IFITM3 protects the heart during influenza virus infection.

Influenza virus can disseminate from the lungs to the heart in severe infections and can induce cardiac pathology, but this has been difficult to study due to a lack of small animal models. In humans, polymorphisms in the gene encoding the antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) are associated with susceptibility to severe influenza, but whether IFITM3 deficiencies contribute to cardiac dysfunction during infection is unclear. We show that IFITM3 deficiency in a new knockout (KO) mouse model increases weight loss and mortality following influenza virus infections. We investigated this enhanced pathogenesis with the A/PR/8/34 (H1N1) (PR8) influenza virus strain, which is lethal in KO mice even at low doses, and observed increased replication of virus in the lungs, spleens, and hearts of KO mice compared with wild-type (WT) mice. Infected IFITM3 KO mice developed aberrant cardiac electrical activity, including decreased heart rate and irregular, arrhythmic RR (interbeat) intervals, whereas WT mice exhibited a mild decrease in heart rate without irregular RR intervals. Cardiac electrical dysfunction in PR8-infected KO mice was accompanied by increased activation of fibrotic pathways and fibrotic lesions in the heart. Infection with a sublethal dose of a less virulent influenza virus strain (A/WSN/33 [H1N1]) resulted in a milder cardiac electrical dysfunction in KO mice that subsided as the mice recovered. Our findings reveal an essential role for IFITM3 in limiting influenza virus replication and pathogenesis in heart tissue and establish IFITM3 KO mice as a powerful model for studying mild and severe influenza virus-induced cardiac dysfunction.

Butyrate Reprograms Expression of Specific Interferon-Stimulated Genes.

Butyrate is an abundant metabolite produced by gut microbiota. While butyrate is a known histone deacetylase inhibitor that activates expression of many genes involved in immune system pathways, its effects on virus infections and on the antiviral type I interferon (IFN) response have not been adequately investigated. We found that butyrate increases cellular infection with viruses relevant to human and animal health, including influenza virus, reovirus, HIV-1, human metapneumovirus, and vesicular stomatitis virus. Mechanistically, butyrate suppresses levels of specific antiviral IFN-stimulated gene (ISG) products, such as RIG-I and IFITM3, in human and mouse cells without inhibiting IFN-induced phosphorylation or nuclear translocation of the STAT1 and STAT2 transcription factors. Accordingly, we discovered that although butyrate globally increases baseline expression of more than 800 cellular genes, it strongly represses IFN-induced expression of 60% of ISGs and upregulates 3% of ISGs. Our findings reveal that there are differences in the IFN responsiveness of major subsets of ISGs depending on the presence of butyrate in the cell environment, and overall, they identify a new mechanism by which butyrate influences virus infection of cells.IMPORTANCE Butyrate is a lipid produced by intestinal bacteria. Here, we newly show that butyrate reprograms the innate antiviral immune response mediated by type I interferons (IFNs). Many of the antiviral genes induced by type I IFNs are repressed in the presence of butyrate, resulting in increased virus infection and replication. Our research demonstrates that metabolites produced by the gut microbiome, such as butyrate, can have complex effects on cellular physiology, including dampening of an inflammatory innate immune pathway resulting in a proviral cellular environment. Our work further suggests that butyrate could be broadly used as a tool to increase growth of virus stocks for research and for the generation of vaccines.

Interferon-induced transmembrane proteins inhibit cell fusion mediated by trophoblast syncytins.

Type I interferon (IFN) induced by virus infections during pregnancy can cause placental damage, but the mechanisms and identities of IFN-stimulated genes that are involved in this damage remain under investigation. The IFN-induced transmembrane proteins (IFITMs) inhibit virus infections by preventing virus membrane fusion with cells and by inhibiting fusion of infected cells (syncytialization). Fusion of placental trophoblasts via expression of endogenous retroviral fusogens known as syncytins forms the syncytiotrophoblast, a multinucleated cell structure essential for fetal development. We found here that IFN blocks fusion of BeWo human placental trophoblasts. Stably expressed IFITM1, -2, and -3 also blocked fusion of these trophoblasts while making them more resistant to virus infections. Conversely, stable IFITM knockdowns in BeWo trophoblasts increased their spontaneous fusion and allowed fusion in the presence of IFN while also making the cells more susceptible to virus infection. We additionally found that exogenous expression of IFITMs in HEK293T cells blocked fusion with cells expressing syncytin-1 or syncytin-2, confirming the ability of IFITMs to block individual syncytin-mediated fusion. Overall, our data indicate that IFITMs inhibit trophoblast fusion and suggest that there may be a critical balance between these antifusogenic effects and the beneficial antiviral effects of IFITMs in virus infections during pregnancy.

