Lineage A Betacoronavirus NS2 Proteins and the Homologous Torovirus Berne pp1a Carboxy-Terminal Domain Are Phosphodiesterases That Antagonize Activation of RNase L.

Viruses in the family Coronaviridae, within the order Nidovirales, are etiologic agents of a range of human and animal diseases, including both mild and severe respiratory diseases in humans. These viruses encode conserved replicase and structural proteins as well as more diverse accessory proteins, encoded in the 3' ends of their genomes, that often act as host cell antagonists. We previously showed that 2',5'-phosphodiesterases (2',5'-PDEs) encoded by the prototypical Betacoronavirus, mouse hepatitis virus (MHV), and by Middle East respiratory syndrome-associated coronavirus antagonize the oligoadenylate-RNase L (OAS-RNase L) pathway. Here we report that additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses infecting both humans and animals, encode 2',5'-PDEs capable of antagonizing RNase L. We used a chimeric MHV system (MHVMut) in which exogenous PDEs were expressed from an MHV backbone lacking the gene for a functional NS2 protein, the endogenous RNase L antagonist. With this system, we found that 2',5'-PDEs encoded by the human coronavirus HCoV-OC43 (OC43; an agent of the common cold), human enteric coronavirus (HECoV), equine coronavirus (ECoV), and equine torovirus Berne (BEV) are enzymatically active, rescue replication of MHVMut in bone marrow-derived macrophages, and inhibit RNase L-mediated rRNA degradation in these cells. Additionally, PDEs encoded by OC43 and BEV rescue MHVMut replication and restore pathogenesis in wild-type (WT) B6 mice. This finding expands the range of viruses known to encode antagonists of the potent OAS-RNase L antiviral pathway, highlighting its importance in a range of species as well as the selective pressures exerted on viruses to antagonize it.IMPORTANCE Viruses in the family Coronaviridae include important human and animal pathogens, including the recently emerged viruses severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome-associated coronavirus (MERS-CoV). We showed previously that two viruses within the genus Betacoronavirus, mouse hepatitis virus (MHV) and MERS-CoV, encode 2',5'-phosphodiesterases (2',5'-PDEs) that antagonize the OAS-RNase L pathway, and we report here that these proteins are furthermore conserved among additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses, suggesting that they may play critical roles in pathogenesis. As there are no licensed vaccines or effective antivirals against human coronaviruses and few against those infecting animals, identifying viral proteins contributing to virulence can inform therapeutic development. Thus, this work demonstrates that a potent antagonist of host antiviral defenses is encoded by multiple and diverse viruses within the family Coronaviridae, presenting a possible broad-spectrum therapeutic target.

Human Genome-Wide RNAi Screen for Host Factors That Facilitate Salmonella Invasion Reveals a Role for Potassium Secretion in Promoting Internalization.

Salmonella enterica can actively invade the gastro-intestinal epithelium. This frequently leads to diarrheal disease, and also gives the pathogen access to phagocytes that can serve as vehicles for dissemination into deeper tissue. The ability to invade host cells is also important in maintaining the carrier state. While much is known about the bacterial factors that promote invasion, relatively little is known about the host factors involved. To gain insight into how Salmonella enterica serovar Typhimurium is able to invade normally non-phagocytic cells, we undertook a global RNAi screen with S. Typhimurium-infected human epithelial cells. In all, we identified 633 genes as contributing to bacterial internalization. These genes fall into a diverse group of functional categories revealing that cytoskeletal regulators are not the only factors that modulate invasion. In fact, potassium ion transport was the most enriched molecular function category in our screen, reinforcing a link between potassium and internalization. In addition to providing new insights into the molecular mechanisms underlying the ability of pathogens to invade host cells, all 633 host factors identified are candidates for new anti-microbial targets for treating Salmonella infections, and may be useful in curtailing infections with other pathogens as well.

Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation.

UNLABELLED: Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. Like many coronaviruses, MERS-CoV carries genes that encode multiple accessory proteins that are not required for replication of the genome but are likely involved in pathogenesis. Evasion of host innate immunity through interferon (IFN) antagonism is a critical component of viral pathogenesis. The IFN-inducible oligoadenylate synthetase (OAS)-RNase L pathway activates upon sensing of viral double-stranded RNA (dsRNA). Activated RNase L cleaves viral and host single-stranded RNA (ssRNA), which leads to translational arrest and subsequent cell death, preventing viral replication and spread. Here we report that MERS-CoV, a lineage CBetacoronavirus, and related bat CoV NS4b accessory proteins have phosphodiesterase (PDE) activity and antagonize OAS-RNase L by enzymatically degrading 2',5'-oligoadenylate (2-5A), activators of RNase L. This is a novel function for NS4b, which has previously been reported to antagonize IFN signaling. NS4b proteins are distinct from lineage ABetacoronavirusPDEs and rotavirus gene-encoded PDEs, in having an amino-terminal nuclear localization signal (NLS) and are localized mostly to the nucleus. However, the expression level of cytoplasmic MERS-CoV NS4b protein is sufficient to prevent activation of RNase L. Finally, this is the first report of an RNase L antagonist expressed by a human or bat coronavirus and provides a specific mechanism by which this occurs. Our findings provide a potential mechanism for evasion of innate immunity by MERS-CoV while also identifying a potential target for therapeutic intervention. IMPORTANCE: Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV). MERS-CoV, like other coronaviruses, carries genes that encode accessory proteins that antagonize the host antiviral response, often the type I interferon response, and contribute to virulence. We found that MERS-CoV NS4b and homologs from related lineage C bat betacoronaviruses BtCoV-SC2013 (SC2013) and BtCoV-HKU5 (HKU5) are members of the 2H-phosphoesterase (2H-PE) enzyme family with phosphodiesterase (PDE) activity. Like murine coronavirus NS2, a previously characterized PDE, MERS NS4b, can antagonize activation of the OAS-RNase L pathway, an interferon-induced potent antiviral activity. Furthermore, MERS-CoV mutants with deletion of genes encoding accessory proteins NS3 to NS5 or NS4b alone or inactivation of the PDE can activate RNase L during infection of Calu-3 cells. Our report may offer a potential target for therapeutic intervention if NS4b proves to be critical to pathogenesis inin vivomodels of MERS-CoV infection.

