Biology subjects

Compton, A. A.

Publications and source records attributed to Compton, A. A..

9 recordsLinked to original sources

bioRxiv2025

IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes

Interferon-induced transmembrane (IFITM) proteins are potent innate immune factors that restrict an array of viruses at the entry stage of infection. We previously characterized a GxxxG motif in the CD225 domain of human IFITM3 that mediates its multimerization, which is essential for the reduction of membrane fluidity by IFITM3 and for its antiviral activity against Influenza A virus. Here, using an unbiased approach coupling immunoprecipitation with mass spectrometry, we show that the GxxxG motif is also important for the interaction of IFITM3 with other proteins, including IFITM1. While IFITM1 is primarily regarded as a cell surface protein that restricts the entry of viruses fusing at the plasma membrane, this model is based mostly on overexpression studies and is at odds with some studies showing that it can restrict endocytic viruses. Here, we show that endogenous IFITM1 and IFITM3 co-reside in membranes of acidic late endosomes and lysosomes (endolysosomes) and form a protein-protein complex as determined by co-immunoprecipitation and proximity ligation assay. Knockdown of endogenous IFITM3 resulted in enhanced localization of IFITM1 at the plasma membrane, indicating that IFITM3 promotes IFITM1 localization to endolysosomes. To assess the antiviral protection conferred by endogenous IFITM1 and IFITM3 against viruses fusing at endolysosomal membranes, we measured cell entry mediated by the Influenza A fusogen hemagglutinin (HA). While knockdown of IFITM3 significantly boosted HA-mediated entry, combined knockdown of both IFITM3 and IFITM1 boosted entry even further. These results suggest that endogenous IFITM1 restricts Influenza A virus entry in a manner that is non-redundant with IFITM3, and that IFITM1 and IFITM3 inhibit virus entry in a cooperative manner.

microbiology↗

bioRxiv2025

Restriction of HIV-1 infectivity by interferon and IFITM3 is counteracted by Nef

The viral accessory protein Nef is a major determinant of HIV-1 pathogenicity in vivo. Nef is a multifunctional, immunomodulatory protein that downmodulates cell surface proteins, including CD4 and MHC class I (MHC-I) important for T-cell-mediated immunity. In addition, Nef also regulates cell-intrinsic immunity--Nef boosts the infectivity of virions produced and released from HIV-infected cells, at least in part, by counteracting the antiviral activity of transmembrane proteins SERINC3 and SERINC5. However, it has been reported that the enhancement of HIV-1 infectivity by Nef persists in certain cell lines deficient for SERINC3/5, revealing the existence of other Nef-sensitive host factors that impact HIV-1 infectivity. Here, we show that Nef proteins, especially those derived from many primary isolates of HIV-1, restore infectivity in interferon-treated cells and confer resistance to interferon-induced transmembrane protein 3 (IFITM3). IFITM3 is a restriction factor that reduces retroviral infectivity by incorporating into virions, inhibiting Envelope glycoprotein function, and reducing entry into cells. Using a primary isolate of Nef derived from HIV-1 clade C, we found that Nef interacts with IFITM3 in membranes and uses the endocytic adaptor protein AP-2 to counteract it. Furthermore, Nef reduced IFITM3 cell surface levels, increased IFITM3 levels in early endosomes, and reduced IFITM3 incorporation into HIV-1 virions. Nef also impaired IFITM3 oligomerization and restored membrane fluidity in IFITM3-expressing cells. The antiviral activity of IFITM3 and its inhibition by Nef were unaffected by SERINC5 knockdown, suggesting that the counteraction of IFITM3 represents a unique function of Nef. Our findings reveal a previously unrecognized immunomodulatory role for Nef in the setting of the interferon-induced antiviral state during HIV-1 infection.

microbiology↗

bioRxiv2024

SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

The CD225/Dispanins superfamily consists of membrane proteins that regulate vesicular transport and membrane fusion events driving neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking was unknown. We reveal that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins, and mutations therein disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against Influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against Influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins.

