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Assays detecting viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here we present Rolosense, a virus sensing platform that transduces the motion of synthetic DNA-based motors transporting 5-micron particles on RNA fuel chips. Motors and chips are modified with virus-binding aptamers that lead to stalling of motion. Therefore, motors perform a “mechanical test” of viral target and stall in the presence of whole virions which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 103copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as we demonstrated one-pot detection of influenza A and SARS-CoV-2. As a proof-of-concept, we show readout can be achieved using a smartphone camera in as little as 15 mins without any sample preparation steps. Taken together, mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost, point-of-care screening of circulating viruses.

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 “leaky” LASV fusion using single pseudoparticle 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.Authors summaryFusion of enveloped virus with host cell membranes is mediated by extensive conformational changes in viral glycoproteins, triggered by binding to cognate receptors and/or by exposure to acidic pH in the maturing endosome. We have previously reported that, unlike many other viral glycoproteins, pH-triggered endosomal fusion by the Lassa virus viral glycoprotein complex (GPc) is preceded by a mild permeabilization of the viral membrane. Here, we provide evidence that this activity is associated with early fusogenic changes in the GPc and is markedly enhanced by virus-cell contact. Permeabilization is induced by the glycoproteins of diverse arenaviruses and occurs in multiple target cell types. Based on these observations, we propose that, by analogy to the influenza virus M2 channel, membrane permeabilization and the resultant acidification of the arenavirus interior facilitate viral fusion and/or post-fusion steps of infection.

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., Author summary Pathogenic arenaviruses pose a serious health threat. The viral envelope glycoprotein GPC mediates attachment to host cells and drives virus entry via endocytosis and low pH-dependent fusion within late endosomes. Understanding the host factors and processes that are essential for arenavirus fusion may identify novel therapeutic targets. To delineate the mechanism of arenavirus entry, we examined cell-cell fusion induced by the Old World Lassa virus GPC proteins at low pH. Lassa GPC-mediated fusion was augmented by the human LAMP1 receptor and progressed through lipid curvature-sensitive intermediates, such as hemifusion (merger of contacting leaflets of viral and cell membrane without the formation of a fusion pore). We found that most GPC-mediated fusion events were off-path hemifusion structures and that the transition from hemifusion to full fusion and fusion pore enlargement were specifically promoted by an anionic lipid, bis(monoacylglycero)phosphate, which is highly enriched in late endosomes. This lipid also specifically promotes fusion of unrelated New World Junin arenavirus. Our results imply that arenaviruses evolved to use bis(monoacylglycero)phosphate to enter cells from late endosomes.

Interferon-induced transmembrane protein 3 (IFITM3) potently inhibits entry of diverse enveloped viruses by trapping the viral fusion at a hemifusion stage, but the underlying mechanism remains unclear. Here, we show that recombinant IFITM3 reconstituted into lipid vesicles induces negative membrane curvature and that this effect maps to its small amphipathic helix (AH). We demonstrate that AH: (i) partitions into lipid-disordered domains where IAV fusion occurs, (ii) induces negative membrane curvature, and (iii) increases lipid order and membrane stiffness. Additionally, replacing the IFITM3 AH with AH from an unrelated protein did not compromise its antiviral activity. These effects on membrane properties correlate with the fusion-inhibitory activity, as exogenous addition of AH to insulin-producing cells reduces glucose-stimulated insulin secretion. Our results thus imply that IFITM3 inhibits the transition from hemifusion to full fusion by imposing an unfavorable membrane curvature and increasing the order and stiffness of the cytoplasmic leaflet of endosomal membranes.