bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Na+‐dependent citrate transport in a strongly metastatic human prostate cancer PC‐3M cell line: regulation by voltage‐gated Na+ channel activity
Authors: Mycielska Maria E. , Palmer Christopher P. , Brackenbury William J. , Djamgoz Mustafa B. A.
Published: December 2004

Prostate is a unique organ which synthesizes and releases large amounts of citrate. It has been shown that in metastatic prostate cancer, the amount of citrate in prostatic fluid is significantly reduced, approaching the level normally found in blood. In our previous study, we characterized electrophysiologically the mechanism of citrate transport in a normal prostatic epithelial (PNT2‐C2) cell line. It was concluded that the cells expressed a novel transporter carrying 1 citrate3− together with 4 K+, primarily out of cells. In the present study, we aimed similarly to characterize the mechanism(s) of citrate transport in a strongly metastatic human prostate cancer (PC‐3M) cell line and to compare this with the previous data. Citrate transport in PC‐3M cells was found to be both Na+ and K+ dependent. Intracellular application of citrate produced an outward current that was primarily K+ dependent whilst extracellular citrate elicited an inward current that was mainly Na+ dependent. The electrophysiological and pharmacological characteristics of the citrate outward current were similar to the K+‐dependent citrate transporter found in the PNT2‐C2 cells. On the other hand, the inward citrate current had a markedly different reversal potential, ionic characteristics, inhibitor profile and pH sensitivity. Preincubation of the PC‐3M cells (24 or 48 h) with the voltage‐gated Na+ channel (VGSC) blocker tetrodotoxin (TTX) significantly reduced the Na+ sensitivity of the citrate current, up‐regulated VGSC mRNA expression but did not change the partial permeability of the membrane to Na+. It was concluded (a) that PC‐3M cells express a K+‐dependent transporter (carrying citrate outward), similar to that found in normal prostate epithelial cells, as well as (b) a Na+‐dependent transporter (carrying citrate inward). The molecular nature of the latter was investigated by RT‐PCR; the three known Na+‐dependent citrate/dicarboxylate transporters could not be detected. VGSC activity, which itself has been associated with metastatic prostate cancer, had a differential effect on the two citrate transporters, down‐regulating the expression of the Na+‐dependent component whilst enhancing the K+‐dependent citrate transporter.
Metabotropic glutamate receptor activity induces a novel oscillatory pattern in neonatal rat hypoglossal motoneurones
Authors: Sharifullina Elina , Ostroumov Konstantin , Nistri Andrea
Published: December 2004

Tongue muscles innervated by the hypoglossal nerves play a crucial role to ensure airway patency and milk suckling in the neonate. Using a slice preparation of the neonatal rat brain, we investigated the electrophysiological characteristics of hypoglossal motoneurones in the attempt to identify certain properties potentially capable of synchronizing motor commands to the tongue. Bath‐applied DHPG, a selective agonist of group I metabotropic glutamate receptors (mGluRs), generated persistent, regular electrical oscillations (4–8 Hz) recorded from patch‐clamped motoneurones. Under voltage clamp, oscillations were biphasic events, comprising large outward slow currents alternated with fast, repeated inward currents. Electrical oscillations had amplitude and period insensitive to cell membrane potential, and required intact glutamatergic transmission via AMPA receptors. Oscillations were mediated by subtype 1 receptors of group I mGluRs (mGluR1s), and were routinely observed during pharmacological block of glycinergic and GABAergic inhibition, although they could also be recorded in standard saline. Simultaneous recordings from pairs of motoneurones within the same hypoglossal nucleus demonstrated that oscillations were due to their strong electrical coupling and were blocked by the gap junction blocker carbenoxolone. Pacing of slow oscillations apparently depended on the operation of KATP channels in view of the block by tolbutamide or glibenclamide. Under current clamp, oscillations generated more regular spike firing of motoneurones and facilitated glutamatergic excitatory inputs. These data suggest that neonatal motoneurones of the nucleus hypoglossus possess a formerly undisclosed ability to express synchronous electrical oscillations, unveiled by activation of mGluR1s.
Rhythmic changes in spike coding in the rat suprachiasmatic nucleus
Authors: Bhumbra G. S. , Inyushkin A. N. , Saeb‐Parsy K. , Hon A. , Dyball R. E. J.
Published: December 2004

