Influenza D virus utilizes both 9-O-acetylated N-acetylneuraminic and 9-O-acetylated N-glycolylneuraminic acids as functional entry receptors.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac2 and Neu5Gc9Ac in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9-O-acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac2 or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac2 or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac2- or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac2.IMPORTANCEInfluenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and non-human 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9-O-acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac2 is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.

Fusogenic structural changes in arenavirus glycoproteins are associated with viroporin activity.

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.

Human LAMP1 accelerates Lassa virus fusion and potently promotes fusion pore dilation upon forcing viral fusion with non-endosomal membrane.

Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry.

Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.

HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes.

Enveloped viruses that rely on a low pH-dependent step for entry initiate infection by fusing with acidic endosomes, whereas the entry sites for pH-independent viruses, such as HIV-1, have not been defined. These viruses have long been assumed to fuse directly with the plasma membrane. Here we used population-based measurements of the viral content delivery into the cytosol and time-resolved imaging of single viruses to demonstrate that complete HIV-1 fusion occurred in endosomes. In contrast, viral fusion with the plasma membrane did not progress beyond the lipid mixing step. HIV-1 underwent receptor-mediated internalization long before endosomal fusion, thus minimizing the surface exposure of conserved viral epitopes during fusion and reducing the efficacy of inhibitors targeting these epitopes. We also show that, strikingly, endosomal fusion is sensitive to a dynamin inhibitor, dynasore. These findings imply that HIV-1 infects cells via endocytosis and envelope glycoprotein- and dynamin-dependent fusion with intracellular compartments.

The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion.

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins-a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion-the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.

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. Here, we constructed and validated a functional IFITM3 tagged with EGFP or other fluorescent proteins. This breakthrough allowed live cell imaging of virus co-trafficking and fusion with endosomal compartments in cells expressing fluorescent IFITM3. Three-color single virus and endosome tracking revealed that sensitive (IAV), but not resistant (LASV), viruses become trapped within IFITM3-positive endosomes where they underwent hemifusion but failed to release their content into the cytoplasm. IAV fusion with IFITM3-containing compartments could be rescued by amphotericin B treatment, which has been previously shown to antagonize the antiviral activity of this protein. By comparison, virtually all LASV particles trafficked and fused with endosomes lacking detectable levels of fluorescent IFITM3, implying that this virus escapes restriction by utilizing endocytic pathways that are distinct from the IAV entry pathways. The importance of virus uptake and transport pathways is further reinforced by the observation that LASV glycoprotein-mediated cell-cell fusion is inhibited by IFITM3 and other members of the IFITM family expressed in target cells. Together, our results strongly support a model according to which IFITM3 accumulation at the sites of virus fusion is a prerequisite for its antiviral activity and that this protein traps viral fusion at a hemifusion stage by preventing the formation of fusion pores. We conclude that the ability to utilize alternative endocytic pathways for entry confers IFITM3-resistance to otherwise sensitive viruses.

Facile autofluorescence suppression enabling tracking of single viruses in live cells.

Live cell fluorescence imaging is the method of choice for studying dynamic processes, such as nuclear transport, vesicular trafficking, and virus entry and egress. However, endogenous cellular autofluorescence masks a useful fluorescence signal, limiting the ability to reliably visualize low-abundance fluorescent proteins. Here, we employed synchronously amplified fluorescence image recovery (SAFIRe), which optically alters ground versus photophysical dark state populations within fluorescent proteins to modulate and selectively detect their background-free emission. Using a photoswitchable rsFastLime fluorescent protein combined with a simple illumination and image-processing scheme, we demonstrate the utility of this approach for suppressing undesirable, unmodulatable fluorescence background. Significantly, we adapted this technique to different commercial wide-field and spinning-disk confocal microscopes, obtaining >10-fold improvements in signal to background. SAFIRe allowed visualization of rsFastLime targeted to mitochondria by efficiently suppressing endogenous autofluorescence or overexpressed cytosolic unmodulatable EGFP. Suppression of the overlapping EGFP signal provided a means to perform multiplexed imaging of rsFastLime and spectrally overlapping fluorophores. Importantly, we used SAFIRe to reliably visualize and track single rsFastLime-labeled HIV-1 particles in living cells exhibiting high and uneven autofluorescence signals. Time-lapse SAFIRe imaging can be performed for an extended period of time to visualize HIV-1 entry into cells. SAFIRe should be broadly applicable for imaging live cell dynamics with commercial microscopes, even in strongly autofluorescent cells or cells expressing spectrally overlapping fluorescent proteins.

A Novel Phenotype Links HIV-1 Capsid Stability to cGAS-Mediated DNA Sensing.

