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The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64’752 unique siRNAs targeting 21’584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51’128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies., Author summary The cellular prion protein (PrPC) acts as both, the substrate for prion formation and mediator of prion toxicity during the progression of all prion diseases. Suppressing the levels of PrPC is a viable therapeutic strategy as PRNP null animals are resistant to prion disease and the knockout of PRNP is not associated with any severe phenotypes. Motivated by the scarcity of knowledge regarding the molecular regulators of PrPC biosynthesis and degradation, which might serve as valuable targets to control its expression, here, we present a cell-based genome wide RNAi screen in arrayed format. The screening effort led to the identification of 54 regulators, nine of which were confirmed by an independent CRISPR-based method. Among the final nine targets, we identified PUM1 as a regulator of PRNP mRNA by acting on the 3’UTR promoting its degradation. The newly identified factors involved in the life cycle of PrPC provided by our study may also represent themselves as therapeutic targets for the intervention of prion diseases.

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication., Author summary Replication of positive-strand RNA viruses, which infect plants and animals, depends on many cellular resources. These viruses subvert cellular membranes and co-opt host proteins to build replication compartments that produce the viral progeny. The viral replication process also requires cellular metabolites and energy in the form of ATP. Using plant host as well as the model host yeast, the authors discovered that tomato bushy stunt tombusvirus (TBSV) co-opts the fermentation pathway to facilitate the local generation of ATP within the replication compartments. TBSV replication induces high expression of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes. With the help of the viral replication protein, TBSV recruits Pdc1 and Adh1 into the viral replication compartment. The authors propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses by facilitating rapid and efficient replication in the infected hosts. The authors also showed that the replication of five other plant viruses depends on the fermentation pathway in plants.

Geminiviruses constitute a group of plant viruses, with a ssDNA genome, whose replication in the nucleus of an infected cell requires the function of geminivirus-encoded replication initiator protein (Rep). Our results suggest that monoubiquitinated histone 2B (H2B-ub) promotes tri-methylation of histone 3 at lysine 4 (H3-K4me3) on the promoter of Chilli leaf curl virus (ChiLCV). We isolated homologues of two major components of the monoubiquitination machinery: UBIQUITIN-CONJUGATING ENZYME2 (NbUBC2) and HISTONE MONOUBIQUITINATION1 (NbHUB1) from N. benthamiana. ChiLCV failed to cause disease in NbUBC2-, and NbHUB1-silenced plants, at the same time, H2B-ub and H3-K4me3 modifications were decreased, and the occupancy of RNA polymerase II on the viral promoter was reduced as well. In further investigations, Rep protein of ChiLCV was found to re-localize NbUBC2 from the cytoplasm to the nucleoplasm, like NbHUB1, the cognate partner of NbUBC2. Rep was observed to interact and co-localize with NbHUB1 and NbUBC2 in the nuclei of the infected cells. In summary, the current study reveals that the ChiLCV Rep protein binds the viral genome and interacts with NbUBC2 and NbHUB1 for the monoubiquitination of histone 2B that subsequently promotes trimethylation of histone 3 at lysine 4 on ChiLCV mini-chromosomes and enhances transcription of the viral genes., Author summary Histone post translation modification plays vital role in regulation of gene expression in eukaryotes. In plants, Ubiquitin-Conjugating Enzyme 2 and Histone Monoubiquitination1 monoubiquitinates histone 2B that promotes trimethylation of histone H3 at lysine 4 and both modifications eventually activates host transcription. The chromatinized genome (viral DNA associated with host histone proteins) of geminiviruses serves as a potential template for histone modification enzymes. Our study reveals the role of Chilli leaf curl virus (Geminiviridae) encoded Replication initiator protein (Rep) in stimulation of viral transcription via histone modifications on the viral minichromosome. Furthermore, the Rep protein redirects NbUBC2 from cytoplasm to the nucleus to form monoubiquitination machinery in association with NbHUB1 and recruits the machinery to accelerate the H2B-ub deposition on the viral promoter. The silencing of NbUBC2 and NbHUB1 did not alter Rep localization in the nucleus but affect ChiLCV pathogenesis. Taken together, our results showed for the first time that geminivirus Rep protein recruits the NbUBC2 and NbHUB1 on the viral promoter for monoubiquitination of H2B and stimulates viral gene expression.

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach., Author summary Recent progress in isolation of mAbs from survivors of filovirus infections suggests that the human adaptive immune system is capable of producing strong antibody responses. However, the effects of mAbs with different epitope specificity on individual steps of filovirus infection are still unclear. We evaluated a panel of mAbs obtained from survivors of natural filovirus infections, specific for the glycan cap or stem region of GP, for their effects on the attachment of viral particles to the cell surface, intracellular traffic of viral particles, proteolytic processing of GP, its interaction with the NPC1 receptor, cell-to-cell virus transmission, virus egress from infected cells, activation of natural killer cells and antibody-dependent cellular phagocytosis through Fc-mediated mechanisms. We found that antiviral activity of glycan cap-specific antibodies results from inhibition of attachment, cell-to-cell transmission and inhibition of virion budding. In contrast, the antiviral mechanisms of stem-specific antibodies were found to be inhibition of virus release from endosomal network to the cytoplasm, and also activation of natural killer cells and phagocytosis mediated by monocytes and neutrophils. The data provide new insight into the development of immune protective mechanisms during natural human infection, and have important implications for the treatment of filovirus infections by passively-transferred antibodies and vaccine design.

