Hepatoma polarization limits CD81 and hepatitis C virus dynamics.

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real-time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C-terminus, we studied the dynamics of a mutant CD81 lacking a C-terminal tail (CD81(ΔC)) and its effect(s) on HCVpp mobility and infection. CD81(ΔC) showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild-type protein in non-polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81(ΔC) expressing non-polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization-dependent manner.

A novel interactive tool for rigid-body modeling of multi-domain macromolecules using residual dipolar couplings.

Residual dipolar couplings (RDC), measured by dissolving proteins in dilute liquid crystal media, or by studying naturally paramagnetic molecules, have rapidly become established as routine measurements in the investigation of the structure of macromolecules by NMR. One of the most obvious applications of the previously inaccessible long-range angular information afforded by RDC is the accurate definition of domain orientation in multi-module macromolecules or complexes. In this paper we describe a novel program developed to allow the determination of alignment tensor parameters for individual or multiple domains in macromolecules from residual dipolar couplings and to facilitate their manipulation to construct low-resolution models of macromolecular structure. For multi-domain systems the program determines the relative orientation of individual structured domains, and provides graphical user-driven rigid-body modeling of the different modules relative to the common tensorial frame. Translational freedom in the common frame, and equivalent rotations about the diagonalized (x,y,z) axes are used to position the different modules in the common frame to find a model in best agreement with experimentally measured couplings alone or in combination with additional experimental or covalent information.

Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data.

A novel program has been developed for the interpretation of 15N relaxation rates in terms of macromolecular anisotropic rotational diffusion. The program is based on a highly efficient simulated annealing/minimization algorithm, designed specifically to search the parametric space described by the isotropic, axially symmetric and fully anisotropic rotational diffusion tensor models. The high efficiency of this algorithm allows extensive noise-based Monte Carlo error analysis. Relevant statistical tests are systematically applied to provide confidence limits for the proposed tensorial models. The program is illustrated here using the example of the cytochrome c' from Rhodobacter capsulatus, a four-helix bundle heme protein, for which data at three different field strengths were independently analysed and compared.