BPIFB3 interacts with ARFGAP1 and TMED9 to regulate non-canonical autophagy and RNA virus infection.

Autophagy is a degradative cellular pathway that targets cytoplasmic contents and organelles for turnover by the lysosome. Various autophagy pathways play key roles in the clearance of viral infections, and many families of viruses have developed unique methods for avoiding degradation. Some positive-stranded RNA viruses, such as enteroviruses and flaviviruses, usurp the autophagic pathway to promote their own replication. We previously identified the endoplasmic reticulum (ER)-localized protein BPIFB3 as an important negative regulator of non-canonical autophagy that uniquely impacts the replication of enteroviruses and flaviviruses. Here, we find that many components of the canonical autophagy machinery are not required for BPIFB3 depletion-induced autophagy and identify the host factors that facilitate its role in the replication of enteroviruses and flaviviruses. Using proximity-dependent biotinylation (BioID) followed by mass spectrometry, we identify ARFGAP1 and TMED9 as two cellular components that interact with BPIFB3 to regulate autophagy and viral replication. Importantly, our data demonstrate that non-canonical autophagy in mammalian cells can be controlled outside of the traditional pathway regulators and define the role of two proteins in BPIFB3 depletion mediated non-canonical autophagy.

A Naturally Occurring Polymorphism in the Base of Sudan Virus Glycoprotein Decreases Glycoprotein Stability in a Species-Dependent Manner.

Sudan virus (SUDV) is one of five filoviruses that compose the genus Ebolavirus that has been responsible for episodic outbreaks in Central Africa. While the SUDV glycoprotein (GP) structure has been solved, GP residues that affect SUDV entry have not been extensively examined; many of the entry characteristics of SUDV GP are inferred from studies with the Zaire Ebola virus (EBOV) GP. Here, we investigate the effect on virus entry of a naturally occurring polymorphism in SUDV GP. Two of the earliest SUDV isolates contain glutamine at residue 95 (Q95) within the base region of GP1, whereas more recent SUDV isolates and GPs from all other ebolaviruses carry lysine at this position (K95). A K95Q change dramatically decreased titers of pseudovirions bearing SUDV GP, whereas the K95Q substitution in EBOV GP had no effect on titer. We evaluated virus entry to identify SUDV GP Q95-specific entry defects. The presence of Q95 in either EBOV or SUDV GP resulted in enhanced sensitivity of GP to proteolytic processing, yet this could not account for the SUDV-specific decrease in GP Q95 infectivity. We found that SUDV GP Q95 pseudovirions were more sensitive to imipramine, a GP-destabilizing antiviral. In contrast, SUDV GP K95 was more stable, requiring elevated temperatures to inhibit virus infection. Thus, the residue present at GP 95 has a critical role in stabilizing the SUDV glycoprotein, whereas this polymorphism has no effect on EBOV GP stability. These results provide novel insights into filovirus species-specific GP structure that affects virus infectivity. IMPORTANCE Filovirus outbreaks are associated with significant morbidity and mortality. Understanding the structural constraints of filoviral GPs that control virus entry into cells is critical for rational development of novel antivirals to block infection. Here, we identify a naturally occurring glutamine (Q) to lysine (K) polymorphism at residue 95 as a critical determinant of Sudan virus GP stability but not Zaire Ebola virus GP stability. We propose that glutamine at residue 95 in Sudan virus GP mediates decreased virus entry, thereby reducing infectivity. Our findings highlight a unique structural characteristic of Sudan virus GP that affects GP-mediated functionality. Further, it provides a cautionary note for the development of future broad-spectrum filovirus antivirals.

BPIFB3 Regulates Endoplasmic Reticulum Morphology To Facilitate Flavivirus Replication.

Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), rely heavily on the availability of endoplasmic reticulum (ER) membranes throughout their life cycle, and degradation of ER membranes restricts flavivirus replication. Accordingly, DENV and ZIKV restrict ER turnover by protease-mediated cleavage of reticulophagy regulator 1 (RETREG1), also known as FAM134B, an autophagy receptor responsible for targeted ER sheet degradation. Given that the induction of autophagy may play an important role in flavivirus replication, the antiviral role of RETREG1 suggests that specialized autophagic pathways may have differential effects on the flavivirus life cycle. We previously identified BPI fold-containing family B member 3 (BPIFB3) as a regulator of autophagy that negatively controls enterovirus replication. Here, we show that in contrast to enteroviruses, BPIFB3 functions as a positive regulator of DENV and ZIKV infection and that its RNA interference-mediated silencing inhibits the formation of viral replication organelles. Mechanistically, we show that depletion of BPIFB3 enhances RETREG1-dependent reticulophagy, leading to enhanced ER turnover and the suppression of viral replication. Consistent with this, the antiviral effects of BPIFB3 depletion can be reversed by RETREG1 silencing, suggesting a specific role for BPIFB3 in regulating ER turnover. These studies define BPIFB3 as a required host factor for both DENV and ZIKV replication and further contribute to our understanding of the requirements for autophagy during flavivirus infection.IMPORTANCE Flaviviruses and other arthropod-transmitted viruses represent a widespread global health problem, with limited treatment options currently available. Thus, a better understanding of the cellular requirements for their infection is needed. Both DENV and ZIKV rely on expansion of the endoplasmic reticulum (ER) and the induction of autophagy to establish productive infections. However, little is known regarding the interplay between the requirements for autophagy initiation during infection and the mechanisms used by these viruses to avoid clearance through the autophagic pathway. Our study highlights the importance of the host factor BPIFB3 in regulating flavivirus replication and further confirms that the RETREG1-dependent reticulophagy pathway is antiviral to both DENV and ZIKV.

Vesicular Stomatitis Virus Pseudotyped with Ebola Virus Glycoprotein Serves as a Protective, Noninfectious Vaccine against Ebola Virus Challenge in Mice.

The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success.IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVΔG pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSVΔG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

UNLABELLED: Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE: Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication.

ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry.

Interferon stimulated genes (ISGs) target viruses at various stages of their infectious life cycles, including at the earliest stage of viral entry. Here we identify ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2) as a gene upregulated by type I IFN treatment in a STAT1-dependent manner. ADAP2 functions as a GTPase-activating protein (GAP) for Arf6 and binds to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and PI(3,4)P2. We show that overexpression of ADAP2 suppresses dengue virus (DENV) and vesicular stomatitis virus (VSV) infection in an Arf6 GAP activity-dependent manner, while exerting no effect on coxsackievirus B (CVB) or Sendai virus (SeV) replication. We further show that ADAP2 expression induces macropinocytosis and that ADAP2 strongly associates with actin-enriched membrane ruffles and with Rab8a- and LAMP1-, but not EEA1- or Rab7-, positive vesicles. Utilizing two techniques--light-sensitive neutral red (NR)-containing DENV and fluorescence assays for virus internalization--we show that ADAP2 primarily restricts DENV infection at the stage of virion entry and/or intracellular trafficking and that incoming DENV and VSV particles associate with ADAP2 during their entry. Taken together, this study identifies ADAP2 as an ISG that exerts antiviral effects against RNA viruses by altering Arf6-mediated trafficking to disrupt viral entry.

The Role of Conserved N-Linked Glycans on Ebola Virus Glycoprotein 2.

BACKGROUND: N-linked glycosylation is a common posttranslational modification found on viral glycoproteins (GPs) and involved in promoting expression, cellular attachment, protection from proteases, and antibody evasion. The GP subunit GP2 of filoviruses contains 2 completely conserved N-linked glycosylation sites (NGSs) at N563 and N618, suggesting that they have been maintained through selective pressures. METHODS: We assessed mutants lacking these glycans for expression and function to understand the role of these sites during Ebola virus entry. RESULTS: Elimination of either GP2 glycan individually had a modest effect on GP expression and no impact on antibody neutralization of vesicular stomatitis virus pseudotyped with Ebola virus GP. However, loss of the N563 glycan enhanced entry by 2-fold and eliminated GP detection by a well-characterized monoclonal antibody KZ52. Loss of both sites dramatically decreased GP expression and abolished entry. Surprisingly, a GP that retained a single NGS at N563, eliminating the remaining 16 NGSs from GP1 and GP2, had detectable expression, a modest increase in entry, and pronounced sensitivity to antibody neutralization. CONCLUSIONS: Our findings support the importance of the GP2 glycans in GP expression/structure, transduction efficiency, and antibody neutralization, particularly when N-linked glycans are also removed from GP1.