The suprachiasmatic nucleus is regarded as the main mammalian circadian pacemaker but evidence for rhythmic firing of single units in vivo has been obtained only recently. The present study was undertaken to determine if rhythms could be seen using measures of activity in addition to the mean spike frequency. We investigated whether there were changes in the irregularity of cell activity measured by the disorder of the interspike interval distribution for neurones recorded in vivo and in vitro. By plotting the entropy of the log interval histogram that quantifies the coding capacity for each action potential against the respective zeitgeber time, we describe oscillations of spike activity in vivo. Entropy measures have the advantage over variances in that they quantify aspects of the shape of the distribution and not just the dispersion. One hundred and sixty‐six cell recordings from the suprachiasmatic nucleus showed a significant rhythm in entropy with an oscillatory trend in the data (P < 0.001) showing a trough towards the end of the light period and a peak in the mid‐dark period. There was a similar rhythm for the cells recorded from the peripheral zone (n= 209, P= 0.037). In separate experiments in vitro, to investigate the relationship between mean spike frequency and entropy, potassium‐induced depolarization of cells recorded during the subjective night was correlated with a significant increase in mean spike frequency (r= 0.259, P= 0.011) and a decrease in entropy (r=−0.296, P= 0.004). The negative correlation between the entropy and mean spike frequency of cells recorded in vitro was significantly different from that seen in vivo (F= 15.5, P < 0.001), which may reflect differences in the balance between deterministic and stochastic influences on spike occurrence. The study shows that while there is a rhythm of mean spike frequency, parameters based on the variability of interspike interval distributions also display rhythmic changes over the day–night cycle.
Broad‐based proteomic strategies: a practical guide to proteomics and functional screening
Authors: Graham David R. M. , Elliott Steven T. , Van Eyk Jennifer E.
Published: December 2004

Proteomics, the study of the proteome (the collection of all the proteins expressed from the genome in all isoforms, polymorphisms and post‐translational modifications), is a rapidly developing field in which there are numerous new and often expensive technologies, making it imperative to use the most appropriate technology for the biological system and hypothesis being addressed. This review provides some guidelines on approaching a broad‐based proteomics project, including strategies on refining hypotheses, choosing models and proteomic approaches with an emphasis on aspects of sample complexity (including abundance and protein characteristics), and separation technologies and their respective strengths and weaknesses. Finally, issues related to quantification, mass spectrometry and informatics strategies are discussed. The goal of this review is therefore twofold: the first section provides a brief outline of proteomic technologies, specifically with respect to their applications to broad‐based proteomic approaches, and the second part provides more details about the application of these technologies in typical scenarios dealing with physiological and pathological processes. Proteomics at its best is the integration of carefully planned research and complementary techniques with the advantages of powerful discovery technologies that has the potential to make substantial contributions to the understanding of disease and disease processes.
Using models of the myocyte for functional interpretation of cardiac proteomic data
Authors: Winslow Raimond L. , Cortassa Sonia , Greenstein Joseph L.
Published: December 2004

There has been significant progress towards the development of highly integrative computational models of the cardiac myocyte over the past decade. Models now incorporate descriptions of voltage‐gated ionic currents and membrane transporters, mechanisms of calcium‐induced calcium release and intracellular calcium cycling, mitochondrial ATP production and its coupling to energy‐requiring membrane transport processes and mechanisms of force generation. There is an extensive literature documenting both the reconstructive and predictive abilities of these models and there is no question that an interplay between quantitative modelling and experimental investigation has become a central component of modern cardiovascular research. As data regarding the cardiovascular proteome in both health and disease emerge, integrative models of the myocyte are becoming useful tools for interpreting the functional significance of changes in protein expression and post‐translational modifications (PTMs). Data of particular importance include information on: (a) changes of expressed protein level, (b) changes of protein PTMs, (c) protein localization, and (d) protein–protein interactions, as it is often possible to incorporate and interpret the functional significance of such findings using computational models. We provide two examples of how models may be used in this fashion. In the first example, we show how information on altered expression of the sarcoplasmic reticulum Ca2+‐ATPase, when interpreted through the use of a computational model, has provided key insights into fundamental mechanisms regulating cardiac action potential duration. In the second example, we show how information on the effects of phosphorylation of L‐type Ca2+ channels, when interpreted through the use of a model, provides insights on how this post‐translational modification alters the properties of excitation–contraction coupling and risk for arrhythmia.
Opening of Size-Selective Pores in Endosomes during Human Rhinovirus Serotype 2 In Vivo Uncoating Monitored by Single-Organelle Flow Analysis
Authors: Brabec Marianne , Schober Daniela , Wagner Ernst , Bayer Nora , Murphy Robert F. , Blaas Dieter , Fuchs Renate
Published: December 2004

