A Poxvirus Decapping Enzyme Colocalizes with Mitochondria To Regulate RNA Metabolism and Translation and Promote Viral Replication.

Decapping enzymes remove the 5' cap of eukaryotic mRNA, leading to accelerated RNA decay. They are critical in regulating RNA homeostasis and play essential roles in many cellular and life processes. They are encoded in many organisms and viruses, including vaccinia virus, which was used as the vaccine to eradicate smallpox. Vaccinia virus encodes two decapping enzymes, D9 and D10, that are necessary for efficient viral replication and pathogenesis. However, the underlying molecular mechanisms regulating vaccinia decapping enzymes' functions are still largely elusive. Here, we demonstrated that vaccinia D10 almost exclusively colocalized with mitochondria. As mitochondria are highly mobile cellular organelles, colocalization of D10 with mitochondria can concentrate D10 locally and mobilize it to efficiently decap mRNAs. Mitochondria were barely observed in "viral factories," where viral transcripts are produced, suggesting that mitochondrial colocalization provides a spatial mechanism to preferentially decap cellular mRNAs over viral mRNAs. We identified three amino acids at the N terminus of D10 that are required for D10's mitochondrial colocalization. Loss of mitochondrial colocalization significantly impaired viral replication, reduced D10's ability to remove the RNA 5' cap during infection, and diminished D10's gene expression shutoff and mRNA translation promotion abilities. IMPORTANCE Decapping enzymes comprise many members from various organisms, ranging from plants, animals, and viruses. The mechanisms regulating their functions vary and are still largely unknown. Our study provides evidence that a vaccinia virus-encoded decapping enzyme, D10, colocalizes with mitochondria. Loss of mitochondrial colocalization significantly impairs viral replication, D10's gene expression shutoff, and mRNA translation promotion ability. Overall, our results suggest that mitochondrial colocalization is a spatial mechanism to concentrate D10 locally and mobilize it to efficiently and preferentially target cellular mRNAs for decapping and promote viral mRNA translation. Our results have broad impacts for understanding the functions and regulatory mechanisms of decapping enzymes.

Rapid and quantitative evaluation of vaccinia virus-induced host shutoff using newly generated cell lines stably expressing secreted Gaussia luciferase.

Host shutoff, characterized by a global decline of cellular protein synthesis, is commonly observed in many viral infections, including vaccinia virus (VACV). Classic methods measuring host shutoff include the use of radioactive or nonradioactive probes to label newly synthesized proteins followed by radioautography or sodium dodecyl-sulfate polyacrylamide gel electrophoresis to resolve the proteins for follow-up detection. Although these are highly reliable methods, they are time- and labor-consuming. Here, we generated two cell lines stably expressing secreted Gaussia luciferase. These reporter cells allow rapid, quantitative, and consecutive monitoring of host shutoff from a single infection sample. We evaluated host shutoff induced by wild-type and various mutant VACVs using the reporter cell lines. The results validated the utilities of the reporter cells and quantitatively characterized VACV-induced host shutoff at different stages of replication. Notably, the results also indicated additional major unidentified VACV shutoff factors. Our study provides new tools to study host shutoff. The reporter cells are also suitable for high throughput settings and rapid testing of clinically isolated viruses. In combination with classical methods, these tools will greatly facilitate understanding of virus-induced host shutoff, and protein synthesis shutoff caused by other physiologically relevant stresses.

Vaccinia Virus as a Master of Host Shutoff Induction: Targeting Processes of the Central Dogma and Beyond.

The synthesis of host cell proteins is adversely inhibited in many virus infections, whereas viral proteins are efficiently synthesized. This phenomenon leads to the accumulation of viral proteins concurrently with a profound decline in global host protein synthesis, a phenomenon often termed "host shutoff." To induce host shutoff, a virus may target various steps of gene expression, as well as pre- and post-gene expression processes. During infection, vaccinia virus (VACV), the prototype poxvirus, targets all major processes of the central dogma of genetics, as well as pre-transcription and post-translation steps to hinder host cell protein production. In this article, we review the strategies used by VACV to induce host shutoff in the context of strategies employed by other viruses. We elaborate on how VACV induces host shutoff by targeting host cell DNA synthesis, RNA production and processing, mRNA translation, and protein degradation. We emphasize the topics on VACV's approaches toward modulating mRNA processing, stability, and translation during infection. Finally, we propose avenues for future investigations, which will facilitate our understanding of poxvirus biology, as well as fundamental cellular gene expression and regulation mechanisms.

Autophagy-lysosome pathway alterations and alpha-synuclein up-regulation in the subtype of neuronal ceroid lipofuscinosis, CLN5 disease.

Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN5 deficiency causes a subtype of NCL, referred to as CLN5 disease. CLN5 is a soluble lysosomal protein with an unclear function in the cell. Increased levels of the autophagy marker protein LC3-II have been reported in several subtypes of NCLs. In this report, we examine whether autophagy is altered in CLN5 disease. We found that the basal level of LC3-II was elevated in both CLN5 disease patient fibroblasts and CLN5-deficient HeLa cells. Further analysis using tandem fluorescent mRFP-GFP-LC3 showed the autophagy flux was increased. We found the alpha-synuclein (α-syn) gene SNCA was highly up-regulated in CLN5 disease patient fibroblasts. The aggregated form of α-syn is well known for its role in the pathogenicity of Parkinson's disease. Higher α-syn protein levels confirmed the SNCA up-regulation in both patient cells and CLN5 knockdown HeLa cells. Furthermore, α-syn was localized to the vicinity of lysosomes in CLN5 deficient cells, indicating it may have a lysosome-related function. Intriguingly, knocking down SNCA reversed lysosomal perinuclear clustering caused by CLN5 deficiency. These results suggest α-syn may affect lysosomal clustering in non-neuronal cells, similar to its role in presynaptic vesicles in neurons.

Neuronal ceroid lipofuscinosis related ER membrane protein CLN8 regulates PP2A activity and ceramide levels.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3ß, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin, suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.

Glutamine-dependent lysosome homeostatic changes induced by starvation and lysosome inhibition.

Lysosomes are a major organelle for degrading macromolecules. When deprived of nutrients, cells activate the autophagy and lysosome biogenesis pathways to recycle cytoplasmic materials and to increase lysosomal degradation capacity for survival, respectively. We have identified a condition in which cells accumulated enlarged lysosomes upon starvation and lysosome inhibition. Selective autophagy and inhibition of the mechanistic target of rapamycin (mTOR) in combination with lysosome inhibition were not able to induce this phenomenon. Conversely, knocking out autophagy genes, ATG5 or ATG7, had no effects on the enlarged lysosome formation. This suggests that the enlarged lysosome formation is an autophagy independent process. Remarkably, adding glutamine to the treatment can prevent formation of the enlarged lysosomes and dissipate the pre-existing ones. Furthermore, the nucleus/cytoplasm translocation of the transcription factor EB (TFEB), but not mTOR activity, correlates with the formation/dissipation of enlarged lysosomes. Knockdown of TFEB, however, suggests that TFEB-mediated lysosome biogenesis is not directly involved in the process. These results indicate that there is a novel mechanism by which lysosome homeostasis can be regulated under certain stress conditions.