bluuebunny/crossref_metadata_2025 huggingface dataset:
Virtual connections: representation and commercial qualitative research
Authors: Thorpe Mark
Published: June 2003

This paper explores the question of representation within commercial qualitative research in the context of a broader “crisis of representation”. It will be argued that a comparison between academic and commercial qualitative research highlights very different contexts in which to raise and deal with questions of representation. Further, it is suggested that the comparison between commercial and academic qualitative research reveals a tension between virtualism and the iconography of authenticity. The paper ends by arguing that, within commercial qualitative research, virtualism is a key route towards bringing a more pragmatic and insightful approach to research.
Institutional theory of police: a review of the state of the art
Authors: Crank John P.
Published: June 2003

One of the important developments in police theory and research is the recognition of the institutional contexts in which departments participate. A body of theory, organized under the rubric of the “theory of institutionalized organizations”, provides a theoretical framework for the conceptualization and empirical assessment of policing contexts and their relationship to police organizational structures and practices. The first part of this paper provides an overview of institutional theory and reviews writings on the application of institutional theory to the police. The second section reviews research conducted on institutional theory across the field of criminal justice. The third part contrasts the competing notions of institutions and utility in institutional theory, and locates police organizations within Giddens’ model of human agency.
Bending granite revisited: the command rank structure of American police organizations
Authors: King William R.
Published: June 2003

For 30 years, critics have noted a number of ailments caused by police rank structure. This paper adds to this literature by exploring the command rank structure of American police organizations in four parts. First, police rank structure is defined. Second, the hypothesized problems and benefits of tall rank structures are culled from the prior literature and described. A review of these contentions finds little reason to believe that tall rank structures are either beneficial or detrimental. Third, a systematic review of empirical studies of a range of organizations reveals that the impact of rank structure on three classes of organizational attributes is not wholly clear. This article closes with suggestions for conducting research to better ascertain the impact of hierarchy on police agencies, and how police executives can apply this knowledge.
Jordan v. The City of New London, police hiring and IQ: “When all the answers they don’t amount to much”
Authors: “Tad” Hughes Tom
Published: June 2003

In the recent case of Jordan v. The City of New London a police applicant was denied employment because he scored too well on the cognitive ability portion of his written application test. The importance of the case stems from its potential impact on three areas. First, in a time of shrinking applications to police forces, legal decisions related to the selection process would appear significant. Second, the rejection of an applicant by a police department because he was thought too intelligent appears to create or reinforce negative stereotypes of police in the USA. Third, the case involves employment law, an area that has proven fertile ground for suits against the police. The article explores the case in detail as well as reporting the results of a survey of police mid‐level managers concerning the impact of intelligence on various police administrative concerns.
Police employee organizations
Authors: Kadleck Colleen
Published: June 2003

Police employee organizations and unions have been identified as obstacles to police management as well as to policy implementation. While much has been made of the behavior of these organizations, little empirical work has addressed the nature of these organizations in terms of their structure, membership, and perspectives on police labor relationships. These organizations have mainly been examined by the use of police chief perspectives, or case studies of large police unions. This article addresses these issues using a national sample of police employee organizations and unions.
Strange bedfellows: civil liability and aggressive policing
Authors: Novak Kenneth J. , Smith Brad W. , Frank James
Published: June 2003

Shaping and monitoring the behavior of street‐level officers has continued to be a difficult task for police managers, and this task may prove to be more difficult as modern departments increasingly rely on proactive law enforcement for the delivery of police services. A popular method to shape police behavior is holding officers, departments and municipalities civilly liable for street‐level behavior. While it may be assumed fear of civil litigation influences the manner in which the police interact with the public, there is little empirical evidence for this claim; in fact, the frequent use of civil liability claims is poised to have an unanticipated side effect on contemporary policing. Officers may engage in fewer proactive law enforcement activities as a way to insulate them from litigation. This study examines whether experience with and attitudes toward civil liability influence the behavior of police officers, with particular attention on officer propensity to make arrests, use force, conduct searches and initiate encounters with suspects. Multivariate results indicate attitudes toward civil liability are weak and inconsistent predictors of behavior.
Time weighted portfolio optimisation
Authors: Lee Stephen , Stevenson Simon
Published: June 2003