The mode of mitosis is dramatically modified by deletion of a single nuclear pore complex gene in Aspergillus nidulans.

Nuclear pore complex (NPC) proteins (Nups) play multiple roles during mitosis. In this study we expand these roles and reveal that in Aspergillus nidulans, compromising the core Nup84-120 subcomplex of the NPC modifies the mitotic behavior of the nuclear envelope (NE). In wildtype cells, the NE undergoes simultaneous double pinching events to separate daughter nuclei during mitotic exit, whereas in Nup84-120 complex mutants, only one restriction of the NE is observed. Investigating the basis for this modified behavior of the NE in Nup deleted cells uncovered previously unrealized roles for core Nups in mitotic exit. During wildtype anaphase, the NE surrounds the two separating daughter DNA masses which typically flank the central nucleolus, to form three distinct nuclear compartments. In contrast, deletion of core Nups frequently results in early nucleolar eviction from the mitotic nucleus, in turn causing an uncharacteristic dumbbell-shaped NE morphology of anaphase nuclei with a nuclear membrane bridge connecting the two forming G1 nuclei. Importantly, the absence of the nucleolus between the separating daughter nuclei during anaphase delays chromosome segregation and progression into G1 as nuclei remain connected by chromatin bridges. Proteins localizing to late segregating chromosome arms are observed between forming daughter nuclei, and the mitotic spindle fails to resolve in a timely manner. These chromatin bridges are occupied by the Aurora kinase until nuclei have fully separated, suggesting involvement of Aurora in monitoring mitotic spindle and nuclear membrane resolution during mitotic exit. Our findings thus reveal a novel requirement for core Nups in mediating nucleolar positioning during mitosis, which dictates the pattern of NE fissions during karyokinesis and facilitates normal chromosome segregation. The findings additionally demonstrate that the mode of mitosis can be dramatically modified by deletion of a single NPC gene and reveals surprising fluidity in mitotic mechanisms.

IFITM3 Restricts Human Metapneumovirus Infection.

Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein toll-like receptor 7 (TLR7), and that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells.

The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity.

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular endosome- and lysosome-localized protein that restricts numerous virus infections. IFITM3 is activated by palmitoylation, a lipid posttranslational modification. Palmitoylation of proteins is primarily mediated by zinc finger DHHC domain-containing palmitoyltransferases (ZDHHCs), but which members of this enzyme family can modify IFITM3 is not known. Here, we screened a library of human cell lines individually lacking ZDHHCs 1-24 and found that IFITM3 palmitoylation and its inhibition of influenza virus infection remained strong in the absence of any single ZDHHC, suggesting functional redundancy of these enzymes in the IFITM3-mediated antiviral response. In an overexpression screen with 23 mammalian ZDHHCs, we unexpectedly observed that more than half of the ZDHHCs were capable of increasing IFITM3 palmitoylation with ZDHHCs 3, 7, 15, and 20 having the greatest effect. Among these four enzymes, ZDHHC20 uniquely increased IFITM3 antiviral activity when both proteins were overexpressed. ZDHHC20 colocalized extensively with IFITM3 at lysosomes unlike ZDHHCs 3, 7, and 15, which showed a defined perinuclear localization pattern, suggesting that the location at which IFITM3 is palmitoylated may influence its activity. Unlike knock-out of individual ZDHHCs, siRNA-mediated knockdown of both ZDHHC3 and ZDHHC7 in ZDHHC20 knock-out cells decreased endogenous IFITM3 palmitoylation. Overall, our results demonstrate that multiple ZDHHCs can palmitoylate IFITM3 to ensure a robust antiviral response and that ZDHHC20 may serve as a particularly useful tool for understanding and enhancing IFITM3 activity.