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.
Natural History of a Recurrent Feline Coronavirus Infection and the Role of Cellular Immunity in Survival and Disease
Authors: de Groot-Mijnes Jolanda D. F. , van Dun Jessica M. , van der Most Robbert G. , de Groot Raoul J.
Published: December 2004

We describe the natural history, viral dynamics, and immunobiology of feline infectious peritonitis (FIP), a highly lethal coronavirus infection. A severe recurrent infection developed, typified by viral persistence and acute lymphopenia, with waves of enhanced viral replication coinciding with fever, weight loss, and depletion of CD4+and CD8+T cells. Our combined observations suggest a model for FIP pathogenesis in which virus-induced T-cell depletion and the antiviral T-cell response are opposing forces and in which the efficacy of early T-cell responses critically determines the outcome of the infection. Rising amounts of viral RNA in the blood, consistently seen in animals with end-stage FIP, indicate that progression to fatal disease is the direct consequence of a loss of immune control, resulting in unchecked viral replication. The pathogenic phenomena described here likely bear relevance to other severe coronavirus infections, in particular severe acute respiratory syndrome, for which multiphasic disease progression and acute T-cell lymphopenia have also been reported. Experimental FIP presents a relevant, safe, and well-defined model to study coronavirus-mediated immunosuppression and should provide an attractive and convenient system for in vivo testing of anticoronaviral drugs.
Diabetes Acceleration or Prevention by a Coxsackievirus B4 Infection: Critical Requirements for both Interleukin-4 and Gamma Interferon
Authors: Serreze David V. , Wasserfall Clive , Ottendorfer Eric W. , Stalvey Michael , Pierce Melissa A. , Gauntt Charles , O'Donnell Brian , Flanagan James B. , Campbell-Thompson Martha , Ellis Tamir M. , Atkinson Mark A.
Published: December 2004

Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets, and in the absence of this insulitic threshold, CVB4 infection leads to long-term disease protection. To understand this acceleration and protection process, we challenged 8- and 12-week-old NOD mice containing a disruption in interleukin-4 (IL-4) or gamma interferon (IFN-γ) genes (NOD IL-4−/−and NOD IFN-γ−/−, respectively) with a diabetogenic, pancreatropic Edwards strain of CVB4. The elimination of IL-4 did not alter the rate of insulitis or diabetes development in NOD mice, while the elimination of IFN-γ delayed these events several weeks. CVB4 infection in 8-week-old mice only significantly accelerated the onset of diabetes in a subset of standard, but not IL-4- or IFN-γ-deficient, NOD mice. Long-term diabetes protection was established in standard NOD mice as well as in the NOD IFN-γ−/−mice that did not rapidly develop disease following CVB4 infection at 8 weeks of age. When mice were infected at 12 weeks of age, the onset of diabetes was accelerated in NOD IL-4−/−mice, while neither acceleration nor long-term protection was elicited in NOD IFN-γ−/−mice. No differences were observed in the kinetics of CVB4 clearance in pancreases from NOD, NOD IL-4−/−, and NOD IFN-γ−/−mice. Collectively, these results suggest that at the insulitis threshold at which CVB4 infection can first accelerate the onset of diabetes in NOD mice, IL-4 as well as IFN-γ contributes to this pathogenic process. The protective mechanism against diabetes elicited in NOD mice infected with CVB4 prior to the development of a critical threshold level of insulitis requires neither IL-4 nor IFN-γ.
Genomic Variation of the Fibropapilloma-Associated Marine Turtle Herpesvirus across Seven Geographic Areas and Three Host Species
Authors: Greenblatt Rebecca J. , Quackenbush Sandra L. , Casey Rufina N. , Rovnak Joel , Balazs George H. , Work Thierry M. , Casey James W. , Sutton Claudia A.
Published: December 2004

