bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential role of the persistent sodium current in spike initiation during slowly rising inputs in mouse spinal neurones
Authors: Kuo J. J. , Lee R. H. , Zhang L. , Heckman C. J.
Published: May 2006

Spinal motoneurons, like many neurons, respond with repetitive spiking to sustained inputs. The afterhyperpolarization (AHP) that follows each spike, however, decays relatively slowly in motoneurons. The slow depolarization during this decay should allow sodium (Na+) channel inactivation to keep up with its activation and thus should prevent initiation of the next spike. We hypothesized that the persistent component of the total Na+current provides the mechanism that generates a rate of rise sufficiently rapid to generate a spike. In large cultured spinal neurons, presumed to be primarily motoneurons, inhibition of persistent sodium current (NaP) by the drug riluzole at low concentrations resulted in a loss of repetitive firing. However, cells remained fully capable of producing spikes to transient inputs. These effects of riluzole were not due to insufficient depolarization, enhancement of the AHP, or sustained Na+channel inactivation. To further test this hypothesis, computer simulations were performed with a kinetic Na+channel model that provided greater independent control of NaPrelative to transient Na+current (NaT) than that provided by riluzole administration. The model was tuned to generate substantial NaPand exhibited good repetitive firing to slowly rising inputs. When NaPwas sharply reduced without significantly altering NaT, the model reproduced the effects of riluzole administration, inducing failure of repetitive firing but allowing single spikes in response to sharp transients. These results strongly support the essential role of NaPin spike initiation to slow inputs in spinal neurons. NaPmay play a fundamental role in determining how a neuron responds to sustained inputs.
Contribution of reverse Na+–Ca2+exchange to spontaneous activity in interstitial cells of Cajal in the rabbit urethra
Authors: Bradley E. , Hollywood M. A. , Johnston L. , Large R. J. , Matsuda T. , Baba A. , McHale N. G. , Thornbury K. D. , Sergeant Gerard P.
Published: May 2006

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+–Ca2+exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+concentration [Na+]ofrom 130 to 13 mm, the frequency of global Ca2+oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB‐R7943 and SEA0400 decreased the frequency of Ca2+oscillations and STICs. Application of caffeine (10 mm) and noradrenaline (10 μm) induced transient Ca2+‐activated chloride currents (IClCa) at −60 mV due to release of Ca2+from ryanodine‐ and inositol trisphosphate (IP3)‐sensitive Ca2+stores, respectively, but these responses were not blocked by KB‐R7943 or SEA0400 suggesting that neither drug blocked Ca2+‐activated chloride channels or Ca2+release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration‐dependent fashion. Finally, single cell RT‐PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+–Ca2+exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+entry via reverse NCX.
Studying properties of neurotransmitter receptors by non‐stationary noise analysis of spontaneous synaptic currents
Authors: Hartveit Espen , Veruki Margaret Lin
Published: May 2006

The properties of neurotransmitter receptor channels are important for determining synaptic transmission in the nervous system. The presence of quantal variability complicates the use of conventional non‐stationary noise analysis for determining the unitary conductance and number of channels involved in synaptic currents. Peak‐scaled non‐stationary noise analysis has been used to compensate for quantal variability, but there is evidence that the resulting variance versus mean relationships can be transformed from parabolic to skewed. We have used computer modelling based on experimentally derived kinetic schemes to investigate such relationships and demonstrate that their shape is a consequence of the temporal structure of the fluctuations during synaptic responses. Covariance analysis showed that peak‐scaling generates a skewed relationship when the covariance function decays rapidly (compared to the average response waveform), corresponding to a low correlation between fluctuations at the peak and in neighbouring regions of the decay phase. A parabolic relationship is obtained when the covariance function decays more slowly, corresponding to a higher correlation. Irrespective of a skewed or parabolic relationship, we demonstrate that the unitary current can be reliably estimated, with a coefficient of variation (CV) as low as 0.05 and bias as low as ±2% under ideal conditions. While the shape of the variance versus mean curve after peak‐scaled non‐stationary noise analysis is ultimately a consequence of the kinetic properties of the channels, inadequate alignment of individual waveforms can transform the relationship from parabolic to skewed, and low‐pass filtering can transform the relationship from skewed to parabolic. These findings have important implications for analysis of experimental data.
Developmental mouse brain gene expression maps
Authors: Brumwell Craig L. , Curran Tom
Published: May 2006

