Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.

Nucleos(t)ide analogs are standard-of-care for the treatment of chronic hepatitis B and can effectively reduce hepatitis B virus (HBV) replication but rarely leads to cure. Nucleos(t)ide analogs do not directly eliminate the viral episome, therefore treatment cessation typically leads to rapid viral rebound. While treatment is effective, HBV DNA is still detectable (although not quantifiable) in the periphery of the majority of nucleos(t)ide analog treated HBV patients, even after prolonged treatment. Addressing whether the detectable HBV DNA represents infectious virus is a key unknown and has important implications for the development of a curative treatment for HBV. The minimum HBV genome equivalents required to establish infection in human liver chimeric mice was determined by titration of HBV patient sera and the infectivity in chimeric mice of serum from patients (n = 7) suppressed to the limit of detection on nucleos(t)ide analog therapy was evaluated. A minimum of 5 HBV genome equivalents were required to establish infection in the chimeric mice, confirming this model has sufficient sensitivity to determine whether serum from virally suppressed patients contains infectious virus. Strikingly, serum from 75% (n = 3 out of 4) of nucleos(t)ide-treated HBV patients with DNA that was detectable, but below the lower limit of quantitation, also established infection in the chimeric mice. These results demonstrate that infectious virus is still present in some HBV patients on suppressive nucleos(t)ide therapy. This residual virus may support viral persistence via continuous infection and explain the ongoing risk for HBV-related complications despite long-term suppression on therapy. Thus, additional treatment intensification may facilitate HBV cure.

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses.

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

Vibrio effector protein VopQ inhibits fusion of V-ATPase-containing membranes.

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V(o) domain of the conserved V-type H(+)-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.

STING-dependent recognition of cyclic di-AMP mediates type I interferon responses during Chlamydia trachomatis infection.

UNLABELLED: STING (stimulator of interferon [IFN] genes) initiates type I IFN responses in mammalian cells through the detection of microbial nucleic acids. The membrane-bound obligate intracellular bacterium Chlamydia trachomatis induces a STING-dependent type I IFN response in infected cells, yet the IFN-inducing ligand remains unknown. In this report, we provide evidence that Chlamydia synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite not previously identified in Gram-negative bacteria, and that this metabolite is a prominent ligand for STING-mediated activation of IFN responses during infection. We used primary mouse lung fibroblasts and HEK293T cells to compare IFN-ß responses to Chlamydia infection, c-di-AMP, and other type I IFN-inducing stimuli. Chlamydia infection and c-di-AMP treatment induced type I IFN responses in cells expressing STING but not in cells expressing STING variants that cannot sense cyclic dinucleotides but still respond to cytoplasmic DNA. The failure to induce a type I IFN response to Chlamydia and c-di-AMP correlated with the inability of STING to relocalize from the endoplasmic reticulum to cytoplasmic punctate signaling complexes required for IFN activation. We conclude that Chlamydia induces STING-mediated IFN responses through the detection of c-di-AMP in the host cell cytosol and propose that c-di-AMP is the ligand predominantly responsible for inducing such a response in Chlamydia-infected cells. IMPORTANCE: This study shows that the Gram-negative obligate pathogen Chlamydia trachomatis, a major cause of pelvic inflammatory disease and infertility, synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite that thus far has been described only in Gram-positive bacteria. We further provide evidence that the host cell employs an endoplasmic reticulum (ER)-localized cytoplasmic sensor, STING (stimulator of interferon [IFN] genes), to detect c-di-AMP synthesized by Chlamydia and induce a protective IFN response. This detection occurs even though Chlamydia is confined to a membrane-bound vacuole. This raises the possibility that the ER, an organelle that innervates the entire cytoplasm, is equipped with pattern recognition receptors that can directly survey membrane-bound pathogen-containing vacuoles for leaking microbe-specific metabolites to mount type I IFN responses required to control microbial infections.

The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING.

