Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

Background: SCARB2 is the cellular receptor for EV71. Results: Seven highly variable amino acids on SCARB2 and a region in proximity to residue Gln-172 of VP1 are critical for EV71 infection. Conclusion: EV71 might adopt the canyon receptor engagement mode for viral entry. Significance: Elucidation of receptor and viral determinants revealed a possible target for anti-EV71 interventions.

Efficient mechanism-based design of antibiotics that are not susceptible to β-lactamases is hindered by the lack of comprehensive knowledge on the energetic landscapes for the hydrolysis of various β-lactams. Herein,...

The bacterial enzyme class of β-lactamases are involved in benzylpenicillin acylation reactions, which are currently being revisited using hybrid quantum mechanical molecular mechanical (QM/MM) chain-of-states pathway optimizations. Minimum energy pathways are sampled by reoptimizing pathway geometry under different representative protein environments obtained through constrained molecular dynamics simulations. Predictive potential energy surface models in the reaction space are trained with machine-learning regression techniques. Herein, using TEM-1/benzylpenicillin acylation reaction as the model system, we introduce two model-independent criteria for delineating the energetic contributions and correlations in the predicted reaction space. Both methods are demonstrated to effectively quantify the energetic contribution of each chemical process and identify the rate limiting step of enzymatic reaction with high degrees of freedom. The consistency of the current workflow is tested under seven levels of quantum chemistry theory and three non-linear machine-learning regression models. The proposed approaches are validated to provide qualitative compliance with experimental mutagenesis studies.

Resistance to carbapenem β-lactams presents major clinical and economical challenges for the treatment of pathogen infections. The fast hydrolysis of carbapenems by carbapenemase-producing bacterial strains enables the effective deactivation of...