Brain gene expression databases are providing an increasing amount of information to the neuroscience community. Most databases are focused on the adult mouse rather than embryonic development. Here we survey the major mouse gene expression databases for the developing brain. The high throughput in situ hybridization approach generates large volumes of gene expression data that can be compiled and examined in a relatively short period of time. It is of increasing importance to compare gene expression patterns of neurodevelopment in the brain in relation to the adult. Often clues to adult gene expression and gene function can be determined by examining embryonic development. It is our hope that once all genes are mapped in the brain from the embryo to the adult, studies can be conducting based on information derived from such databases in conjunction with other bioinformatics sources.
Design Modification in a Collaborative Assembly Design Environment
Authors: Lu C. , Fuh J. Y. H. , Wong Y. S. , Qiu Z. M. , Li W. D. , Lu Y. Q.
Published: May 2006

This paper discusses the design modification issue in a collaborative assembly (co-assembly) design environment, which enables multiple geographically dispersed designers to design and assemble parts collaboratively and synchronously through the Internet. An assembly representation model, viz. feature-based hierarchical co-assembly representation, is proposed to resolve the co-assembly design issues. In order to realize the design modification, a design modification propagation control mechanism is proposed. A system framework that is suitable for realizing the design modification is also proposed and developed. Finally, the detailed design modification propagation control mechanism is demonstrated through a case study.
Intellectual Property Protection in Collaborative Design through Lean
Information Modeling and Sharing
Authors: Wang Yan , Ajoku Pamela N. , Brustoloni José C. , Nnaji Bart O.
Published: May 2006

Establishing efficient, effective, and trustworthy engineering collaboration while protecting intellectual property is vital to maintain organizational competence in today’s global business environment. In this paper, a lean information modeling and sharing framework is described to support engineering data security management in a peer-to-peer collaborative environment. It allows for selective and interoperable data sharing with fine-grained access control at both the server and client sides, thus securing different levels of design information dissemination for intellectual property protection purposes. The considerations of time and value-adding activity with roles, policy delegation relation in a distributed context, and fine-grained control at data set level in the model are to adhere to the general least privilege principle in access control. Heterogeneous design data are exchanged selectively through an eXtensible Markup Language common interface, which provides a neutral format to enhance data interoperability and prevents reverse engineering.
Document-Driven Design for Distributed CAD Services in Service-Oriented Architecture
Authors: Wang Yan , Nnaji Bart O.
Published: May 2006

Current computer-aided design (CAD) systems only support interactive geometry generation, which is not ideal for distributed engineering services in enterprise-to-enterprise collaboration with a generic thin-client service-oriented architecture. This paper proposes a new feature-based modeling mechanism—document-driven design—to enable batch mode geometry construction for distributed CAD systems. A semantic feature model is developed to represent informative and communicative design intent. Feature semantics is explicitly captured as a trinary relation, which provides good extensibility and prevents semantics loss. Data interoperability between domains is enhanced by schema mapping and multiresolution semantics. This mechanism aims to enable asynchronous communication in distributed CAD environments with ease of design alternative evaluation and reuse, reduced human errors, and improved system throughput and utilization.
A Socio-Technical Framework for Collaborative Product Development
Authors: Lu Stephen C.-Y. , Li Qingfeng , Case Michael , Grobler Francois
Published: May 2006

Human behavior dynamics impact technical decisions that cause societal changes, which, in turn, shape social dynamics to influence future technical decisions. This paper presents a socio-technical framework, based on the Engineering as Collaborative Negotiation paradigm. Collaborative product development is viewed as a socially mediated technical activity aiming to achieve a human purpose and modeled as a dynamic co-construction process, where stakeholders’ perspectives continuously evolve to form a share reality through collaborative negotiations. The paper introduces the socio-technical framework, the socio-technical co-construction process, and its software implementation. It also reports the applications of this research in the facility planning and development domain.
Scalable Vector Graphics (SVG) Based Multi-Level Graphics Representation for Engineering Rich-Content Exchange in Mobile Collaboration Computing Environments
Authors: Su Xiaoyong , Prabhu B. S. , Chu Chi-Cheng , Gadh Rajit
Published: May 2006

