Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.

A standardised Phase III clinical trial framework to assess therapeutic interventions for Lassa fever.

BACKGROUND: Only one recommendation currently exists for the treatment of Lassa fever (LF), which is ribavirin administered in conjunction with supportive care. This recommendation is primarily based on evidence generated from a single clinical trial that was conducted more than 30 years ago-the methodology and results of which have recently come under scrutiny. The requirement for novel therapeutics and reassessment of ribavirin is therefore urgent. However, a significant amount of work now needs to be undertaken to ensure that future trials for LF can be conducted consistently and reliably to facilitate the efficient generation of evidence. METHODOLOGY: We convened a consultation group to establish the position of clinicians and researchers on the core components of future trials. A Core Eligibility Criteria (CEC), Core Case Definition (CCD), Core Outcome Set (COS) and Core Data Variables (CDV) were developed through the process of a multi-stakeholder consultation that took place using a modified-Delphi methodology. RESULTS: A consensus position was achieved for each aspect of the framework, which accounts for the inclusion of pregnant women and children in future LF clinical trials. The framework consists of 8 core criteria, as well as additional considerations for trial protocols. CONCLUSIONS: This project represents the first step towards delineating the clinical development pathway for new Lassa fever therapeutics, following a period of 40 years without advancement. Future planned projects will bolster the work initiated here to continue the advancement of LF clinical research through a regionally-centred, collaborative methodology, with the aim of delineating a clear pathway through which LF clinical trials can progress efficiently and ensure sustainable investments are made in research capacity at a regional level.

Screening and identification of Lassa virus endonuclease-targeting inhibitors from a fragment-based drug discovery library.

Lassa virus (LASV) belongs to the Old World genus Mammarenavirus, family Arenaviridae, and order Bunyavirales. Arenavirus contains a segmented negative-sense RNA genome, which is in line with the bunyavirus and orthomyxoviruses. The segmented negative-sense RNA viruses utilize a cap-snatching strategy to provide primers cleavaged from the host capped mRNA for viral mRNA transcription. As a similar strategy and the conformational conservation shared with these viruses, the endonuclease (EN) would serve as an attractive target for developing broad-spectrum inhibitors. Using the LASV minigenome (MG) system, we screened a fragment-based drug discovery library and found that two hits, F1204 and F1781, inhibited LASV MG activity. Both hits also inhibited the prototype arenavirus Lymphocytic choriomeningitis virus (LCMV) MG activity. Furthermore, both hits effectively inhibited authentic LCMV and severe fever with thrombocytopenia syndrome virus (SFTSV) infections. Similarly, both hits could inhibit the activity of LASV, LCMV, and SFTSV EN. The combination of either compound with an arenavirus entry inhibitor had significant synergistic antiviral effects. Moreover, both hits were found to be capable of binding to LASV EN with a binding affinity at the micromolar level. These findings provide a basis for developing the hits as potential candidates for the treatment of segmented negative-sense RNA virus infections.

CP100356 Hydrochloride, a P-Glycoprotein Inhibitor, Inhibits Lassa Virus Entry: Implication of a Candidate Pan-Mammarenavirus Entry Inhibitor.

Lassa virus (LASV)-a member of the family Arenaviridae-causes Lassa fever in humans and is endemic in West Africa. Currently, no approved drugs are available. We screened 2480 small compounds for their potential antiviral activity using pseudotyped vesicular stomatitis virus harboring the LASV glycoprotein (VSV-LASVGP) and a related prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). Follow-up studies confirmed that CP100356 hydrochloride (CP100356), a specific P-glycoprotein (P-gp) inhibitor, suppressed VSV-LASVGP, LCMV, and LASV infection with half maximal inhibitory concentrations of 0.52, 0.54, and 0.062 µM, respectively, without significant cytotoxicity. Although CP100356 did not block receptor binding at the cell surface, it inhibited low-pH-dependent membrane fusion mediated by arenavirus glycoproteins. P-gp downregulation did not cause a significant reduction in either VSV-LASVGP or LCMV infection, suggesting that P-gp itself is unlikely to be involved in arenavirus entry. Finally, our data also indicate that CP100356 inhibits the infection by other mammarenaviruses. Thus, our findings suggest that CP100356 can be considered as an effective virus entry inhibitor for LASV and other highly pathogenic mammarenaviruses.

