Syntaxin 11 Contributes to the Interferon-Inducible Restriction of Coxiella burnetii Intracellular Infection.

There is a limited understanding of host defense mechanisms targeting intracellular pathogens that proliferate in a lysosome. Coxiella burnetii is a model bacterial pathogen capable of replicating in the hydrolytic and acidic environment of the lysosome. It has been shown that gamma interferon (IFNγ)-stimulated host cells restrict C. burnetii replication by a mechanism that involves host IDO1 depletion of tryptophan. Host cells deficient in IDO1 activity, however, retain the ability to restrict C. burnetii replication when stimulated with IFNγ, which suggests additional mechanisms of host defense. This study identified syntaxin 11 (STX11) as a host protein that contributes to IFNγ-mediated suppression of C. burnetii replication. STX11 is a SNARE protein; SNARE proteins are proteins that mediate fusion of host vesicles with specific subcellular organelles. Depletion of STX11 using either small interfering RNA (siRNA)- or CRISPR-based approaches enhanced C. burnetii replication intracellularly. Stable expression of STX11 reduced C. burnetii replication in epithelial cells and macrophages, which indicates that this STX11-dependent cell-autonomous response is operational in multiple cell types and can function independently of other IFNγ-induced factors. Fluorescently tagged STX11 localized to the Coxiella-containing vacuole (CCV), and STX11 restriction was found to involve an interaction with STX8. Thus, STX11 regulates a vesicle fusion pathway that limits replication of this intracellular pathogen in a lysosome-derived organelle. IMPORTANCE Cell intrinsic defense mechanisms are used by eukaryotic cells to restrict the replication and dissemination of pathogens. This study identified a human protein called syntaxin 11 (STX11) as a host restriction factor that inhibits the intracellular replication of Coxiella burnetii. Syntaxins regulate the delivery of cargo inside vesicles by promoting specific membrane fusion events between donor and acceptor vesicles. Data presented here demonstrate that STX11 regulates an immunological defense pathway that controls replication of pathogens in lysosome-derived organelles, which provides new insight into the function of this SNARE protein.

Finding the Middle Ground for Autophagic Fusion Requirements.

Autophagosome/amphisome-lysosome fusion is a highly regulated process at the protein, lipid, and biochemical level. Each primary component of fusion, such as the core SNAREs, HOPS complex, or physical positioning by microtubule-associated dynein motors, are regulated at multiple points to ensure optimum conditions for autophagic flux to proceed. With the complexity of the membrane fusion system, it is not difficult to imagine how autophagic flux defect-related disorders, such as Huntington's disease, non-familial Alzheimer's disease, and Vici syndrome develop. Each membrane fusion step is regulated at the protein, lipid, and ion level. This review aims to discuss the recent developments toward understanding the regulation of autophagosome, amphisome, and lysosome fusion requirements for successful autophagic flux.

Oh, SNAP! How enteroviruses redirect autophagic traffic away from degradation.

Picornaviruses, one of the major causes of human diseases ranging from the common cold to acute flaccid paralysis, have a short cytosolic lifecycle that, in cultured cells, ends in cell lysis. For years, the prevailing model was that these viruses exit from cells exclusively through cell lysis. However, over the last several years it has become apparent that for some picornaviruses, a macroautophagy/autophagy-related pathway can result in release of virus particles wrapped in a membrane containing autophagic markers. It has been proposed that this enveloped release predominates within hosts, allowing cell-to-cell movement of virus while minimizing exposure to the immune system. One reason that picornaviruses induce the autophagy pathway is to provide membrane scaffolds for RNA replication complexes. Perhaps more importantly, acidified autophagosomes (known as amphisomes) provide havens for maturation of new viral particles into infectious viruses. In back-to-back papers recently published in Cell Reports, our labs investigated a basic question: if picornavirus particles are maturing inside amphisomes, then how are they avoiding the typical degradative fate of autophagic cargo and exiting the cell intact?

Poliovirus induces autophagic signaling independent of the ULK1 complex.

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway. ABBREVIATIONS: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1.

Enteroviruses Remodel Autophagic Trafficking through Regulation of Host SNARE Proteins to Promote Virus Replication and Cell Exit.

Enterovirus D68 (EV-D68) is a medically important respiratory plus-strand RNA virus of children that has been linked to acute flaccid myelitis. We have determined that EV-D68 induces autophagic signaling and membrane formation. Autophagy, a homeostatic degradative process that breaks down protein aggregates and damaged organelles, promotes replication of multiple plus-strand viruses. Induction of autophagic signals promotes EV-D68 replication, but the virus inhibits the downstream degradative steps of autophagy in multiple ways. EV-D68 proteases cleave a major autophagic cargo adaptor and the autophagic SNARE SNAP29, which reportedly regulates fusion between autophagosome to amphisome/autolysosome. Although the virus inhibits autophagic degradation, SNAP29 promotes virus replication early in infection. An orphan SNARE, SNAP47, is shown to have a previously unknown role in autophagy, and SNAP47 promotes the replication of EV-D68. Our study illuminates a mechanism for subversion of autophagic flux and redirection of the autophagic membranes to benefit EV-D68 replication.