A two-dimensional (2D) graphics hierarchical representation framework and an on-demand content delivery mechanism for facilitating mobile engineering collaboration are presented in this paper. Multi-level graphics content sub-division is utilized to transform large engineering graphics into multiple levels of Scalable Vector Graphics (SVG) content. The hierarchical structure of the SVG content that maintains the relationship between the sub-divided content is formed during the process of sub-division. The divided content is selectively delivered and rendered on the mobile devices in an on-demand fashion. A prototypical system of the proposed approach is implemented and the performance of the framework is evaluated.
Reuse of Manufacturing Knowledge to Facilitate Platform-Based Product Realization
Authors: Alizon Fabrice , Shooter Steven B. , Simpson Timothy W.
Published: May 2006

Product platforming is a technique for exploiting commonality across a family of products. While utilizing a common platform can have many advantages when developing and manufacturing products, the approach places greater demands on collaboration, in particular, the sharing and reuse of knowledge and information. Repositories are intended to facilitate information sharing across organizational groups and geographically distributed collaborators. A particular challenge in utilizing repositories is culling a search for the most appropriate information for the problem at hand. The Reuse Existing Unit for Shape and Efficiency (R.E.U.S.E.) method facilitates the search of information in a repository through three stages that consider similarity, efficiency, and configuration. Automated search and filter techniques are implemented with interaction with the user to effectively obtain the desired results. The similarity study uses thresholds to clarify different opportunities for reuse. The user can then select alternatives for further examination based on efficiency of satisfaction of desired characteristics. The degree of modification of the similar alternatives is reported to assist in the configuration of the new design. This method contributes to the field by (a) accounting for the variety of the product family during the reuse of existing process design information; (b) integrating an efficiency assessment for retrieval by considering characteristics beyond cost; and (c) addressing the search with a multicriteria method. The implementation of the R.E.U.S.E method is supported with an example of assembly line design for an air conditioner module in automobile production.
Empirical Studies on Inter-Organizational Collaborative Product Development
Authors: Chu Chih-Hsing , Chang Chia-Jung , Cheng Han-Chung
Published: May 2006

Collaborative product development (CPD) has become an imperative for companies to strengthen their niche values and remain competitive in the global market. However, most scholars may lack knowledge of deploying CPD in current industrial settings, and their research efforts may thus fail to fulfill practical needs. This paper presents a series of empirical case studies on distributed product development in Asia-Pacific Region. Engineering collaborations among brand-owner, manufacturer, and supplier in six product design chains (desktop PC, IC substrate, LCD monitor, bicycle, mold design, and CPU cooler) are under investigation. A systematic approach is developed that categorizes CPD into three modes based on the number of engineering BOM items and the difficulty of modularization in product design. It provides a preliminary but structured template for CPD deployment by characterizing the implications and software features of each mode. The cases also reveal values which CPD creates for the downstream supplier of a global value chain and new IT technologies enabling the collaboration process. This work is one of the early studies that analyze collaborative product development from the perspective of a manufacturer. The findings complement and corroborate theoretical studies in related research.
Controlling Electromagnetic Fields
Authors: Pendry J. B. , Schurig D. , Smith D. R.
Published: May 2006

