Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

Mammalian reovirus is a naturally benign virus that preferentially replicates in cancer cells (reovirus oncolysis) and has been tested as a potential cancer therapy in vitro, in vivo and in clinical trials for treating cancers from a wide range of origins. Reovirus-induced apoptosis has been shown to be important for reovirus oncolysis. The tumor suppressor protein p53 plays vital roles in mediating host apoptosis but its effect on reovirus oncolysis has not been fully understood and hence investigated. Data here show that p53 does not affect reovirus replication or reovirus-induced apoptosis in human cancer cells. However, significant enhancement of the reovirus-mediated apoptosis is induced by addition of p53 accumulators/activators such as a MDM2 antagonist Nutlin-3a or sub-lethal concentrations of chemotherapy drugs. This enhanced cell death is p53-dependent, requires NF-kappaB activation and p53 target genes p21 and bax. Furthermore, a combination of reovirus and p53 accumulators/activators directly results in significantly higher level of reovirus dissemination and spread. One of the hurdles for current chemotherapy in patients is the side effects caused by high concentrations of cytotoxic drugs. Hence, a therapeutic regime using the combination of reovirus and sub-lethal (lower concentrations of) chemotherapeutics that induce p53 activation/accumulation potentially can both enhance tumor regression and reduce the side effects in patients. Ras mutation, one of the most prevalent mutations in human cancer, has been implicated to determine the susceptibility of cancer cells to reovirus infection. However, the underlying mechanism of reovirus preferential replication needs to be further delineated. Quantitative analysis was used to compare individual steps of reovirus replication between non-transformed and Ras-transformed NIH 3T3 cells. Contrary to previous reports, reoviral protein synthesis is shown to be comparable between non-Ras and Ras-transformed cells. Meanwhile, although reovirus binding and internalization is not affected by Ras-transformation, reovirus uncoating is enhanced in Ras-transformed cells. Ras-transformation also enables reovirus to better spread to neighboring cells through apoptosis. Furthermore, reovirus infection of Ras effector mutant cells that activate specific Ras effector pathways indicate that sub-Ras pathways play different roles in enhancing reovirus preferential replication in Ras-transformed cells and therefore provide additional targets for cancer therapy.

Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

The S1 genome segment of avian reovirus is functionally tricistronic, encoding three independent protein products (named p10, p17 and ?C) from three sequential, partially overlapping open reading frames (ORFs). The dogma of translation initiation, the cap-dependent scanning model, suggests that ribosomes would normally only translate the 5?-proximal ORF. Four alternate mechanisms of translation initiation could account for translation of the downstream ?C ORF; an IRES element, reinitiation, ribosome shunting, and leaky scanning. The objective of my doctoral research was to investigate the translation initiation mechanisms that are operative on the S1 mRNA. Translation of the p10 and p17 ORFs was revealed to be coordinated via standard leaky scanning, while none of the known mechanisms of translation initiation could account for expression of the ?C ORF. Further investigation determined that two alternate cap-dependent mechanisms contribute to translation initiation at the ?C AUG codon. The first mechanism involves a modified version of enhanced leaky scanning. Although insertion of upstream elements known to impede scanning ribosomal subunits dramatically inhibited translation of the downstream ORF in the context of other mRNAs, the same elements only marginally reduced ?C translation. Specific features of the S1 mRNA therefore function to promote leaky scanning and translation of the ?C ORF. The inability to eliminate ?C expression beyond a threshold retention level of ~20-30%, despite the presence of eight upstream start codons that should eliminate leaky scanning, strongly suggests that ribosomes must also utilize a scanning-independent means to access the internal ?C start site. This mechanism for ?C translation initiation, which I termed ribosome handoff, allows ribosomes to bypass upstream elements, and requires a sequence-dependent translation enhancer element present within S1 nucleotides 366-392 that may function to mediate handoff via complementarity with 18S ribosomal RNA. Translation initiation at the ?C start site is therefore made possible by two alternative mechanisms, enhanced leaky scanning and ribosome handoff from the 5?-cap. The novelty of these two mechanisms highlights the complexity of the translation initiation process and the potential heterogeneity of cellular ribosomes, which raises the possibility that internal initiation may be far more common than currently appreciated.