In estimating the inputs into the modern portfolio theory (MPT) portfolio optimisation problem, it is usual to use equal weighted historic data. Equal weighting of the data, however, does not take account of the current state of the market. Consequently this approach is unlikely to perform well in any subsequent period as the data is still reflecting market conditions that are no longer valid. The need for some return weighting scheme that gives greater weight to the most recent data would seem desirable. Therefore, this study uses returns data which are weighted to give greater weight to the most recent observations to see if such a weighting scheme can offer improved ex ante performance over that based on unweighted data.
A bounded rationality framework for property investment behaviour
Authors: de Bruin Anne , Flint‐Hartle Susan
Published: June 2003

This paper attempts to explain the motivations of residential rental property investors in New Zealand in terms of the behavioural assumption of bounded rationality. Commencing with a rejection of the more standard neo‐classical economics view of rationality as an explanation of investment behaviour, the paper seeks to both examine the extent to which bounded rationality applies to the investment behaviour encountered and to elaborate on that behaviour. The discussion is underpinned by the findings of a postal survey of a large nationwide sample of private residential rental property owners, and is directly based on a study of a smaller sample of investors using in‐depth interview techniques. Qualitative analysis overlays the quantitative data, to enable better exploration of the constraints within which individual investors operate.
Keynes and the future
Authors: Fontela Emilio
Published: June 2003

In 1930, John Maynard Keynes explored long‐term economic growth, anticipating that the economic problem of scarcity was likely to vanish in a century’s time. The Keynes’ scenario is confronted with current developments related to the growth of services and to the distributional and environmental aspects of sustainable development.
Fourastié’s foresight after 50 years
Authors: Hospers Gert‐Jan
Published: June 2003

More than half a century ago the French futurist Jean Fourastié (1907‐1990) presented an interesting view of society in the year 2000. His vision of the future has remained relatively unfamiliar in the Anglo‐Saxon world. However Fourastié’s “hypothesis” that the fundamental “laws of production and consumption” would lead to the rise of a service‐oriented society would seem largely to have come true. Fourastié’s vision is still relevant because it also warns policy‐makers not to allow themselves to be blinded by exaggerated expectations of the future. He states that the true task of the authorities is to focus on the training and guidance of entrepreneurs and citizens in the difficult transition process from the existing socio‐economic structure to the new.
Socially responsible leadership
Authors: Karp Tom
Published: June 2003

This article examines the role of business leadership in the coming decade with respect to social responsibility. It is argued, herein, that the successful leadership agenda in the coming decade will, to a greater degree than today, be shaped by the leader’s ability to take an active and constructive part in the society in which the business operates. The premises for putting forward this hypothesis are that the excesses of the 1990s are over, and the geopolitical, the economic, and the ecological environments offer challenges not seen for a long time in business. Socially responsible leadership in the coming decade will not only be about doing business, but also about questioning how this business is done and how value is created. In an increasingly complex environment, the integrity of the single business leader will matter, as will his or her ability to see the overall role of his or her company in the society in which it operates. Leaders are, to some degree, reflections of what their societies want from them. This paper points to a number of trends where public expectations today call for more social responsibility from commercial players. There are companies showing the way and taking the lead in meeting those expectations, and thereby setting new requirements for business leadership in the coming decade. Even though it is a debated issue, this article concludes that socially responsible leadership will be the answer in meeting those growing expectations. It is also concluded herein that most business leaders will be able to rise to the challenges in the coming decade, as they have before.
Through a glass darkly: the future and business revisited
Authors: Loveridge Denis , Woodling Geoffrey
Published: June 2003

This article revisits a paper written by Denis Loveridge 15 years ago about the merit or otherwise of the long view versus its short‐term counterpart. The paper revisits the notions set out in 1988 and enlarges them, by making use of the authors’ practical experience. Two notions lay at the heart of the paper: the question of what people will value in their lives, and Maxwell’s notion of a philosophy of “wisdom”. The authors contrast this view with those seeking an epistemological basis for foresight, concluding that the latter does not have much to offer those who conduct foresight for prosaic aims in business and the public sphere.
International foresight’s contribution to globalisation
Authors: Jewell Tamsin
Published: June 2003

