkansas for Medical Sciences Medical Research Endowment. Author Contributions: E.J.S. and K.W.B conceived and designed the experiments; E.J.S., M.J.H., J.D.S., and K.W.B. performed the experiments; E.J.S., M.J.H., J.D.S., and K.W.B. analyzed the data; E.J.S., M.J.H., J.D.S., and K.W.B. wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Viruses 2017, 9, 140 16 of 19

References

1. Russell, S.J.; Peng, K.W.; Bell, J.C. Oncolytic virotherapy. Nat. Biotechnol. 2012, 30, 658–670. [CrossRef][PubMed] 2. Miest, T.S.; Cattaneo, R. New viruses for cancer therapy: Meeting clinical needs. Nat. Rev. Microbiol. 2014, 12, 23–34. [CrossRef][PubMed] 3. Duncan, M.R.; Stanish, S.M.; Cox, D.C. Differential sensitivity of normal and transformed human cells to reovirus infection. J. Virol. 1978, 28, 444–449. [PubMed] 4. Coffey, M.C.; Strong, J.E.; Forsyth, P.A.; Lee, P.W. Reovirus therapy of tumors with activated Ras pathway. Science 1998, 282, 1332–1334. [CrossRef][PubMed] 5. Hirasawa, K.; Nishikawa, S.G.; Norman, K.L.; Alain, T.; Kossakowska, A.; Lee, P.W. Oncolytic reovirus against ovarian and colon cancer. Cancer Res. 2002, 62, 1696–1701. [PubMed] 6. Norman, K.L.; Coffey, M.C.; Hirasawa, K.; Demetrick, D.J.; Nishikawa, S.G.; DiFrancesco, L.M.; Strong, J.E.; Lee, P.W. Reovirus oncolysis of human breast cancer. Hum. Gene. Ther. 2002, 13, 641–652. [CrossRef][PubMed] 7. Strong, J.E.; Coffey, M.C.; Tang, D.; Sabinin, P.; Lee, P.W. The molecular basis of viral oncolysis: Usurpation of the Ras signaling pathway by reovirus. EMBO J. 1998, 17, 3351–3362. [CrossRef][PubMed] 8. Wilcox, M.E.; Yang, W.; Senger, D.; Rewcastle, N.B.; Morris, D.G.; Brasher, P.M.; Shi, Z.Q.; Johnston, R.N.; Nishikawa, S.; Lee, P.W.; et al. Reovirus as an oncolytic agent against experimental human malignant gliomas. J. Natl. Cancer Inst. 2001, 93, 903–912. [CrossRef][PubMed] 9. Comins, C.; Heinemann, L.; Harrington, K.; Melcher, A.; De Bono, J.; Pandha, H. Reovirus: Viral therapy for cancer “as nature intended”. Clin. Oncol. 2008, 20, 548–554. [CrossRef][PubMed] 10. Comins, C.; Spicer, J.; Protheroe, A.; Roulstone, V.; Twigger, K.; White, C.M.; Vile, R.; Melcher, A.; Coffey, M.C.; Mettinger, K.L.; et al. REO-10: A phase I study of intravenous reovirus and docetaxel in patients with advanced cancer. Clin. Cancer Res. 2010, 16, 5564–5572. [CrossRef][PubMed] 11. Turnbull, S.; West, E.J.; Scott, K.J.; Appleton, E.; Melcher, A.; Ralph, C. Evidence for Oncolytic Virotherapy: Where Have We Got to and Where Are We Going? Viruses 2015, 7, 6291–6312. [CrossRef][PubMed] 12. Pol, J.; Kroemer, G.; Galluzzi, L. First oncolytic virus approved for melanoma immunotherapy. Oncoimmunology 2016, 5, e1115641. [CrossRef][PubMed] 13. Liu, B.L.; Robinson, M.; Han, Z.Q.; Branston, R.H.; English, C.; Reay, P.; McGrath, Y.; Thomas, S.K.; Thornton, M.; Bullock, P.; et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther. 2003, 10, 292–303. [CrossRef][PubMed] 14. Vacchelli, E.; Eggermont, A.; Fridman, W.H.; Galon, J.; Zitvogel, L.; Kroemer, G.; Galluzzi, L. Trial Watch: Immunostimulatory cytokines. Oncoimmunology 2013, 2, e24850. [CrossRef][PubMed] 15. Stanford, M.M.; Barrett, J.W.; Nazarian, S.H.; Werden, S.; McFadden, G. Oncolytic virotherapy synergism with signaling inhibitors: Rapamycin increases myxoma virus tropism for human tumor cells. J. Virol. 