The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with aminoterminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to assemble further. In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells. The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein. The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.

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Glucose enhances mitochondrial function, insulin secretion and Myc expression in β-cells. β-cell-specific Myc knockout mice show glucose intolerance, hypoinsulinemia and lack of adaptive β-cell mass expansion following high-fat diet feeding. However, whether Myc regulates GSIS and mitochondrial function in β-cells is unknown. Here, we tested the effects of the Myc activity inhibitor 10058-F4 (1RH) in GSIS, mitochondrial function and metabolism in islets using islet perifusion, Seahorse and metabolomics/transcriptomics approaches. Mouse and human islets incubated 6h with 40µM 1RH displayed impaired phase 1 and phase 2 insulin secretion induced by 11mM glucose. Insulin secretion was not affected in the presence of 25mM KCl suggesting that Myc is required for glucose- but not membrane depolarization-induced insulin secretion. Since adequate mitochondrial function is essential for GSIS, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to analyze mitochondrial respiration and glycolytic flux in these islets. 1RH significantly reduced OCR, ECAR and glucose-induced ATP production. Conversely, the Myc inducer harmine (10µM), increased basal OCR, ATP production, and ECAR and partially reversed 1RH effects on mitochondrial function. RNAseq of mouse islets treated with 1RH in 11mM glucose revealed reduced expression of both oxidative phosphorylation and β-cell signature genes but enhanced gene expression of glycolysis, ROS, fatty acid metabolism and autophagy/mitophagy pathways. Metabolomics analysis of mouse islets treated with 1RH in 11mM glucose confirmed the reduction in ATP and highlighted the accumulation of L-palmitoylcarnitine and palmitic acid suggesting inefficient β-oxidation. In conclusion, Myc is required for GSIS and mitochondrial function in β-cells. Impaired Myc action can lead to unbalanced metabolism and autophagy/mytophagy leading to β-cell dysfunction. Disclosure G.Lu: None. R.Kang: None. P.Diaz-pozo: None. J.Lee: None. M.Li: None. V.M.Victor: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding National Institutes of Health (R01DK126450)