bluuebunny/crossref_metadata_2025 huggingface dataset:
Assignment of the [4Fe‐4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site‐directed mutants
Authors: Forzi Lucia , Koch Jürgen , Guss Adam M. , Radosevich Carl G. , Metcalf William W. , Hedderich Reiner
Published: September 2005

Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane‐bound [NiFe] hydrogenases with sequence similarity to energy‐conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe‐4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 105 Pa of H2 at pH 7 giving rise to two distinct S½ electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH‐dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH‐dependence of these two [4Fe‐4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton‐translocating machinery.
Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase‐dependent cross‐linking
Authors: Iijima Manabu , Hashimoto Tomonori , Matsuda Yasuyuki , Nagai Taku , Yamano Yuichiro , Ichi Tomohiko , Osaki Tsukasa , Kawabata Shun‐ichiro
Published: September 2005

Arthropod cuticles play an important role as the first barrier against invading pathogens. We extensively determined the sequences of horseshoe crab cuticular proteins. Proteins extracted from a part of the ventral side of the cuticle were purified by chitin‐affinity chromatography, and separated by two‐dimensional SDS/PAGE. Proteins appearing on the gel were designated high molecular mass chitin‐binding proteins, and these proteins were then grouped into classes based on their approximate isoelectric points and predominant amino acid compositions. Members of groups designated basic G, basic Y, and acidic S groups contained a so‐called Rebers and Riddiford consensus found in arthropod cuticular proteins. Proteins designated acidic DE25 and DE29 each contained a Cys‐rich domain with sequences similar to those of insect peritrophic matrix proteins and chitinases. In contrast, basic QH4 and QH10 contained no consensus sequences found in known chitin‐binding proteins. Alternatively, a low molecular mass chitin‐binding fraction was prepared by size exclusion chromatography, and 15 low molecular mass chitin‐binding proteins, named P1 through P15, were isolated. With the exception of P9 and P15, all were found to be identical to known antimicrobial peptides. P9 consisted of a Kunitz‐type chymotrypsin inhibitor sequence, and P15 contained a Cys‐rich motif found in insulin‐like growth factor‐binding proteins. Interestingly, we observed transglutaminase‐dependent polymerization of nearly all high molecular mass chitin‐binding proteins, a finding suggests that transglutaminase‐dependent cross‐linking plays an important role in host defense in the arthropod cuticle, analogous to that observed in the epidermal cornified cell envelope in mammals.
Light‐harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions
Authors: Kargul Joanna , Turkina Maria V. , Nield Jon , Benson Sam , Vener Alexander V. , Barber James
Published: September 2005

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light‐harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper‐phosphorylated form of the LHCII‐like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1‐exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI–PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox‐dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.
Hydrogen independent expression of hupSL genes in Thiocapsa roseopersicina BBS
Authors: Kovács Ákos T. , Rákhely Gábor , Balogh Judit , Maróti Gergely , Cournac Laurent , Carrier Patrick , Mészáros Lívia S. , Peltier Gilles , Kovács Kornél L.
Published: September 2005

The expression of many membrane bound [NiFe] hydrogenases is regulated by their substrate molecule, hydrogen. The HupSL hydrogenase, encoded in the hupSLCDHIR operon, probably plays a role in hydrogen recycling in the phototrophic purple bacterium, Thiocapsa roseopersicina BBS. RpoN, coding for sigma factor 54, was shown to be important for expression, suggesting a regulated biosynthsis from the hup gene cluster. The response regulator gene, hupR, has been identified in the hup operon and expression of hupSL was reduced in a chromosomal hupR mutant, which indicated that HupR was implicated in the activation process. The hupT and hupUV genes were isolated, and show similarity to the histidine kinase element of the H2‐driven signal transduction system and to the regulatory hydrogenases of Ralstonia eutropha and Rhodobacter capsulatus, respectively. Although the genes of the entire H2 sensing and regulation system were present, the expression of the hupSL genes was not affected by the presence or absence of H2. Using reverse transcription PCR, we could not detect any mRNA specific to the hupTUV genes in cells grown under diverse conditions. The hupT and hupUV mutant strains had the same phenotype as the wild‐type strains. The hupT gene product, expressed from a plasmid, repressed HupSL synthesis as expected while introduction of actively expressed hupTUV genes together derepressed the HupSL activity in T. roseopersicina. The gene product of hupUV behaves similarly to other regulatory hydrogenases and shows H–D exchange activity.
Adolescents with Anorexia Nervosa: Multiple Perspectives of Discharge Readiness
Authors: Turrell Sheri L. , Davis Ron , Graham Heather , Weiss Iris
Published: September 2005