Fibropapillomatosis (FP) of marine turtles is an emerging neoplastic disease associated with infection by a novel turtle herpesvirus, fibropapilloma-associated turtle herpesvirus (FPTHV). This report presents 23 kb of the genome of an FPTHV infecting a Hawaiian green turtle (
Chelonia mydas
). By sequence homology, the open reading frames in this contig correspond to herpes simplex virus genes U
L
23 through U
L
36. The order, orientation, and homology of these putative genes indicate that FPTHV is a member of the
Alphaherpesvirinae
. The U
L
27-, U
L
30-, and U
L
34-homologous open reading frames from FPTHVs infecting nine FP-affected marine turtles from seven geographic areas and three turtle species (
C. mydas
,
Caretta caretta
, and
Lepidochelys olivacea
) were compared. A high degree of nucleotide sequence conservation was found among these virus variants. However, geographic variations were also found: the FPTHVs examined here form four groups, corresponding to the Atlantic Ocean, West pacific, mid-Pacific, and east Pacific. Our results indicate that FPTHV was established in marine turtle populations prior to the emergence of FP as it is currently known.
Antigenicity and Immunogenicity of a Synthetic Human Immunodeficiency Virus Type 1 Group M Consensus Envelope Glycoprotein
Authors: Gao Feng , Weaver Eric A. , Lu Zhongjing , Li Yingying , Liao Hua-Xin , Ma Benjiang , Alam S. Munir , Scearce Richard M. , Sutherland Laura L. , Yu Jae-Sung , Decker Julie M. , Shaw George M. , Montefiori David C. , Korber Bette T. , Hahn Beatrice H. , Haynes Barton F.
Published: December 2004

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensusenvgene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 subtypes. When used as a DNA vaccine followed by a recombinant vaccinia virus boost in BALB/c mice, CON6envgp120 and gp140CF elicited gamma interferon-producing T-cell responses that recognized epitopes within overlapping peptide pools from three HIV-1 Env proteins, CON6, MN (subtype B), and Chn19 (subtype C). Sera from guinea pigs immunized with recombinant CON6 Env gp120 and gp140CF glycoproteins weakly neutralized selected HIV-1 primary isolates. Thus, the computer-generated “consensus”envgenes are capable of expressing envelope glycoproteins that retain the structural, functional, and immunogenic properties of wild-type HIV-1 envelopes.
A Surface Loop of the Potato Leafroll Virus Coat Protein Is Involved in Virion Assembly, Systemic Movement, and Aphid Transmission
Authors: Lee Lawrence , Kaplan Igor B. , Ripoll Daniel R. , Liang Delin , Palukaitis Peter , Gray Stewart M.
Published: December 2004

Two acidic domains of thePotato leafroll virus(PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants byAgrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.
Distinct Subsets of Primary Effusion Lymphoma Can Be Identified Based on Their Cellular Gene Expression Profile and Viral Association
Authors: Fan Wen , Bubman Darya , Chadburn Amy , Harrington William J. , Cesarman Ethel , Knowles Daniel M.
Published: December 2004

Primary effusion lymphomas (PELs) are specifically associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection and most frequently occur in human immunodeficiency virus (HIV)-positive individuals as lymphomatous effusions in the serous cavities without a detectable solid tumor mass. Most PELs have concomitant Epstein-Barr virus (EBV) infection, suggesting that EBV is an important pathogenetic cofactor, although other as yet unidentified cofactors, such as cellular genetic alterations, are also likely to play a role. Lymphomatous effusions that lack KSHV also occur; these are frequently EBV associated in the setting of HIV infection. Here we used gene expression profile analysis to determine the viral impact on cellular gene expression and the pathogenesis of these lymphomatous effusions. Our results show that many genes, including cell cycle and signal transduction regulators, are differentially expressed between KSHV-positive PELs and KSHV-negative lymphomatous effusions and also between KSHV-positive, EBV-positive and KSHV-positive, EBV-negative PELs. Our results confirm that KSHV plays an important role in the pathogenesis of PELs, as its presence selects for a very distinct cellular gene expression category and a clearly different lymphoma type. Within the KSHV-positive PELs, the effect of EBV is more subtle but nevertheless clear.
Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Antibodies 2F5 and 4E10 Require Surprisingly Few Crucial Residues in the Membrane-Proximal External Region of Glycoprotein gp41 To Neutralize HIV-1
Authors: Zwick Michael B. , Jensen Richard , Church Sarah , Wang Meng , Stiegler Gabriela , Kunert Renate , Katinger Hermann , Burton Dennis R.
Published: December 2004

