We preincubated HEK293F cells with (A) amiloride hydrochloride, an inhibitor of Na+/H+ exchanger activity, (B) bafilomycin A1, a specific and potent inhibitor of vacuolar H+ ATPase in early endosomes, (C) monensin, an inhibitor of both endosomal acidification and receptor recycling, and (D) dynasore, a potent and specific dynamin inhibitor (which is involved in clathrin- and caveolin-mediated endocytosis) in 5% MBL-deficient serum in the absence or presence of rhMBL at 37C for 1 hour. p 0.005) compared with O/O or O/A haplotypes (O refers to B,C or D alleles).(EPS) pone.0060838.s002.eps (711K) GUID:?EADD60C8-C1AE-484F-812A-50A2599A9363 Figure S3: Endoglycosidases cleave Hybrid Lectin (1:500) followed by streptavidin-horseradish peroxidase (HRP; 1:5000). Membranes were then incubated with Immobilon Western Chemiluminescent AP substrate and exposed to autoradiography film. High-mannose moieties were preserved only in untreated virus confirming that PNGase F and endo H effectively cleaved high-mannose residues on GPs of HIV-EBOV virion-like particles.(EPS) pone.0060838.s003.eps (1.6M) GUID:?0C4788FB-5606-4E3B-BB12-6EC4DDB1B126 Figure S4: Thermolysin treatment of HIV-EBOV GP abrogates enhancement of infection by MI-1061 rhMBL in a thermolysin-concentration dependent manner. We preincubated HIV-EBOV GP virion-like particles (1200 pg p24/100 L) without or with thermolysin (31.3, 62.5, 125, 250 g/ml) in buffer (40 mM HEPES, 40 mM MES, 50 mM NaCl, 0.1 mM CaCl2) shaking for 30 minutes at 37C. The reactions were then terminated with 0.1 mM EDTA. HEK293F cells were infected with thermolysin-treated virion-like particles mixed with MBL-deficient ( 0.6 ng/ml) 5% human serum alone or with the same supplemented with 10 g/ml rhMBL as described in Materials and Methods. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). Shown are average adjusted luciferase results from two experiments each with four replicates. The maximal reduction of viral infection was 17-fold in the presence of rhMBL, whereas there was no inhibition of infection in the absence of MBL.(EPS) pone.0060838.s004.eps (696K) GUID:?A6E9B114-1C61-4274-9C81-49F275A12377 Figure S5: MBL-mediated MI-1061 macropinocytosis of HIV-EBOV GP may bypass early endosomes and is dynamin independent. We preincubated HEK293F cells with (A) amiloride hydrochloride, an inhibitor of Na+/H+ exchanger activity, (B) bafilomycin A1, a specific and potent inhibitor of vacuolar H+ ATPase in early endosomes, (C) monensin, an inhibitor of both endosomal acidification and receptor recycling, and (D) dynasore, a potent and specific dynamin inhibitor (which is involved in clathrin- and caveolin-mediated endocytosis) in 5% MBL-deficient serum in the absence or presence of rhMBL at 37C for 1 hour. We then infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 l). Percentages of infected cells are relative to DMSO controls (a methanol control was used for monensin). Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). Significant differences are shown. Experiments were performed twice in quadruplicate.(EPS) pone.0060838.s005.eps (988K) GUID:?C50440E9-59E5-4769-AC81-E3CF1C4966A1 Figure S6: shRNA knockdown of cathepsin-L (CTSL1) and MGL (CLEC10A). We performed RNA interference with a pLK0.1 vector encoding shRNAs that targeted (A) CTSL1 MI-1061 and (B) MGL (CLEC10A) as described for Figure 6. Luciferase values were adjusted for cell viability. Percentage change in infection was normalized to an empty pLK0.1 control vector.(EPS) pone.0060838.s006.eps (713K) GUID:?B0849C2A-7B10-4E05-8F4C-CEEC2BABD42C Figure S7: rhMBL targets gene which disrupt assembly of MBL high-order oligomers and lead to reduced MBL concentrations and activity. Single nucleotide polymorphisms in the promoter region (allele) evolved into multiple low MBL-producing haplotypes because of heterotic balanced selection in which individuals carrying structural and regulatory heterozygous polymorphisms had a survival advantage [11]. It is not known whether viral infections exerted such selective pressure over time. EBOV is a non-segmented, negative-strand RNA virus of the order and family Rabbit polyclonal to UGCGL2 that can cause rapidly fatal viral hemorrhagic fevers in part by dysregulating the innate immune system..