ABSTRACT
Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin-proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed 'induced protein aggregation motif'. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism.
Subject(s)
Herpesviridae/immunology , Intracellular Signaling Peptides and Proteins/immunology , Receptor-Interacting Protein Serine-Threonine Kinases/immunology , Animals , Autophagy/immunology , Autophagy-Related Protein 5/deficiency , Autophagy-Related Protein 5/genetics , Cells, Cultured , HEK293 Cells , Herpesviridae/metabolism , Herpesviridae/pathogenicity , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/metabolism , Herpesvirus 1, Human/pathogenicity , Host Microbial Interactions/immunology , Humans , Immune Evasion , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred C57BL , Models, Biological , Muromegalovirus/immunology , Muromegalovirus/metabolism , Muromegalovirus/pathogenicity , Protein Aggregates/immunology , Proteolysis , Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Ribonucleotide Reductases/genetics , Ribonucleotide Reductases/immunology , Ribonucleotide Reductases/metabolism , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolismABSTRACT
Clostridium pasteurianum produces industrially valuable chemicals such as n-butanol and 1,3-propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3-propanediol-deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl-CoA to butyryl-CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl-CoA to butyryl-CoA and butanol, indicating a new, 1,3-propanediol-independent pattern of glycerol fermentation in Clostridium pasteurianum.