PROBLEM:  Little is known about the conditions that must be in place to help adolescent patients and their families gain the confidence needed to continue recovery at home, following the adolescents’ hospitalization for anorexia nervosa.

METHODS:  Beliefs about discharge readiness were obtained through an open‐ended questionnaire following the patients’ first weekend pass home from an in‐patient unit. The perceptions of patients, parents, and registered nurses were obtained using parallel versions of a questionnaire.

FINDINGS:  An examination of the responses revealed four themes; medical stability, education, psychological changes, and community resource planning, that were common to all respondents, as well as themes specific to adolescents and to nurses.

CONCLUSIONS:  The findings suggest that each group of respondents has unique discharge readiness needs and that registered nurses have an important role to play in helping patients and families make the transition home as successful as possible. Implications for nursing practice are highlighted.
Grade‐Level Invariance of a Theoretical Causal Structure Predicting Reading Comprehension With Vocabulary and Oral Reading Fluency
Authors: Yovanoff Paul , Duesbery Luke , Alonzo Julie , Tindal Gerald
Published: September 2005

This research investigates the relative importance of vocabulary and oral reading fluency as measurement dimensions of reading comprehension as the student passes from elementary to high school. Invariance of this model over grades 4 through 8 is tested using two independent student samples reading grade‐level appropriate passages. Results from structural equation modeling indicate that the model is not invariant across grade levels. Vocabulary knowledge is a significant and constant predictor of overall reading comprehension irrespective of grade level. While significant, fluency effects diminish over grades, especially in the later grades. Lack of grade level invariance was obtained with both samples. Results are discussed in light of vertically linked reading assessments, adequate yearly progress, and instruction.
EFFECT OF CENTRIFUGAL FORCE ON THE AQUEOUS EXTRACTION OF SOLUTE FROM SUGAR BEET TISSUE PRETREATED BY A PULSED ELECTRIC FIELD
Authors: EL‐BELGHITI KAMAL , RABHI ZIED , VOROBIEV EUGENE
Published: September 2005

In this article, the centrifugal aqueous extraction of solute from sugar beet tissue is investigated at ambient temperature after a pulsed electric field (PEF) treatment. Two kinds of samples of fresh sugar beet were used: a sample with a determined discoid shape and gratings. Both samples were pretreated by a PEF with 250 rectangular pulses of 100 µS each. The PEF intensity was fixed at 940 V/cm for the disk samples and 670 V/cm for gratings. The pretreated samples were placed in distilled water at ambient temperature with a water‐to‐solid ratio equal to 3 and subjected to different centrifugal accelerations. The centrifugal field significantly enhanced the kinetics of extraction from the electrically pretreated tissues of sugar beet. However, the increase of centrifugal acceleration was only effective up to a certain value (5430 × g for disk samples and 600 × g for gratings). The centrifugal extraction can be assumed to proceed in two stages: a first rapid washing followed by a slow diffusion stage. A two‐exponential kinetics model taking into account these two stages was applied and correctly described the centrifugal extraction from beet samples (disks and gratings).
A Multiscale Investigation of Ground Water Flow at Clear Lake, Iowa
Authors: Simpkins William W.
Published: September 2005