Broad-spectrum antivirals of protoporphyrins inhibit the entry of highly pathogenic emerging viruses.

Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91 ± 0.25 µM to 1.88 ± 0.34 µM. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.

Lassa viral dynamics in non-human primates treated with favipiravir or ribavirin.

Lassa fever is an haemorrhagic fever caused by Lassa virus (LASV). There is no vaccine approved against LASV and the only recommended antiviral treatment relies on ribavirin, despite limited evidence of efficacy. Recently, the nucleotide analogue favipiravir showed a high antiviral efficacy, with 100% survival obtained in an otherwise fully lethal non-human primate (NHP) model of Lassa fever. However the mechanism of action of the drug is not known and the absence of pharmacokinetic data limits the translation of these results to the human setting. Here we aimed to better understand the antiviral effect of favipiravir by developping the first mathematical model recapitulating Lassa viral dynamics and treatment. We analyzed the viral dynamics in 24 NHPs left untreated or treated with ribavirin or favipiravir, and we put the results in perspective with those obtained with the same drugs in the context of Ebola infection. Our model estimates favipiravir EC50 in vivo to 2.89 µg.mL-1, which is much lower than what was found against Ebola virus. The main mechanism of action of favipiravir was to decrease virus infectivity, with an efficacy of 91% at the highest dose. Based on our knowledge acquired on the drug pharmacokinetics in humans, our model predicts that favipiravir doses larger than 1200 mg twice a day should have the capability to strongly reduce the production infectious virus and provide a milestone towards a future use in humans.

Methanol Fixation, but not Giemsa Staining, Inactivates Ebola and Lassa Viruses in Peripheral Blood Smears Made on Plastic Microscope Slides.

Diseases caused by many highly pathogenic viruses, including Ebola virus (EBOV) and Lassa virus (LASV), present with nonspecific signs and symptoms that overlap with common tropical diseases such as malaria. Initial diagnostic tests performed on patients under investigation for viral hemorrhagic fevers routinely include analysis of peripheral blood smears to detect and quantify Plasmodium species. In light of recent and ongoing Ebola virus disease and Lassa fever epidemics, clinical laboratories around the world require protocols for dealing with highly infectious specimens from patients with suspected or confirmed high-consequence diseases. Few validated protocols for safe analysis of peripheral blood smears are available, revealing a need for further research. In this study, we evaluated the performance of two plastic microscope slide types that offer safe alternatives to glass slides, determined the temporal parameters required to inactivate EBOV and LASV in thin blood smears by methanol fixation, and assessed the virucidal activity of Giemsa stain. Both types of plastic microscope slides performed optimally; there were no significant differences in blood cell morphology or tinctorial properties nor were differences noted in Plasmodium ovale morphology or staining, when compared with glass slides. For both EBOV and LASV, viable viruses were not detected in thin blood smears following fixation in absolute methanol for at least 2 minutes. By contrast, viable EBOV and LASV were recovered from all Giemsa-stained thick blood smears.

The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry.