T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus.

The glycoproteins (GP) of enveloped viruses facilitate entry into the host cell by interacting with specific cellular receptors. Despite extensive study, a cellular receptor for the deadly filoviruses Ebolavirus and Marburgvirus has yet to be identified and characterized. Here, we show that T-cell Ig and mucin domain 1 (TIM-1) binds to the receptor binding domain of the Zaire Ebola virus (EBOV) glycoprotein, and ectopic TIM-1 expression in poorly permissive cells enhances EBOV infection by 10- to 30-fold. Conversely, reduction of cell-surface expression of TIM-1 by RNAi decreased infection of highly permissive Vero cells. TIM-1 expression within the human body is broader than previously appreciated, with expression on mucosal epithelia from the trachea, cornea, and conjunctiva--tissues believed to be important during in vivo transmission of filoviruses. Recognition that TIM-1 serves as a receptor for filoviruses on these mucosal epithelial surfaces provides a mechanistic understanding of routes of entry into the human body via inhalation of aerosol particles or hand-to-eye contact. ARD5, a monoclonal antibody against the IgV domain of TIM-1, blocked EBOV binding and infection, suggesting that antibodies or small molecules directed against this cellular receptor may provide effective filovirus antivirals.

Modification of N-Linked Glycan Sites in Viral Glycoproteins.

Post-translational modification of proteins by the addition of sugar chains, or glycans, is a functionally important hallmark of proteins trafficked through the secretory system. These proteins are termed glycoproteins. Glycans are known to be important for initiating signaling through binding of cell surface receptors, facilitating protein folding, and maintaining protein stability. For pathogens, glycans can also mask vulnerable protein regions from neutralizing antibodies. Thus, there is a need to develop methods to decipher the role of specific glycans attached to proteins in order to understand their biological role. Here, we describe established methods for identifying glycosylated residues and understanding their role in protein synthesis and function using viral glycoproteins as a model.

Construction and Rescue of a DNA-Launched DENV2 Infectious Clone.

Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, the production of Zika virus has been successful from CMV promoter-driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued viruses maintained the ability to form plaques and replicate efficiently in commonly used cell lines. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.

Dual-fluorescent reporter for live-cell imaging of the ER during DENV infection.

Infection by flaviviruses leads to dramatic remodeling of the endoplasmic reticulum (ER). Viral replication occurs within virus-induced vesicular invaginations in the ER membrane. A hallmark of flavivirus infection is expansion of the ER membrane which can be observed at specific time points post infection. However, this process has not been effectively visualized in living cells throughout the course of infection at the single cell resolution. In this study, we developed a plasmid-based reporter system to monitor flavivirus infection and simultaneous virus-induced manipulation of single cells throughout the course of infection in real-time. This system requires viral protease cleavage to release an ER-anchored fluorescent protein infection reporter that is fused to a nuclear localization signal (NLS). This proteolytic cleavage allows for the translocation of the infection reporter signal to the nucleus while an ER-specific fluorescent marker remains localized in the lumen. Thus, the construct allows for the visualization of virus-dependent changes to the ER throughout the course of infection. In this study, we show that our reporter was efficiently cleaved upon the expression of multiple flavivirus proteases, including dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). We also found that the DENV protease-dependent cleavage of our ER-anchored reporter exhibited more stringent cleavage sequence specificity than what has previously been shown with biochemical assays. Using this system for long term time-lapse imaging of living cells infected with DENV, we observed nuclear translocation of the reporter signal beginning approximately 8 hours post-infection, which continued to increase throughout the time course. Interestingly, we found that increased reporter signal translocation correlated with increased ER signal intensity, suggesting a positive association between DENV infection and ER expansion in a time-dependent manner. Overall, this report demonstrates that the FlavER platform provides a useful tool for monitoring flavivirus infection and simultaneously observing virus-dependent changes to the host cell ER, allowing for study of the temporal nature of virus-host interactions.

Enhancement of respiratory mucosal antiviral defenses by the oxidation of iodide.

