Characterization of a novel nucleoside analog for dengue virus infection Open Access

Dimitrova, Silvia D. (2016)

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Dengue virus (DENV) causes a febrile disease prevalent in tropical and subtropical regions of the world that affects approximately 390 million persons annually. This mosquito-borne single-stranded RNA virus is a member of the family Flaviviridae and is classified into four serotypes, DENV 1-4. Currently, there are no approved antiviral agents that are available to treat dengue infections. The development of a therapeutic agent is necessary and could decrease morbidity and mortality rates among dengue-infected persons. One specific target for an antiviral agent is the DENV RNA-dependent RNA polymerase (RdRp) that replicates the viral genome and is essential for virus survival. An approach to inhibit the activity of the RdRp enzyme is to utilize ribonucleoside triphosphate (NTP) analog inhibitors that act as chain terminators during the synthesis of new genomic RNAs and prevent viral replication. Our laboratory previously discovered that the ribonucleoside analog 7-deaza-7-fluoro-2'-C-methyl-adenosine (DFMA) synthesized by our chemists inhibits dengue virus replication. The focus of this thesis work was to characterize the anti-DENV potency of DFMA utilizing both cell biological and biochemical assays. In addition, the phosphorylation of DFMA in the megakaryoblastic cell line Meg-01 was studied. Finally, the anti-DENV mechanism of action of DFMA was studied by selecting DFMA drug-resistant virus in vitro in a tissue culture system. This thesis work will advance anti-DENV drug development, as well as increase our knowledge with regards to mechanisms of DENV inhibition and viral resistance to ribonucleoside analog drugs.

Table of Contents

Abstract. 1

Title Page. 2

Acknowledgements. 3

Chapter I: Introduction


Global burden of dengue disease. 5

The dengue virus. 6

Immune Responses to DENV. 7

Vaccines. 8

Nucleoside analog inhibitors. 9

DFMA. 11

Aims and goals of project. 11

Figure 1. 12

Chapter II: Potency of DFMA in the Megakaryoblastic Cell Line Meg-01

Introduction. 13

Materials and Methods. 14

Results. 16

Discussion. 17

Figures 2-6. 18-21

Table 1. 21

Chapter III: Cellular Pharmacology of DFMA in the Cell Line Meg-01

Introduction. 22

Materials and Methods. 22

Results. 24

Discussion. 25

Figures 7-9. 26-27

Table 2. 27

Chapter IV: Dengue Virus Resistance to DFMA

Introduction. 28

Materials and Methods. 28

Results. 31

Discussion. 33

Figures 10-13. 34-36

Table 3. 36

Chapter V: Purification and Activity of Dengue Virus NS5 Protein

Introduction. 37

Materials and Methods. 37

Results. 40

Discussion. 42

Figures 14-18. 43-45

Table 4. 46

Chapter VI: Conclusion

Future Directions. 47

Concluding Remarks. 48

Addendum. 51

References. 52-57

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