Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy.

The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individual single particles depends on molecular mass, as expected theoretically since the information in each particle image increases with molecular mass. The angular uncertainty is less than 1° for particles of high molecular mass (~50 MDa), several degrees for particles in the range 1-5 MDa, and tens of degrees for particles below 1 MDa. Orientational uncertainty may be the major contributor to the effective temperature factor (B-factor) describing contrast loss and therefore the maximum resolution of a structure determination. We also made two unexpected observations. Single particles that are known to be flexible showed a wider spread in orientation accuracy, and the orientations of the largest particles examined changed by several degrees during typical low-dose exposures. Smaller particles presumably also reorient during the exposure; hence, specimen movement is a second major factor that limits resolution. Tilt pairs thus enable assessment of orientation accuracy, map quality, specimen motion, and conformational heterogeneity. A convincing tilt-pair parameter plot, where 60% of the particles show a single cluster around the expected tilt axis and tilt angle, provides confidence in a structure determined using electron cryomicroscopy.

Impact of hepatitis B virus surface protein mutations on the diagnosis of occult hepatitis B virus infection.

UNLABELLED: Genotype D occult hepatitis B virus (HBV) infections (OBIs) have a high frequency of amino acid substitutions in the major hydrophilic region of the small surface protein (S protein). This possibly reflects an escape mutation mechanism to evade detection by the host immune system. Mutations may also impact the detection of hepatitis B surface antigen (HBsAg) by commercial assays. To test these hypotheses, 20 recombinant HBV genotype D surface proteins from OBI carriers with or without antibody to hepatitis B surface antigen (anti-HBs) were expressed in yeast. Recombinant surface protein (rS protein) variants were nonreactive with autologous anti-HBs but reacted weakly with vaccine-induced anti-HBs supporting an immune escape mechanism. rS protein variants tested with a wide range of HBs antibodies, and HBsAg commercial assays showed significantly lower antigenic reactivity in anti-HBs carriers than in donors with antibody to hepatitis B core antigen (anti-HBc) only. Eight out of 10 recombinant variants from anti-HBs carriers reacted weakly or were nonreactive with antibodies to HBs as well as with qualitative and quantitative commercial HBsAg assays, whereas eight out of 10 anti-HBc-only plasmas were fully reactive. rS proteins with substitutions of wild-type cysteine at positions 121, 124, and 137 were nonreactive or showed poor reactivity. However, mutation of cysteine 147 did not alter reactivity compared with controls. Restoration of cysteines 124 and 137 by site-directed mutagenesis improved antigenic reactivity. CONCLUSION: Escape mutation is a mechanism associated with OBI, which also leads to decreased reactivity in HBsAg detection assays. Performance of commercial assays would be improved by the incorporation of OBI mutants in reagent development.

Structure and disassembly of filaments formed by the ESCRT-III subunit Vps24.

The ESCRT machinery mediates sorting of ubiquitinated transmembrane proteins to lysosomes via multivesicular bodies (MVBs) and also has roles in cytokinesis and viral budding. The ESCRT-III subunits are metastable monomers that transiently assemble on membranes. However, the nature of these assemblies is unknown. Among the core yeast ESCRT-III subunits, Snf7 and Vps24 spontaneously form ordered polymers in vitro. Single-particle EM reconstruction of helical Vps24 filaments shows both parallel and head-to-head subunit arrangements. Mutations of regions involved in intermolecular assembly in vitro result in cargo-sorting defects in vivo, suggesting that these homopolymers mimic interactions formed by ESCRT-III heteropolymers during MVB biogenesis. The C terminus of Vps24 is at the surface of the filaments and is not required for filament assembly. When this region is replaced by the MIT-interacting motif from the Vps2 subunit of ESCRT-III, the AAA-ATPase Vps4 can both bundle and disassemble the chimeric filaments in a nucleotide-dependent fashion.

Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity.

BACKGROUND: Retroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5' UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation in vivo and its implication in viral replication. RESULTS: Using a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores. CONCLUSION: In addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target.