Global ubiquitination analysis reveals extensive modification and proteasomal degradation of cowpox virus proteins, but preservation of viral cores

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.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 . As a member of the Poxviridae family the genus Orthopoxvirus (OPV) comprises complex enveloped DNA viruses, including the smallpox-causing Variola virus (VARV), Vaccinia virus (VACV), which was used for smallpox eradication 7 , and several animal-borne viruses with zoonotic potential like Cowpox virus (CPXV) [8][9][10] . Having caused the death of hundreds of millions of people, VARV is the most prominent member of OPV. Although VARV has been eradicated in 1979, it is still a global health threat because of its possible release in the context of a bioterrorist attack and the emergence of VARV-like viruses from circulating OPV by natural evolution 11 . CPXV are well suited to fill the niche that was created by VARV eradication, since it has the largest OPV genome including homologous open reading frames of all VARV genes. Currently, the number of zoonotic infections in Europe caused by CPXV is increasing, illustrating the need for a comprehensive understanding of the virus biology, especially the interaction with the host cell 12,13 .

Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

“…Indeed, we found that MG132 rescues 69% of host proteins from degradation including HDAC5, suggesting that many of the downregulated proteins may be specifically targeted to the proteasome. This finding is consistent with a recent analysis of protein ubiquitylation during cowpox virus infection ( Grossegesse et al., 2018 ). Transcriptional mechanisms may nevertheless play an important role in protein downregulation.…”

Ubiquitin-Modified Proteome of SARS-CoV-2-Infected Host Cells Reveals Insights into Virus–Host Interaction and Pathogenesis

“…Modification of ubiquitination can be important for antiviral defense and virus replication, and viral proteins can also be modified. 17 , 22 , 23 The extent of ubiquitination of SARS-CoV-2 proteins is unclear. Therefore, we analyzed SARS-CoV-2 proteins for the presence of ubiquitinated residues.…”

Pleiotropic roles of the ubiquitin-proteasome system during viral propagation

“…To date, many viral proteins have been reported to be ubiquitinated, including influenza A virus-encoded NP, M2 and PB1 [25][26][27][28], Dengue Virus-encoded NP [29], and 54 viral proteins encoded by the wellcharacterised cowpox virus strain [30]. In ubiquitinated proteins, Ub is frequently found to be covalently conjugated to Lys residues, but non-Lys residues of substrates such as serine (Ser), threonine (Thr) and cysteine (Cys) are also modified in rare cases.…”