The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. However, immunogens that bear MPER epitopes so far have not elicited neutralizing antibodies in laboratory animals. One explanation is that the immunogens fail to recreate the proper molecular environment in which the epitopes of 2F5 and 4E10 are presented on the virus. To explore this molecular environment, we used alanine-scanning mutagenesis across residues 660 to 680 in the MPER of a pseudotyped variant of HIV-1
JR-FL
, designated HIV-1
JR2
, and examined the ability of 2F5 and 4E10 to neutralize the Ala mutant viruses. The results show that the only changes to produce neutralization resistance to 2F5 occurred in residue D, K, or W of the core epitope (LEL
DKW
ANL). Likewise, 4E10 resistance arose by replacing one of three residues; two (W and F) were in the core epitope, and one (W) was seven residues C-terminal to these two (N
WF
DISNWL
W
). Importantly, no single substitution resulted in resistance of virus to both 2F5 and 4E10. Surprisingly, 8 out of 21 MPER Ala mutants were more sensitive than the parental pseudovirus to 2F5 and/or 4E10. At most, only small differences in neutralization sensitivity to anti-gp120 monoclonal antibody b12 and peptide T20 were observed with the MPER Ala mutant pseudoviruses. These data suggest that MPER substitutions can act locally and enhance the neutralizing activity of antibodies to this region and imply a distinct role of the MPER of gp41 during HIV-1 envelope-mediated fusion. Neutralization experiments showing synergy between and T20 and 4E10 against HIV-1 are also presented. The data presented may aid in the design of antigens that better present the MPER of gp41 to the immune system.
Influenza A Viruses with Mutations in the M1 Helix Six Domain Display a Wide Variety of Morphological Phenotypes
Authors: Burleigh Laura M. , Calder Lesley J. , Skehel John J. , Steinhauer David A.
Published: December 2004

Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated “helix six.” This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.
Molecular Determinants for Subcellular Localization of Hepatitis C Virus Core Protein
Authors: Suzuki Ryosuke , Sakamoto Shinichiro , Tsutsumi Takeya , Rikimaru Akiko , Tanaka Keiko , Shimoike Takashi , Moriishi Kohji , Iwasaki Takuya , Mizumoto Kiyohisa , Matsuura Yoshiharu , Miyamura Tatsuo , Suzuki Tetsuro
Published: December 2004

Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic α-helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-α. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.
Potential Nectin-1 Binding Site on Herpes Simplex Virus Glycoprotein D
Authors: Connolly Sarah A. , Landsburg Daniel J. , Carfi Andrea , Whitbeck J. Charles , Zuo Yi , Wiley Don C. , Cohen Gary H. , Eisenberg Roselyn J.
Published: December 2004

Four glycoproteins (gD, gB, gH, and gL) are essential for herpes simplex virus (HSV) entry into cells. An early step of fusion requires gD to bind one of several receptors, such as nectin-1 or herpesvirus entry mediator (HVEM). We hypothesize that a conformational change in gD occurs upon receptor binding that triggers the other glycoproteins to mediate fusion. Comparison of the crystal structures of gD alone and gD bound to HVEM reveals that upon HVEM binding, the gD N terminus transitions from a flexible stretch of residues to a hairpin loop. To address the contribution of this transition to the ability of gD to trigger fusion, we attempted to “lock” the gD N terminus into a looped conformation by engineering a disulfide bond at its N and C termini. The resulting mutant (gD-A3C/Y38C) failed to trigger fusion in the absence of receptor, suggesting that formation of the loop is not the sole fusion trigger. Unexpectedly, although gD-A3C/Y38C bound HVEM, it failed to bind nectin-1. This was due to the key role played by Y38 in interacting with nectin-1. Since tyrosines are often “hot spot” residues at the center of protein-protein interfaces, we mutated residues that surround Y38 on the same face of gD and tested their binding and functional properties. Our results suggest that this region of gD is important for nectin-1 interaction and is distinct from but partially overlaps the site of HVEM binding. Unique gD mutants with altered receptor usage generated in this study may help dissect the roles played by various HSV receptors during infection.
Terminal Differentiation into Plasma Cells Initiates the Replicative Cycle of Epstein-Barr Virus In Vivo
Authors: Laichalk Lauri L. , Thorley-Lawson David A.
Published: December 2004