cell biology↗

bioRxiv2023

Syncytia Formation Promotes Virus Resistance to Interferon and Neutralizing Antibodies

SARS-CoV-2, like many viruses, generates syncytia but the role of syncytia formation in viral evolution remains unknown. Using SARS-CoV-2 and SARS-CoV-2 Spike (S) replacement vesicular stomatitis (VSV), we show that S-mediated syncytia impair the antiviral effects of interferons in cultured cells, human lung cell cultures, and hACE2 transgenic mice. Amino acid substitutions that modulate syncytia formation in Delta- and Omicron-encoded S have parallel effects on viral interferon resistance. S-mediated syncytia compromise antibody-mediated virus neutralization in cultured cells. We recapitulate interferon and neutralizing antibody resistance in syncytia generated by the orthoreovirus p14 fusion-associated small transmembrane (FAST) protein in VSV, influenza virus, and seasonal coronavirus OC43 infections. These findings explain selection of SARS-CoV-2 fusogenic variants in humans and, more generally, the evolution of fusogenic viruses driven by adaptive and innate immunity.

microbiology↗

bioRxiv2023

Alternative splicing expands the antiviral IFITM repertoire in Chinese horseshoe bats

The interferon response is shaped by the evolutionary arms race between hosts and the pathogens they carry. The human interferon-induced transmembrane protein (IFITM) family consists of three antiviral IFITM genes that arose by gene duplication, they restrict virus entry and are key players of the interferon response. Yet, little is known about IFITMs in other mammals. Here, we identified an IFITM gene in Chinese horseshoe bat, a natural host of SARS-coronaviruses, that is alternatively spliced to produce two IFITM isoforms. These bat IFITMs have conserved structures in vitro and differential antiviral activities against influenza A virus and coronaviruses including SARS- and MERS-coronavirus. In parallel with human IFITM1-3, the bat IFITM isoforms localize to distinct cellular compartments. Further analysis of IFITM repertoires in 205 mammals reveals that alternative splicing is a ubiquitous strategy for IFITM diversification, albeit less widely adopted than gene duplication. These findings showcase an example of convergent evolution where species-specific selection pressures led to expansion of the IFITM family through multiple means, underscoring the importance of IFITM diversity as a component of innate immunity.

microbiology↗

bioRxiv2022

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.

immunology↗

bioRxiv2021

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-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 increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers 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. SignificanceRapamycin is an immunosuppressant used in humans to treat cancer, autoimmunity, and other disease states. Here, we show that rapamycin and related compounds promote the first step of the SARS-CoV-2 infection cycle--entry into cells--by disarming cell-intrinsic immune defenses. We outline the molecular basis for this effect by identifying a rapamycin derivative that is inactive, laying the foundation for improved mTOR inhibitors that do not suppress intrinsic immunity. We find that rapamycin analogs that promote SARS-CoV-2 entry are those that activate TFEB, a transcription factor that triggers the degradation of antiviral membrane proteins inside of cells. Finally, rapamycin administration to rodents prior to SARS-CoV-2 challenge results in enhanced viral disease, revealing that its use in humans may increase susceptibility to infection.

microbiology↗

bioRxiv2020

Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

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. We identified a GxxxG motif in many CD225 proteins, including IFITM3 and proline rich transmembrane protein 2 (PRRT2). Mutation of 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 in IFITM3 disrupted its oligomerization and reduced its antiviral activity against Influenza A and HIV-1. The oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-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.

evolutionary biology↗

bioRxiv2019

Single-cell glycolytic activity regulates membrane tension 1 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 viruses 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. As glycolytic activity sets membrane tension levels by altering cellular cholesterol surface levels, our results suggest additional previously unknown benefits of cholesterol-lowering medication in HIV-1 infection.

immunology↗