Multifunctional roles of a bacteriophage phi 29 morphogenetic factor in assembly and infection

Low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. The Bacillus subtilis double-stranded DNA bacteriophage phi 29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. Western blots and immuno-electron microscopy detected a few copies of gp13 in phi 29, DNA-free particles, purified tails, and defective particles produced in suppressor-sensitive (sus) mutant sus13(330) infections. Particles assembled in the absence of intact gp13 (sus13(342) and sus13(330)) had the gross morphology of phi 29 but were not infectious. gp13 has predicted structural homology and sequence similarity to the M23 metalloprotease LytM. Poised at the tip of the phi 29 tail knob, gp13 may serve as a plug to help restrain the highly pressurized packaged genome. Also, in this position, gp13 may be the first virion protein to contact the cell wall in infection, acting as a pilot protein to depolymerize the cell wall. gp13 may facilitate juxtaposition of the tail knob onto the cytoplasmic membrane and the triggering of genome injection.     

The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.     

Enhancement of tomogram interpretability using the locked self-rotation function

The interpretation of cryo-electron tomograms of macromolecular complexes can be difficult because of the large amount of noise and because of the missing wedge effect. Here it is shown how the presence of rotational symmetry in a sample can be utilized to enhance the quality of a tomographic analysis. The orientation of symmetry axes in a sub-tomogram can be determined using a locked self-rotation function. Given this knowledge, the sub-tomogram density can then be averaged to improve its interpretability. Sub-tomograms of the icosahedral bacteriophage phiX174 are used to demonstrate the procedure.     

Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1

Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.     

Review: rhinoviruses and their ICAM receptors

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen or macrophage-1 antigen. However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses.     

Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host

The contractile tail of bacteriophage T4 undergoes major structural transitions when the virus attaches to the host cell surface. The baseplate at the distal end of the tail changes from a hexagonal to a star shape. This causes the sheath around the tail tube to contract and the tail tube to protrude from the baseplate and pierce the outer cell membrane and the cell wall before reaching the inner cell membrane for subsequent viral DNA injection. Analogously, the T4 tail can be contracted by treatment with 3 M urea. The structure of the T4 contracted tail, including the head-tail joining region, has been determined by cryo-electron microscopy to 17 A resolution. This 1200 A-long, 20 MDa structure has been interpreted in terms of multiple copies of its approximately 20 component proteins. A comparison with the metastable hexagonal baseplate of the mature virus shows that the baseplate proteins move as rigid bodies relative to each other during the structural change.     

Combining electron microscopic with x-ray crystallographic structures

Analgorithm has been developed for placing three-dimensional atomic structures into appropriately scaled cryoelectron microscopy maps. The first stage in this process is to conduct a three-dimensional angular search in which the center of gravity of an X-ray crystallographically determined structure is placed on a selected position in the cryoelectron microscopy map. The quality of the fit is measured by the sum of the density at each atomic position. The second stage is to refine the three angles and three translational parameters for the best (usually 25 to 100) fits. Useful criteria for this refinement include the sum of densities at atomic sites, the lack of atoms in negative or low density, the absence of atomic clashes between symmetry-related positions of the atomic structure, and the distances between identifiable features in the map and their positions on the fitted atomic structure. These refinements generally lead to a convergence of the originally chosen, top scoring fits to just a few (about 3 to 8) acceptable possibilities. Usually, the best remaining fit is clearly superior to any of the others.     

Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria

Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.