In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38
hi
, CD10
−
, CD19
+
, CD20
lo
, surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.
Simian Immunodeficiency Virus Infection in Wild-Caught Chimpanzees from Cameroon
Authors: Nerrienet Eric , Santiago Mario L. , Foupouapouognigni Yacouba , Bailes Elizabeth , Mundy Nicolas I. , Njinku Bernadette , Kfutwah Anfumbom , Muller-Trutwin Michaela C. , Barre-Sinoussi Françoise , Shaw George M. , Sharp Paul M. , Hahn Beatrice H. , Ayouba Ahidjo
Published: December 2004

Simian immunodeficiency viruses (SIVcpz) infecting chimpanzees (Pan troglodytes) in west central Africa are the closest relatives to all major variants of human immunodeficiency virus type 1 ([HIV-1]; groups M, N and O), and have thus been implicated as the source of the human infections; however, information concerning the prevalence, geographic distribution, and subspecies association of SIVcpz still remains limited. In this study, we tested 71 wild-caught chimpanzees from Cameroon for evidence of SIVcpz infection. Thirty-nine of these were of the central subspecies (Pan troglodytes troglodytes), and 32 were of the Nigerian subspecies (Pan troglodytes vellerosus), as determined by mitochondrial DNA analysis. Serological analysis determined that oneP. t. troglodytesape (CAM13) harbored serum antibodies that cross-reacted strongly with HIV-1 antigens; all other apes were seronegative. To characterize the newly identified virus, 14 partially overlapping viral fragments were amplified from fecal virion RNA and concatenated to yield a complete SIVcpz genome (9,284 bp). Phylogenetic analyses revealed that SIVcpzCAM13 fell well within the radiation of the SIVcpzPttgroup of viruses, as part of a clade including all other SIVcpzPttstrains as well as HIV-1 groups M and N. However, SIVcpzCAM13 clustered most closely with SIVcpzGAB1 from Gabon rather than with SIVcpzCAM3 and SIVcpzCAM5 from Cameroon, indicating the existence of divergent SIVcpzPttlineages within the same geographic region. These data, together with evidence of recombination among ancestral SIVcpzPttlineages, indicate long-standing endemic infection of central chimpanzees and reaffirm a west central African origin of HIV-1. WhetherP. t. vellerosusapes are naturally infected with SIVcpz requires further study.
Three-Dimensional Localization of the Smallest Capsid Protein in the Human Cytomegalovirus Capsid
Authors: Yu Xuekui , Shah Sanket , Atanasov Ivo , Lo Pierrette , Liu Fenyong , Britt William J. , Zhou Z. Hong
Published: December 2004

The smallest capsid proteins (SCPs) of the human herpesviruses differ substantially in size and sequence and are thought to impart some unique aspects of infection to their respective viruses. We used electron cryomicroscopy and antibody labeling to show that the 8-kDa SCP of human cytomegalovirus is attached only to major capsid protein subunits of the hexons, not the pentons. Thus, the SCPs of different herpesviruses illustrate that a protein can evolve significantly in sequence, structure, and function, while preserving its role in the architecture of the virus by binding to a specific partner in a specific oligomeric state.
Virus-Like Particles as Carriers for T-Cell Epitopes: Limited Inhibition of T-Cell Priming by Carrier-Specific Antibodies
Authors: Ruedl Christiane , Schwarz Katrin , Jegerlehner Andrea , Storni Tazio , Manolova Vania , Bachmann Martin F.
Published: December 2004

