The poxvirus encoded ubiquitin ligase, p28, is regulated by proteasomal degradation and autoubiquitination

Abstract

Virus manipulation of the ubiquitin-proteasome system has become increasingly apparent. Ubiquitin is a 76 amino acid protein that is post-translationally conjugated to target proteins, while poly-ubiquitination subsequently leads to degradation via the 26S proteasome. Target specificity is determined by a large family of ubiquitin ligases. Poxviruses encode p28, a highly conserved ubiquitin ligase expressed in a wide range of poxviruses (J. Virol. 79:597). Here we investigate the relationship between p28 and ubiquitination. Confocal microscopy indicated that orthologs of p28 co-localized with ubiquitin at the virus factory. Flow cytometry assays further demonstrated that p28 was regulated by proteasomal degradation. Moreover, when the ubiquitin ligase activity of p28 was disrupted by mutating the RING domain conjugated ubiquitin still localized to the viral factories, indicating that an unknown ubiquitin ligase(s) was responsible for regulating p28. Our observations indicate that p28 is a ubiquitin ligase that is regulated by ubiquitination and proteasomal degradation.

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

“…CPXV encode one E3 ubiquitin ligase, the C7 protein (p28), which catalyzes K63-linked polyubiquitination in vitro 15 . Also the accumulation of K48-linked polyubiquitin in VF has been shown to be promoted by p28 37 . Additionally, it is assumed that at least one cellular ubiquitin ligase recruits ubiquitin to VF 37 .…”

“…Also the accumulation of K48-linked polyubiquitin in VF has been shown to be promoted by p28 37 . Additionally, it is assumed that at least one cellular ubiquitin ligase recruits ubiquitin to VF 37 . P28 is expressed early in infection and locates with ubiquitin to VF 16 , 37 , making the K48-linked polyubiquitination of CPXV mature virion proteins catalyzed by p28 an attractive hypothesis.…”

“…Additionally, it is assumed that at least one cellular ubiquitin ligase recruits ubiquitin to VF 37 . P28 is expressed early in infection and locates with ubiquitin to VF 16 , 37 , making the K48-linked polyubiquitination of CPXV mature virion proteins catalyzed by p28 an attractive hypothesis. However, p28 has been shown to be essential only for replication of ectromelia virus 38 in mouse macrophages, suggesting the compensative activity of another ubiquitin ligase in cells other than macrophages.…”

Poxviral Ankyrin Proteins

“…The poxviral specific p28 ligases are known virulence factors found in the majority of poxviral genera, and contain a DNA binding domain and a RING domain [ 19 , 138 ]. The p28 ligases co-localise to the viral factory with ubiquitin and are regulated by proteasomal degradation [ 140 ]. Poxviral versions of MARCH (Membrane associated RING-CH) ligases are also present in the LSG group, where a key example is the myxoma viral M153R protein that enables the internalization and lysosomal degradation of CD4 and removal of cell surface MHC class I [ 138 , 141 ].…”

Replication of cowpox virus in macrophages is dependent on the host range factor p28/N1R

“…The full-length p28 protein consists of 242 amino acids and combines an N-terminal KilA-N domain, which acts as DNA-binding domain [ 13 ], and a C-terminal RING domain [ 6 ]. The protein is translated in the early phase of viral replication, is present throughout the viral life cycle and localizes to cytoplasmic virus factories facilitated by its KilA-N domain [ 9 , 10 , 14 ]. The mechanism of how p28 contributes to viral host range is currently not understood.…”