2007, 81, 1251–1260. [CrossRef][PubMed] 16. Bischoff, J.R.; Kirn, D.H.; Williams, A.; Heise, C.; Horn, S.; Muna, M.; Ng, L.; Nye, J.A.; Sampson-Johannes, A.; Fattaey, A.; et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 1996, 274, 373–376. [CrossRef][PubMed] 17. Au, G.G.; Lindberg, A.M.; Barry, R.D.; Shafren, D.R. Oncolysis of vascular malignant human melanoma tumors by Coxsackievirus A21. Int. J. Oncol. 2005, 26, 1471–1476. [CrossRef][PubMed] 18. Grote, D.; Russell, S.J.; Cornu, T.I.; Cattaneo, R.; Vile, R.; Poland, G.A.; Fielding, A.K. Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice. Blood 2001, 97, 3746–3754. [CrossRef][PubMed] 19. Stojdl, D.F.; Lichty, B.; Knowles, S.; Marius, R.; Atkins, H.; Sonenberg, N.; Bell, J.C. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat. Med. 2000, 6, 821–825. [PubMed] 20. Mohamed, A.; Johnston, R.N.; Shmulevitz, M. Potential for Improving Potency and Specificity of Reovirus Oncolysis with Next-Generation Reovirus Variants. Viruses 2015, 7, 6251–6278. [CrossRef][PubMed] 21. Sabin, A.B. Reoviruses: A new group of respiratory and enteric viruses formerly classified as ECHO type 10 is described. Science 1959, 130, 1387–1389. [CrossRef][PubMed] 22. Galanis, E.; Markovic, S.N.; Suman, V.J.; Nuovo, G.J.; Vile, R.G.; Kottke, T.J.; Nevala, W.K.; Thompson, M.A.; Lewis, J.E.; Rumilla, K.M.; et al. Phase II trial of intravenous administration of Reolysin (Reovirus Serotype-3- dearing Strain) in patients with metastatic melanoma. Mol. Ther. 2012, 20, 1998–2003. [CrossRef][PubMed] Viruses 2017, 9, 140 17 of 19

23. Morris, D.G.; Feng, X.; DiFrancesco, L.M.; Fonseca, K.; Forsyth, P.A.; Paterson, A.H.; Coffey, M.C.; Thompson, B. REO-001: A phase I trial of percutaneous intralesional administration of reovirus type 3 dearing (Reolysin) in patients with advanced solid tumors. Investig. New Drugs 2013, 31, 696–706. [CrossRef] [PubMed] 24. Shmulevitz, M.; Gujar, S.A.; Ahn, D.G.; Mohamed, A.; Lee, P.W. Reovirus variants with mutations in genome segments S1 and L2 exhibit enhanced virion infectivity and superior oncolysis. J. Virol. 2012, 86, 7403–7413. [CrossRef][PubMed] 25. Dermody, T.S.; Parker, J.S.L.; Sherry, B. Orthoreovirus. In Fields Virology, 6th ed.; Knipe, D.M., Howley, P.M., Eds.; Lippincott, Williams & Wilkins: Philadelphia, PA, USA, 2013; Volume 2, pp. 1304–1346. 26. Greaves, M.; Maley, C.C. Clonal evolution in cancer. Nature 2012, 481, 306–313. [CrossRef][PubMed] 27. Meacham, C.E.; Morrison, S.J. Tumour heterogeneity and cancer cell plasticity. Nature 2013, 501, 328–337. [CrossRef][PubMed] 28. Kobayashi, T.; Antar, A.A.; Boehme, K.W.; Danthi, P.; Eby, E.A.; Guglielmi, K.M.; Holm, G.H.; Johnson, E.M.; Maginnis, M.S.; Naik, S.; et al. A plasmid-based reverse genetics system for animal double-stranded RNA viruses. Cell Host Microbe 2007, 1, 147–157. [CrossRef][PubMed] 29. Kobayashi, T.; Ooms, L.S.; Ikizler, M.; Chappell, J.D.; Dermody, T.S. An improved reverse genetics system for mammalian . Virology 2010, 398, 194–200. [CrossRef][PubMed] 30. Komoto, S.; Kawagishi, T.; Kobayashi, T.; Ikizler, M.; Iskarpatyoti, J.; Dermody, T.S.; Taniguchi, K. A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase. J. Virol. Methods 2014, 196, 36–39. [CrossRef][PubMed] 31. Kanai, Y.; Komoto, S.; Kawagishi, T.; Nouda, R.; Nagasawa, N.; Onishi, M.; Matsuura, Y.; Taniguchi, K.; Kobayashi, T. Entirely plasmid-based reverse genetics system for rotaviruses. Proc. Natl. Acad. Sci. USA 2017, 114, 2349–2354. [CrossRef][PubMed] 32. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer Statistics, 2017. CA Cancer J. Clin. 2017, 67, 7–30. [CrossRef] [PubMed] 33. American Lung Association. Available online: http://www.lung.org (accessed on 29 May 2017). 34. U.S. National Institutes of Health; National Cancer Institute. SEER Cancer Statistics Review, 1975–2013. Available online: https://seer.cancer.gov/archive/csr/1975_2013/ (accessed on 29 May 2017). 35. American Cancer Society. Available online: http://www.cancer.org (accessed on 29 May 2017). 36. Danthi, P.; Kobayashi, T.; Holm, G.H.; Hansberger, M.W.; Abel, T.W.; Dermody, T.S. Reovirus apoptosis and virulence are regulated by host cell membrane penetration efficiency. J. Virol. 2008, 82, 161–172. [CrossRef] [PubMed] 37. Roner, M.R.; Mutsoli, C. The use of monoreassortants and reverse genetics to map reovirus lysis of a ras-transformed cell line. J. Virol. Methods 2007, 139, 132–142. [CrossRef][PubMed] 38. Yang, W.Q.; Senger, D.L.; Lun, X.Q.; Muzik, H.; Shi, Z.Q.; Dyck, R.H.; Norman, K.; Brasher, P.M.; Rewcastle, N.B.; George, D.; et al. Reovirus as an experimental therapeutic for brain and leptomeningeal metastases from breast cancer. Gene Ther. 2004, 11, 1579–1589. [CrossRef][PubMed] 39. Kawaguchi, K.; Etoh, T.; Suzuki, K.; Mitui, M.T.; Nishizono, A.; Shiraishi, N.; Kitano, S. Efficacy of oncolytic reovirus against human gastric cancer with peritoneal metastasis in experimental animal model. Int. J. Oncol. 2010, 37, 1433–1438. [CrossRef][PubMed] 40. Travis, W.D.; Brambilla, E.; Noguchi, M.; Nicholson, A.G.; Geisinger, K.; Yatabe, Y.; Ishikawa, Y.; Wistuba, I.; Flieder, D.B.; Franklin, W.; et al. Diagnosis of lung cancer in small biopsies and cytology: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Arch. Pathol. Lab. Med. 2013, 137, 668–684. [CrossRef][PubMed] 41. Travis, W.D.; Brambilla, E.; Noguchi, M.; Nicholson, A.G.; Geisinger, K.; Yatabe, Y.; Ishikawa, Y.; Wistuba, I.; Flieder, D.B.; Franklin, W.; et al. Diagnosis of lung adenocarcinoma in resected specimens: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Arch. Pathol. Lab. Med. 2013, 137, 685–705. [CrossRef][PubMed] 42. Sei, S.; Mussio, J.K.; Yang, Q.E.; Nagashima, K.; Parchment, R.E.; Coffey, M.C.; Shoemaker, R.H.; Tomaszewski, J.E. Synergistic antitumor activity of oncolytic reovirus and chemotherapeutic agents in non-small cell lung cancer cells. Mol. Cancer 2009, 8, 47. [CrossRef][PubMed] 43. Etoh, T.; Himeno, Y.; Matsumoto, T.; Aramaki, M.; Kawano, K.; Nishizono, A.; Kitano, S. Oncolytic viral therapy for human pancreatic cancer cells by reovirus. Clin. Cancer Res. 2003, 9, 1218–1223. [PubMed] Viruses 2017, 9, 140 18 of 19