Murine AKAP7 has a 2',5'-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene.

Viral 2',5'-phosphodiesterases (2',5'-PDEs) help disparate RNA viruses evade the antiviral activity of interferon (IFN) by degrading 2',5'-oligoadenylate (2-5A) activators of RNase L. A kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) to localize and organize cyclic AMP (cAMP) signaling during diverse physiological processes. Among more than 43 AKAP isoforms, AKAP7 appears to be unique in its homology to viral 2',5'-PDEs. Here we show that mouse AKAP7 rapidly degrades 2-5A with kinetics similar to that of murine coronavirus (mouse hepatitis virus [MHV]) strain A59 ns2 and human rotavirus strain WA VP3 proteins. To determine whether AKAP7 could substitute for a viral 2',5'-PDE, we inserted AKAP7 cDNA into an MHV genome with an inactivated ns2 gene. The AKAP7 PDE domain or N-terminally truncated AKAP7 (both lacking a nuclear localization motif), but not full-length AKAP7 or a mutant, AKAP7(H185R), PDE domain restored the infectivity of ns2 mutant MHV in bone marrow macrophages and in livers of infected mice. Interestingly, the AKAP7 PDE domain and N-terminally deleted AKAP7 were present in the cytoplasm (the site of MHV replication), whereas full-length AKAP7 was observed only in nuclei. We suggest the possibility that viral acquisition of the host AKAP7 PDE domain might have occurred during evolution, allowing diverse RNA viruses to antagonize the RNase L pathway. Importance: Early virus-host interactions determine whether an infection is established, highlighting the need to understand fundamental mechanisms regulating viral pathogenesis. Recently, our laboratories reported a novel mode of regulation of the IFN antiviral response. We showed that the coronavirus MHV accessory protein ns2 antagonizes the type I IFN response, promoting viral replication and hepatitis. ns2 confers virulence by cleaving 2',5'-oligoadenylate (2-5A) activators of RNase L in macrophages. We also reported that the rotavirus VP3 C-terminal domain (VP3-CTD) cleaves 2-5A and that it may rescue ns2 mutant MHV. Here we report that a cellular protein, AKAP7, has an analogous 2',5'-phosphodiesterase (2',5'-PDE) domain that is able to restore the growth of chimeric MHV expressing inactive ns2. The proviral effect requires cytoplasmic localization of the AKAP7 PDE domain. We speculate that AKAP7 is the ancestral precursor of viral proteins, such as ns2 and VP3, that degrade 2-5A to evade the antiviral activity of RNase L.

A naturally occurring single nucleotide polymorphism in the Salmonella SPI-2 type III effector srfH/sseI controls early extraintestinal dissemination.

CD18 expressing phagocytes associated with the gastro-intestinal (GI) epithelium can shuttle Salmonella directly into the bloodstream within a few minutes following microbial ingestion. We have previously demonstrated that Salmonella controls the CD18 pathway to deeper tissue, manipulating the migratory properties of infected cells as an unappreciated component of its pathogenesis. We have observed that one type III effector, SrfH (also called SseI) that Salmonella secretes into infected phagocytes manipulates the host protein TRIP6 to stimulate their migration. Paradoxically, SrfH was shown in another study to subvert a different host protein, IQGAP1, in a manner that inhibits the productive motility of such cells, perhaps to avoid interactions with T cells. Here, we resolve the discrepancy. We report that one naturally occurring allele of srfH promotes the migration of infected phagocytes into the bloodstream, while another naturally occurring allele that differs by only a single nucleotide polymorphism (SNP) does not. This SNP determines if the protein contains an aspartic acid or a glycine residue at position 103 and may determine if SrfH binds TRIP6. SrfH Gly103 is a rare allele, but is present in the highly invasive strain Salmonella enterica serovar Typhimurium UK-1 (stands for universal killer). It is also present in the genome of the only sequenced strain belonging to the emerging pandemic Salmonella enterica serovar 4, [5],12,i:-, which is frequently associated with septicemia. Finally, we present evidence that suggests that Gifsy-2, the bacteriophage upon which srfH resides, is present in a clinical isolate of the human-specific pathogen, Salmonella enterica serovar Typhi. These observations may have interesting implications for our understanding of Salmonella pathogenesis.

Human genome-wide RNAi screen for host factors that modulate intracellular Salmonella growth.

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.