This paper presents and discusses the APEC Center for Technology Foresight’s experiences with its first five international foresight projects. The projects are described in some detail to illuminate the kinds of topics addressed, the rationale for their selection and the kinds of conclusions which emerged. The impact of the projects on policy is considered, and important additional benefits are demonstrated, especially their contribution to the creation of a “forward‐thinking” culture among scientists and policy‐makers. Thus, the projects are seen to address critical issues of regional and global concern and, at the same time, to build foresight capacity within the Asia‐Pacific region.
Crystal complexes of a predicted S‐adenosylmethionine‐dependent methyltransferase reveal a typical AdoMet binding domain and a substrate recognition domain
Authors: Miller Darcie J. , Ouellette Nancy , Evdokimova Elena , Savchenko Alexei , Edwards Aled , Anderson Wayne F.
Published: June 2003

S‐adenosyl‐L‐methionine‐dependent methyltransferases (MTs) are abundant, and highly conserved across phylogeny. These enzymes use the cofactor AdoMet to methylate a wide variety of molecular targets, thereby modulating important cellular and metabolic activities. Thermotoga maritima protein 0872 (TM0872) belongs to a large sequence family of predicted MTs, ranging phylogenetically from relatively simple bacteria to humans. The genes for many of the bacterial homologs are located within operons involved in cell wall synthesis and cell division. Despite preliminary biochemical studies in E. coli and B. subtilis, the substrate specificity of this group of more than 150 proteins is unknown. As part of the Midwest Center for Structural Genomics initiative (www.mcsg.anl.gov), we have determined the structure of TM0872 in complexes with AdoMet and with S‐adenosyl‐L‐homocysteine (AdoHcy). As predicted, TM0872 has a typical MT domain, and binds endogenous AdoMet, or co‐crystallized AdoHcy, in a manner consistent with other known MT structures. In addition, TM0872 has a second domain that is novel among MTs in both its location in the sequence and its structure. The second domain likely acts in substrate recognition and binding, and there is a potential substrate‐binding cleft spanning the two domains. This long and narrow cleft is lined with positively charged residues which are located opposite the S+‐CH3 bond, suggesting that a negatively charged molecule might be targeted for catalysis. However, AdoMet and AdoHcy are both buried, and access to the methyl group would presumably require structural rearrangement. These TM0872 crystal structures offer the first structural glimpses at this phylogenetically conserved sequence family.
Measuring the stability of partly folded proteins using TMAO
Authors: Mello Cecilia C. , Barrick Doug
Published: June 2003

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N‐oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte‐induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline‐resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two‐state equilibrium unfolding, we found that ΔG° for unfolding depends linearly on TMAO concentration, and that the sensitivity of ΔG° to urea (the m‐value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline‐resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of ΔG° on each cosolvent are similar to those obtained by standard urea‐induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full‐length Notch ankyrin domain.
Regulation of Hepatitis B Virus Replication by the Ras-Mitogen-Activated Protein Kinase Signaling Pathway
Authors: Zheng Yanyan , Li Jie , Johnson Deborah L. , Ou Jing-hsiung
Published: June 2003

The replication of hepatitis B virus (HBV) can be regulated by a variety of factors, including hormones, growth factors, and cytokines. However, the molecular mechanisms of these regulations are largely unknown. Ras is a small GTPase that responds to many of these external stimuli. In this study, we investigated the possible effect of Ras on the replication of HBV. Our results indicated that activated Ras could suppress the replication of HBV in both Huh7 and HepG2 cells. This suppression was independent of the X protein and most likely occurred at the transcriptional level. Deletion-mapping analysis of the HBV core promoter and its upstream ENI and ENII enhancers revealed multiple elements responsive to activated Ras. This suppression of HBV replication by activated Ras was apparently mediated by the mitogen-activated protein (MAP) kinase pathway, as it was accompanied by activation of ERK1/2 and abolished by the MEK1/2 inhibitor U0126. Our results thus indicate that external stimuli may suppress HBV replication through the Ras-MAP kinase pathway.
Activation and Inhibition of Cellular Calcium and Tyrosine Kinase Signaling Pathways Identify Targets of the HBx Protein Involved in Hepatitis B Virus Replication
Authors: Bouchard Michael J. , Puro Robyn J. , Wang Lihua , Schneider Robert J.
Published: June 2003

