Opposing activities of IFITM proteins in SARS-CoV-2 infection.

Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain- and loss-of-function approaches. Mechanistically, SARS-CoV-2 restriction occurred independently of IFITM3 S-palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis-promoting YxxФ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal SARS-CoV-2 restriction.

CD225 Proteins: A Family Portrait of Fusion Regulators.

The CD225 superfamily regulates vesicular membrane fusion events essential to neurotransmission, immunity, development, and metabolism. Its importance to physiology is reinforced by the identification of polymorphisms associated with disease. This article highlights the shared features that drive the function of CD225 proteins such as interferon-inducible transmembrane proteins 3 (IFITM3) and proline-rich transmembrane protein 2 (PRRT2) and is intended to catalyze efforts towards characterizing the lesser-known family members.

Correction: Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion.

[This corrects the article DOI: 10.1371/journal.ppat.1008359.].

The Indirect Antiviral Potential of Long Noncoding RNAs Encoded by IFITM Pseudogenes.

The interferon-induced transmembrane (IFITM) gene family performs multiple functions in immunity, including inhibition of virus entry into cells. The IFITM repertoire varies widely between species and consists of protein-coding genes and pseudogenes. The selective forces driving pseudogenization within gene families are rarely understood. In this issue, the human pseudogene IFITM4P is characterized as a virus-induced, long noncoding RNA that contributes to restriction of influenza A virus by regulating mRNA levels of IFITM1, IFITM2, and IFITM3.

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion.

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.

Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells.

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3.

Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are well-established immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus-cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes.

IFITM3 requires an amphipathic helix for antiviral activity.

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3-dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin-mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion.

Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity.

The interferon-induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV-1 virions and inhibit virus fusion and cell-to-cell spread, with IFITM3 showing the greatest potency. Here, we report that amino-terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. This suggests that "runaway" IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade-off in antiviral specificity, as variants exhibiting enhanced activity against HIV-1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.

More than meets the I: the diverse antiviral and cellular functions of interferon-induced transmembrane proteins.

The first responders of human antiviral immunity are components of the intrinsic immune response that reside within each and every one of our cells. This cell-autonomous arsenal consists of nucleic acid sensors and antiviral effectors strategically placed by evolution to detect and restrict invading viruses. While some factors are present at baseline to allow for constant surveillance of the cell interior, others are upregulated by cytokines (such as interferons) that signal a viral infection underway in neighboring cells. In this review, we highlight the multiple roles played by the interferon-induced transmembrane (IFITM) proteins during viral infection, with focuses on IFITM3 and HIV-1. Moreover, we discuss the cellular pathways in which IFITM proteins are intertwined and the various functions they have been ascribed outside the context of infection. While appreciated as broadly-acting, potent restriction factors that prevent virus infection and pathogenesis in cell culture and in vivo, questions remain regarding their precise mode of action and importance in certain viral contexts. Continued efforts to study IFITM protein function will further cement their status as critical host determinants of virus susceptibility and prioritize them in the development of new antiviral therapies.

Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control.

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

Convergence and divergence in the evolution of the APOBEC3G-Vif interaction reveal ancient origins of simian immunodeficiency viruses.

Naturally circulating lentiviruses are abundant in African primate species today, yet their origins and history of transmitting between hosts remain obscure. As a means to better understand the age of primate lentiviruses, we analyzed primate genomes for signatures of lentivirus-driven evolution. Specifically, we studied the adaptive evolution of host restriction factor APOBEC3G (A3G) in Old World Monkey (OWM) species. We find recurrent mutation of A3G in multiple primate lineages at sites that determine susceptibility to antagonism by the lentiviral accessory protein Vif. Using a broad panel of SIV Vif isolates, we demonstrate that natural variation in OWM A3G confers resistance to Vif-mediated degradation, suggesting that adaptive variants of the host factor were selected upon exposure to pathogenic lentiviruses at least 5-6 million years ago (MYA). Furthermore, in members of the divergent Colobinae subfamily of OWM, a multi-residue insertion event in A3G that arose at least 12 MYA blocks the activity of Vif, suggesting an even more ancient origin of SIV. Moreover, analysis of the lentiviruses associated with Colobinae monkeys reveal that the interface of the A3G-Vif interaction has shifted and given rise to a second genetic conflict. Our analysis of virus-driven evolution describes an ancient yet ongoing genetic conflict between simian primates and lentiviruses on a million-year time scale.

Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue.

Omicron emerged following COVID-19 vaccination campaigns, displaced previous SARS-CoV-2 variants of concern worldwide, and gave rise to lineages that continue to spread. Here, we show that Omicron exhibits increased infectivity in primary adult upper airway tissue relative to Delta. Using recombinant forms of SARS-CoV-2 and nasal epithelial cells cultured at the liquid-air interface, we show that mutations unique to Omicron Spike enable enhanced entry into nasal tissue. Unlike earlier variants of SARS-CoV-2, our findings suggest that Omicron enters nasal cells independently of serine transmembrane proteases and instead relies upon metalloproteinases to catalyze membrane fusion. Furthermore, we demonstrate that this entry pathway unlocked by Omicron Spike enables evasion from constitutive and interferon-induced antiviral factors that restrict SARS-CoV-2 entry following attachment. Therefore, the increased transmissibility exhibited by Omicron in humans may be attributed not only to its evasion of vaccine-elicited adaptive immunity, but also to its superior invasion of nasal epithelia and resistance to the cell-intrinsic barriers present therein.

Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue.

Omicron emerged following COVID-19 vaccination campaigns, displaced previous SARS-CoV-2 variants of concern worldwide, and gave rise to lineages that continue to spread. Here, we show that Omicron exhibits increased infectivity in primary adult upper airway tissue relative to Delta. Using recombinant forms of SARS-CoV-2 and nasal epithelial cells cultured at the liquid-air interface, enhanced infectivity maps to the step of cellular entry and evolved recently through mutations unique to Omicron Spike. Unlike earlier variants of SARS-CoV-2, Omicron enters nasal cells independently of serine transmembrane proteases and instead relies upon metalloproteinases to catalyze membrane fusion. This entry pathway unlocked by Omicron Spike enables evasion of constitutive and interferon-induced antiviral factors that restrict SARS-CoV-2 entry following attachment. Therefore, the increased transmissibility exhibited by Omicron in humans may be attributed not only to its evasion of vaccine-elicited adaptive immunity, but also to its superior invasion of nasal epithelia and resistance to the cell-intrinsic barriers present therein.

Lessons in self-defence: inhibition of virus entry by intrinsic immunity.

Virus entry, consisting of attachment to and penetration into the host target cell, is the first step of the virus life cycle and is a critical 'do or die' event that governs virus emergence in host populations. Most antiviral vaccines induce neutralizing antibodies that prevent virus entry into cells. However, while the prevention of virus invasion by humoral immunity is well appreciated, considerably less is known about the immune defences present within cells (known as intrinsic immunity) that interfere with virus entry. The interferon-induced transmembrane (IFITM) proteins, known for inhibiting fusion between viral and cellular membranes, were once the only factors known to restrict virus entry. However, the progressive development of genetic and pharmacological screening platforms and the onset of the COVID-19 pandemic have galvanized interest in how viruses infiltrate cells and how cells defend against it. Several host factors with antiviral potential are now implicated in the regulation of virus entry, including cholesterol 25-hydroxylase (CH25H), lymphocyte antigen 6E (LY6E), nuclear receptor co-activator protein 7 (NCOA7), interferon-γ-inducible lysosomal thiol reductase (GILT), CD74 and ARFGAP with dual pleckstrin homology domain-containing protein 2 (ADAP2). This Review summarizes what is known and what remains to be understood about the intrinsic factors that form the first line of defence against virus infection.

Restriction of Influenza A Virus by SERINC5.

Serine incorporator 5 (Ser5), a transmembrane protein, has recently been identified as a host antiviral factor against human immunodeficiency virus (HIV)-1 and gammaretroviruses like murine leukemia viruses (MLVs). It is counteracted by HIV-1 Nef and MLV glycogag. We have investigated whether it has antiviral activity against influenza A virus (IAV), as well as retroviruses. Here, we demonstrated that Ser5 inhibited HIV-1-based pseudovirions bearing IAV hemagglutinin (HA); as expected, the Ser5 effect on this glycoprotein was antagonized by HIV-1 Nef protein. We found that Ser5 inhibited the virus-cell and cell-cell fusion of IAV, apparently by interacting with HA proteins. Most importantly, overexpressed and endogenous Ser5 inhibited infection by authentic IAV. Single-molecular fluorescent resonance energy transfer (smFRET) analysis further revealed that Ser5 both destabilized the pre-fusion conformation of IAV HA and inhibited the coiled-coil formation during membrane fusion. Ser5 is expressed in cultured small airway epithelial cells, as well as in immortal human cell lines. In summary, Ser5 is a host antiviral factor against IAV which acts by blocking HA-induced membrane fusion. IMPORTANCE SERINC5 (Ser5) is a cellular protein which has been found to interfere with the infectivity of HIV-1 and a number of other retroviruses. Virus particles produced in the presence of Ser5 are impaired in their ability to enter new host cells, but the mechanism of Ser5 action is not well understood. We now report that Ser5 also inhibits infectivity of Influenza A virus (IAV) and that it interferes with the conformational changes in IAV hemagglutinin protein involved in membrane fusion and virus entry. These findings indicate that the antiviral function of Ser5 extends to other viruses as well as retroviruses, and also provide some information on the molecular mechanism of its antiviral activity.

Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity.

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with an increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increased susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. We identified 1 rapalog (ridaforolimus) that was less potent in this regard and demonstrated that rapalogs promote spike-mediated entry into cells, by triggering the degradation of the antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increased virus entry inhibited mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitated its nuclear translocation and triggered microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.