SAS: Split antibiotic selection for identifying chaperones that improve protein solubility.

Background: Heterologous expression of active, native-folded protein in Escherichia coli is critical in both academic research and biotechnology settings. When expressing non-native recombinant proteins in E. coli, obtaining soluble and active protein can be challenging. Numerous techniques can be used to enhance a proteins solubility, and largely focus on either altering the expression strain, plasmid vector features, growth conditions, or the protein coding sequence itself. However, there is no one-size-fits-all approach for addressing issues with protein solubility, and it can be both time and labor intensive to find a solution. An alternative approach is to use the co-expression of chaperones to assist with increasing protein solubility. By designing a genetic system where protein solubility is linked to viability, the appropriate protein folding factor can be selected for any given protein of interest. To this end, we developed a Split Antibiotic Selection (SAS) whereby an insoluble protein is inserted in-frame within the coding sequence of the hygromycin B resistance protein, aminoglycoside 7″-phosphotransferase-Ia (APH(7″)), to generate a tripartite fusion. By creating this tripartite fusion with APH(7″), the solubility of the inserted protein can be assessed by measuring the level of hygromycin B resistance of the cells. Results: We demonstrate the functionality of this system using a known protein and co-chaperone pair, the human mitochondrial Hsp70 ATPase domain (ATPase70) and its co-chaperone human escort protein (Hep). Insertion of the insoluble ATPase70 within APH(7'') renders the tripartite fusion insoluble and results in sensitivity to hygromycin B. Antibiotic resistance can be rescued by expression of the co-chaperone Hep which assists in the folding of the APH(7'')-ATPase70-APH(7'') tripartite fusion and find that cellular hygromycin B resistance correlates with the total soluble fusion protein. Finally, using a diverse chaperone library, we find that SAS can be used in a pooled genetic selection to identify chaperones capable of improving client protein solubility. Conclusions: The tripartite APH(7'') fusion links the in vivo solubility of the inserted protein of interest to hygromycin B resistance. This construct can be used in conjunction with a chaperone library to select for chaperones that increase the solubility of the inserted protein. This selection system can be applied to a variety of client proteins and eliminates the need to individually test chaperone-protein pairs to identify those that increase solubility.

Tracing the evolutionary origins of antiviral immunity.

Animal and bacterial cells use shared mechanisms to defend against viruses. Analyzing 3 families of immune genes, a new study in PLOS Biology illuminates this evolutionary connection and traces the emergence of antiviral signaling across domains of life.

Production of antibodies in SHuffle Escherichia coli strains.

Antibodies are globally important macromolecules, used for research, diagnostics, and as therapeutics. The common mammalian antibody immunoglobulin G (IgG) is a complex glycosylated macromolecule, composed of two heavy chains and two light chains held together by multiple disulfide bonds. For this reason, IgG and related antibody fragments are usually produced through secretion from mammalian cell lines, such as Chinese Hamster Ovary cells. However, there is growing interest in production of antibodies in prokaryotic systems due to the potential for rapid and cheap production in a highly genetically manipulable system. Research on oxidative protein folding in prokaryotes has enabled engineering of Escherichia coli strains capable of producing IgG and other disulfide bonded proteins in the cytoplasm, known as SHuffle. In this protocol, we provide a review of research on prokaryotic antibody production, guidelines on cloning of antibody expression constructs, conditions for an initial expression and purification experiment, and parameters which may be optimized for increased purification yields. Last, we discuss the limitations of prokaryotic antibody production, and highlight potential future avenues for research on antibody expression and folding.

Molecular basis of CD-NTase nucleotide selection in CBASS anti-phage defense.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are signaling proteins that initiate antiviral immunity in animal cells and cyclic-oligonucleotide-based anti-phage signaling system (CBASS) phage defense in bacteria. Upon phage recognition, bacterial CD-NTases catalyze synthesis of cyclic-oligonucleotide signals, which activate downstream effectors and execute cell death. How CD-NTases control nucleotide selection to specifically induce defense remains poorly defined. Here, we combine structural and nucleotide-analog interference-mapping approaches to identify molecular rules controlling CD-NTase specificity. Structures of the cyclic trinucleotide synthase Enterobacter cloacae CdnD reveal coordinating nucleotide interactions and a possible role for inverted nucleobase positioning during product synthesis. We demonstrate that correct nucleotide selection in the CD-NTase donor pocket results in the formation of a thermostable-protein-nucleotide complex, and we extend our analysis to establish specific patterns governing selectivity for each of the major bacterial CD-NTase clades A-H. Our results explain CD-NTase specificity and enable predictions of nucleotide second-messenger signals within diverse antiviral systems.

Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host-pathogen conflict.