The zoonotic Old World mammarenavirus Lassa (LASV) causes severe hemorrhagic fever with high mortality and morbidity in humans in endemic regions. The development of effective strategies to combat LASV infections is of high priority, given the lack of a licensed vaccine and restriction on available treatment to off-label use of ribavirin. A better understanding of the fundamental aspects of the virus's life cycle would help to improve the development of novel therapeutic approaches. Host cell entry and restriction factors represent major barriers for emerging viruses and are promising targets for therapeutic intervention. In addition to the LASV main receptor, the extracellular matrix molecule dystroglycan (DG), the phosphatidylserine-binding receptors of the Tyro3/Axl/Mer (TAM), and T cell immunoglobulin and mucin receptor (TIM) families are potential alternative receptors of LASV infection. Therefore, the relative contributions of candidate receptors to LASV entry into a particular human cell type are a complex function of receptor expression and functional DG availability. Here, we describe the role of two receptor tyrosine kinases (RTKs), Axl and hepatocyte growth factor receptor (HGFR), in the presence and absence of glycosylated DG for LASV entry. We found that both RTKs participated in the macropinocytosis-related LASV entry and, regardless of the presence or absence of functional DG, their inhibition resulted in a significant antiviral effect.

The antifungal isavuconazole inhibits the entry of lassa virus by targeting the stable signal peptide-GP2 subunit interface of lassa virus glycoprotein.

Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever in humans, and the limited therapeutic treatment for Lassa fever poses significant threat to public health in West Africa. Using an HIV based pseudovirus platform, we identified isavuconazole, a triazole antifungal for systemic use, as a LASV entry inhibitor with an EC50 of 1.2 µM. Isavuconazole inhibits Lassa virus entry by blocking the pH dependent viral fusion mediated by the Lassa virus surface glycoprotein. Fragment replacement mutational study indicated that isavuconazole targets the stable signal peptide (SSP)-membrane fusion subunit (GP2) interface of Lassa glycoprotein. Further mutational study of the SSP-GP2 region of LASV glycoprotein revealed that S27 in the N-terminal transmembrane region of SSP and V431, F434 and V435 in the transmembrane domain of GP2 affect anti-LASV activity of isavuconazole. Isavuconazole also displays antiviral activity to five New World (NW) mammarenaviruses that cause hemorrhagic fever. This study facilitates the potential repurposing of isavuconazole for therapeutic intervention against human-pathogenic arenaviruses, and provides the basis for further structural optimization of arenavirus fusion inhibitors based on the predicted structural characteristics of the unique SSP-GP2 interface.

A cell-based, infectious-free, platform to identify inhibitors of lassa virus ribonucleoprotein (vRNP) activity.

The mammarenavirus Lassa (LASV) is highly prevalent in West Africa where it infects several hundred thousand individuals annually resulting in a high number of Lassa fever (LF) cases, a febrile disease associated with high morbidity and significant mortality. Mounting evidence indicates that the worldwide-distributed prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. There are not Food and Drug Administration (FDA) licensed vaccines and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective and can cause significant side effects. Therefore, there is an unmet need for novel antiviral drugs to combat LASV. This task would be facilitated by the implementation of high throughput screens (HTS) to identify inhibitors of the activity of the virus ribonucleoprotein (vRNP) responsible for directing virus RNA genome replication and gene transcription. The use of live LASV for this purpose is jeopardized by the requirement of biosafety level 4 (BSL4) containment. We have developed a virus-free cell platform, where expression levels of reporter genes serve as accurate surrogates of vRNP activity, to develop cell-based assays compatible with HTS to identify inhibitors of LASV and LCMV mammarenavirus vRNP activities.

Lassa fever in pregnancy with a positive maternal and fetal outcome: A case report.