Human hepatitis B virus (HBV) HBx protein is a multifunctional protein that activates cellular signaling pathways and is thought to be essential for viral infection. Woodchuck HBV mutants that lack HBx are unable to replicate in vivo or are severely impaired. HBV replication in HepG2 cells, a human hepatoblastoma cell line, is stimulated 5- to 10-fold by HBx protein. We have utilized the HepG2, HBx-dependent HBV replication system to study the effects of activators and inhibitors of cytosolic calcium and tyrosine kinase signaling pathways on viral replication. By transfecting either a wild-type HBV genome or an HBV genome that does not express HBx and then treating transfected cells with activators or inhibitors of signaling pathways, we identified compounds that either impair wild-type HBV replication or rescue HBx-deficient HBV replication. Geldanamycin or herbimycin A, tyrosine kinase inhibitors, blocked HBV replication. Derivatives of cyclosporine, i.e., cyclosporine A, cyclosporine H, and SDZ NIM811, which block cytosolic calcium signaling and specifically the mitochondrial permeability transition pore (SDZ NIM811), also impaired HBV replication. Treatment of cells with compounds that increase cytosolic calcium levels by a variety of mechanisms rescued replication of an HBx-deficient HBV mutant. Transcription of viral RNA and production of viral capsids were only minimally affected by these treatments. These results define a functional signaling circuit for HBV replication that includes calcium signaling and activation of cytosolic signaling pathways involving Src kinases, and they suggest that these pathways are stimulated by HBx acting on the mitochondrial transition pore.
Tax and Overlapping Rex Sequences Do Not Confer the Distinct Transformation Tropisms of Human T-Cell Leukemia Virus Types 1 and 2
Authors: Ye Jianxin , Xie Li , Green Patrick L.
Published: June 2003

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are distinct oncogenic retroviruses that infect several cell types but display their biological and pathogenic activity only in T cells. Previous studies have indicated that in vivo HTLV-1 has a preferential tropism for CD4
+
T cells, whereas HTLV-2 in vivo tropism is less clear but appears to favor CD8
+
T cells. Both CD4
+
and CD8
+
T cells are susceptible to HTLV-1 and HTLV-2 infection in vitro, and HTLV-1 has a preferential immortalization and transformation tropism of CD4
+
T cells, whereas HTLV-2 immortalizes and transforms primarily CD8
+
T cells. The molecular mechanism that determines this tropism of HTLV-1 and HTLV-2 has not been determined. HTLV-1 and HTLV-2 carry the
tax
and
rex
transregulatory genes in separate but partially overlapping reading frames. Since Tax has been shown to be critical for cellular transformation in vitro and interacts with numerous cellular processes, we hypothesized that the viral determinant of transformation tropism is encoded by
tax
. Using molecular clones of HTLV-1 (Ach) and HTLV-2 (pH6neo), we constructed recombinants in which
tax
and overlapping
rex
genes of the two viruses were exchanged. p19 Gag expression from proviral clones transfected into 293T cells indicated that both recombinants contained functional Tax and Rex but with significantly altered activity compared to the wild-type clones. Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with peripheral blood mononuclear cells. Both recombinants were competent to transform T lymphocytes with an efficiency similar to that of the parental viruses. Flow cytometry analysis indicated that HTLV-1 and HTLV-1/TR2 had a preferential tropism for CD4
+
T cells and that HTLV-2 and HTLV-2/TR1 had a preferential tropism for CD8
+
T cells. Our results indicate that
tax/rex
in different genetic backgrounds display altered functional activity but ultimately do not contribute to the different in vitro transformation tropisms. This first study with recombinants between HTLV-1 and HTLV-2 is the initial step in elucidating the different pathobiologies of HTLV-1 and HTLV-2.
Infectivity and Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1 Variants Isolated during Primary Infection
Authors: Simon Viviana , Padte Neal , Murray Deya , Vanderhoeven Jeroen , Wrin Terri , Parkin Neil , Di Mascio Michele , Markowitz Martin
Published: June 2003