From APOBEC to ZAP: Diverse mechanisms used by cellular restriction factors to inhibit virus infections.

Antiviral restriction factors are cellular proteins that inhibit the entry, replication, or spread of viruses. These proteins are critical components of the innate immune system and function to limit the severity and host range of virus infections. Here we review the current knowledge on the mechanisms of action of several restriction factors that affect multiple viruses at distinct stages of their life cycles. For example, APOBEC3G deaminates cytosines to hypermutate reverse transcribed viral DNA; IFITM3 alters membranes to inhibit virus membrane fusion; MXA/B oligomerize on viral protein complexes to inhibit virus replication; SAMHD1 decreases dNTP intracellular concentrations to prevent reverse transcription of retrovirus genomes; tetherin prevents release of budding virions from cells; Viperin catalyzes formation of a nucleoside analogue that inhibits viral RNA polymerases; and ZAP binds virus RNAs to target them for degradation. We also discuss countermeasures employed by specific viruses against these restriction factors, and mention secondary functions of several of these factors in modulating immune responses. These important examples highlight the diverse strategies cells have evolved to combat virus infections.

A mitotic nuclear envelope tether for Gle1 also impacts nuclear and nucleolar architecture.

During Aspergillus nidulans mitosis peripheral nuclear pore complex (NPC) proteins (Nups) disperse from the core NPC structure. Unexpectedly, one predicted peripheral Nup, Gle1, remains at the mitotic NE via an unknown mechanism. Gle1 affinity purification identified MtgA ( M: itotic T: ether for G: le1), which tethers Gle1 to the NE during mitosis, but not during interphase when Gle1 is at NPCs. MtgA is the ortholog of the Schizosaccharomyces pombe telomere-anchoring inner nuclear membrane protein Bqt4. Like Bqt4, MtgA has meiotic roles but is functionally distinct from Bqt4 as MtgA is not required for tethering telomeres to the NE. Domain analyses revealed MtgA targeting to the NE requires its C-terminal transmembrane domain and a nuclear localization signal. Importantly, MtgA functions beyond Gle1 mitotic targeting and meiosis and impacts nuclear and nucleolar architecture when deleted or overexpressed. Deletion of MtgA generates small, round nuclei whereas overexpressing MtgA generates larger nuclei with altered nuclear compartmentalization resulting from NE expansion around the nucleolus. The accumulation of MtgA around the nucleolus promotes a similar accumulation of the endoplasmic reticulum (ER) protein Erg24 lowering its levels in the ER. This study extends the functions of Bqt4-like proteins to include mitotic Gle1 targeting and modulation of nuclear and nucleolar architecture.

Reciprocal regulation of activating and inhibitory Fc{gamma} receptors by TLR7/8 activation: implications for tumor immunotherapy.

PURPOSE: Activation of Toll-like receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcgamma receptor (FcgammaR) function, as they are critical mediators of antibody therapy. EXPERIMENTAL DESIGN: The effect of the TLR7/8 agonist R-848 on cytokine production and antibody-dependent cellular cytotoxicity by human peripheral blood monocytes was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848 and Western blots were done to measure protein levels of FcgammaR. Murine bone marrow-derived macrophages from WT and knockout mice were examined to determine the downstream pathway involved with regulating FcgammaR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model. RESULTS: Overnight incubation with R-848 increased FcgammaR-mediated cytokine production and antibody-dependent cellular cytotoxicity in human peripheral blood monocytes. Expression of FcgammaRI, FcgammaRIIa, and the common gamma-subunit was increased. Surprisingly, expression of the inhibitory FcgammaRIIb was almost completely abolished. In bone marrow-derived macrophage, this required TLR7 and MyD88, as R-848 did not increase expression of the gamma-subunit in TLR7(-/-) nor MyD88(-/-) cells. In a mouse solid tumor model, R-848 treatment superadditively enhanced the effects of antitumor antibody. CONCLUSIONS: These results show an as-yet-undiscovered regulatory and functional link between the TLR7/8 and FcgammaR pathways. This suggests that TLR7/8 agonists may be especially beneficial during antibody therapy.