Recent reports postulate that the dual oxidase (DUOX) proteins function as part of a multicomponent oxidative pathway used by the respiratory mucosa to kill bacteria. The other components include epithelial ion transporters, which mediate the secretion of the oxidizable anion thiocyanate (SCN(-)) into airway surface liquid, and lactoperoxidase (LPO), which catalyzes the H(2)O(2)-dependent oxidation of the pseudohalide SCN(-) to yield the antimicrobial molecule hypothiocyanite (OSCN(-)). We hypothesized that this oxidative host defense system is also active against respiratory viruses. We evaluated the activity of oxidized LPO substrates against encapsidated and enveloped viruses. When tested for antiviral properties, the LPO-dependent production of OSCN(-) did not inactivate adenovirus or respiratory syncytial virus (RSV). However, substituting SCN(-) with the alternative LPO substrate iodide (I(-)) resulted in a marked reduction of both adenovirus transduction and RSV titer. Importantly, well-differentiated primary airway epithelia generated sufficient H(2)O(2) to inactivate adenovirus or RSV when LPO and I(-) were supplied. The administration of a single dose of 130 mg of oral potassium iodide to human subjects increased serum I(-) concentrations, and resulted in the accumulation of I(-) in upper airway secretions. These results suggest that the LPO/I(-)/H(2)O(2) system can contribute to airway antiviral defenses. Furthermore, the delivery of I(-) to the airway mucosa may augment innate antiviral immunity.

Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.

Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals.

The exoribonuclease Xrn1 is a post-transcriptional negative regulator of autophagy.

Macroautophagy/autophagy is a conserved catabolic process that promotes survival during stress. Autophagic dysfunction is associated with pathologies such as cancer and neurodegenerative diseases. Thus, autophagy must be strictly modulated at multiple levels (transcriptional, post-transcriptional, translational and post-translational) to prevent deregulation. Relatively little is known about the post-transcriptional control of autophagy. Here we report that the exoribonuclease Xrn1/XRN1 functions as a negative autophagy factor in the yeast Saccharomyces cerevisiae and in mammalian cells. In yeast, chromosomal deletion of XRN1 enhances autophagy and the frequency of autophagosome formation. Loss of Xrn1 results in the upregulation of autophagy-related (ATG) transcripts under nutrient-replete conditions, and this effect is dependent on the ribonuclease activity of Xrn1. Xrn1 expression is regulated by the yeast transcription factor Ash1 in rich conditions. In mammalian cells, siRNA depletion of XRN1 enhances autophagy and the replication of 2 picornaviruses. This work provides insight into the role of the RNA decay factor Xrn1/XRN1 as a post-transcriptional regulator of autophagy.

Chromosome 19 microRNAs exert antiviral activity independent from type III interferon signaling.

INTRODUCTION: Cultured primary human trophoblasts (PHT), derived from term placentas, are relatively resistant to infection by diverse viruses. The resistance can be conferred to non-trophoblastic cells by pre-exposing them to medium that was conditioned by PHT cells. This antiviral effect is mediated, at least in part, by microRNAs (miRNA) expressed from the chromosome 19 microRNA cluster (C19MC). Recently we showed that PHT cells and cells pre-exposed to PHT medium are also resistant to infection by Zika virus (ZIKV), an effect mediated by the constitutive release of the type III interferons (IFN) IFN lambda-1 and IFN lambda-2 in trophoblastic medium. We hypothesized that trophoblastic C19MC miRNA are active against ZIKV, and assessed the interaction of this pathway with IFN lambda-1 - mediated resistance. METHODS: Term PHT cells were cultured using standard techniques. An osteosarcoma cell line (U2OS) was used as non-trophoblastic cells, which were infected with either ZIKV or vesicular stomatitis virus (VSV). Trophoblastic extracellular vesicles (EVs) were produced by gradient ultracentrifugation. RT-qPCR was used to determine viral infection, cellular or medium miRNA levels and the expression of interferon-stimulated genes. RESULTS: We showed that C19MC miRNA attenuate infection of U2OS cells by ZIKV, and that C19MC miRNA or exosomes that contain C19MC miRNA did not influence the type III IFN pathway. Similarly, cell exposure to recombinant IFN lambda-1 had no effect on miRNA expression, and these pathways did not exhibit synergistic interaction. DISCUSSION: PHT cells exert antiviral activity by at least two independent mechanisms, mediated by C19MC miRNA and by type III IFNs.

