Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies-broad-spectrum antivirals-are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly ␣-helical structures with small amounts of ␤-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCEThe viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of muchneeded new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV. Hepatitis B virus (HBV) is a highly infectious, species-specific pathogen that causes serious liver diseases, including cancer, and causes 600,000 deaths annually (1). While excellent prophylactic HBV vaccines exist, these are ineffective against extant chronic HBV infections which affect 350 million people worldwide. Approved therapies for chronic HBV infections include immuno-modulators (e.g., interferon therapies) and nucleoside analogues that inhibit the reverse transcriptase domain of hPOL, the multifunctional viral polymerase of human HBV. These reverse transcriptase inhibitors, currently our best weapons against HBV, are not complete cures and have unwanted side effects (2, 3). While reverse transcriptase inhibitors are initially very effective at...

The aim of this study was to investigate the inhibitory effect of various dairy powders and milk constituents on the adhesion of a clinical isolate of Streptococcus mutans to hydroxylapatite (HA), an analogue of tooth enamel. Adhesion of a microorganism to a cell surface such as epithelial cells or tooth enamel is considered to be the first step in pathogenesis. Inhibiting this process may have therapeutic effects in vivo. The adherence assays were performed by incubating S. mutans with HA in the presence of each test material for 45 min, followed by centrifugal separation of the HA. Unbound bacteria were then quantified using a fluorescent dye. Sweet and Acid WPC80, buttermilk powder and cream powder were found to very effectively inhibit adherence of S. mutans to phosphate-buffered saline coated HA (PBS-HA). Sodium caseinate and the casein fractions a-,b-and j-casein were also found to show high levels of anti-adhesive activity. A selection of test materials were assessed using saliva-coated HA (S-HA), and similar trends were observed. The results suggest commercial dairy powders, and certain milk proteins, can inhibit adhesion of S. mutans to HA and may have potential to control dental caries.