It is believed that replication capacity is an important determinant of human immunodeficiency virus type 1 (HIV-1) pathogenicity and transmissibility. To explore this, we conducted a comprehensive analysis of the replication properties of nine drug-resistant and nine drug-susceptible viral isolates derived from patients with primary HIV-1 infection. Viral isolates were tested for single-cycle infectivity in the GHOST cell line. The infectivity of isolates carrying resistance-associated mutations was significantly higher than that of drug-susceptible isolates. Additionally, the growth kinetics of these isolates were determined in CD4
+
T lymphocytes. Drug-resistant isolates replicated as well as drug-susceptible viruses. Insertion of the resistance-conferring regions into an NL4-3-based molecular background resulted in chimeras that displayed a modest but significant reduction in replication capacity compared to the drug-susceptible chimeric viruses. Of note, two multidrug-resistant isolates and one protease inhibitor-resistant isolate displayed higher rates of infectivity and growth kinetics than the other drug-resistant or drug-susceptible isolates. These distinct replicative features, however, were not seen in the corresponding chimeras, indicating that changes within the C-terminal region of Gag as well as within the protease and reverse transcriptase genes contribute to but are not sufficient for the level of compensatory adaptation observed. These findings suggest that some drug-resistant viruses isolated during primary infection possess unique adaptive changes that allow for both high viral replication capacity and resistance to one or more classes of antiretroviral drugs. Further studies are needed to elucidate the precise regions that are essential for these characteristics.
Quantitative Analysis of Long-Term Virus-Specific CD8+-T-Cell Memory in Mice Challenged with Unrelated Pathogens
Authors: Liu Haiyan , Andreansky Samita , Diaz Gabriela , Turner Stephen J. , Wodarz Dominik , Doherty Peter C.
Published: June 2003

The consequences for the long-term maintenance of virus-specific CD8+-T-cell memory have been analyzed experimentally for sequential respiratory infections with readily eliminated (influenza virus) and persistent (gammaherpesvirus 68 [γHV68]) pathogens. Sampling a broad range of tissue sites established that the numbers of CD8+T cells specific for the prominent influenza virus DbNP366epitope were reduced by about half in mice that had been challenged 100 days previously with γHV68, though the prior presence of a large CD8+DbNP366+population caused no selective defect in the γHV68-specific CD8+Kbp79+response. Conversely, mice that had been primed and boosted to generate substantial γHV68-specific CD8+Dbp56+populations did not show any decrease in prevalence for this set of CD8+memory cytotoxic T lymphocytes (CTL) at 200 days after respiratory exposure to an influenza A virus. However, in both experiments, the total magnitude of the CD8+-T-cell pool was significantly diminished in those that had been infected with γHV68 and the influenza A virus. The broader implications of these findings, especially under conditions of repeated exposure to unrelated pathogens, are explored with a mathematical model which emphasizes that the immune effector and memory “phenome” is a function of the overall infection experience of the individual.
Mutations within the ADP (E3-11.6K) Protein Alter Processing and Localization of ADP and the Kinetics of Cell Lysis of Adenovirus-Infected Cells
Authors: Tollefson Ann E. , Scaria Abraham , Ying Baoling , Wold William S. M.
Published: June 2003

ADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus infection and is required for efficient lysis of infected cells and release of viral progeny at the end of the viral replication cycle. ADP is a type III bitopic NendoCexonuclear membrane and Golgi glycoprotein that is produced at high levels in late adenovirus infection (>24 h postinfection). We show pulse-chase and other studies indicating that ADP undergoes a complex process of N- and O-linked glycosylation and proteolytic cleavage. In order to further characterize ADP, a series of 23 deletion and point mutations has been constructed in the adenovirus serotype 2adpgene and then built into a wild-type adenovirus background. These mutants were analyzed for processing and intracellular localization of ADP. Mutation of the single predicted N glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich in serine and threonine residues reduced O glycosylation. In general, mutations within the lumenal domain of ADP resulted in lower protein stability; immunofluorescence assays indicated that these ADPs were primarily present in the Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic domain of ADP showed normal glycosylation patterns and protein abundance for ADP, but the protein was often found throughout cellular membranes rather than being localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus mutants were analyzed by cell viability assays to determine the kinetics of cell lysis following infection of human A549 cells. In general, viruses with mutations within the lumenal domain of ADP display greatly reduced efficiencies of cell lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of ADP retain much of their ability to lyse infected cells.
Kunjin Virus Replicon Vectors for Human Immunodeficiency Virus Vaccine Development
Authors: Harvey Tracey J. , Anraku Itaru , Linedale Richard , Harrich David , Mackenzie Jason , Suhrbier Andreas , Khromykh Alexander A.
Published: June 2003

