Abstract
The emergence of Variola virus-like viruses by natural evolution of zoonotic Orthopoxviruses, like Cowpox virus (CPXV), is a global health threat. The proteasome is essential for poxvirus replication, making the viral components interacting with the ubiquitin-proteasome system attractive antiviral targets. We show that proteasome inhibition impairs CPXV replication by prevention of uncoating, suggesting that uncoating is mediated by proteasomal degradation of viral core proteins. Although Orthopoxvirus particles contain considerable amounts of ubiquitin, distinct modification sites are largely unknown. Therefore, for the first time, we analyzed globally ubiquitination sites in CPXV mature virion proteins using LC-MS/MS. Identification of 137 conserved sites in 54 viral proteins among five CPXV strains revealed extensive ubiquitination of structural core proteins. Moreover, since virions contained primarily K48-linked polyubiquitin, we hypothesized that core proteins are modified accordingly. However, quantitative analysis of ubiquitinated CPXV proteins early in infection showed no proteasomal degradation of core proteins. Instead, our data indicate that the recently suggested proteasomal regulation of the uncoating factor E5 is a prerequisite for uncoating. Expanding our understanding of poxvirus uncoating and elucidating a multitude of novel ubiquitination sites in poxvirus proteins, the present study verifies the major biological significance of ubiquitin in poxvirus infection.
Highlights
The ubiquitin-proteasome system (UPS) is exploited by members of most virus families[1] and is essential for the replication of different virus families, e.g. Pox-2–4, Reo-5 and Coronaviridae[6]
After the viral DNA is released by core uncoating, replication as well as intermediate, and late viral gene expression proceed in delimited cytoplasmic areas called virus factories (VF)[21,22]
The UPS plays an important role during OPV infection[2,3,23] and during infection of viruses belonging to diverse other families[5,6], demonstrating the general regulatory function of ubiquitin in viral infection
Summary
The ubiquitin-proteasome system (UPS) is exploited by members of most virus families[1] and is essential for the replication of different virus families, e.g. Pox-2–4, Reo-5 and Coronaviridae[6]. Treatment of CPXV-infected cells with proteasome inhibitors prevents late protein expression and reduces viral titers[3] but it remains unknown to what extent results generated with VACV apply to CPXV. We showed that proteasome inhibition impairs CPXV replication by prevention of viral core degradation, resulting in early but not in late viral protein expression. We hypothesized that proteasomal degradation of K48-linked polyubiquitinated core proteins is the mechanism underlying CPXV uncoating, as previously suggested for VACV4. Since this hypothesis has not been verified yet, we aimed to prove it by conducting the first global LC-MS/MS-based ubiquitinome analysis of OPV IMV proteins. We detected the proteasomal degradation of the uncoating factor E5 (VACV D5), which is likely a prerequisite for OPV genome uncoating[23]
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