Using the freedom of design that metamaterials provide, we show how electromagnetic fields can be redirected at will and propose a design strategy. The conserved fields—electric displacement field
D
, magnetic induction field
B
, and Poynting vector
B
—are all displaced in a consistent manner. A simple illustration is given of the cloaking of a proscribed volume of space to exclude completely all electromagnetic fields. Our work has relevance to exotic lens design and to the cloaking of objects from electromagnetic fields.
Chimpanzee Reservoirs of Pandemic and Nonpandemic HIV-1
Authors: Keele Brandon F. , Van Heuverswyn Fran , Li Yingying , Bailes Elizabeth , Takehisa Jun , Santiago Mario L. , Bibollet-Ruche Frederic , Chen Yalu , Wain Louise V. , Liegeois Florian , Loul Severin , Ngole Eitel Mpoudi , Bienvenue Yanga , Delaporte Eric , Brookfield John F. Y. , Sharp Paul M. , Shaw George M. , Peeters Martine , Hahn Beatrice H.
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1), the cause of human acquired immunodeficiency syndrome (AIDS), is a zoonotic infection of staggering proportions and social impact. Yet uncertainty persists regarding its natural reservoir. The virus most closely related to HIV-1 is a simian immunodeficiency virus (SIV) thus far identified only in captive members of the chimpanzee subspecies
Pan troglodytes troglodytes
. Here we report the detection of SIVcpz antibodies and nucleic acids in fecal samples from wild-living
P. t. troglodytes
apes in southern Cameroon, where prevalence rates in some communities reached 29 to 35%. By sequence analysis of endemic SIVcpz strains, we could trace the origins of pandemic (group M) and nonpandemic (group N) HIV-1 to distinct, geographically isolated chimpanzee communities. These findings establish
P. t. troglodytes
as a natural reservoir of HIV-1.
Identification of an N-Terminal Trimeric Coiled-Coil Core within Arenavirus Glycoprotein 2 Permits Assignment to Class I Viral Fusion Proteins
Authors: Eschli Bruno , Quirin Katharina , Wepf Alexander , Weber Jacqueline , Zinkernagel Rolf , Hengartner Hans
Published: May 2006

The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal α-helix. An α-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second α-helical peptide corresponding to a predicted C-terminal α-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of ∼230 and ∼440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.
The Hypervariable Immunodominant NP418-426Epitope from the Influenza A Virus Nucleoprotein Is Recognized by Cytotoxic T Lymphocytes with High Functional Avidity
Authors: Boon Adrianus C. M. , de Mutsert Gerrie , Fouchier Ron A. M. , Osterhaus Albert D. M. E. , Rimmelzwaan Guus F.
Published: May 2006

Recently it was shown that influenza A viruses can accumulate mutations in epitopes associated with escape from recognition by human virus-specific cytotoxic T lymphocytes (CTL). It is unclear what drives diversification of CTL epitopes and why certain epitopes are variable and others remain conserved. It has been shown that simian immunodeficiency virus-specific CTL that recognize their epitope with high functional avidity eliminate virus-infected cells efficiently and drive diversification of CTL epitopes. T-cell functional avidity is defined by the density of major histocompatibility complex class I peptide complexes required to activate specific CTL. We hypothesized that functional avidity of CTL contributes to epitope diversification and escape from CTL also for influenza viruses. To test this hypothesis, the functional avidity of polyclonal CTL populations specific for nine individual epitopes was determined. To this end, peripheral blood mononuclear cells from HLA-A- and -B-genotyped individuals were stimulated in vitro with influenza virus-infected cells to allow expansion of virus-specific CTL, which were used to determine the functional avidity of CTL specific for nine individual epitopes in enzyme-linked immunospot assays. We found that the functional avidity for the respective epitopes varied widely. Furthermore, the functional avidity of CTL specific for the hypervariable NP418-426epitope was significantly higher than that of CTL recognizing other epitopes (P< 0.01). It was speculated that the high functional avidity of NP418-426-specific CTL was responsible for the diversification of this influenza A virus CTL epitope.
A Bicistronic Subgenomic mRNA Encodes both the ORF2 and ORF3 Proteins of Hepatitis E Virus
Authors: Graff Judith , Torian Udana , Nguyen Hanh , Emerson Suzanne U.
Published: May 2006

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.
Comparing Phylogenetic Codivergence between Polyomaviruses and Their Hosts
Authors: Pérez-Losada Marcos , Christensen Ryan G. , McClellan David A. , Adams Byron J. , Viscidi Raphael P. , Demma James C. , Crandall Keith A.
Published: May 2006