A 2'FY-RNA Motif Defines an Aptamer for Ebolavirus Secreted Protein.

With properties such as stability to long-term storage and amenability to repetitive use, nucleic acid aptamers are compatible with many sensing/transducing platforms intended for use in remote locations. Sensors with these properties are important for quickly identifying ebolavirus outbreaks, which frequently start in locations that lack sophisticated equipment. Soluble glycoprotein (sGP), an excellent biomarker for ebolaviruses, is produced from the same gene as the ebolavirus glycoprotein GP1,2 that decorates the surface of the viral particle and is secreted in abundance into the blood stream even during the early stages of infection. Here, we report the selection and properties of a 2'fluoro pyrimidine (2'FY)-modified RNA aptamer, 39SGP1A, that specifically binds sGP. We demonstrate by computational and biochemical analysis that the recognition motif of 39SGP1A is a novel polypyrimidine-rich sequence. Replacement of -F by -OH in the 2' position of the ribose resulted in complete loss of affinity for sGP. The protein motif to which the aptamer binds requires an intact sGP dimer and binds to an epitope conserved between Ebola virus (EBOV) and Sudan virus (SUDV) sGP, the most divergent Ebolavirus species. This identifies 39SGP1A as an excellent option for integration on a sensor platform to detect ebolavirus infections.

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B.

The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.

A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier.

The blood-brain barrier (BBB) comprises the foremost protective barrier in the brain and is composed in part of a layer of microvascular endothelial cells that line the capillaries surrounding the brain. Here, we describe a human three-dimensional (3-D) cell-based model of the BBB microvascular endothelium that recapitulates properties of these cells in vivo, including physiologically relevant transcriptional profiles, the capacity to induce potent antimicrobial innate immune signaling, and the ability to resist infection by diverse RNA viruses, including members of the enterovirus (coxsackievirus B, echovirus 11, enterovirus 71, poliovirus) and flavivirus (dengue virus, Zika virus [ZIKV]) families. We show that disruption of apical tight junctions by proinflammatory cytokine tumor necrosis factor alpha (TNF-α) sensitizes 3-D-cultured BBB cells to ZIKV infection and that 3-D derived BBB cells can be used to model the transmigration of ZIKV-infected monocytes across the endothelial barrier to access underlying astrocytes. Taken together, our findings show that human BBB microvascular endothelial cells cultured in 3-D can be used to model the mechanisms by which RNA viruses access the central nervous system (CNS), which could be used for the development and screening of therapeutics to limit this event. IMPORTANCE Neurotropic viral infections are significant sources of global morbidity and mortality. The blood-brain barrier (BBB) is composed in part of a layer of microvascular endothelial cells and functions to restrict viral access to the brain. In vitro models that recapitulate many of the properties of the human BBB endothelium are lacking, particularly with respect to the unique cellular and immunological mechanisms by which these cells restrict viral infections of the brain. Here, we developed a three-dimensional cell culture model that recapitulates many of the morphological and functional properties of the BBB microvasculature and apply this model to the study of RNA virus infections. The model we describe can therefore be used to study a variety of aspects of BBB physiology, including the mechanisms by which viruses might access the CNS, and could be used for the development and screening of antiviral therapeutics to limit this important step in viral pathogenesis.

Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection.

During mammalian pregnancy, the placenta acts as a barrier between the maternal and fetal compartments. The recently observed association between Zika virus (ZIKV) infection during human pregnancy and fetal microcephaly and other anomalies suggests that ZIKV may bypass the placenta to reach the fetus. This led us to investigate ZIKV infection of primary human trophoblasts (PHTs), which are the barrier cells of the placenta. We discovered that PHT cells from full-term placentas are refractory to ZIKV infection. In addition, medium from uninfected PHT cells protects non-placental cells from ZIKV infection. PHT cells constitutively release the type III interferon (IFN) IFNλ1, which functions in both a paracrine and autocrine manner to protect trophoblast and non-trophoblast cells from ZIKV infection. Our data suggest that for ZIKV to access the fetal compartment, it must evade restriction by trophoblast-derived IFNλ1 and other trophoblast-specific antiviral factors and/or use alternative strategies to cross the placental barrier.