Document Type

Dissertation - Open Access

Award Date

2024

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Xiuqing Wang

Abstract

In this study, we studied the role of IFITM3 in SVA replication in vitro by silencing the endogenous IFITM3 in NCI-H1299 cells. An average of 1.94-fold increase in SVA VP2 expression and an average of 4.4-fold increase in supernatant virus titer was observed after 24h SVA isolate SD15-26 infection. We have indeed observed an increase in viral mRNA copies by an average of 5-fold in IFITM3 silenced NCI-H1299 cells at 8 hpi indicating that IFITM3 restricts SVA replication post virus attachment and entry. A positive correlation between interferon-alpha induced IFITM3 upregulation and reduced SVA replication was observed. To further confirm the antiviral role of IFITM3 in SVA replication, we employed the ectopic over-expression of exogenous IFITM3 approach. Surprisingly, an average of 2.5-fold and 1.42-fold increase in SVA VP2 expression and a significant increase in supernatant virus titer was observed in NCI-H1299 cells and PK-15 cells after 24h and 16h SVA isolate SD15-26 infection respecitively. Furthermore, overexpression of exogenous IFITM3 induces autophagy in NCI-H1299 cells and PK-15 cells. An average of 3.1-fold and 1.28-fold increase in LC3II/LC3I ratios was observed in IFITM3 over-expressing NCI-H1299 cells and PK-15 cells respectively when compared to control vector pQCXIP. An average of 14.3% colocalization of EGFPLC3 with LAMP1 was observed in IFITM3 overexpressing NCI -H1299 cells whereas, pQCXIP group only showed 1.9% colocalization. Moreover, a significant reduction in supernatant virus titer was observed in 3-MA treated NCI-H1299 cells compared to DMSO treated cells. Overall, our results indicate an antiviral role of endogenous IFITM3 against SVA replication. The molecular mechanisms by which endogenous IFITM3 restrict SVA replication and the role of autophagy in enhancing SVA replication in IFITM3 over-expressing NCI-H1299 cells remain to be determined in future studies. The role of NEDD4 in SVA replication was also studied. Overexpression of NEDD4 in NCI-H1299 cells reduced SVA VP2 expression by an average of 25% and supernatant virus titer by an average of 6.38-folds. We have indeed observed a decrease in endogenous IFITM3 expression by an average of 23% in NEDD4 overexpressed cells compared to empty vector control indicating that NEDD4 is involved in degradation of IFITM3. No change in SVA VP2 expression and IFITM3 expression was observed in NEDD4 mutant (C867A) overexpressed NCI-H1299 cells when compared to empty vector control suggesting that catalytic domain is required by NEDD4 for IFITM3 degradation and SVA restriction. We have also silenced the endogenous NEDD4 and observed a slight increase in SVA VP2 expression and supernatant virus titer after 24h SVA isolate SD15-26 infection. Interestingly, an average of 28% increase in endogenous IFITM3 expression was observed in endogenous NEDD4 silenced cells compared to negative control. Futher experiments have to be repeated and new studies are needed to establish an antiviral role of NEDD4 in SVA replication. Also, additional experiments are needed to investigate the contradictory effects of endogenous IFITM3 in SVA replication under the influence of NEDD4.

Library of Congress Subject Headings

Swine vesicular disease.
Swine -- Virus diseases.
Viruses -- Reproduction.
Membrane proteins.
Interferon.

Publisher

South Dakota State University

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Rights Statement

In Copyright