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8
+
T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8
+
T-cell responses. Two immunizations with KUN
gag
replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of ≥1:10,000. Immunization with KUN
gag
replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8
+
T-cell responses, with one immunization of KUN
gag
VLPs inducing 4.5-fold-more CD8
+
T cells than the number induced after immunization with recombinant vaccinia virus carrying the
gag
gene (rVV
gag
). Two immunizations with KUN
gag
VLPs also provided significant protection against challenge with rVV
gag
. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8
+
T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.
Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication
Authors: Liu Wen Jun , Chen Hua Bo , Khromykh Alexander A.
Published: June 2003

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in
trans
by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in
trans
by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.
An Infectious Clone of Woolly Monkey Hepatitis B Virus
Authors: Lanford Robert E. , Chavez Deborah , Barrera Azeneth , Brasky Kathleen M.
Published: June 2003

Members of the
Hepadnaviridae
family have been isolated from birds, rodents, and primates. A new hepadnavirus isolated from the woolly monkey, a New World primate, is phylogenetically distinct from other primate isolates. An animal model has been established for woolly monkey hepatitis B virus (WMHBV) by using spider monkeys, since woolly monkeys are endangered. In this study, a greater-than-genome length construct was prepared without amplification by using covalently closed circular DNA extracted from the liver of an infected woolly monkey. Transfection of the human liver cell line Huh7 with WMHBV DNA resulted in the production of viral transcripts, DNA replicative intermediates, and secreted virions at levels similar to those obtained with an infectious human HBV clone, demonstrating that the host range restriction of WMHBV is not at the level of genome replication. WMHBV particles from the medium of transfected cultures initiated an infection in a spider monkey similar to that obtained with virions derived from woolly monkey serum. In an attempt to adapt the virus for higher levels of replication in spider monkeys, immunosuppressed and newborn animals were inoculated. Neither procedure produced persistent infections, and the level of viral replication remained several logs lower than that observed in persistently infected woolly monkeys. These data demonstrate the production of an infectious clone for WMHBV and extend the characterization of the spider monkey animal model.
Drug Resistance Patterns of Recombinant Herpes Simplex Virus DNA Polymerase Mutants Generated with a Set of Overlapping Cosmids and Plasmids
Authors: Bestman-Smith Julie , Boivin Guy
Published: June 2003

Herpes simplex virus (HSV) DNA polymerase (Pol) mutations can confer resistance to all currently available antiherpetic drugs. However, discrimination between mutations responsible for drug resistance and those that are part of viral polymorphism can be difficult with current methodologies. A new system is reported for rapid generation of recombinant HSV type 1 (HSV-1) DNA Pol mutants based on transfection of a set of overlapping viral cosmids and plasmids. With this approach, twenty HSV-1 recombinants with single or dual mutations within the DNA
pol
gene were successfully generated and subsequently evaluated for their susceptibilities to acyclovir (ACV), foscarnet (FOS), cidofovir (CDV), and adefovir (ADV). Mutations within DNA Pol conserved regions II (A719T and S724N), VI (L778M, D780N, and L782I), and I (F891C) were shown to induce cross-resistance to ACV, FOS, and ADV, with two of these mutations (S724N and L778M) also conferring significant reduction in CDV susceptibility. Mutant F891C was associated with the highest levels of resistance towards ACV and FOS and was strongly impaired in its replication capacity. One mutation (D907V) lying outside of the conserved regions was also associated with this ACV-, FOS-, and ADV-resistant phenotype. Some mutations (K522E and Y577H) within the δ-region C were lethal, whereas others (P561S and V573M) induced no resistance to any of the drugs tested. Recombinants harboring mutations within conserved regions V (N961K) and VII (Y941H) were resistant to ACV but susceptible to FOS. Finally, mutations within conserved region III were associated with various susceptibility profiles. This new system allows a rapid and accurate evaluation of the functional role of various DNA Pol mutations, which should translate into improved management of drug-resistant HSV infections.
Involvement of Lipids in Different Steps of the Flavivirus Fusion Mechanism
Authors: Stiasny Karin , Koessl Christian , Heinz Franz X.
Published: June 2003

