The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU

ABSTRACT Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo . The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-β expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor. IMPORTANCE The Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies

A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns

[ENG] Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defenseSIGenotyping of cowpea accessions was supported by the Feed the Future Innovation Laboratory for Climate Resilient Cowpea (US Agency for International Development Cooperative Agreement AID-OAA-A-13-00070). OS analyses were supported by European Research Council Advanced Grant 788949. Research in the C.Z. laboratory was supported by The Gatsby Charitable Foundation and the Biotechnology and Biological Research Council (BB/P012574/1

Recognition and Activation Domains Contribute to Allele-Specific Responses of an Arabidopsis NLR Receptor to an Oomycete Effector Protein

In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function

Recognition and Activation Domains Contribute to Allele-Specific Responses of an Arabidopsis NLR Receptor to an Oomycete Effector Protein

In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function

Chimeric RPP1 constructs implicate LRRs and ARC2 domains in specificity.

<p><b>(A)</b> Amino acid numbering for chimeric exchanges. Colors (light blue for RPP1-NdA, dark orange for RPP1-WsB) indicate domains with either RPP1-NdA or RPP1-WsB amino acid identity. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004665#ppat.1004665.s004" target="_blank">S4 Fig.</a> for chimeric constructs in context of pairwise sequence alignment. <b>(B)</b> Chimeric constructs of RPP1-NdA and RPP1-WsB with complete LRR exchanges tested against ATR1 alleles with conserved recognition. <b>(C)</b> N-terminal LRRs mediate recognition of ATR1-Cala2 WsB-GOF. Tobacco HR phenotypes against ATR1-WsB-GOF are shown, with additional chimeric exchanges within LRRs. <b>(D)</b> RPP1-NdA ARC2 domain is required for activation by ATR1-Cala2 NdA GOF. Tobacco HR phenotypes against ATR1-Cala2 NdA-GOF are shown, with additional chimeric exchanges N-terminal to the LRRs. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004665#ppat.1004665.s005" target="_blank">S5 Fig.</a> for additional non-functional chimeric constructs and visible mixed (+/-) recognition phenotype by WsB⁵⁹⁸NdA. Inoculations were performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004665#ppat.1004665.g002" target="_blank">Fig. 2A</a>, with RPP1 construct indicated on the right. Relative strength of HR is indicated with symbols, +++, strong HR, ++, moderate HR, +, weak HR, +/-, mixed HR, -, no HR.</p

The RPP1-NdA ARC2 subdomain contributes to specificity by facilitating receptor activation.

<p><b>(A)</b> RPP1-NdA ARC2 is insufficient for allele-specific recognition. Chimeric constructs, including a double chimera with RPP1-NdA ARC2 in a RPP1-WsB context were tested against NdA-specific (ATR1-Cala2 NdA-GOF) and conserved recognized (ATR1-Emoy2) ATR1 alleles. <b>(B)</b> RPP1-NdA ARC2 is dispensable for allele-specific recognition through association. FLAG-tagged ATR1 alleles were immunoprecipitated from <i>N</i>. <i>benthamiana</i> tissue extracts and probed for association with HA-tagged RPP1 by Western blot. HR phenotype is indicated with + or - in table. <b>(C)</b> RPP1-NdA ARC2 domain sensitizes the receptor to low inoculum of ATR1-Emoy2. Density of inoculum of Agrobacterium strain for ATR1-Emoy2 expression is indicated at top. Inoculations were performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004665#ppat.1004665.g002" target="_blank">Fig. 2A</a>, with RPP1 construct indicated on the right. Relative strength of HR is indicated with symbols, +++, strong HR, ++, moderate HR, -, no HR.</p

Model for RPP1 specificity involving recognition and activation domains.

<p><b>(A)</b> Wild-type ATR1-Cala2 is unrecognized by RPP1, maintaining an “off” state conformation, potentially mediated by intramolecular ARC2-LRR interactions. <b>(B)</b> Specific amino acid substitutions in ATR1-Cala2 confer allele-specific recognition by RPP1-NdA (light blue). RPP1-NdA LRRs are sufficient for association with ATR1, but not for full-strength activation of the receptor. Further activation specificity is conferred by inclusion of an RPP1-NdA ARC2 domain. Full receptor activation strength occurs with wild-type RPP1-NdA, with full intramolecular compatibility.</p

Delivery of alleles and mutants of ATR1 by <i>Pseudomonas syringae</i> pv. <i>tomato</i> strain DC3000 leads to allele specific growth inhibition.

<p>Plant lines were inoculated with pEDV3 constructs containing ATR1 variants and inoculated at OD = 0.0001. *, p<0.05, **, p<0.01, Student’s T-test.</p