The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING) that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2'-5')pA(3'-5')p], which contains a single 2'-5' phosphodiester bond. Cyclic [G(2'-5')pA(3'-5')p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3'-5') cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS.

Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid.

Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) represent a novel approach for cancer treatment. DMXAA has potent antitumor activity in mice and, despite significant preclinical promise, failed human clinical trials. The antitumor activity of DMXAA has been linked to its ability to induce type I IFNs in macrophages, although the molecular mechanisms involved are poorly understood. In this study, we identify stimulator of IFN gene (STING) as a direct receptor for DMXAA leading to TANK-binding kinase 1 and IFN regulatory factor 3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anticancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.

STING and the innate immune response to nucleic acids in the cytosol.

Cytosolic detection of pathogen-derived nucleic acids is critical for the initiation of innate immune defense against diverse bacterial, viral and eukaryotic pathogens. Conversely, inappropriate responses to cytosolic nucleic acids can produce severe autoimmune pathology. The host protein STING has been identified as a central signaling molecule in the innate immune response to cytosolic nucleic acids. STING seems to be especially critical for responses to cytosolic DNA and the unique bacterial nucleic acids called 'cyclic dinucleotides'. Here we discuss advances in the understanding of STING and highlight the many unresolved issues in the field.

STING is a direct innate immune sensor of cyclic di-GMP.

The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1-IRF3 signalling axis. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria, have also been shown to induce a STING-dependent type I IFN response. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

Vibrio VopQ induces PI3-kinase-independent autophagy and antagonizes phagocytosis.

Vibrio parahaemolyticus is a Gram-negative bacterium responsible for gastroenteritis acquired from the consumption of contaminated shellfish. This bacterium harbours two type III secretion systems, one on each chromosome. The type III secretion system on chromosome I induces cell death by a temporally controlled sequence of events that is caspase-independent and first involves induction of autophagy, followed by cellular rounding, and finally cellular lysis. VopQ is a type III secreted effector that is necessary for the induction of autophagy as mutant strains lacking VopQ are attenuated in their ability to induce autophagy during infection. VopQ is sufficient to induce rapid autophagy as demonstrated by microinjection of recombinant VopQ into GFP-LC3 HeLa cells. Our results demonstrate that VopQ is both necessary and sufficient for induction of autophagy during V. parahaemolyticus-mediated cell death and this effect is independent of phosphatidylinositol-3-kinases but requires Atg5. Furthermore, induction of VopQ-mediated autophagy prevents recruitment of the necessary cellular machinery required for phagocytosis of V. parahaemolyticus during infection. These data provide important insights into the mechanism used by V. parahaemolyticus to cause disease.

Not without cause: Vibrio parahaemolyticus induces acute autophagy and cell death.

Vibrio parahaemolyticus (V. parahaemolyticus) is a gram-negative halophillic bacterium that causes worldwide seafood-borne gastroenteritis. The prevalence of V. parahaemolyticus in the environment and incidence of infection have been linked to rising water temperatures caused by global warming. Among its virulence factors, V. parahaemolyticus harbors two type III secretion systems (T3SS). Recently, we have shown that T3SS1 induces rapid cellular death that initiates with acute autophagy, as measured by LC3 lipidation and accumulation of early autophagosomal vesicles. While not the first characterized pathogen to usurp autophagy, this is the first example of an extracellular pathogen that exploits this pathway for its own benefit. Here we discuss possible roles for the induction of autophagy during infection and discuss how V. parahaemolyticus-induced autophagy provides insight into key regulatory steps that govern the decision between apoptosis and autophagy.

Vibrio parahaemolyticus orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and then cell lysis.