The HIV-1 capsid executes essential functions that are regulated by capsid stability and host factors. In contrast to increasing knowledge on functional roles of capsid-interacting host proteins during postentry steps, less is known about capsid stability and its impact on intracellular events. Here, using the antiviral compound PF-3450074 (PF74) as a probe for capsid function, we uncovered a novel phenotype of capsid stability that has a profound effect on innate sensing of viral DNA by the DNA sensor cGAS. A single mutation, R143A, in the capsid protein conferred resistance to high concentrations of PF74, without affecting capsid binding to PF74. A cell-free assay showed that the R143A mutant partially counteracted the capsid-destabilizing activity of PF74, pointing to capsid stabilization as a resistance mechanism for the R143A mutant. In monocytic THP-1 cells, the R143A virus, but not the wild-type virus, suppressed cGAS-dependent innate immune activation. These results suggest that capsid stabilization improves the shielding of viral DNA from innate sensing. We found that a naturally occurring transmitted founder (T/F) variant shares the same properties as the R143A mutant with respect to PF74 resistance and DNA sensing. Imaging assays revealed delayed uncoating kinetics of this T/F variant and the R143A mutant. All these phenotypes of this T/F variant were controlled by a genetic polymorphism located at the trimeric interface between capsid hexamers, thus linking these capsid-dependent properties. Overall, this work functionally connects capsid stability to innate sensing of viral DNA and reveals naturally occurring phenotypic variation in HIV-1 capsid stability.IMPORTANCE The HIV-1 capsid, which is made from individual viral capsid proteins (CA), is a target for a number of antiviral compounds, including the small-molecule inhibitor PF74. In the present study, we utilized PF74 to identify a transmitted/founder (T/F) strain that shows increased capsid stability. Interestingly, PF74-resistant variants prevented cGAS-dependent innate immune activation under a condition where the other T/F strains induced type I interferon. These observations thus reveal a new CA-specific phenotype that couples capsid stability to viral DNA recognition by cytosolic DNA sensors.

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

HIV-1 particles assemble and bud from the plasma membrane of infected T lymphocytes. Infected macrophages, in contrast, accumulate particles within an apparent intracellular compartment known as the virus-containing compartment or VCC. Many aspects of the formation and function of the VCC remain unclear. Here we demonstrate that VCC formation does not actually require infection of the macrophage, but can be reproduced through the exogenous addition of non-infectious virus-like particles or infectious virions to macrophage cultures. Particles were captured by Siglec-1, a prominent cell surface lectin that attaches to gangliosides on the lipid envelope of the virus. VCCs formed within infected macrophages were readily targeted by the addition of ganglioside-containing virus-like particles to the extracellular media. Depletion of Siglec-1 from the macrophage or depletion of gangliosides from viral particles prevented particle uptake into the VCC and resulted in substantial reductions of VCC volume. Furthermore, Siglec-1-mediated virion capture and subsequent VCC formation was required for efficient trans-infection of autologous T cells. Our results help to define the nature of this intracellular compartment, arguing that it is a compartment formed by particle uptake from the periphery, and that this compartment can readily transmit virus to target T lymphocytes. Inhibiting or eliminating the VCC may be an important component of strategies to reduce HIV transmission and to eradicate HIV reservoirs.

How entry inhibitors synergize to fight HIV.

HIV fusion with the cell membrane can be inhibited by blocking coreceptor binding or by preventing fusion-inducing conformational changes in the Env protein. Logically, inhibitors that act by these two mechanisms should act synergistically, but previous studies have reported conflicting results. A new study by Ahn and Root reconciles these discordant reports by demonstrating that synergy emerges when Env engages multiple coreceptors prior to inducing fusion and when high-affinity inhibitory peptides are used, a condition that may not be satisfied in vivo.

SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins.

The host proteins, SERINC3 and SERINC5, have been recently shown to incorporate into HIV-1 particles and compromise their ability to fuse with target cells, an effect that is antagonized by the viral Nef protein. Envelope (Env) glycoproteins from different HIV-1 isolates exhibit a broad range of sensitivity to SERINC-mediated restriction, and the mechanism by which SERINCs interfere with HIV-1 fusion remains unclear. Here, we show that incorporation of SERINC5 into virions in the absence of Nef inhibits the formation of small fusion pores between viruses and cells. Strikingly, we found that SERINC5 promotes spontaneous functional inactivation of sensitive but not resistant Env glycoproteins. Although SERINC5-Env interaction was not detected by co-immunoprecipitation, incorporation of this protein enhanced the exposure of the conserved gp41 domains and sensitized the virus to neutralizing antibodies and gp41-derived inhibitory peptides. These results imply that SERINC5 restricts HIV-1 fusion at a step prior to small pore formation by selectively inactivating sensitive Env glycoproteins, likely through altering their conformation. The increased HIV-1 sensitivity to anti-gp41 antibodies and peptides suggests that SER5 also delays refolding of the remaining fusion-competent Env trimers.

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. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells.

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. Here we designed a labeling strategy based on the bifunctional mCherry-2xCL-YFP-Vpr construct, in which 2xCL denotes a tandem cleavage site for the viral protease. This bifunctional marker was efficiently cleaved during virus maturation, producing free mCherry and the core-associated YFP-Vpr. A nearly perfect colocalization of these two markers in virions and their fixed 1:1 ratio enabled automated detection of single-particle fusion in both fixed and live cells based on loss of the mCherry signal. Furthermore, a drop in FRET efficiency between YFP and mCherry because of cleavage of the bifunctional marker, which manifested as a marked shift in the normalized YFP/mCherry fluorescence ratio, reliably predicted viral protease activity in single virions. This feature could discriminate between the particles containing free mCherry, and therefore likely representing mature viruses, and immature particles whose fusion cannot be detected. In summary, our new labeling strategy offers several advantages compared with previous approaches, including increased reliability and throughput of detection of viral fusion. We anticipate that our method will have significant utility for studying viral fusion and maturation.

Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.

Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process.

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.

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. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4(+) T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes.

P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions.

UNLABELLED: HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE: Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF279, is due to the blocking of virus interactions with both the CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by the respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and a mechanism of action that are distinct from those of known HIV-1 coreceptor antagonists.

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. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.

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. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. CONCLUSIONS: The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.