Virus-like particles (VLPs) are able to induce cytotoxic T-cell responses in the absence of infection or replication. This makes VLPs promising candidates for the development of recombinant vaccines. However, VLPs are also potent inducers of B-cell responses, and it is generally assumed that such VLP-specific antibodies interfere with the induction of protective immune responses, a phenomenon summarized as carrier suppression. In this study, we investigated the impact of preexisting VLP-specific antibodies on the induction of specific cytotoxic T-cell and Th-cell responses in mice. The data show that VLP-specific antibodies did not measurably reduce antigen presentation in vitro or in vivo. Nevertheless, T-cell priming was slightly reduced by antigen-specific antibodies; however, the overall reduction was limited and vaccination with VLPs in the presence of VLP-specific antibodies still resulted in protective T-cell responses. Thus, carrier suppression is unlikely to be a limiting factor for VLP-based T-cell vaccines.
Integrating Reptilian Herpesviruses into the Family
Herpesviridae
Authors: McGeoch Duncan J. , Gatherer Derek
Published: December 2004

The phylogeny of reptilian herpesviruses (HVs) relative to mammalian and avian HVs was investigated by using available gene sequences and by alignment of encoded amino acid sequences and derivation of trees by maximum-likelihood and Bayesian methods. Phylogenetic loci were obtained for green turtle HV (GTHV) primarily on the basis of DNA polymerase (POL) and DNA binding protein sequences, and for lung-eye-trachea disease-associated HV (LETV) primarily from its glycoprotein B sequence; both have nodes on the branch leading to recognized species in the
Alphaherpesvirinae
subfamily and should be regarded as new members of that subfamily. A similar but less well defined locus was obtained for an iguanid HV based on a partial POL sequence. On the basis of short POL sequences (around 60 amino acid residues), it appeared likely that GTHV and LETV belong to a private clade and that three HVs of gerrhosaurs (plated lizards) are associated with the iguanid HV. Based on phylogenetic branching patterns for mammalian HV lineages that mirror those of host lineages, we estimated a date for the HV tree's root of around 400 million years ago. Estimated dates for branching events in the development of reptilian, avian, and mammalian
Alphaherpesvirinae
lineages could plausibly be accounted for in part but not completely by ancient coevolution of these virus lines with reptilian lineages and with the development of birds and mammals from reptilian progenitors.
Involvement of Influenza Virus PA Subunit in Assembly of Functional RNA Polymerase Complexes
Authors: Kawaguchi Atsushi , Naito Tadasuke , Nagata Kyosuke
Published: December 2004

The RNA-dependent RNA polymerase of influenza virus consists of three subunits, PB1, PB2, and PA, and synthesizes three kinds of viral RNAs, vRNA, cRNA, and mRNA. PB1 is a catalytic subunit; PB2 recognizes the cap structure for generation of the primer for transcription; and PA is thought to be involved in viral RNA replication. However, the process of polymerase complex assembly and the exact nature of polymerase complexes involved in synthesis of the three different RNA species are not yet clear.
ts
53 virus is a temperature-sensitive (
ts
) mutant derived from A/WSN/33 (A. Sugiura, M. Ueda, K. Tobita, and C. Enomoto, Virology
65:
363-373, 1975). We confirmed that the mRNA synthesis level of
ts
53 remains unaffected at the nonpermissive temperature, whereas vRNA synthesis is largely reduced. Sequencing of the gene encoding
ts
53 PA and recombinant virus rescue experiments revealed that an amino acid change from Leu to Pro at amino acid position 226 is causative of temperature sensitivity. By glycerol density gradient analyses of nuclear extracts prepared from wild-type virus-infected cells, we found that polymerase proteins sediment in three fractions: one (H fraction) consists of RNP complexes, another (M fraction) contains active polymerases but not viral RNA, and the other (L fraction) contains inactive forms of polymerases. Pulse-chase experiments showed that polymerases in the L fraction are converted to those in the M fraction. In
ts
53-infected cells, polymerases accumulated in the L fraction. These results strongly suggest that PA is involved in the assembly of functional viral RNA polymerase complexes from their inactive intermediates.
Functional Analysis of Vif Protein Shows Less Restriction of Human Immunodeficiency Virus Type 2 by APOBEC3G
Authors: Ribeiro Ana Clara , Maia e Silva Alexandra , Santa-Marta Mariana , Pombo Ana , Moniz-Pereira José , Goncalves Joao , Barahona Isabel
Published: December 2004