BACKGROUND: The signs and symptoms of Lassa fever are initially indistinguishable from other febrile illnesses common in the tropics and complications of pregnancy. Surviving Lassa fever during pregnancy is rare. Only few cases have been documented. The antiviral drug of choice is ribavirin. CASE DESCRIPTION: A 25-year-old multigravida farmer with fever who was initially thought to have malaria in pregnancy at 29 weeks gestation. Further changes in her clinical state and laboratory tests led to a confirmation of Lassa fever. The Liver enzymes were markedly deranged and the packed cell volume was 27%. She commenced on ribavirin and subsequently was delivered of a live male neonate who was RT PCR negative for Lassa fever virus. Her clinical state improved, repeat RT PCR on day 15 was negative and she made full recovery. DISCUSSION: The case reported had similar clinical features of fever and abdominal pain and resulted in the initial diagnoses of Malaria in pregnancy. When she failed to respond to antimalarial and antibiotics treatments, a strong suspicion of viral hemorrhagic fever was made. At this time the patient was in advanced stage of the disease with bleeding from vagina and puncture sites. On the third day of admission she was delivered of a live male neonate who remained negative after 2 consecutive RT PCR tests for Lassa fever virus. Lassa fever carries a high risk of death to the fetus throughout pregnancy and to the mother in the third trimester. Mothers with Lassa fever improved rapidly after evacuation of the uterus by spontaneous abortion, or normal delivery. She was clinically stable following delivery. Her laboratory investigations were essentially normal. Throughout her management transmission based precautions were observed. None of the six close contacts developed symptoms after been followed up for 21 days. CONCLUSION: This report adds to the body of literature that individuals can survive Lassa fever during pregnancy with good maternal and fetal outcome.

The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors.

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections.

Ribavirin for the treatment of Lassa fever: A systematic review and meta-analysis.

OBJECTIVES: Lassa fever (LF) causes annual outbreaks in endemic regions with high mortality of symptomatic patients. Ribavirin is recommended as standard treatment for LF in national and international guidelines but the evidence base for this recommendation has been questioned recently. METHODS: We conducted a systematic review and included 6 studies providing efficacy data of ribavirin treatment for LF (PROSPERO protocol CRD42018103994). RESULTS: Besides retrospective case series, the evidence mostly relies on a single prospective clinical trial with critical risk of bias. In this trial, LF associated mortality is reduced for patients with elevated aspartate aminotransferase (AST) when treated with ribavirin (OR 0.41, 95% CI 0.23-0.73), while mortality is higher for patients without elevated AST (OR 2.37, 95% CI 1.07-5.25). CONCLUSIONS: Based on the available data, current treatment guidelines may therefore put patients with mild LF at increased risk of death. The role of ribavirin in the treatment of LF requires urgent reassessment.

Identification of a clinical compound losmapimod that blocks Lassa virus entry.

Lassa virus (LASV) causes Lassa hemorrhagic fever in humans and poses a significant threat to public health in West Africa. Current therapeutic treatments for Lassa fever are limited, making the development of novel countermeasures an urgent priority. In this study, we identified losmapimod, a p38 mitogen-activated protein kinase (MAPK) inhibitor, from 102 screened compounds as an inhibitor of LASV infection. Losmapimod exerted its inhibitory effect against LASV after p38 MAPK down-regulation, and, interestingly, had no effect on other arenaviruses capable of causing viral hemorrhagic fever. Mechanistic studies showed that losmapimod inhibited LASV entry by affecting the stable signal peptide (SSP)-GP2 subunit interface of the LASV glycoprotein, thereby blocking pH-dependent viral fusion. As an aryl heteroaryl bis-carboxyamide derivative, losmapimod represents a novel chemical scaffold with anti-LASV activity, and it provides a new lead structure for the future development of LASV fusion inhibitors.

Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion.

Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses.IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.

Use of ribavirin in viruses other than hepatitis C. A review of the evidence. / Uso de ribavirina en virus distintos de la hepatitis C. Una revisión de la evidencia.

Ribavirin is a molecule with antiviral activity against different viruses. In clinical practice, it has made its niche almost exclusively for the treatment of the hepatitisC virus. However, there are other diseases in which it could be of benefit and it has the advantage of being suitable for oral, intravenous and inhaled administration. We conducted a review of the indications of the main drug agencies (Spanish, European and American) and other possible indications, mainly haemorrhagic fevers and coronavirus.

A potent Lassa virus antiviral targets an arenavirus virulence determinant.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses.

Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays.

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000⁻300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

Critical role for cholesterol in Lassa fever virus entry identified by a novel small molecule inhibitor targeting the viral receptor LAMP1.

Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.