Seventy-two
full genomes corresponding to nine mammalian (67 strains) and two avian
(5 strains) polyomavirus species were analyzed using maximum likelihood
and Bayesian methods of phylogenetic inference. Our fully resolved and
well-supported (bootstrap proportions > 90%; posterior
probabilities = 1.0) trees separate the bird polyomaviruses
(avian polyomavirus and goose hemorrhagic polyomavirus) from the
mammalian polyomaviruses, which supports the idea of spitting the genus
into two subgenera. Such a split is also consistent with the different
viral life strategies of each group. Simian (simian virus 40, simian
agent 12 [Sa12], and lymphotropic polyomavirus) and rodent (hamster
polyomavirus, mouse polyomavirus, and murine pneumotropic polyomavirus
[MPtV]) polyomaviruses did not form monophyletic groups. Using our best
hypothesis of polyomavirus evolutionary relationships and established
host phylogenies, we performed a cophylogenetic reconciliation analysis
of codivergence. Our analyses generated six optimal cophylogenetic
scenarios of coevolution, including 12 codivergence events (
P
< 0.01), suggesting that
Polyomaviridae
coevolved with
their avian and mammal hosts. As individual lineages, our analyses
showed evidence of host switching in four terminal branches leading to
MPtV, bovine polyomavirus, Sa12, and BK virus, suggesting a combination
of vertical and horizontal transfer in the evolutionary history of the
polyomaviruses.
Antigenic and Immunogenic Characterization of Recombinant Baculovirus-Expressed Severe Acute Respiratory Syndrome Coronavirus Spike Protein: Implication for Vaccine Design
Authors: He Yuxian , Li Jingjing , Heck Susanne , Lustigman Sara , Jiang Shibo
Published: May 2006

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.
Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus
Authors: Codoñer Francisco M. , Fares Mario A. , Elena Santiago F.
Published: May 2006

The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions.
Recovery of Avian Metapneumovirus Subgroup C from cDNA: Cross-Recognition of Avian and Human Metapneumovirus Support Proteins
Authors: Govindarajan Dhanasekaran , Buchholz Ursula J. , Samal Siba K.
Published: May 2006

Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with “swollen head syndrome” in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.
Second-Site Revertants of a Semliki Forest Virus Fusion-Block Mutation Reveal the Dynamics of a Class II Membrane Fusion Protein
Authors: Chanel-Vos Chantal , Kielian Margaret
Published: May 2006

The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced membrane fusion mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 inserts into target membranes via its hydrophobic fusion loop and refolds to form a stable E1 homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the fusion loop was previously shown to block SFV fusion and infection, although the mutant E1 protein still inserts into target membranes and forms a homotrimer. Here we report on second-site mutations in E1 that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the membrane-interacting tip, and within the groove where the E1 stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the fusion protein refolding reaction.
Trimethylation of Histone H3 Lysine 4 by Set1 in the Lytic Infection of Human Herpes Simplex Virus 1
Authors: Huang Jing , Kent Jennifer R. , Placek Brandon , Whelan Kelly A. , Hollow Charles M. , Zeng Ping-Yao , Fraser Nigel W. , Berger Shelley L.
Published: May 2006

Human herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that causes facial, ocular, and encephalitic disease in humans. Previous work showed that the genome of HSV-1 is associated with acetylated and methylated histones during lytic infection. However, the physiological role of histone modifications in lytic infection of HSV-1 is unclear. We examined the role of protein methylation in lytic infection of HSV-1 using a protein methylation inhibitor, 5′-deoxy-5′-methylthioadenosine (MTA). We found that MTA strongly reduces the transcription and replication of HSV-1. Moreover, MTA treatment decreases the level of trimethylation of lysine 4 in histone H3 (H3K4me3) on the HSV-1 genome. These results suggest that protein methylation, and in particular, histone methylation, is involved in the lytic infection of HSV-1. To delineate the underlying mechanism, we investigated the role of two H3K4 methyltransferases, Set1 and Set7/9, in the lytic infection of HSV-1. Using small interference RNA, we found that the reduction of Set1, but not Set7/9, reduces the transcription and replication of HSV-1 and specifically decreases H3K4me3 on the virus genome. These results indicate that H3K4me3 mediated by Set1 is required for optimal gene expression and replication of HSV-1 during lytic infection and suggest that this pathway could be a potential point of pharmacological intervention during HSV-1 infection.
The NS2 Protein of Human Respiratory Syncytial Virus Suppresses the Cytotoxic T-Cell Response as a Consequence of Suppressing the Type I Interferon Response
Authors: Kotelkin Alexander , Belyakov Igor M. , Yang Lijuan , Berzofsky Jay A. , Collins Peter L. , Bukreyev Alexander
Published: May 2006