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2
vif
-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1
vif
-minus nonpermissive cell line H9 does not show a similar phenotype for
vif
-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.
Directed Evolution of Retrovirus Envelope Protein Cytoplasmic Tails Guided by Functional Incorporation into Lentivirus Particles
Authors: Merten Christoph A. , Stitz Jörn , Braun Gundula , Poeschla Eric M. , Cichutek Klaus , Buchholz Christian J.
Published: December 2004

In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.
Epstein-Barr Virus gp42 Is Posttranslationally Modified To Produce Soluble gp42 That Mediates HLA Class II Immune Evasion
Authors: Ressing Maaike E. , van Leeuwen Daphne , Verreck Frank A. W. , Keating Sinéad , Gomez Raquel , Franken Kees L. M. C. , Ottenhoff Tom H. M. , Spriggs Melanie , Schumacher Ton N. , Hutt-Fletcher Lindsey M. , Rowe Martin , Wiertz Emmanuel J. H. J.
Published: December 2004

Epstein-Barr virus (EBV) resides as a persistent infection in human leukocyte antigen (HLA) class II+B lymphocytes and is associated with a number of malignancies. The EBV lytic-phase protein gp42 serves at least two functions: gp42 acts as the coreceptor for viral entry into B cells and hampers T-cell recognition via HLA class II molecules through steric hindrance of T-cell receptor-class II-peptide interactions. Here, we show that gp42 associates with class II molecules at their various stages of maturation, including immature αβIi heterotrimers and mature αβ-peptide complexes. When analyzing the biosynthesis and maturation of gp42 in cells stably expressing the viral protein, we found that gp42 occurs in two forms: a full-length type II membrane protein and a truncated soluble form. Soluble gp42 is generated by proteolytic cleavage in the endoplasmic reticulum and is secreted. Soluble gp42 is sufficient to inhibit HLA class II-restricted antigen presentation to T cells. In an almost pure population of Burkitt's lymphoma cells in the EBV lytic cycle, both transmembrane and soluble forms of gp42 are detected. These results imply that soluble gp42 is generated during EBV lytic infection and could contribute to undetected virus production by mediating evasion from T-cell immunity.
Selection for Virulent Dengue Viruses Occurs in Humans and Mosquitoes
Authors: Cologna Raymond , Armstrong Philip M. , Rico-Hesse Rebeca
Published: December 2004

Dengue is the most common mosquito-borne viral disease in humans. The spread of both mosquito vectors and viruses has led to the resurgence of epidemic dengue fever (a self-limited flu-like syndrome) and the emergence of dengue hemorrhagic fever (severe dengue with bleeding abnormalities) in urban centers of the tropics. There are no animal or laboratory models of dengue disease; indirect evidence suggests that dengue viruses differ in virulence, including their pathogenicities for humans and epidemic potential. We developed two assay systems (using human dendritic cells and
Aedes aegypti
mosquitoes) for measuring differences in virus replication that correlate with the potential to cause hemorrhagic dengue and increased virus transmission. Infection and growth experiments showed that dengue serotype 2 viruses causing dengue hemorrhagic fever epidemics (Southeast Asian genotype) can outcompete viruses that cause dengue fever only (American genotype). This fact implies that Southeast Asian genotype viruses will continue to displace other viruses, causing more hemorrhagic dengue epidemics.
Human Immunodeficiency Virus Type 1 Clade B Superinfection: Evidence for Differential Immune Containment of Distinct Clade B Strains
Authors: Yang Otto O. , Daar Eric S. , Jamieson Beth D. , Balamurugan Arumugam , Smith Davey M. , Pitt Jacqueline A. , Petropoulos Christos J. , Richman Douglas D. , Little Susan J. , Brown Andrew J. Leigh
Published: December 2004

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (∼1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (∼40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of
pol
genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by
pol
, and
env
sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8
+
-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.