Inhibition of viral membrane fusion by interferon-induced proteins

Interferon-induced transmembrane proteins (IFITMs) inhibit fusion of diverse enveloped viruses with host cells through a poorly understood mechanism. At least two modes of restriction have been described for IFITMs – protection from incoming viruses when expressed in target cells and reduction of infectivity of progeny virions upon incorporation into the viral membrane (dubbed “negative imprinting”). According to the currently accepted “tough membrane” model, IFITMs block viral fusion by increasing the negative curvature, lipid order and bending modulus of cell membranes and, thereby, arresting fusion at a hemifusion stage. However, our pilot results challenge this model by revealing that, whereas IFITM expression in cells increases the lipid order of intracellular compartments, the membrane of HIV-1 particles produced by IFITM-expressing cells has lower lipid order and membrane tension. Another critical new finding is that IFITM mutants that lack antiviral activity when expressed in target cells incorporate into and negatively imprint progeny virions. These results highlight key differences between the effects of IFITMs on cellular vs viral membranes and warrant further investigation. Our central hypothesis is that IFITMs inhibit viral entry through a multifaceted mechanism that involves: (a) rigidification of cell membranes at the sites of virus entry that arrests fusion at a hemifusion stage; and (b) “negative imprinting” of virions through reducing the order and tension of viral membrane and/or disrupting its nanoscale organization. To test this hypothesis, we propose the following Specific Aims. Specific Aim 1 will focus on ultrastructural basis for IFITM-mediated inhibition of viral fusion with endosomes, using correlative light-electron microscopy, electron tomography, and immunogold EM to elucidate the primary mechanism of the influenza A virus restriction by IFITM3 and the mechanism of fusion rescue by cyclosporine A treatment. Specific Aim 2 will define the determinants of stability of IFITM-arrested hemifusion intermediate through experimental manipulations that alter the propensity of arrested hemifusion to progress to full fusion. Specific Aim 3 will identify the IFITM residues that are critical for negative imprinting of virions, which are distinct from residues regulating the protection of target cells from infection; this will be accomplished by constructing and testing chimeras between active and inactive IFITM orthologs. Specific Aim 4 will delineate the effects of IFITMs on viral membrane in the context of negative imprinting of virions, which we hypothesize to occur through disruption of nanoscale organization of viral membrane and/or reduction of viral membrane tension. Successful completion of these Specific Aims will provide critical paradigm-shifting insights into the mechanism(s) of antiviral activity of IFITMs expressed in target cells and incorporated into progeny virions. Project Number: 1R01AI190198-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Gregory Melikian | Institution: EMORY UNIVERSITY, ATLANTA, GA | Award Amount: $2,297,892 | Activity Code: R01 | Study Section: Biochemistry and Biophysics of Membranes Study Section[BBM] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19019801