Ground water flow was investigated at Clear Lake, a 1468‐ha glacial lake in north‐central Iowa, as part of a comprehensive water quality study. A multiscale approach, consisting of seepage meters (and a potentiomanometer), Darcy's law, and an analytic element (AE) model, was used to estimate ground water inflow to and outflow from the lake. Estimates from the three methods disagreed. Seepage meters recorded a median‐specific discharge of 0.25 μm/s, which produced a lake inflow rate between 90,750 and 138,200 m3/d, but no detectable outflow. A wave‐induced Bernoulli effect probably compromised both inflow and outflow measurements. Darcy's law was applied to 11 zones around the lake, producing inflow and outflow values of 10,500 and 5000 m3/d, respectively. The AE model, GFLOW, coupled with the parameter estimation model, UCODE, simulated ground water flow in a 700‐km2 region using 31 hydraulic head and base flow measurements as calibration targets. The model produced ground water inflow and outflow rates of 14,300 and 9200 m3/d, respectively. Although not a substitute for field data, the model's ability to simulate ground water flow to the lake and the region, estimate uncertainty for model parameters, and calculate a lake stage and associated lake water balance makes it a powerful tool for water quality management and an attractive alternative to the traditional methods of ground water/lake investigation.
Leveraging FDA Health Claims
Authors: WANSINK BRIAN , CHENEY MATTHEW M.
Published: September 2005

Although past research has investigated different aspects of various health claims, relatively little effort has been directed toward synthesizing the reasons why some Food and Drug Administration (FDA) health claims are more successful than others. After outlining two necessary conditions for a health claim's success, currently approved FDA health claims are examined to explain both the diet–health relationship and its effect on the widespread awareness and adoption of the claim. Understanding what makes health claims successful provides useful nutrition education and product‐labeling lessons for policy makers in all countries.
Consumer Economics and Family Economics: The Charge, the Response
Authors: GEISTFELD LOREN V
Published: September 2005

A “Timeless” charge to consumer economics and family economics is identified. In light of the charge, the area is examined with particular emphasis on scholarly activity. It is suggested that scholarship in the area is diffuse and often addresses parts of issues. This creates concerns as to whether the historical charge is being met.
Effects of Plasma Parameters on the Temperature Field in a Workpiece Experiencing Solid-Liquid Phase Transition
Authors: Yeh F. B. , Wei P. S.
Published: September 2005

The heat transfer characteristics of a workpiece subject to plasma heating and melting are theoretically and systematically studied. Plasma etching, spray deposition, sputtering, cutting and surface treatment, etc., are usually controlled by energy transfer from plasma to workpieces. In this work, the one-dimensional unsteady conduction equation accounting for solid-liquid phase transition with distinct thermal properties in a workpiece is solved. The plasma is composed of a collisionless presheath and sheath on an electrically floating workpiece that partially reflects or secondarily emits ions and electrons. The energy transport from plasma to the surface is kinetically, analytically and exactly calculated from self-consistent velocity distributions of the ions and electrons. The results show that the predicted surface temperature and energy transmission factor agree well with experimental data. The effects of plasma characteristics and thermal parameters of the workpiece on unsteady temperature profiles and thickness of the molten layer in the workpiece are quantitatively provided in this work. Energy released from recombination of the ions and electrons on the surface is found to play the most important role on heating the workpiece. The deviation of surface temperature contributed by recombination energy can be 1000 K.
Frost Growth in Regenerative Wheels
Authors: Shang Wei , Chen Hong , Besant Robert W.
Published: September 2005

An experimental investigation was carried out for frost growth in a desiccant-coated regenerative wheel. The test facility was set up following ASHRAE Standard 84-1991R. Temperature, relative humidity, mass flow rate, and pressure drops were measured at each measuring station. Photos of frost within energy wheel flow channels show frost accumulation. The problem of frost growth within the narrow parallel flow passages of a regenerative heat or energy rotary wheel is formulated for a very cold-temperature ventilation application. Frost growth is assumed to grow as a porous media while the wheel is exposed to warm humid airflow on the exhaust side. While the wheel is exposed to cold dry airflow on the supply side, the frost is cooled but no frost grows. This cyclic frost growth and cooling process is continued with each wheel rotation. An analytical/numerical model is developed to simulate these frost properties over the depth of the wheel and as a function of time. Simulation results are used to interpret experimental data for the early stage of frost growth on a typical energy wheel with a cold supply air temperature of −40°C, a warm exhaust temperature of 20 °C and 40% relative humidity. Pressure drop measurements across a wheel taken for constant mass flow conditions revealed some very significant fluctuations of up to 100% of original pressure drop with a period ranging from 2 to 4 min for a wheel speed of 20 rpm. Each fluctuation in pressure drop is interpreted to imply a catastrophic failure of the outer frost layer sequenced over 1–2 min throughout the wheel followed by another frost growth period on top of a slightly thicker frost base.
Energy Transmission by Photon Tunneling in Multilayer Structures Including Negative Index Materials
Authors: Fu C. J. , Zhang Z. M. , Tanner D. B.
Published: September 2005

