Replication of positive-stranded RNA viruses requires the activity of proteases that cleave the viral replicase polyproteins. For Middle East respiratory coronavirus (MERS-CoV), the virus-encoded... Show moreReplication of positive-stranded RNA viruses requires the activity of proteases that cleave the viral replicase polyproteins. For Middle East respiratory coronavirus (MERS-CoV), the virus-encoded papain-like protease (PLpro) is one of such proteases. This protease also functions as a deubiquitinating enzyme (DUB) that removes ubiquitin from substrates, most likely to suppress the ubiquitin-dependent activation of the innate immune response. The work described in this thesis provides novel insights in the interaction between PLpro and ubiquitin. The crystal structure of the PLpro-ubiquitin complex facilitated the design of substitutions in PLpro that selectively disrupted its DUB activity. DUB-negative MERS-CoV induced enhanced immune responses compared to wild-type virus, while showing similar replication in infected cells. Relative to wild-type virus, the virulence of DUB-negative MERS-CoV was reduced in mice and earlier, better-regulated immune responses were measured in their lungs. In the search for novel antivirals, ubiquitin sequence variants were selected that bound with very high affinity to MERS-CoV PLpro. Expression of those ubiquitin variants affected the activity of PLpro and concomitantly inhibited virus replication resulting in severely less virus progeny. Collectively, the gained knowledge can be used to design novel coronavirus vaccines or further develop ubiquitin variants as antiviral agents against viruses that encode DUBs. Show less
The research described in this thesis aimed focused on CHIKV replication and on the identification of much-needed inhibitors of CHIKV infection. Following the development of an in vitro assay to... Show moreThe research described in this thesis aimed focused on CHIKV replication and on the identification of much-needed inhibitors of CHIKV infection. Following the development of an in vitro assay to study CHIKV replication, this tool was used to characterize the mode of action (MoA) of antiviral compounds and suramin was identified as a potent inhibitor of viral RNA synthesis. However, we discovered that in cell culture, suramin’s antiviral activity was mainly due to inhibition of CHIKV binding/entry, and to a lesser extent of virus release. Suramin was also found to inhibit binding/entry and virion biogenesis of Zika virus (ZIKV), a recently emerged flavivirus. Due to its ability to form electrostatic interactions with positive charges on proteins, suramin may block the contact between virions and their (co)receptors, by interacting with either virus or receptor, or with both. Using radioactively-labelled suramin, it was clearly shown that the compound interacts with CHIKV particles, more specifically with their envelope proteins. Additionally, suramin could interfere with cell attachment and/or the structural changes required for fusion. Suramin-resistant CHIKV variants were selected, which contained mutations in the E2 envelope protein (involved in receptor interactions), supporting the idea that suramin blocks the early steps of the infectious cycle. Show less
