Capsid conformational sampling in HK97 maturation visualized by X-ray crystallography and cryo-EM

Maturation of the bacteriophage HK97 capsid from a precursor (Prohead II) to the mature state (Head II) involves a 60 A radial expansion. The mature particle is formed by 420 copies of the major capsid protein organized on a T = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. Well-characterized pH 4 expansion intermediates make HK97 valuable for investigating quaternary structural dynamics. Here, we use X-ray crystallography and cryo-EM to demonstrate that in the final transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 A translations and 6 degrees rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.     

Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.     

Nucleic acid visualization with UCSF Chimera

With the increase in the number of large, 3D, high-resolution nucleic acid structures, particularly of the 30S and 50S ribosomal subunits and the intact bacterial ribosome, advancements in the visualization of nucleic acid structural features are essential. Large molecular structures are complicated and detailed, and one goal of visualization software is to allow the user to simplify the display of some features and accent others. We describe an extension to the UCSF Chimera molecular visualization system for the purpose of displaying and highlighting nucleic acid characteristics, including a new representation of sugar pucker, several options for abstraction of base geometries that emphasize stacking and base pairing, and an adaptation of the ribbon backbone to accommodate the nucleic acid backbone. Molecules are displayed and manipulated interactively, allowing the user to change the representations as desired for small molecules, proteins and nucleic acids. This software is available as part of the UCSF Chimera molecular visualization system and thus is integrated with a suite of existing tools for molecular graphics.     

The acute effects of a caffeine-containing supplement on bench press strength and time to running exhaustion

The purpose of the present study was to examine the acute effects of a caffeine-containing supplement (SUPP) on one-repetition maximum (1-RM) bench press strength and time to running exhaustion (TRE) at a velocity that corresponded to 85% of the peak oxygen uptake ([latin capital V with dot above]O2peak). The study used a double-blinded, placebo-controlled, crossover design. Thirty-one men (mean +/- SD age = 23.0 +/- 2.6 years) were randomly assigned to take either the SUPP or placebo (PLAC) first. The SUPP contained 201 mg of caffeine, and the PLAC was microcrystalline cellulose. All subjects were tested for 1-RM bench press strength and TRE at 45 minutes after taking either the SUPP or PLAC. After 1 week of rest, the subjects returned to the laboratory and ingested the opposite substance (SUPP or PLAC) from what was taken during the previous visit. The 1-RM bench press and TRE tests were then performed in the same manner as before. The results indicated that the SUPP had no effect on 1-RM bench press strength or TRE at 85% [latin capital V with dot above]O2peak. It is possible that the acute effects of caffeine are affected by differences in training status and/or the relative intensity of the exercise task. Future studies should examine these issues, in addition to testing the acute effects of various caffeine doses on performance during maximal strength, power, and aerobic activities. These findings do not, however, support the use of caffeine as an ergogenic aid in untrained to moderately trained individuals.     

Cell architecture comes to phage biology

Nothing is more iconic of the early days of molecular biology than the image of a bacteriophage infecting an Escherichia coli cell. It is perhaps surprising therefore that more than 50 years later, it is still possible to learn something entirely new and unexpected about how phage infection works, as we see in the paper by Edgar et al. of this issue of Molecular Microbiology. The results give fundamental new insight into the way these viruses infect their hosts and promise to open new windows on the virus-host interactions that have shaped the evolution of both.     

Virus capsid expansion driven by the capture of mobile surface loops

The capsids of tailed-DNA bacteriophages first assemble as procapsids, which mature by converting into a new form that is strong enough to contain a densely packed viral chromosome. We demonstrate that the intersubunit crosslinking that occurs during maturation of HK97 capsids actually promotes the structural transformation. Small-angle X-ray scattering and crosslinking assays reveal that a shift in the crosslink pattern accompanies conversion of a semimature particle, Expansion Intermediate-I/II, to a more mature state, Balloon. This transition occurs in a switch-like fashion. We find that crosslink formation shifts the global conformational balance to favor the balloon state. A pseudoatomic model of EI-I/II derived from cryo-EM provides insight into the relationship between crosslink formation and conformational switching.     

Evolution: the long evolutionary reach of viruses

The structure of a phage capsid protein provides good evidence this phage shares ancestry with an animal virus. In this and similar cases, either the viral lineages predate the emergence of the three contemporary domains of life, or viruses have been leaping the phylogenetic chasms that separate the domains.     

Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization

In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.     

Relationship between Aphis gossypii (Homoptera: Aphididae) and sticky lint in cotton

The study was conducted in the northern Texas Rolling Plains in 1999 to define the relationship between number of cotton aphids, Aphis gossypii Glover, and resulting contamination of cotton lint by honeydew. Whole-plot treatments were three furrow irrigation management treatments: cotton grown without supplemental irrigation (dryland), irrigated cotton with last irrigation in mid August, and irrigated cotton with last irrigation in late August. Subplots within each irrigation treatment included an untreated check, a plot treated with lambda-cyhalothrin to stimulate aphid population increase, a plot treated with lambda-cyhalothrin followed by pymetrozine after aphids began to increase, and a plot treated with lambda-cyhalothrin followed by thiamethoxam after aphids began to increase. Cotton aphids were counted on leaves picked from the top and bottom half of the plant. Cotton lint was analyzed for contamination by glucose, fructose, sucrose, and melezitose secreted by cotton aphids, and percentage leaf moisture and nitrogen and leaf sucrose concentrations were determined. The manual sticky cotton thermodetector was used to determine degree of lint stickinesss. There was a significant relationship between thermodetector counts and melezitose contamination on lint, and a melezitose concentration of 90.9 microg/g of lint was associated with a thermodetector count of 10, the threshold for sticky lint problems in textile mills. An equation was developed to estimate melezitose concentration on lint as a function of average numbers of aphids per leaf and the interaction between percentage leaf moisture and nitrogen. The number of aphids per leaf associated with a melezitose concentration of 90.9 microg/g of lint ranged from 11.1 to 50.1, depending on percentage leaf moisture and nitrogen. The threshold for sticky lint problems occurred when aphid numbers ranged between 11.1 and 50.1 per leaf after bolls open.     

Corrected sequence of the bacteriophage p22 genome

We report the first accurate genome sequence for bacteriophage P22, correcting a 0.14% error rate in previously determined sequences. DNA sequencing technology is now good enough that genomes of important model systems like P22 can be sequenced with essentially 100% accuracy with minimal investment of time and resources.