The phenomenon of photon tunneling, which depends on evanescent waves for radiative transfer, has important applications in microscale energy conversion devices and near-field optical microscopy. In recent years, there has been a surge of interest in the so-called negative index materials (NIMs), which have simultaneously negative electric permittivity and negative magnetic permeability. The present work investigates photon tunneling in multilayer structures consisting of positive index materials (PIMs) and NIMs. Some features, such as the enhancement of radiative transfer by the excitation of surface polaritons for both polarizations, are observed in the predicted transmittance spectra. The influence of the number of layers on the transmittance is also examined. The results suggest that the enhanced tunneling transmittance by polaritons also depends on the NIM layer thickness and that subdividing the PIM/NIM layers to enhance polariton coupling can reduce the effect of material loss on the tunneling transmittance.
From Birth to Death: The Complex Lives of Eukaryotic mRNAs
Authors: Moore Melissa J.
Published: September 2005

Recent work indicates that the posttranscriptional control of eukaryotic gene expression is much more elaborate and extensive than previously thought, with essentially every step of messenger RNA (mRNA) metabolism being subject to regulation in an mRNA-specific manner. Thus, a comprehensive understanding of eukaryotic gene expression requires an appreciation for how the lives of mRNAs are influenced by a wide array of diverse regulatory mechanisms.
Controlling the Kondo Effect of an Adsorbed Magnetic Ion Through Its Chemical Bonding
Authors: Zhao Aidi , Li Qunxiang , Chen Lan , Xiang Hongjun , Wang Weihua , Pan Shuan , Wang Bing , Xiao Xudong , Yang Jinlong , Hou J. G. , Zhu Qingshi
Published: September 2005

We report that the Kondo effect exerted by a magnetic ion depends on its chemical environment. A cobalt phthalocyanine molecule adsorbed on an Au(111) surface exhibited no Kondo effect. Cutting away eight hydrogen atoms from the molecule with voltage pulses from a scanning tunneling microscope tip allowed the four orbitals of this molecule to chemically bond to the gold substrate. The localized spin was recovered in this artificial molecular structure, and a clear Kondo resonance was observed near the Fermi surface. We attribute the high Kondo temperature (more than 200 kelvin) to the small on-site Coulomb repulsion and the large half-width of the hybridized d-level.
An mRNA Is Capped by a 2', 5' Lariat Catalyzed by a Group I-Like Ribozyme
Authors: Nielsen Henrik , Westhof Eric , Johansen Steinar
Published: September 2005

Twin-ribozyme introns are formed by two ribozymes belonging to the group I family and occur in some ribosomal RNA transcripts. The group I-like ribozyme, GIR1, liberates the 5′ end of a homing endonuclease messenger RNA in the slime mold
Didymium iridis
. We demonstrate that this cleavage occurs by a transesterification reaction with the joining of the first and the third nucleotide of the messenger by a 2′,5′-phosphodiester linkage. Thus, a group I-like ribozyme catalyzes an RNA branching reaction similar to the first step of splicing in group II introns and spliceosomal introns. The resulting short lariat, by forming a protective 5′ cap, might have been useful in a primitive RNA world.
The Ultrasmoothness of Diamond-like Carbon Surfaces
Authors: Moseler Michael , Gumbsch Peter , Casiraghi Cinzia , Ferrari Andrea C. , Robertson John
Published: September 2005

