Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens

Preexisting and de novo humoral immunity to SARS-CoV-2 in humans

Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detect preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable by a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the IgG class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection

Biochemical Characterization of Arterivirus Nonstructural Protein 11 Reveals the Nidovirus-Wide Conservation of a Replicative Endoribonuclease▿ †

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses

Evolutionary Conserved Function of Barley and Arabidopsis 3-KETOACYL-CoA SYNTHASES in Providing Wax Signals for Germination of Powdery Mildew Fungi

For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matter of vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible. We investigated a barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildew fungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screen and identified to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required for synthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has significantly fewer aliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcame germination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigation of Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powdery mildew fungi. Our findings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly related powdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before the monocot-dicot split took place approximately 150 million years ago

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

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Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family

The ≈30-kb coronavirus (+)RNA genome is replicated and transcribed by a membrane-bound replicase complex made up of 16 viral nonstructural proteins (nsp) with multiple enzymatic activities. The complex includes an RNA endonuclease, NendoU, that is conserved among nidoviruses but no other RNA virus, making it a genetic marker of this virus order. NendoU (nsp15) is a Mn2+-dependent, uridylate-specific enzyme, which leaves 2′–3′-cyclic phosphates 5′ to the cleaved bond. Neither biochemical nor sequence homology criteria allow a classification of nsp15 into existing endonuclease families. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus nsp15 at 2.6-Å resolution. Nsp15 exhibits a unique fold and assembles into a toric hexamer with six potentially active, peripheric catalytic sites. The structure and the spatial arrangement of the catalytic residues into an RNase A-like active site define a separate endonuclease family, endoU, and represent another spectacular example of convergent evolution toward an enzymatic function that is critically involved in the coronavirus replication cycle

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.status: publishe