Avian influenza is a pandemic threat and new anti-virals need to be developed. Studying the pathogenesis of H5N1 infection and focusing on the vital role that host proteins play in influenza replication will offer new therapeutic targets. Host proteins from the coat protein complex (COP) are involved in many cellular pathways such as endocytosis, protein transport between the endoplasmic reticulum and the Golgi apparatus, and protein glycosylation. COP proteins have been shown to play a role in the viral replication cycle and their role in H5N1 pathogenesis is of great interest. In this study, we aimed at modulating the expression of a COP protein and measure its impact on H5N1 replication.
H5N1 infections of HeLa cells were done in a BSL3 facility. Viral replication was evaluated by TCID50. Short interfering RNAs (siRNAs) were designed against COP subunit alpha (COPA). siRNA-mediated gene expression knockdown was determined by quantitative real-time PCR. Cytotoxicity was assessed using the high throughput imager CellInsight. Golgi disruption was quantified using the CellInsight and confocal microscopy. Changes in endocytosis of H5N1 in HeLa cells were evaluated by confocal microscopy.
In this study, we successfully identified a concentration of COPA siRNA showing efficient downregulation of COPA expression without any associated cytotoxicity. We found that blocking COPA results in significant Golgi disruption but does not impair H5N1 entry (endocytosis). Furthermore, we showed that COPA knockdown inhibits H5N1 replication, possibly by limiting the number of virions produced by each infected cells.
This demonstrates that transient downregulation of host proteins can reduce avian influenza replication in human cells. Modulating host proteins offers new antiviral strategies against H5N1 avian influenza. Such strategies can also be applied to a wide range of viruses requiring common host proteins for their replication.