The ultrasmoothness of diamond-like carbon coatings is explained by an atomistic/continuum multiscale model. At the atomic scale, carbon ion impacts induce downhill currents in the top layer of a growing film. At the continuum scale, these currents cause a rapid smoothing of initially rough substrates by erosion of hills into neighboring hollows. The predicted surface evolution is in excellent agreement with atomic force microscopy measurements. This mechanism is general, as shown by similar simulations for amorphous silicon. It explains the recently reported smoothing of multilayers and amorphous transition metal oxide films and underlines the general importance of impact-induced downhill currents for ion deposition, polishing, and nanopattering.
The Effect of Diurnal Correction on Satellite-Derived Lower Tropospheric Temperature
Authors: Mears Carl A. , Wentz Frank J.
Published: September 2005

Satellite-based measurements of decadal-scale temperature change in the lower troposphere have indicated cooling relative to Earth's surface in the tropics. Such measurements need a diurnal correction to prevent drifts in the satellites' measurement time from causing spurious trends. We have derived a diurnal correction that, in the tropics, is of the opposite sign from that previously applied. When we use this correction in the calculation of lower tropospheric temperature from satellite microwave measurements, we find tropical warming consistent with that found at the surface and in our satellite-derived version of middle/upper tropospheric temperature.
Structural Evidence for a Two-Metal-Ion Mechanism of Group I Intron Splicing
Authors: Stahley Mary R. , Strobel Scott A.
Published: September 2005

We report the 3.4 angstrom crystal structure of a catalytically active group I intron splicing intermediate containing the complete intron, both exons, the scissile phosphate, and all of the functional groups implicated in catalytic metal ion coordination, including the 2′-OH of the terminal guanosine. This structure suggests that, like protein phosphoryltransferases, an RNA phosphoryltransferase can use a two-metal-ion mechanism. Two Mg
2+
ions are positioned 3.9 angstroms apart and are directly coordinated by all six of the biochemically predicted ligands. The evolutionary convergence of RNA and protein active sites on the same inorganic architecture highlights the intrinsic chemical capacity of the two-metal-ion catalytic mechanism for phosphoryl transfer.
Single-Molecule Torsional Pendulum
Authors: Meyer Jannik C. , Paillet Matthieu , Roth Siegmar
Published: September 2005

We have built a torsional pendulum based on an individual single-walled carbon nanotube, which is used as a torsional spring and mechanical support for the moving part. The moving part can be rotated by an electric field, resulting in large but fully elastic torsional deformations of the nanotube. As a result of the extremely small restoring force associated with the torsional deformation of a single molecule, unusually large oscillations are excited by the thermal energy of the pendulum. By diffraction analysis, we are able to determine the handedness of the molecule in our device. Mechanical devices with molecular-scale components are potential building blocks for nanoelectromechanical systems and may also serve as sensors or actuators.
Fewer Genes, More Noncoding RNA
Authors: Claverie Jean-Michel
Published: September 2005

Recent studies showing that most “messenger” RNAs do not encode proteins finally explain the long-standing discrepancy between the small number of protein-coding genes found in vertebrate genomes and the much larger and ever-increasing number of polyadenylated transcripts identified by tag-sampling or microarray-based methods. Exploring the role and diversity of these numerous noncoding RNAs now constitutes a main challenge in transcription research.
It's a Small RNA World, After All
Authors: Vaughn Matthew W. , Martienssen Rob
Published: September 2005

Small RNAs (sRNAs) can regulate transcript and protein abundance. Previously, they have been identified using traditional cloning approaches, which has limited how many could be characterized. Now, the Meyers and Green laboratories have used massively parallel signature sequencing technology to find over 1.5 million sRNAs in
Arabidopsis thaliana
. These new sRNAs reveal a greater-than-expected potential role for sRNAs in gene regulation, preferential expression or usage of sRNAs in flowers, and the prospect of targeted sRNA-mediated regulation of pseudogenes. In addition, new plant microRNAs have been identified, some of which may be unique to
Arabidopsis.
Capping by Branching: A New Ribozyme Makes Tiny Lariats
Authors: Pyle Anna Marie
Published: September 2005

The number of naturally occurring RNA enzymes has just been expanded by the discovery of a new branching ribozyme. But this ribozyme has unexpected relatives: group I introns.
Emperors on the Ice
Authors: Kennedy Donald
Published: September 2005

March of the Penguins
.
By Luc Jacquet
. Warner Independent Pictures, Burbank, CA, and National Geographic Feature Films, Washington, DC, 2005. 80 minutes.
www.marchofthepenguins.com