The NS1 and NS2 proteins of human respiratory syncytial virus (HRSV) have been shown to antagonize the type I interferon (IFN) response, an effect subject to host range constraints. We have now found that the HRSV NS2 protein strongly controls IFN induction in mouse cells in vitro, validating the use of the mouse model to study the consequences of these gene deletions on host immunity. We evaluated the effects of deleting the NS1 and/or NS2 gene on the induction of HRSV-specific pulmonary cytotoxic T lymphocytes (CTL) in BALB/c and 129S6 mice in response to intranasal infection with HRSV lacking the NS1 and/or NS2 gene and subsequent challenge with wild-type (wt) HRSV. In mice infected with HRSV lacking the NS2 gene (ΔNS2) or lacking the NS2 gene in combination with the NS1 gene (ΔNS1/2 HRSV), the magnitude of the pulmonary CTL response was substantially elevated compared to that of mice infected with wt HRSV or the ΔNS1 mutant, whether measured by binding of CD8+cells to an HRSV-specific major histocompatibility complex class I tetramer, by measurement of CD8+cells secreting gamma interferon (IFN-γ) in response to specific in vitro stimulation, or by a standard chromium release cell-killing assay. In contrast, in STAT1 knockout mice, which lack responsiveness to type I IFN, the level of IFN-γ-secreting CD8+cells was not significantly different for HRSV lacking the NS2 gene, suggesting that the increase in CTL observed in IFN-responsive mice is type I IFN dependent. Thus, the NS2 protein of HRSV suppresses the CTL component of the adaptive immune response, and this appears to be a consequence of its suppression of type I IFN.
Receptor (SLAM [CD150]) Recognition and the V Protein Sustain Swift Lymphocyte-Based Invasion of Mucosal Tissue and Lymphatic Organs by a Morbillivirus
Authors: von Messling Veronika , Svitek Nicholas , Cattaneo Roberto
Published: May 2006

Experimental infections of ferrets with canine distemper virus (CDV) recapitulate many hallmarks of measles: rash, high fever, viremia, depression of delayed-type hypersensitivity responses, lowered leukocyte counts, and reduced lymphocyte proliferation activity. To understand how a morbillivirus invades the host and causes immunosuppression, we generated CDV either unable to recognize one of the receptors or incapable of expressing either one or both of the candidate interferon antagonist proteins V and C. Variants of these viruses expressing green fluorescent protein were also generated. Striking similarities between CDV infection of ferrets and human immunodeficiency virus host invasion were documented: first, massive early replication in the gut-associated lymphatic tissue, including intestinal Peyer's patches, followed by extensive infection of lymphatic organs, including thymus and circulating lymphocytes. Moreover, T cells were selectively depleted. Thus, CDV takes advantage of mucosal surfaces for host invasion and lymphocytes for swift dissemination. A CDV unable to recognize the signaling lymphocytic activation molecule (SLAM [CD150]) that is expressed in lymphocytes and other immune cells did not spread. A V-defective CDV multiplied with reduced efficiency in lymphocytes and did not inhibit the interferon and cytokine responses. Protein C affected the severity of rash and digestive symptoms elicited by V-defective CDV, but it was dispensable for the invasion of the lymphatic organs. These findings prove formally that SLAM recognition is necessary for morbillivirus virulence. They also reveal how two viral proteins affect pathogenesis: V sustains the swift lymphocyte-based invasion of mucosal tissue and lymphatic organs, whereas C sustains subsequent infection phases.
Mutational Alteration of Human Immunodeficiency Virus Type 1 Vif Allows for Functional Interaction with Nonhuman Primate APOBEC3G
Authors: Schröfelbauer Bärbel , Senger Tilo , Manning Gerard , Landau Nathaniel R.
Published: May 2006