DNA viruses in the family Poxviridae encode poxin enzymes that degrade the immune second messenger 2'3'-cGAMP to inhibit cGAS-STING immunity in mammalian cells. The closest homologs of poxin exist in the genomes of insect viruses suggesting a key mechanism of cGAS-STING evasion may have evolved outside of mammalian biology. Here we use a biochemical and structural approach to discover a broad family of 369 poxins encoded in diverse viral and animal genomes and define a prominent role for 2'3'-cGAMP cleavage in metazoan host-pathogen conflict. Structures of insect poxins reveal unexpected homology to flavivirus proteases and enable identification of functional self-cleaving poxins in RNA-virus polyproteins. Our data suggest widespread 2'3'-cGAMP signaling in insect antiviral immunity and explain how a family of cGAS-STING evasion enzymes evolved from viral proteases through gain of secondary nuclease activity. Poxin acquisition by poxviruses demonstrates the importance of environmental connections in shaping evolution of mammalian pathogens.

Conserved strategies for pathogen evasion of cGAS-STING immunity.

The cyclic GMP-AMP synthase (cGAS)- Stimulator of Interferon Genes (STING) pathway of cytosolic DNA sensing allows mammalian cells to detect and respond to infection with diverse pathogens. Pathogens in turn encode numerous factors that inhibit nearly all steps of cGAS-STING signal transduction. From masking of cytosolic DNA ligands, to post-translational modification of cGAS and STING, and degradation of the nucleotide second messenger 2'3'-cGAMP, pathogens have evolved convergent mechanisms to evade cGAS-STING sensing. Here we examine pathogen inhibitors of innate immunity in the context of newly discovered regulatory features controlling cellular cGAS-STING activation. Comparative analysis of these strategies provides insight into mechanisms of action and suggests aspects of cGAS-STING regulation and immune evasion that remain to be discovered.

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity.

Publisher Correction: Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling.

In this Letter, Supplementary Fig. 1 was missing. This error has been corrected online.

Bacterial cGAS-like enzymes synthesize diverse nucleotide signals.

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling.

Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)1-4. 2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5-7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp[2'-5']Ap[3']. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.

Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein.

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3.

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.

Discovery of urchin-associated densoviruses (family Parvoviridae) in coastal waters of the Big Island, Hawaii.

Echinoderms are important constituents of marine ecosystems, where they may influence the recruitment success of benthic flora and fauna, and are important consumers of detritus and plant materials. There are currently no described viruses of echinoderms. We used a viral metagenomic approach to examine viral consortia within three urchins - Colobocentrotus atratus, Tripneustes gratilla and Echinometra mathaei - which are common constituents of reef communities in the Hawaiian archipelago. Metagenomic libraries revealed the presence of bacteriophages and densoviruses (family Parvoviridae) in tissues of all three urchins. Densoviruses are known typically to infect terrestrial and aquatic arthropods. Urchin-associated densoviruses were detected by quantitative PCR in all tissues tested, and were also detected in filtered suspended matter (>0.2 µm) from plankton and in sediments at several locations near to where the urchins were collected for metagenomic analysis. To the best of our knowledge, this is the first report of echinoderm-associated viruses, which extends the known host range of parvoviruses.

Temporal dynamics and decay of putatively allochthonous and autochthonous viral genotypes in contrasting freshwater lakes.

Aquatic viruses play important roles in the biogeochemistry and ecology of lacustrine ecosystems; however, their composition, dynamics, and interactions with viruses of terrestrial origin are less extensively studied. We used a viral shotgun metagenomic approach to elucidate candidate autochthonous (i.e., produced within the lake) and allochthonous (i.e., washed in from other habitats) viral genotypes for a comparative study of their dynamics in lake waters. Based on shotgun metagenomes prepared from catchment soil and freshwater samples from two contrasting lakes (Cayuga Lake and Fayetteville Green Lake), we selected two putatively autochthonous viral genotypes (phycodnaviruses likely infecting algae and cyanomyoviruses likely infecting picocyanobacteria) and two putatively allochthonous viral genotypes (geminiviruses likely infecting terrestrial plants and circoviruses infecting unknown hosts but common in soil libraries) for analysis by genotype-specific quantitative PCR (TaqMan) applied to DNAs from viruses in the viral size fraction of lake plankton, i.e., 0.2 µm > virus > 0.02 µm. The abundance of autochthonous genotypes largely reflected expected host abundance, while the abundance of allochthonous genotypes corresponded with rainfall and storm events in the respective catchments, suggesting that viruses with these genotypes may have been transported to the lake in runoff. The decay rates of allochthonous and autochthonous genotypes, assessed in incubations where all potential hosts were killed, were generally lower (0.13 to 1.50% h(-1)) than those reported for marine virioplankton but similar to those for freshwater virioplankton. Both allochthonous and autochthonous viral genotypes were detected at higher concentrations in subsurface sediments than at the water-sediment interface. Our data indicate that putatively allochthonous viruses are present in lake plankton and sediments, where their temporal dynamics reflect active transport to the lake during hydrological events and then decay once there.