Date Published: August 8, 2013
Publisher: Public Library of Science
Author(s): Lénia Rodrigues, Nikita Popov, Kenneth M. Kaye, J. Pedro Simas, Dirk P. Dittmer.
Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC5SmLANA modulates cellular control of Myc turnover by antagonizing SCFFbw7 mediated proteasomal degradation of Myc, mimicking SCFβ-TrCP. The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease.
Myc is a transcription factor that enhances the expression of genes involved in cellular growth and proliferation. Hence, it is not surprising that viruses have evolved mechanisms to modulate Myc to promote their own life cycle. Myc heterodimerizes with Max, through a basic region/helix-loop-helix/leucine-zipper domain, to regulate the transcription of specific E-box-containing genes in response to mitogenic stimuli. Myc functions as a universal amplifier of gene expression by promoting the transcriptional elongation of RNA polymerase II driving biomass accumulation and enhanced cellular bioenergetic pathways , , . The expression of c-myc is tightly regulated with extremely short half-lives for mRNA and protein. In non-transformed cells, Myc is continuously subjected to ubiquitination and proteasomal-degradation, resulting in a highly unstable protein with a half-life of about 15–20 minutes . Several mechanisms of Myc regulation have been identified that operate at the level of protein stability. The best characterized mechanism involves the interplay between phosphorylation at two specific residues and ubiquitination. Phosphorylation at serine (S) 62 by extracellular signal-regulated kinase (ERK) stabilizes Myc resulting in enhancement of its transcription activity. In contrast, phosphorylation of Myc at threonine (T) 58 by glycogen synthase kinase 3 (Gsk-3), which is dependent on previous phosphorylation of Myc at S62, leads to proteasomal degradation of Myc . The mechanism involves the assembly of homotypic poly-ubiquitin chains on Myc specifically dependent on lysine (K) 48 linkage by SCF (Skp1/Cul/Fbox)Fbw7, . Myc turnover by SCFFbw7 is antagonized by polymerization of mixed heterotypic poly-ubiquitination chains via SCFβ-TrCP on the N-terminus of Myc . Thus, SCFFbw7 and SCFβ-TrCP assemble different K-linkage poly-ubiquitin chains with functionally distinct outcomes on Myc stability, i.e., degradation versus stability. The physiological relevance of regulating Myc activity through protein stability is underscored by observations that point mutations at or near T58, which render Myc resistant to proteasomal degradation, occur with high frequency in B-cell lymphomas .
In this study, we describe the first example of a viral protein activating Myc transcriptional activity through increased protein stability by mimicking a physiological post-translational regulatory pathway. Modulation of Myc function was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibit increased expression of known Myc target-genes, and using a genetic approach, virus was found to amplify exclusively in intact Myc GC B-cells. The molecular mechanism involved heterotypic poly-ubiquitination of Myc mediated via the mLANA protein encoded by ORF73. This was reminiscent of a newly described pathway of Myc regulation through poly-ubiquitination. Popov et al. showed that the cellular E3 ubiquitin-ligase SCFβ-TrCP uses UbcH5 ubiquitin-conjugating enzyme to form heterotypic poly-ubiquitin chains on the N-terminus of Myc. Poly-ubiquitination of Myc by SCFβ-TrCP leads to Myc stabilization and was shown to antagonize SCFFbw7-mediated proteasomal turnover of Myc . Like SCFβ-TrCP, EC5SmLANA uses UbcH5 and antagonizes SCFFbw7. Furthermore, as previously demonstrated for SCFβ-TrCP, single substitutions of K33, K48 or K63 of ubiquitin reduced or eliminated the ability of EC5SmLANA to poly-ubiquitinate, stabilize or increase the transcriptional activity of Myc. However, our results suggest that the molecular mechanism of mLANA modulation of Myc activity is not limited to Fbw7 antagonism. This is supported by the fact that mLANA protective effects on Myc stability, in conditions of Fbw7 over expression, are significantly more pronounced than observed for β-TrCP. Moreover, mLANA was able to increase the stability and activity of MycT58A, a Myc version that is not recognized by Fbw7. This contrasts with what has been reported for β-TrCP that albeit being able to poly-ubiquitinate MycT58A it did not impact on its turnover . Thus, the ability of mLANA to increase Myc transcriptional activity through poly-ubiquitination, independently of the phosphorylation status of Myc on S58 and T62, indicates that this novel viral modulatory mechanism does not rely on post-translation cellular regulation.