Human APOBEC3F (hA3F) and APOBEC3G (hA3G) are antiretroviral cytidine deaminases that can be encapsidated during virus assembly to catalyze C→U deamination of the viral reverse transcripts in the next round of infection. Lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have evolved the accessory protein Vif to induce their degradation before packaging. HIV type 1 (HIV-1) Vif counteracts hA3G but not rhesus macaque APOBEC3G (rhA3G) or African green monkey (AGM) APOBEC3G (agmA3G) because of a failure to bind the nonhuman primate proteins. The species specificity of the interaction is controlled by amino acid 128, which is aspartate in hA3G and lysine in rhA3G. With the objective of overcoming this species restriction, mutations were introduced into HIV-1 Vif at amino acid positions that differed in charge between HIV-1 Vif and SIV Vif. The mutant proteins were tested for the ability to counteract hA3G, rhA3G, and agmA3G. Alteration of the conserved sequence at positions 14 to 17 from DRMR to SERQ, which is the sequence in AGM Vif, caused HIV-1 Vif to functionally interact with rhA3G and agmA3G. Mutation of three residues to the sequence SEMQ allowed interaction with rhA3G. SEMQ Vif also counteracted D128K mutant hA3G and wild-type hA3G. Introduction of the sequence into an infectious molecular HIV-1 clone allowed the virus to replicate productively in human cells that expressed rhA3G or hA3G. These findings provide insight into the interaction of Vif with A3G and are a step toward the development of a novel primate model for AIDS.
High-Level Transgene Expression in Nonhuman Primate Liver with Novel Adeno-Associated Virus Serotypes Containing Self-Complementary Genomes
Authors: Gao Guang-Ping , Lu You , Sun Xun , Johnston Julie , Calcedo Roberto , Grant Rebecca , Wilson James M.
Published: May 2006

Adeno-associated virus (AAV) vectors are being considered for in vivo applications of gene therapy in the treatment of a variety of disorders. This study evaluates the biology of second-generation vectors based on the novel serotypes AAV7 and AAV8 and containing self-complementary genomes in the nonhuman primate liver. Stable levels of transgene expression were achieved in cynomolgus macaques and suggest efficiencies at least 2 log higher than what could be achieved with AAV2 vectors using traditional single-stranded genomes. Analysis of DNAs from tissues revealed high levels of vector in the liver that appeared proportional to the relative amounts of transgene expression.
Cellular and Molecular Requirements for Association of the Murine Cytomegalovirus Protein m4/gp34 with Major Histocompatibility Complex Class I Molecules
Authors: Lu Xiuju , Kavanagh Daniel G. , Hill Ann B.
Published: May 2006

The murine cytomegalovirus (MCMV) protein m4/gp34 is unique among known viral genes that target the major histocompatibility complex (MHC) class I pathway of antigen presentation in the following two ways: it is found in association with class I MHC molecules at the cell surface, and it inhibits antigen presentation without reducing cell surface class I levels. The current study was undertaken to define more clearly the structural and cellular requirements for m4/gp34 association with the MHC class I molecule Kb. We first assessed the role of the peptide-loading complex in m4/gp34-Kbassociation, using cell lines lacking TAP, tapasin, or β2m. m4/gp34-Kbcomplexes formed in the absence of TAP or tapasin, although not as efficiently as in wild-type cells. The expression of full-length and truncation mutants of m4/gp34 in a gutless adenovirus vector revealed that the transmembrane region of m4/gp34 was required for efficient association with the Kbheavy chain. However, the peptide-loading complex was not absolutely required for the association, since m4/gp34 readily formed complexes with Kbin detergent lysates. The addition of Kb-binding peptide to the detergent lysates facilitated but was not essential for the formation of the complexes. The ease of complex formation in detergent lysates contrasted with the small fractions of m4/gp34 and Kbthat form complexes in infected cells, suggesting that the endoplasmic reticulum (ER) environment restricts access of m4/gp34 to Kb. Finally, although m4/gp34-Kbcomplexes could form when m4 was carried either by MCMV or by the adenovirus vector, they were only efficiently exported from the ER in MCMV-infected cells, suggesting that MCMV provides additional factors needed for transport of the complexes.
Cellular and Biochemical Differences between Two Attenuated Poxvirus Vaccine Candidates (MVA and NYVAC) and Role of the C7L Gene
Authors: Nájera José Luis , Gómez Carmen Elena , Domingo-Gil Elena , Gherardi María Magdalena , Esteban Mariano
Published: May 2006

