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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.

The capacity to form syncytia in T cell lines was adopted as an early classification system (syncytia-inducing [SI] versus non-syncytia-inducing [NSI]) to distinguish different HIV-1 strains. Since HIV-induced syncytia can harbor large amounts of virus yet undergo lysis soon after their formation, these cell masses were considered to play a part in the characteristic loss of CD4+ helper T cells in HIV/AIDS disease progression [4, 5]. [...]many remained unconvinced that T cell syncytia represented something other than in vitro curiosities, referring to them as \"fusion sinks\" that do not promote virus replication or spread.

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.

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.

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. Shi and Li et al. show that SARS-CoV-2 Omicron subvariants have increased capacity to infect primary human nasal tissue using a distinct entry route that depends on matrix metalloproteinases as opposed to TMPRSS2 used by previous variants, which enables evasion from antiviral factors.

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.

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.Besides neutralizing antibodies, viruses face a range of cell-intrinsic inhibitors that are specialized to limit virus entry into host cells. Majdoul and Compton describe the mechanisms of action of the cellular factors providing this important first line of defence against virus infection, including infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline-rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, Amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

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. 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.

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.