44. Twigger, K.; Roulstone, V.; Kyula, J.; Karapanagiotou, E.M.; Syrigos, K.N.; Morgan, R.; White, C.; Bhide, S.; Nuovo, G.; Coffey, M.; et al. Reovirus exerts potent oncolytic effects in head and neck cancer cell lines that are independent of signalling in the EGFR pathway. BMC Cancer 2012, 12, 368. [CrossRef][PubMed] 45. Marusyk, A.; Polyak, K. Tumor heterogeneity: Causes and consequences. Biochim. Biophys. Acta 2010, 1805, 105–117. [CrossRef][PubMed] 46. Tyler, K.L.; Squier, M.K.; Brown, A.L.; Pike, B.; Willis, D.; Oberhaus, S.M.; Dermody, T.S.; Cohen, J.J. Linkage between reovirus-induced apoptosis and inhibition of cellular DNA synthesis: Role of the S1 and M2 genes. J. Virol. 1996, 70, 7984–7991. [PubMed] 47. Tyler, K.L.; Squier, M.K.; Rodgers, S.E.; Schneider, B.E.; Oberhaus, S.M.; Grdina, T.A.; Cohen, J.J.; Dermody, T.S. Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein sigma 1. J. Virol. 1995, 69, 6972–6979. [PubMed] 48. Fleeton, M.N.; Contractor, N.; Leon, F.; Wetzel, J.D.; Dermody, T.S.; Kelsall, B.L. Peyer’s patch dendritic cells process viral antigen from apoptotic epithelial cells in the intestine of reovirus-infected mice. J. Exp. Med. 2004, 200, 235–245. [CrossRef][PubMed] 49. Danthi, P.; Coffey, C.M.; Parker, J.S.; Abel, T.W.; Dermody, T.S. Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain. PLoS Pathog. 2008, 4, e1000248. [CrossRef][PubMed] 50. Thirukkumaran, C.M.; Nodwell, M.J.; Hirasawa, K.; Shi, Z.Q.; Diaz, R.; Luider, J.; Johnston, R.N.; Forsyth, P.A.; Magliocco, A.M.; Lee, P.; et al. Oncolytic viral therapy for prostate cancer: Efficacy of reovirus as a biological therapeutic. Cancer Res. 2010, 70, 2435–2444. [CrossRef][PubMed] 51. Kelly, K.R.; Espitia, C.M.; Mahalingam, D.; Oyajobi, B.O.; Coffey, M.; Giles, F.J.; Carew, J.S.; Nawrocki, S.T. Reovirus therapy stimulates endoplasmic reticular stress, NOXA induction, and augments bortezomib-mediated apoptosis in multiple myeloma. Oncogene 2012, 31, 3023–3038. [CrossRef][PubMed] 52. Carew, J.S.; Espitia, C.M.; Zhao, W.; Kelly, K.R.; Coffey, M.; Freeman, J.W.; Nawrocki, S.T. Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer. Cell Death Dis. 2013, 4, e728. [CrossRef][PubMed] 53. Berger, A.K.; Danthi, P. Reovirus activates a caspase-independent cell death pathway. mBio 2013, 4, e00178-13. [CrossRef][PubMed] 54. Thirukkumaran, C.M.; Shi, Z.Q.; Luider, J.; Kopciuk, K.; Gao, H.; Bahlis, N.; Neri, P.; Pho, M.; Stewart, D.; Mansoor, A.; et al. Reovirus modulates autophagy during oncolysis of multiple myeloma. Autophagy 2013, 9, 413–414. [CrossRef][PubMed] 55. Ralph, S.J.; Harvey, J.D.; Bellamy, A.R. Subunit structure of the reovirus spike. J. Virol. 1980, 36, 894–896. [PubMed] 56. White, C.K.; Zweerink, H.J. Studies on the structure of reovirus cores: Selective removal of polypeptide lambda 2. Virology 1976, 70, 171–180. [CrossRef] 57. Schiff, L.A.; Nibert, M.L.; Tyler, K.L. Orthoreoviruses and their replication. In Fields Virology, 5th ed.; Knipe, D.M., Howley, P.M., Eds.; Lippincott, Williams & Wilkins: Philadelphia, PA, USA, 2007; Volume 2, pp. 1853–1915. 58. Dryden, K.A.; Wang, G.; Yeager, M.; Nibert, M.L.; Coombs, K.M.; Furlong, D.B.; Fields, B.N.; Baker, T.S. Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: Analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction. J. Cell Biol. 1993, 122, 1023–1041. [CrossRef][PubMed] 59. Cleveland, D.R.; Zarbl, H.; Millward, S. Reovirus guanylyltransferase is L2 gene product lambda 2. J. Virol. 