Document Type

Dissertation - Open Access

Award Date

2022

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Xiuqing Wang

Abstract

In this study, the role of IFITM3 on PRRSV replication was studied in vitro by expressing exogenous IFITM3 in MARC-145 cells. An average of 31% reduction in PRRSV N protein expression and an average of 5.4 fold decrease in virus titer in the supernatant were observed in IFITM3 overexpressing cells as compared to vector control cells at 24 hours post infection (hpi). Moreover, there was a positive correlation between interferon- induced IFITM3 up-regulation and reduced PRRSV replication. To determine the role of endogenous IFITM3 in PRRSV replication, siRNA induced knockdown of IFITM3 was employed. RT-PCR validated the successful silencing of IFITM3 in MARC- 145 cells, with an average knockdown of 50%. PRRSV RNA copies were 1.28-fold higher in IFITM3 silenced cells as compared to control silencing, suggesting that knockdown of endogenous IFITM3 only slightly enhanced PRRSV replication. Taken together, these results suggest antiviral role of IFITM3 against PRRSV in vitro. In this study, we tested if the antifungal drug Amphotericin B restores PRRSV replication in IFITM3 overexpressing MARC-145 cells. Amphotericin B only partially restored PRRSV replication as confirmed by flow cytometry. Interestingly, more colocalization of PRRSV with early endosome marker was observed at 3 hpi (37.9%) than at 1 hpi (23.8%) and 6 hpi (31.8%). To further investigate the stage of PRRSV infection restricted by IFITM3 over-expression, colocalization study was performed at 3 and 24 hpi. Our results showed that IFITM3 expressing cells were positive for PRRSV at both 3 and 24 hpi. The percentage of IFITM3 positive cells with positive PRRSV staining was significantly higher at 3 hpi as compared to 24 hpi. Collectively, our data suggest that IFITM3 may restrict PRRSV via multiple post-entry mechanisms. The role of restriction factor, ZMPSTE24, on PRRSV replication was also studied. The ZMPSTE24 exerted antiviral effect against PRRSV as confirmed by both ZMPSTE24 overexpression and silencing experiments. A reduced expression of PRRSV N protein by approximately 3% and a 146-fold decrease in virus titer in the supernatant in the ZMPSTE24 overexpressing cells compared to vector control were observed. To further determine the role of endogenous ZMPSTE24 in PRRSV replication, we performed siRNA induced silencing of ZMPSTE24 in MARC-145 cells. RT-PCR validated highly successful silencing of ZMPSTE24, with an average knockdown of 74%. Knockdown of endogenous ZMPSTE24 slightly affected PRRSV replication, as only 1.2 fold increase in PRRSV RNA copies was observed. To study the stage of PRRSV infection impeded by ZMPSTE24, colocalization study was performed at 3 and 24 hpi. There were no significant differences in the number of PRRSV positive cells or total viral RNA copies between the vector control and ZMPSTE24 over-expressing cells at 3 hpi. The colocalization of PRRSV with ZMPSTE24 was significantly higher at 3 hpi as compared to 24 hpi, suggesting that ZMPSTE24 does not affect PRRSV entry into endosomes and restriction occurs after 3 hpi. Taken together, our results suggest that IFITM3 and ZMPSTE24 likely restrict PRRSV at multiple post-entry steps.

Number of Pages

110

Publisher

South Dakota State University

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Rights Statement

In Copyright