This nature film follows the winter pilgrimage of emperor penguins across the ice between the Antarctic seas and their inland breeding grounds.
Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions
Authors: Song Chisu , Micoli Keith , Bauerova Helena , Pichova Iva , Hunter Eric
Published: September 2005

Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.
Differential Restriction of Human Immunodeficiency Virus Type 2 and Simian Immunodeficiency Virus SIVmac by TRIM5α Alleles
Authors: Ylinen Laura M. J. , Keckesova Zuzana , Wilson Sam J. , Ranasinghe Srinika , Towers Greg J.
Published: September 2005

Primate lentiviruses have narrow host ranges, due in part to their sensitivities to mammalian intracellular antiviral factors such as APOBEC3G and TRIM5α. Despite the protection provided by this innate immune system, retroviruses are able to transfer between species where they can cause disease. This is true for sooty mangabey simian immunodeficiency virus, which has transferred to humans as HIV-2 and to rhesus macaques as SIVmac, where it causes AIDS. Here we examine the sensitivities of the closely related HIV-2 and SIVmac to restriction by TRIM5α. We show that rhesus TRIM5α can restrict HIV-2 but not the closely related SIVmac. SIVmac has not completely escaped TRIM5α, as shown by its sensitivity to distantly related TRIM5α from the New World squirrel monkey. Squirrel monkey TRIM5α blocks SIVmac infection after DNA synthesis and is not saturable with restriction-sensitive virus-like particles. We map the determinant for TRIM5α sensitivity to the structure in the capsid protein that recruits CypA into HIV-1 virions. We also make an SIV, mutated at this site, which bypasses restriction in all cells tested.
Mutagenesis Analysis of the rGTP-Specific Binding Site of Hepatitis C Virus RNA-Dependent RNA Polymerase
Authors: Cai Zhaohui , Yi MinKyung , Zhang Chen , Luo Guangxiang
Published: September 2005

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 Å away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol
76:
3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3′ untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo.
Simian Virus 40 Large T Antigen's Association with the CUL7 SCF Complex Contributes to Cellular Transformation
Authors: Kasper Jocelyn S. , Kuwabara Hiroshi , Arai Takehiro , Ali Syed Hamid , DeCaprio James A.
Published: September 2005

Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that Δ69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not Δ69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and Δ69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.
In-Depth Analysis of a Heterosexually Acquired Human Immunodeficiency Virus Type 1 Superinfection: Evolution, Temporal Fluctuation, and Intercompartment Dynamics from the Seronegative Window Period through 30 Months Postinfection
Authors: McCutchan F. E. , Hoelscher M. , Tovanabutra S. , Piyasirisilp S. , Sanders-Buell E. , Ramos G. , Jagodzinski L. , Polonis V. , Maboko L. , Mmbando D. , Hoffmann O. , Riedner G. , von Sonnenburg F. , Robb M. , Birx D. L.
Published: September 2005

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multiregion genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/μl at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasispecies, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in
gag
and six were detected in
env
. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.
Sialidase Activity of Influenza A Virus in an Endocytic Pathway Enhances Viral Replication
Authors: Suzuki Takashi , Takahashi Tadanobu , Guo Chao-Tan , Hidari Kazuya I.-P. Jwa , Miyamoto Daisei , Goto Hideo , Kawaoka Yoshihiro , Suzuki Yasuo
Published: September 2005

N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-α-
N
-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-
N
-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.
Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Authors: Elshuber Sigrid , Mandl Christian W.
Published: September 2005

Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The “pr” part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37°C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28°C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.
Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein
Authors: Chen Mingzhou , Cortay Jean-Claude , Logan Ian R. , Sapountzi Vasileia , Robson Craig N. , Gerlier Denis
Published: September 2005

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione
S
-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.
Deletion of gpUL132, a Structural Component of Human Cytomegalovirus, Results in Impaired Virus Replication in Fibroblasts
Authors: Spaderna Simone , Kropff Barbara , Ködel Yvonne , Shen Siyuan , Coley Scott , Lu Shan , Britt William , Mach Michael
Published: September 2005