The poxvirus strains NYVAC and MVA are two candidate vectors for the development of vaccines against a broad spectrum of diseases. Although these attenuated virus strains have proven to be safe in animals and humans, little is known about their comparative behavior in vitro. In contrast with MVA, NYVAC infection triggers greater cytopathic effect in a range of permissive and nonpermissive cell lines. The yields of NYVAC cell-associated virus in permissive cells (BHK-21) were slightly reduced compared with those of MVA infection. During the course of infection in HeLa cells, there is a translational block induced by NYVAC late in infection, which correlated with a marked increase in phosphorylation levels of the initiation factor eIF-2α. In contrast to MVA, the synthesis of certain late viral proteins was only blocked in NYVAC-infected HeLa cells. Electron microscopy (EM) analysis revealed that morphogenesis of NYVAC in HeLa cells was blocked at the stage of formation of immature viral forms. Phase-contrast microscopy, EM, flow cytometry, and rRNA analyses demonstrated that contrary to MVA, NYVAC infection induces potent apoptosis, a phenomenon dependent on activation of caspases and RNase L. Apoptosis induced by NYVAC was prevented when the virus gene C7L was placed back into the NYVAC genome, recovering the ability of NYVAC to replicate in HeLa cells and maintaining the attenuated phenotype in mice. Overall, our findings demonstrate distinct behavior between NYVAC and MVA strains in cultured cells, as well as a new role for the C7L viral gene as an inhibitor of apoptosis in NYVAC infection.
Human Leukocyte Antigen B58 Supertype and Human Immunodeficiency Virus Type 1 Infection in Native Africans
Authors: Lazaryan Aleksandr , Lobashevsky Elena , Mulenga Joseph , Karita Etienne , Allen Susan , Tang Jianming , Kaslow Richard A.
Published: May 2006

Human leukocyte antigen (HLA) class I alleles can be grouped into supertypes according to their shared peptide binding properties. We examined alleles of the HLA-B58 supertype (B58s) in treatment-naïve human immunodeficiency virus type 1 (HIV-1)-seropositive Africans (423 Zambians and 202 Rwandans). HLA-B and HLA-C alleles were resolved to four digits by a combination of molecular methods, and their respective associations with outcomes of HIV-1 infection were analyzed by statistical procedures appropriate for continuous or categorical data. The effects of the individual alleles on natural HIV-1 infection were heterogeneous. In HIV-1 subtype C-infected Zambians, the mean viral load (VL) was lower among B*5703 (P= 0.01) or B*5703-Cw*18 (P< 0.001) haplotype carriers and higher among B*5802 (P= 0.02) or B*5802-Cw*0602 (P= 0.03) carriers. The B*5801-Cw*03 haplotype showed an association with low VL (P= 0.05), whereas B*5801 as a whole did not. Rwandans with HIV-1 subtype A infection showed associations of B*5703 and B*5802 with slow (P= 0.06) and rapid (P= 0.003) disease progression, respectively. In neither population were B*1516-B*1517 alleles associated with more favorable responses. Overall, B58s alleles, individually or as part of an HLA-B-HLA-C haplotype, appeared to have a distinctive impact on HIV-1 infection among native Africans. As presently defined, B58s alleles cannot be considered uniformly protective against HIV/AIDS in every population.
RNA Polymerase II-Controlled Expression of Antigenomic RNA Enhances the Rescue Efficacies of Two Different Members of the
Mononegavirales
Independently of the Site of Viral Genome Replication
Authors: Martin Arnold , Staeheli Peter , Schneider Urs
Published: May 2006

De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the
Mononegavirales
with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the
Mononegavirales
with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5′-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5′ termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.
Characterization of the Capsid Protein Glycosylation of Adeno-Associated Virus Type 2 by High-Resolution Mass Spectrometry
Authors: Murray Sarah , Nilsson Carol L. , Hare Joan T. , Emmett Mark R. , Korostelev Andrei , Ongley Heather , Marshall Alan G. , Chapman Michael S.
Published: May 2006

Adeno-associated virus type 2 (AAV-2) capsid proteins have eight sequence motifs that are potential sites for O- or N-linked glycosylation. Three are in prominent surface locations, close to the sites of cellular receptor attachment and to neutralizing epitopes on or near protrusions surrounding the three-fold axes, raising the possibility that AAV-2 might use glycosylation as a means of immune escape or for preventing reattachment on release of progeny virus. Peptide mapping and structural analysis by Fourier transform ion cyclotron resonance mass spectrometry demonstrates, however, no glycosylation of the capsid protein for virus prepared in cultured HeLa cells.