Memoria para optar al Título Profesional de Médico Veterinario. / Piscine reovirus (PRV) es considerado el agente causal de la enfermedad denominada “inflamación del músculo cardíaco y esquelético” (HSMI), una de las afecciones más comunes en salmones del Atlántico (Salmo salar) criados en jaulas marinas en Noruega. En Chile, recientemente también se ha detectado PRV en salmones del Atlántico cultivados en el mar, especie que no es nativa de nuestro país, aunque sin lesiones compatibles con HSMI. A fin de buscar la presencia de PRV en poblaciones de peces nativos marinos silvestres y asociarla con HSMI, se analizaron quince congrios colorados (Genypterus chilensis) que fueron capturados en la Península de Punta Lengua de Vaca de la Región de Coquimbo de Chile. Cada pez fue sometido a una necropsia para registrar sus lesiones macroscópicas generales, examinar su corazón histopatológicamente y detectar PRV desde muestras de riñón anterior mediante reacción en cadena de la polimerasa con transcriptasa inversa en tiempo real. Se encontraron varias lesiones como ascitis, hidropericardio, epicarditis y, especialmente, infiltración de células mononucleares en tejido cardíaco, las cuales son compatibles con HSMI. Sin embargo, se descartó la presencia de HSMI debido a que todos los peces resultaron negativos a PRV. Como hallazgos no patológicos, se encontró folículos tiroideos localizados de modo adyacente al atrio en uno de los congrios y ausencia del estrato compacto del ventrículo en la mayoría de los peces examinados. Los resultados sugieren que PRV no está presente en congrios colorados de la zona geográfica muestreada. Sería útil extender estos estudios para establecer si este virus realmente aún no se encuentra en poblaciones de peces silvestres nativas de nuestro país. / Piscine reovirus (PRV) is described to be the causative agent of “Heart and Skeletal Muscle Inflammation” (HSMI), a highly prevalent disease of sea-cages reared Atlantic salmon (Salmo salar) in Norway. Rather recently, this virus has also been detected in Atlantic salmon cultured in Chilean sea-water, aquatic habitat in which they are not a native species, but these fish did not show lesions consistent with HSMI. In order to search for PRV presence and its association with HSMI in wild marine fish, fifteen red cusk-eels (Genypterus chilensis), captured in the near shore of the Península de Punta Lengua de Vaca in the Coquimbo Region of Chile, were studied. Necropsy, heart histopathology and PRV testing from head kidney samples using real-time reverse transcription polymerase chain reaction were carried out in each fish. Although several lesions associated to HSMI, such as ascitis, liver paleness, hydropericardium, epicarditis and, particularly, mononuclear cell infiltration in heart, were found, all fish resulted negative to PRV and therefore the presence of HSMI, as described so far, was also discarded. Thyroid follicles close to the atrium in one fish and lack of the ventricle compact stratum in most of them were interesting non-pathological findings. Results suggest that PRV would not be present in red cusk-eels of the sampled area. It would be useful to expand this research to establish if Chilean native wild fish are really PRV free.

The tumor-selective viral replication capacity and pro-apoptotic effects of oncolytic reovirus have been reported to be dependent on the presence of an activated RAS pathway in several solid tumor types. However, the mechanisms of selective anticancer efficacy of the reovirus-based formulation for cancer therapy (Reolysin, pelareorep) have not been rigorously studied in soft tissue sarcomas (STS). Here we report that Reolysin triggered a striking induction of the anti-angiogenic chemokine interferon-gamma-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) in both wild type and RAS mutant STS cells. Further analysis determined that Reolysin treatment possessed significant anti-angiogenic activity irrespective of RAS status. In addition to CXCL10 induction, Reolysin dramatically downregulated the expression of hypoxia inducible factor (HIF)-1 alpha, HIF-2 alpha and inhibited vascular endothelial growth factor (VEGF) secretion. CXCL10 antagonism significantly diminished the anti-angiogenic effects of Reolysin indicating that it is a key driver of this phenomenon. Xenograft studies demonstrated that Reolysin significantly improved the anticancer activity of the anti-angiogenic agents sunitinib, temsirolimus, and bevacizumab in a manner that was associated with increased CXCL10 levels. This effect was most pronounced following treatment with Reolysin in combination with temsirolimus. Further analysis in additional sarcoma xenograft models confirmed the significant increase in CXCL10 and increased anticancer activity of this combination. Our collective results demonstrate that Reolysin possesses CXCL10-driven anti-angiogenic activity in sarcoma models, which can be harnessed to enhance the anticancer activity of temsirolimus and other agents that target the tumor vasculature.