The bacterial pathogen Vibrio parahaemolyticus utilizes a type III secretion system to cause death of host cells within hours of infection. We report that cell death is completely independent of apoptosis and occurs by a mechanism in which injection of multiple type III effectors causes induction of autophagy, cell rounding, and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated light chain 3 (LC3) and by increases in punctae and vacuole formation. Electron microscopy reveals the production of early autophagic vesicles during infection. Consistent with phosphoinositide 3 (PI3) kinase playing a role in autophagy, treatment of infected cells with a PI3 kinase inhibitor attenuates autophagy in infected cells. Because many effectors are injected during a V. parahaemolyticus infection, it is not surprising that the presence of a sole PI3 kinase inhibitor does not prevent inevitable host-cell death. Our studies reveal an infection paradigm whereby an extracellular pathogen uses its type III secretion system to cause at least three parallel events that eventually result in the proinflammatory death of an infected host cell.

Arp2/3-independent assembly of actin by Vibrio type III effector VopL.

Microbial pathogens use a variety of mechanisms to disrupt the actin cytoskeleton during infection. Vibrio parahaemolyticus (V. para) is a Gram-negative bacterium that causes gastroenteritis, and new pandemic strains are emerging throughout the world. Analysis of the V. para genome revealed a type III secretion system effector, VopL, encoding three Wiskott-Aldrich homology 2 domains that are interspersed with three proline-rich motifs. Infection of HeLa cells with V. para induces the formation of long actin fibers in a VopL-dependent manner. Transfection of VopL promotes the assembly of actin stress fibers. In vitro, recombinant VopL potently induces assembly of actin filaments that grow at their barbed ends, independent of eukaryotic factors. Vibrio VopL is predicted to be a bacterial virulence factor that disrupts actin homeostasis during an enteric infection of the host.

The RACK1 homologue from Trypanosoma brucei is required for the onset and progression of cytokinesis.

The receptor for activated C kinase 1 (RACK1) is a conserved scaffold protein that helps regulate a range of cell activities including cell growth, shape, and protein translation. We report that a homologue of RACK1 is required for cytokinesis in pathogenic Trypanosoma brucei. The protein, referred to as TRACK, is comprised of WD repeat elements and can complement cpc2 null mutants of Schizosaccharomyces pombe. TRACK is expressed throughout the trypanosome life cycle and is distributed predominantly in a perinuclear region and the cytoplasm but not along the endoplasmic reticulum, mitochondrion, or cleavage furrow of dividing cells. When tetracycline-inducible RNA interference (RNAi) is used to deplete the cellular content of TRACK, the cells remain metabolically active, but growth is inhibited. In bloodstream forms, growth arrest is due to a delay in the onset of cytokinesis. By contrast, procyclic forms are able to initiate cytokinesis in the absence of TRACK but arrest midway through cell cleavage. The RNAi cells undergo multiple rounds of partial cytokinesis and accumulate nuclei and cytoplasmic extensions with attached flagella. The TRACK RNAi construct is also inducible within infected mice. Under these conditions parasites are eliminated from peripheral blood within 3 days post-infection. Taken as a whole, these data indicate that trypanosomes utilize a RACK1 homologue to regulate the final stages of mitosis. Moreover, disrupting the interaction between TRACK and its partners might be targeted in the design of novel therapies.

Inhibition of MAPK signaling pathways by VopA from Vibrio parahaemolyticus.

During infection, bacterial pathogens utilize a type III secretion system to inject effectors into the cytoplasm of a target cell where they disrupt the defense system of the host cell. Vibrio parahaemolyticus, a causative agent of gastroenteritis endemic in Southeast Asia, has a type III secretion system that encodes a novel member of the YopJ-like protein effector family, VopA (Vibrio outer protein A). Our studies revealed that Vibrio VopA encodes an evolutionarily conserved activity that is extremely potent and requires an intact catalytic site to abrogate signaling pathways in a manner distinct from that of other YopJ-like effectors. We observed that VopA efficiently inhibits the MAPK signaling pathways but not the NFkappaB pathway in mammalian cells. When expressed in yeast, VopA induces a growth arrest phenotype and also blocks yeast MAPK signaling pathways. Our observations provide insight into the immense diversity of targets utilized by YopJ-like effectors to manipulate eukaryotic signaling machineries that are important for the response and survival of the host cell during infection and/or symbiosis.