1986, 60, 307–311. [PubMed] 60. Coombs, K.M. Stoichiometry of reovirus structural proteins in virus, ISVP, and core particles. Virology 1998, 243, 218–228. [CrossRef][PubMed] 61. Noble, S.; Nibert, M.L. Core protein mu2 is a second determinant of nucleoside triphosphatase activities by reovirus cores. J. Virol. 1997, 71, 7728–7735. [PubMed] 62. Noble, S.; Nibert, M.L. Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein lambda1. J. Virol. 1997, 71, 2182–2191. [PubMed] 63. Bisaillon, M.; Bergeron, J.; Lemay, G. Characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus lambda1 protein. J. Biol. Chem. 1997, 272, 18298–18303. [CrossRef] [PubMed] Viruses 2017, 9, 140 19 of 19

64. Bisaillon, M.; Lemay, G. Characterization of the reovirus lambda1 protein RNA 50-triphosphatase activity. J. Biol. Chem. 1997, 272, 29954–29957. [CrossRef][PubMed] 65. Sherry, B.; Baty, C.J.; Blum, M.A. Reovirus-induced acute myocarditis in mice correlates with viral RNA synthesis rather than generation of infectious virus in cardiac myocytes. J. Virol. 1996, 70, 6709–6715. [PubMed] 66. Sherry, B.; Torres, J.; Blum, M.A. Reovirus induction of and sensitivity to beta interferon in cardiac myocyte cultures correlate with induction of myocarditis and are determined by viral core proteins. J. Virol. 1998, 72, 1314–1323. [PubMed] 67. Zurney, J.; Kobayashi, T.; Holm, G.H.; Dermody, T.S.; Sherry, B. Reovirus mu2 protein inhibits interferon signaling through a novel mechanism involving nuclear accumulation of interferon regulatory factor 9. J. Virol. 2009, 83, 2178–2187. [CrossRef][PubMed] 68. Chakrabarty, R.; Tran, H.; Fortin, Y.; Yu, Z.; Shen, S.H.; Kolman, J.; Onions, D.; Voyer, R.; Hagerman, A.; Serl, S.; et al. Evaluation of homogeneity and genetic stability of REOLYSIN (pelareorep) by complete genome sequencing of reovirus after large scale production. Appl. Microbiol. Biotechnol. 2014, 98, 1763–1770. [CrossRef][PubMed] 69. Sandekian, V.; Lim, D.; Prud’homme, P.; Lemay, G. Transient high level mammalian reovirus replication in a bat epithelial cell line occurs without cytopathic effect. Virus Res. 2013, 173, 327–335. [CrossRef][PubMed] 70. Furlong, D.B.; Nibert, M.L.; Fields, B.N. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 1988, 62, 246–256. [PubMed] 71. Smith, R.E.; Zweerink, H.J.; Joklik, W.K. Polypeptide components of virions, top component and cores of reovirus type 3. Virology 1969, 39, 791–810. [CrossRef] 72. Virgin, H.W.T.; Bassel-Duby, R.; Fields, B.N.; Tyler, K.L. Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing). J. Virol. 1988, 62, 4594–4604. [PubMed] 73. Mainou, B.A.; Zamora, P.F.; Ashbrook, A.W.; Dorset, D.C.; Kim, K.S.; Dermody, T.S. Reovirus cell entry requires functional . mBio 2013, 4, e00405-13. [CrossRef][PubMed] 74. Berger, A.K.; Hiller, B.E.; Thete, D.; Snyder, A.J.; Perez, E., Jr.; Upton, J.W.; Danthi, P. Viral RNA at Two Stages of Reovirus Infection Is Required for the Induction of Necroptosis. J. Virol. 2017, 91, e02404-16. [CrossRef] [PubMed] 75. Van den Hengel, S.K.; Balvers, R.K.; Dautzenberg, I.J.; van den Wollenberg, D.J.; Kloezeman, J.J.; Lamfers, M.L.; Sillivis-Smit, P.A.; Hoeben, R.C. Heterogeneous reovirus susceptibility in human glioblastoma stem-like cell cultures. Cancer Gene Ther. 2013, 20, 507–513. [CrossRef][PubMed]

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