The coding capacity of human cytomegalovirus (HCMV) for glycoproteins by far exceeds that of other herpesviruses. Few of these proteins have been characterized so far. We have investigated the gene product of reading frame UL132. The putative protein product of UL132 is a glycoprotein with a theoretical mass of 29.8 kDa. Transcription analysis revealed that the gene is transcribed with a true late kinetics from the laboratory-adapted strain AD169 and the low-passage isolate TB40E. Two proteins of 22 to 28 kDa and 45 to 60 kDa were detected in virus-infected cells as well as in extracellular virions. The larger protein carried N-linked carbohydrates. Both protein forms were present in laboratory-adapted strains as well as in low-passage isolates of HCMV. Recombinant viruses with the UL132 gene deleted were constructed in the low-passage HCMV isolate PAN as well as the high-passage isolate AD169. Deletion of UL132 from either genome resulted in a pronounced replication deficit with a reduction of approximately 100-fold for HCMV strain AD169. Thus, the protein product of the UL132 reading frame represents a structural viral glycoprotein of HCMV that has an important function for viral replication in tissue culture.
Human Immunodeficiency Virus Type 1 Variants Isolated from Single Plasma Samples Display a Wide Spectrum of Neutralization Sensitivity
Authors: Skrabal Katharina , Saragosti Sentob , Labernardière Jean-Louis , Barin Francis , Clavel François , Mammano Fabrizio
Published: September 2005

Individuals infected with human immunodeficiency virus type 1 (HIV-1) harbor a mixture of viral variants with different sequences and in some instances with different phenotypic properties. Major and rapid fluctuations in the proportion of viral variants coexisting in an infected individual can be observed under strong pharmacological and immune selective pressure. Because of the short half-life of HIV-infected cells and of HIV virions in the blood, plasma virus populations are highly relevant to HIV evolution in the face of these selective pressures. Here we analyzed the sensitivity to antibody-mediated neutralization of viral variants coexisting in the plasma virus populations of two infected patients. For each patient, several replication-competent viral clones were constructed that carry primary envelope gene sequences obtained from a single plasma sample. Viral clones differed in their tropism and replicative capacity and in the number and positions of glycosylation sites in the envelope glycoproteins. Viruses were tested against heterologous and autologous sera obtained at different time points. Interestingly, we found that viral variants coexisting in each plasma sample were highly heterogeneous in terms of sensitivity to neutralization. The order of sensitivity depended on the serum used and was not associated with virus tropism. The neutralization potency of sera increased with the duration of the infection for both autologous and heterologous neutralization.
The Carboxy-Terminal Sequence of the Pestivirus Glycoprotein E
rns
Represents an Unusual Type of Membrane Anchor
Authors: Fetzer Christiane , Tews Birke Andrea , Meyers Gregor
Published: September 2005

The E
rns
protein is a structural glycoprotein of pestiviruses that lacks a typical membrane anchor sequence and is known to be secreted from the infected cell. However, major amounts of the protein are retained within the cell and attached to the virion by a so far unknown mechanism. Transient-expression studies with cDNA constructs showed that in a steady-state situation, 16% of the protein is found in the supernatant of the transfected cells while 84% appears as intracellular protein. We show here that E
rns
represents a membrane-bound protein. Membrane binding occurs via the carboxy-terminal region of E
rns
. By fusion of this sequence to the carboxy terminus of green fluorescent protein (GFP), the subcellular localization of the reporter protein switched from cytosolic to membrane bound. A core sequence of 11 amino acids necessary for membrane binding was elicited in truncation experiments with GFP constructs. However, this peptide is not sufficient to confer membrane anchoring but needs either upstream or downstream accessory sequences. Analyses with different extraction procedures showed that E
rns
is neither easily stripped from the membrane, like a peripheral membrane protein, nor as tightly membrane bound as a transmembrane protein.
The Human Herpesvirus 6 G Protein-Coupled Receptor Homolog U51 Positively Regulates Virus Replication and Enhances Cell-Cell Fusion In Vitro
Authors: Zhen Zhu , Bradel-Tretheway Birgit , Sumagin Sarah , Bidlack Jean M. , Dewhurst Stephen
Published: September 2005

Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G protein-coupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.