Host Factors Invovled in the Entry of Coronaviruses into Mammalian Cells

Abstract

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs) and the role of host factors therein. CoVs are widespread in companion and farm animals and may cause large economic losses. The human variants cause roughly 30-50% of the common cold cases. However, the recent emergence of SARS- and MERS-coronaviruses that managed to cross the host species barrier has shown the potential of CoVs to also cause life-threatening disease in humans. The research described in the thesis on hand primarily made use of the mouse hepatitis virus (MHV), which serves as a model for other coronavirus family members. In addition, we studied the entry of MERS-CoV and feline infectious peritonitis virus. Most viral entry studies rely on the measurement of post-entry parameters such as the expression of viral proteins. In order to increase our understanding of the CoV entry process, we developed novel techniques allowing the dissection of viral entry steps including binding, internalization, penetration and proteolytic processing in a replication-independent manner. These novel entry assays either depend on the minimal complementation of otherwise defective b-galactosidase by using genetically modified MHV or on conditional biotinylation of the spike glycoprotein (S) that functions in virus-cell attachment and fusion. While MHV had been demonstrated to enter via clathrin-mediated endocytosis, no other host factors critical for entry of MHV had previously been identified. Using our newly developed entry assays, in combination with other cell biological and biochemical techniques, we characterized the MHV entry process and the host factor requirements in detail. We confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. We furthermore showed that entry of MHV depended on proteolytic processing of its fusion protein S by endo/lysosomal proteases. The identity of a cleavage site directly upstream of the fusion peptide in S was shown to correlate with fusion in early endosomes or lysosomes. In addition, we elucidated the importance of the a-subunit of the Na+,K+-ATPase, encoded by the ATP1A1 gene, for virus entry. By using gene silencing as well as by applying cardiotonic drugs that bind to ATP1A1 we could reveal that Src-signaling mediated via ATP1A1 inhibits CoV entry. These results suggest that the use of cardiotonic steroids, long prescribed in the clinical treatment of heart failure, may be an attractive therapeutic approach against coronavirus infection. The work described in this thesis contributes in a significant way to our understanding of coronavirus cell entry. It provides new observations and insights, and resolves a number of longstanding enigmas in the field.