Cyclosporines Antagonize the Antiviral Activity of IFITMProteins by Redistributing Them toward the Golgi Apparatus.

Interferon-induced transmembrane proteins (IFITMs) block the fusion of diverse enveloped viruses, likely through increasing the cell membrane's rigidity. Previous studies have reported that the antiviral activity of the IFITM family member, IFITM3, is antagonized by cell pretreatment with rapamycin derivatives and cyclosporines A and H (CsA and CsH) that promote the degradation of IFITM3. Here, we show that CsA and CsH potently enhance virus fusion with IFITM1- and IFITM3-expressing cells by inducing their rapid relocalization from the plasma membrane and endosomes, respectively, towards the Golgi.

Interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes.

Late endosome-resident interferon-induced transmembrane protein 3 (IFITM3) inhibits fusion of diverse viruses, including Influenza A virus (IAV), by a poorly understood mechanism. Despite the broad antiviral activity of IFITM3, viruses like Lassa virus (LASV), are fully resistant to its inhibitory effects. It is currently unclear whether resistance arises from a highly efficient fusion machinery that is capable of overcoming IFITM3 restriction or the ability to enter from cellular sites devoid of this factor.

An improved labeling strategy enables automated detection of single-virus fusion and assessment of HIV-1 protease activity in single virions.

Enveloped viruses transfer their genomes into host cells by fusing their membrane to that of the cell. To visualize single-virus fusion in living cells, researchers take advantage of the proteolytic maturation of HIV, type 1 (HIV-1), which can generate free fluorescent proteins within the viral particle. Co-labeling viruses with a content marker and a fluorescently tagged Vpr (a viral core protein) enables detection of single-virus fusions, but a major limitation of this approach is that not all viral particles incorporate both markers.

pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion.

Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.

Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion.

Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion.

Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

Background: HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm.

Results: Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus.

Molecular Details of Olfactomedin Domains Provide Pathway to Structure-Function Studies.

Olfactomedin (OLF) domains are found within extracellular, multidomain proteins in numerous tissues of multicellular organisms. Even though these proteins have been implicated in human disorders ranging from cancers to attention deficit disorder to glaucoma, little is known about their structure(s) and function(s). Here we biophysically, biochemically, and structurally characterize OLF domains from H.

Distinct requirements for HIV-cell fusion and HIV-mediated cell-cell fusion.

Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as "fusion-from-without" (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample.

IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion.

Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry.

Downhill protein folding modules as scaffolds for broad-range ultrafast biosensors.

Conformational switches are macromolecules that toggle between two states (active/inactive or folded/unfolded) upon specific binding to a target molecule. These molecular devices provide an excellent scaffold for developing real-time biosensors. Here we take this concept one step beyond to build high-performance conformational rheostat sensors.

The effect of electrostatics on the marginal cooperativity of an ultrafast folding protein.

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures.