Mechanistic Basis for Enhanced Oncolytic Potency by Mutations in Reovirus Sigma1 and Mu2 proteins

• Author / Creator

  Yip, Wan Kong

• Wild-type mammalian orthoreoviruses (reoviruses) are non-pathogenic viruses infecting hosts via the respiratory and enteric tracts. There are four prototype reovirus strains: the Type 1 Lang (T1L), Type 2 Jones (T2J), Type 3 Abney (T3A), Type 3 Daring (T3D) and Type 4 Ndelle (T4N). A proprietary variant of T3D has been developed as an oncolytic virotherapy under the brand name REOLYSIN® and is currently being evaluated in clinical trials. One major goal of our lab is to improve oncolytic potency of reovirus. We generated a number of reovirus variants by directed evolution that exhibited increased infectivity towards transformed cells while sparing normal cells. My thesis focused on variants that had mutations in the cell-attachment protein σ1 and polymerase-cofactor 2, with the overarching goal to understand how these mutations promote replication of reovirus in tumor cells.
  
  Our lab previously showed that reduction of σ1 levels leads to faster uncoating and it is one of the main mechanisms that augments reovirus infectivity towards transformed cells. In chapter 3 of this thesis, we characterize 6 variants with big-plaque phenotype and provide evidence that all variants have reduced levels of σ1. Previous studies showed that the middle region of the σ1 tail was important for virion encapsidation. I used truncations and single amino acid mutants to investigate how other regions in the σ1 tail mediated virion association. Immunoprecipitations of transfected σ1 showed that residues 22 and 66, as well as residues near the C-terminus of the σ1 tail were important for virion association. Unexpectedly, data from mutant viruses showed that residues 252-286 may inhibit virion encapsidation by a mechanism yet to be identified. Our data revealed that residues 154-251 in the σ1 tail mediate viral factory accumulation and virion association.
  
  All documented variants to date have enhanced oncolytic potency in the binding or uncoating (entry) steps. The second goal of this thesis was to identify novel mechanisms that increased reovirus replication in transformed cells at post-entry steps. Chapter 4 describes 5 variants with mutations in different genes. One variant, named T3v10M1, had a single amino acid change in the µ2 protein. The µ2 is a polymerase co-factor and is known to interact with reovirus non-structural protein µNS and cellular microtubules. Data in chapter 5 demonstrates that the altered µ2 had both infection-hindering and –promoting effects, with an overall net benefit of promoting replication in cancer cells. T3v10M1 bound host-cell and uncoated at similar rates relative to wild-type virus (T3wt), but in vitro phosphate release and RT-qPCR assays showed that T3v10M1 µ2 was less efficient at hydrolyzing ribonucleotide triphosphates (rNTP) and RNA synthesis. As a consequence, T3v10M1 exhibited impaired ability to establish infection by 75% compared to T3wt. However, when L929 cells were exposed to equal cell-bound T3v10M1, the mutant accumulated up to 4-fold more viral proteins and 3-fold more viral RNAs. Data from immunoprecipitation and far-Western blot approaches indicated that T3v10M1 µ2 associated with NS and other viral proteins 2-fold more efficiently than T3wt µ2, which directly enhanced progeny production by 2-fold in one replication cycle. This represents the first example of oncolytic enhancement via a post-entry mechanism. In conclusion, our findings provide insights into the mechanistic basis for enhanced oncolytic potency by mutations in reovirus σ1 and µ2 proteins.  

• Subjects / Keywords

  • Oncolytic virus
  • Reovirus

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-93p4-6n96

• License

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