3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

Direct visualization of the putative portal in the Kaposi's sarcoma-associated herpesvirus capsid by cryoelectron tomography.

Genetic and biochemical studies have suggested the existence of a bacteriophage-like, DNA-packaging/ejecting portal complex in herpesviruses capsids, but its arrangement remained unknown. Here, we report the first visualization of a unique vertex in the Kaposi's sarcoma-associated herpesvirus (KSHV) capsid by cryoelectron tomography, thus providing direct structural evidence for the existence of a portal complex in a gammaherpesvirus. This putative KSHV portal is an internally localized, umbilicated structure and lacks all of the external machineries characteristic of portals in DNA bacteriophages.

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^