Structural organization of pregenomic RNA and the carboxy-terminal domain of the capsid protein of hepatitis B virus.

The Hepatitis B Virus (HBV) double-stranded DNA genome is reverse transcribed from its RNA pregenome (pgRNA) within the virus core (or capsid). Phosphorylation of the arginine-rich carboxy-terminal domain (CTD) of the HBV capsid protein (Cp183) is essential for pgRNA encapsidation and reverse transcription. However, the structure of the CTD remains poorly defined. Here we report sub-nanometer resolution cryo-EM structures of in vitro assembled empty and pgRNA-filled Cp183 capsids in unphosphorylated and phosphorylation-mimic states. In empty capsids, we found unexpected evidence of surface accessible CTD density partially occluding pores in the capsid surface. We also observed that CTD organization changed substantively as a function of phosphorylation. In RNA-filled capsids, unphosphorylated CTDs favored thick ropes of RNA, while the phosphorylation-mimic favored a mesh of thin, high-density strands suggestive of single stranded RNA. These results demonstrate that the CTD can regulate nucleic acid structure, supporting the hypothesis that the HBV capsid has a functional role as a nucleic acid chaperone.

Differential assembly of Hepatitis B Virus core protein on single- and double-stranded nucleic acid suggest the dsDNA-filled core is spring-loaded.

Hepatitis B Virus (HBV) cores assemble on viral RNA, which is reverse transcribed within the core to the partially dsDNA genome of mature HBV. However, constraining dsDNA, a stiff polymer, within a core necessarily requires far greater capsid stability than constraining ssRNA. We hypothesized that, unlike ssRNA, dsDNA would be a poor substrate for assembly. We examined titrations of ssDNA and dsDNA with purified HBV core protein, Cp183, by EMSA, EM, DLS, and etheno-DNA fluorescence. Cp183 bound ssDNA with high affinity to form virus-like capsids. However, Cp183 bound dsDNA poorly, forming a mixture of irregular complexes. Nonetheless, we observed some normal cores in dsDNA assembly reactions, indicating that the energy required to bend DNA could be similar to the protein-protein association energy. This similarity of energies suggests that dsDNA stresses mature HBV cores, in agreement with calculation, which may be the basis for the virus maturation signal and DNA release.

Cellular recognition of Mycobacterium tuberculosis ESAT-6 and KatG peptides in systemic sarcoidosis.

Sarcoidosis is an enigmatic disease with a pathology similar to that of tuberculosis. We detected Th-1 immune responses to Mycobacterium tuberculosis ESAT-6 and KatG peptides from peripheral blood mononuclear cells from 15/26 sarcoidosis, 1/24 purified-protein-derivative-negative (PPD-) (P < 0.0001, Fisher's exact test), and 7/8 PPD-positive (PPD+) subjects (P = 0.21). This finding provides immunologic links between mycobacteria and systemic sarcoidosis.

Zebrafish atoh1 genes: classic proneural activity in the inner ear and regulation by Fgf and Notch.

Hair cells of the inner ear develop from an equivalence group marked by expression of the proneural gene Atoh1. In mouse, Atoh1 is necessary for hair cell differentiation, but its role in specifying the equivalence group (proneural function) has been questioned and little is known about its upstream activators. We have addressed these issues in zebrafish. Two zebrafish homologs, atoh1a and atoh1b, are together necessary for hair cell development. These genes crossregulate each other but are differentially required during distinct developmental periods, first in the preotic placode and later in the otic vesicle. Interactions with the Notch pathway confirm that atoh1 genes have early proneural function. Fgf3 and Fgf8 are upstream activators of atoh1 genes during both phases, and foxi1, pax8 and dlx genes regulate atoh1b in the preplacode. A model is presented in which zebrafish atoh1 genes operate in a complex network leading to hair cell development.