The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication.

Bats host a large number of zoonotic viruses, including several viruses that are highly pathogenic to other mammals. The mechanisms underlying this rich viral diversity are unknown, but they may be linked to unique immunological features that allow bats to act as asymptomatic viral reservoirs. Vertebrates respond to viral infection by inducing IFNs, which trigger antiviral defenses through IFN-stimulated gene (ISG) expression. Although the IFN system of several bats is characterized at the genomic level, less is known about bat IFN-mediated transcriptional responses. In this article, we show that IFN signaling in bat cells from the black flying fox () consists of conserved and unique ISG expression profiles. In IFN-stimulated cells, bat ISGs comprise two unique temporal subclusters with similar early induction kinetics but distinct late-phase declines. In contrast, human ISGs lack this decline phase and remained elevated for longer periods. Notably, in unstimulated cells, bat ISGs were expressed more highly than their human counterparts. We also found that the antiviral effector 2-5A-dependent endoribonuclease, which is not an ISG in humans, is highly IFN inducible in black flying fox cells and contributes to cell-intrinsic control of viral infection. These studies reveal distinctive innate immune features that may underlie a unique virus-host relationship in bats.

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Flaviviruses such as Zika virus and West Nile virus have the potential to cause severe neuropathology if they invade the central nervous system. The type I interferon response is well characterized as contributing to control of flavivirus-induced neuropathogenesis. However, the interferon-stimulated gene (ISG) effectors that confer these neuroprotective effects are less well studied. Here, we used an ISG expression screen to identify Shiftless (SHFL, C19orf66) as a potent inhibitor of diverse positive-stranded RNA viruses, including multiple members of the Flaviviridae (Zika, West Nile, dengue, yellow fever, and hepatitis C viruses). In cultured cells, SHFL functions as a viral RNA-binding protein that inhibits viral replication at a step after primary translation of the incoming genome. The murine ortholog, Shfl, is expressed constitutively in multiple tissues, including the central nervous system. In a mouse model of Zika virus infection, Shfl−/− knockout mice exhibit reduced survival, exacerbated neuropathological outcomes, and increased viral replication in the brain and spinal cord. These studies demonstrate that Shfl is an important antiviral effector that contributes to host protection from Zika virus infection and virus-induced neuropathological disease.

Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.

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Bats are reservoirs for a number of highly pathogenic zoonotic viruses, yet they remain relatively asymptomatic during infection. Whether this viral resistance is due to a unique innate immune system is unknown. An evolutionarily conserved feature of vertebrate antiviral immunity is the interferon (IFN) response, which triggers cellular defenses through interferon-stimulated gene (ISG) expression. While bats encode an intact IFN system, global ISG expression patterns in bat cells are not well characterized. Here, we used RNA-Seq to assess the transcriptional response to IFNα in cells derived from the bat Pteropus alecto (black flying fox). We show induction of more than 100 transcripts, most of which are canonical ISGs observed in other species. Kinetic gene profiling revealed that P. alecto ISGs fall into two unique temporal subclusters with similar early induction kinetics but distinct late-phase declines. In contrast to bat ISGs, human ISGs generally remained elevated for longer periods following IFN treatment, suggesting host-based differences in gene regulatory mechanisms. Notably, we also identified a small group of non-canonical bat ISGs, including an enzymatically active RNASEL that plays a role in controlling viral infection. These studies provide insight into the innate immune response of an important viral reservoir and lay a foundation for studies into the immunological features that may underlie unique virus-host relationship in bats.

Cigarette smoking is a significant risk factor for atherosclerosis, which involves the invasion of vascular smooth muscle cells (VSMCs) from the media to intima. Many invasive cells remodel the actin cytoskeleton to form podosomes, which regulate metalloproteinase (MMP) release for extracellular matrix (ECM) degradation. We tested the hypothesis that cigarette smoke extract (CSE) modulates the structure and function of podosomes in VSMCs. We found that, in response to PKC activation by phorbol dibutyrate (PDBu), untreated A7r5 VSMCs formed podosomes, whereas CSE, nicotine, and carbachol-treated cells formed actin-rich rings. The nicotinic acetylcholine receptor (nAChR) antagonist a-bungarotoxin abolished the effects of nicotine and carbachol on actin-rich ring formation. Immunofluorescence labeling experiments localized MMP-2 and nAChRs at the actin-rich rings. Nicotineinduced actin-rich ring formation required 6-12 hr exposure, and was sensitive to the protein synthesis inhibitor cycloheximide. Conditioned media collected from cell culture of nicotine-treated cells induced podosome remodeling in untreated cells. When cells were cultured on DQ-gelatin, untreated cells exhibited a fibrillar network of fluorescence from DQ-gelatin degradation, which, upon PDBu stimulation, reorganized into peripheral dots and migrated towards the perinuclear region. In contrast, nicotine-treated cells, upon PDBu stimulation, reorganized the fluorescence into perinuclear fibrils, which dispersed into small dots and disappeared rapidly over time. Results from this study suggest that nicotine, by activating nAChRs and inducing phenotypic modulation, may prime VSMCs to become hyperresponsive to PKC activation with an enhanced ability to remodel the actin cytoskeleton and release MMP for invasion of the ECM.