Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the β-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.
Human adenoviruses (HAdVs) are fatal to immuno-suppressed individuals, but no effective anti-HAdV therapy is available. Here, we present a novel image-based high-throughput screening (HtS) platform, which scores the full viral replication cycle from virus entry to dissemination of progeny and second-round infections. We analysed 1,280 small molecular weight compounds of the Prestwick Chemical Library (PCL) for interference with HAdV-C2 infection in a quadruplicate, blinded format, and performed robust image analyses and hit filtering. We present the entire set of the screening data including all images, image analyses and data processing pipelines. the data are made available at the Image Data Resource (IDR, idr0081). Our screen identified Nelfinavir mesylate as an inhibitor of HAdV-C2 multi-round plaque formation, but not single round infection. Nelfinavir has been FDA-approved for anti-retroviral therapy in humans. Our results underscore the power of image-based full cycle infection assays in identifying viral inhibitors with clinical potential.
Rhinoviruses (RVs) replicate on cytoplasmic membranes derived from the Golgi apparatus. They encode membrane-targeted proteins 2B, 2C, and 3A, which control trafficking and lipid composition of the replication membrane. The virus recruits host factors for replication, such as phosphatidylinositol 4 (PI4)-kinase 3beta (PI4K3b), which boosts PI4-phosphate (PI4P) levels and drives lipid countercurrent exchange of PI4P against cholesterol at endoplasmic reticulum-Golgi membrane contact sites through the lipid shuttling protein oxysterol binding protein 1 (OSBP1). We identified a PI4K3b inhibitor-resistant RV-A16 variant with a single point mutation in the conserved 2B protein near the cytosolic carboxy terminus, isoleucine 92 to threonine (termed 2B[I92T]). The mutation did not confer resistance to cholesterol-sequestering compounds or OSBP1 inhibition, suggesting invariant dependency on the PI4P/cholesterol lipid countercurrents. In the presence of PI4K3b inhibitor, Golgi reorganization and PI4P lipid induction occurred in RV-A16 2B[I92] but not in wild-type infection. The knockout of PI4K3b abolished the replication of both the 2B[I92T] mutant and the wild type. Doxycycline-inducible expression of PI4K3b in PI4K3b knockout cells efficiently rescued the 2B[I92T] mutant and, less effectively, wild-type virus infection. Ectopic expression of 2B[I92T] or 2B was less efficient than that of 3A in recruiting PI4K3b to perinuclear membranes, suggesting a supportive rather than decisive role of 2B in recruiting PI4K3b. The data suggest that 2B tunes the recruitment of PI4K3b to the replication membrane and allows the virus to adapt to cells with low levels of PI4K3b while still maintaining the PI4P/cholesterol countercurrent for establishing Golgi-derived RV replication membranes. Human rhinoviruses (RVs) are the major cause of the common cold worldwide. They cause asthmatic exacerbations and chronic obstructive pulmonary disease. Despite recent advances, the development of antivirals and vaccines has proven difficult due to the high number and variability of RV types. The identification of critical host factors and their interactions with viral proteins and membrane lipids for the establishment of viral replication is a basis for drug development strategies. Our findings here shed new light on the interactions between nonstructural viral membrane proteins and class III phosphatidylinositol 4 kinases from the host and highlight the importance of phosphatidylinositol 4 phosphate for RV replication.
Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. The human AdV (HAdV) species B and C, such as HAdV-C2, C5 and B14, cause respiratory disease, and constitute a health threat for immuno-compromised individuals. HAdV-Cs are well known for lysing cells, owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening frame-work and identified an inhibitor of HAdV-C2 multi-round infection, Nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of the human immuno-deficiency virus (HIV) aspartyl protease, and used to treat acquired immuno-deficiency syndrome (AIDS). It is not effective against single round HAdV infections. Here, we show that Nelfinavir inhibits the lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based trans-mission of cell-free viral particles from an infected cell to neighbouring uninfected cells. HAdV lacking ADP was insensitive to Nelfinavir, but gave rise to comet-shaped foci indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and B14p1 lacking ADP were highly sensitive to Nelfinavir, although HAdV-A31, B3, B7, B11, B16, B21, D8, D30 or D37 were less sensitive. Conspicuously, Nelfinavir unco-vered slow-growing round-shaped HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of non-lytic cell-to-cell transmission. Our study demonstrates the repurposing potential of Nelfinavir with post-exposure efficacy against different HAdVs, and describes an alternative non-lytic cell-to-cell transmission mode of HAdV.
14 15Human adenoviruses (HAdVs) are fatal to immune-suppressed people, but no effective 16 anti-HAdV therapy is available. Here, we present a novel image-based high-throughput 17 screening (HTS) platform, which scores the full viral replication cycle from virus entry to 18 dissemination of progeny. We analysed 1,280 small molecular compounds of the Prestwick 19Chemical Library (PCL) for interference with HAdV-C2 infection in a quadruplicate blinded 20 format, followed by robust image analyses, and hit identification. We present the entire set 21 of image-based screening data including all the images, and the image analysis and data 22 processing pipelines, as deposited at the Image Data repository (IDR) 1 , accession number 23 idr0081. We identified Nelfinavir mesylate as an inhibitor of HAdV plaque formation, in 24 agreement with the previous notion that Nelfinavir is ineffective in single round HAdV 25 infection assays. Nelfinavir has been FDA-approved for anti-retroviral therapy in humans. 26Our results underscore the power of image-based multi-round infection assays in 27 identifying viral inhibitors with clinical potential. 28 29
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