The intricate interactions between viruses and hosts include exploitation of host cells for viral replication by using many cellular resources, metabolites and energy. Tomato bushy stunt virus (TBSV), similar to other (+)RNA viruses, induces major changes in infected cells that lead to the formation of large replication compartments consisting of aggregated peroxisomal and ER membranes. Yet, it is not known how TBSV obtains the energy to fuel these energy-consuming processes. In the current work, the authors discovered that TBSV co-opts the glycolytic ATP-generating Pgk1 phosphoglycerate kinase to facilitate the assembly of new viral replicase complexes. The recruitment of Pgk1 into the viral replication compartment is through direct interaction with the viral replication proteins. Altogether, we provide evidence that the ATP generated locally within the replication compartment by the co-opted Pgk1 is used to fuel the ATP-requirement of the co-opted heat shock protein 70 (Hsp70) chaperone, which is essential for the assembly of new viral replicase complexes and the activation of functional viral RNA-dependent RNA polymerase. The advantage of direct recruitment of Pgk1 into the virus replication compartment could be that the virus replicase assembly does not need to intensively compete with cellular processes for access to ATP. In addition, local production of ATP within the replication compartment could greatly facilitate the efficiency of Hsp70-driven replicase assembly by providing high ATP concentration within the replication compartment., Author summary Positive-strand (+)RNA viruses build replication organelles with the help of many co-opted cellular factors and by usurping cellular metabolites and energy, which frequently lead to disease states in plants, animals and humans. The authors discovered that tomato bushy stunt virus (TBSV) co-opts the glycolytic ATP-generating Pgk1 phosphoglycerate kinase to facilitate the intracellular assembly of new viral replicase complexes. The ATP generated by co-opted Pgk1 within the replication compartment is used to fuel the ATP requirement of co-opted cellular heat shock protein 70 chaperone, which is essential for the assembly of new TBSV replicase complexes. Direct recruitment of Pgk1 by TBSV might provide easy access to ATP. Metabolic reprogramming of the glycolytic pathway might be a widespread phenomenon during various viral infections.

Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe. We found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of lipopolysaccharide O antigen enables vesicles to bypass clathrin-mediated endocytosis, which enhances both their entry rate and efficiency into host cells. Collectively, our findings highlight the composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections., Author summary All Gram negative species of bacteria, including those that cause significant disease, release small vesicles from their cell membrane. These vesicles deliver toxins and other virulence factors to host cells during infection. Current methods for studying host cell entry are limited due to the nanometer size and rapid uptake kinetics of vesicles. Here we developed a method to monitor the rapid vesicle entry into host cells in real-time. This method highlighted differences in kinetics and entry route of vesicles into host cells, which varied with the bacterial cell wall composition and thus, the vesicle surface. Increased understanding of vesicular entry mechanisms could identify targets which may allow us to combat infections by inhibiting delivery of vesicle-associated toxins to host cells.

Cyclic GMP-AMP (cGAMP) synthase (cGAS, a.k.a. MB21D1), a cytosolic DNA sensor, catalyzes formation of the second messenger 2’3’-cGAMP that activates the stimulator of interferon genes (STING) signaling. How the cGAS activity is modulated remains largely unknown. Here, we demonstrate that sentrin/SUMO-specific protease 7 (SENP7) interacted with and potentiated cGAS activation. The small ubiquitin-like modifier (SUMO) was conjugated onto the lysine residues 335, 372 and 382 of cGAS, which suppressed its DNA-binding, oligomerization and nucleotidyl-transferase activities. SENP7 reversed this inhibition via catalyzing the cGAS de-SUMOylation. Consistently, silencing of SENP7 markedly impaired the IRF3-responsive gene expression induced by cGAS-STING axis. SENP7-knockdown mice were more susceptible to herpes simplex virus 1 (HSV-1) infection. SENP7 was significantly up-regulated in patients with SLE. Our study highlights the temporal modulation of the cGAS activity via dynamic SUMOylation, uncovering a novel mechanism for fine-tuning the STING signaling in innate immunity., Author Summary The Cyclic GMP-AMP (cGAMP) synthase (cGAS, a.k.a. MB21D1) is critical for monitoring the pathogen-derived DNA upon microbial infection. Its activity should be dynamically modulated in case the inadvertent recognition of the aberrant self nucleic acids in cytosol leads to severe autoimmune diseases. Protein posttranslational modifications dynamically shape the strength and duration of the immune signaling pathways. It is intriguing to explore whether SUMOylation could modulate the cGAS-initiated signaling. In this study, we characterized sentrin/SUMO-specific protease 7 (SENP7) to specifically potentiate the cGAS activation. Upon microbial DNA challenge, the small ubiquitin-like modifier (SUMO) was conjugated onto cGAS, which suppressed its DNA-binding, oligomerization and nucleotidyl-transferase activities. SENP7 reversed this inhibition via catalyzing the de-SUMOylation of cGAS. Our study sheds new light on the dynamic function of the SUMOylation in cytosolic DNAs-triggered innate immunity response.