Flavivirus membrane fusion is triggered by acidic pH and mediated by the major envelope protein E. A structurally very similar fusion protein is found in alphaviruses, and these molecules are designated class II viral fusion proteins. In contrast to that of flaviviruses, however, alphavirus fusion has been shown to be absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane, suggesting significant differences in the fusion protein-membrane interactions that lead to fusion. With the flavivirus tick-borne encephalitis virus (TBEV), we have therefore conducted a study on the lipid requirements of viral fusion with liposomes and on the processes preceding fusion, specifically, the membrane-binding step and the fusion-associated oligomeric switch from E protein dimers to trimers. As with alphaviruses, cholesterol had a strong promoting effect on membrane binding and trimerization of the fusion protein, and—as shown by the use of cholesterol analogs—the underlying interactions involve the 3β-hydroxyl group at C-3 in both viral systems. In contrast to alphaviruses, however, these effects are much less pronounced with respect to the overall fusion of TBEV and can only be demonstrated when fusion is slowed down by lowering the temperature. The data presented thus suggest the existence of structurally related interactions of the flavivirus and alphavirus fusion proteins with cholesterol in the molecular processes required for fusion but, at the same time, point to significant differences between the class II fusion machineries of these viruses.
The Tailless Icosahedral Membrane Virus PRD1 Localizes the Proteins Involved in Genome Packaging and Injection at a Unique Vertex
Authors: Gowen Brent , Bamford Jaana K. H. , Bamford Dennis H. , Fuller Stephen D.
Published: June 2003

The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection.
Increased Thymic Output during Acute Measles Virus Infection
Authors: Permar Sallie R. , Moss William J. , Ryon Judith J. , Douek Daniel C. , Monze Mwaka , Griffin Diane E.
Published: June 2003

Measles virus infects thymic epithelia, induces a transient lymphopenia, and impairs cell-mediated immunity, but thymic function during measles has not been well characterized. Thirty Zambian children hospitalized with measles were studied at entry, hospital discharge, and at 1-month follow-up and compared to 17 healthy children. During hospitalization, percentages of naïve (CD62L+, CD45RA+) CD4+and CD8+T lymphocytes decreased (P= 0.01 for both), and activated (HLA-DR+, CD25+, or CD69+) CD4+and CD8+T lymphocytes increased (P= 0.02 and 0.03, respectively). T-cell receptor rearrangement excision circles (TRECs) in measles patients were increased in CD8+T cells at entry compared to levels at hospital discharge (P =0.02) and follow-up (P =0.04). In CD4+T cells, the increase in TRECS occurred later but was more sustained. At discharge, TRECs in CD4+T cells (P= 0.05) and circulating levels of interleukin-7 (P= 0.007) were increased compared to control values and remained elevated for 1 month, similar to observations in two measles virus-infected rhesus monkeys. These findings suggest that a decrease in thymic output is not the cause of the lymphopenia and depressed cellular immunity associated with measles.
Fitness of a Turnip Crinkle Virus Satellite RNA Correlates with a Sequence-Nonspecific Hairpin and Flanking Sequences That Enhance Replication and Repress the Accumulation of Virions
Authors: Sun Xiaoping , Simon Anne E.
Published: June 2003

satC, a satellite RNA associated with
Turnip crinkle virus
(TCV), enhances the ability of the virus to colonize plants by interfering with stable virion accumulation (F. Zhang and A. E. Simon, unpublished data). Previous results suggested that the motif1-hairpin (M1H), a replication enhancer on minus strands, forms a plus-strand hairpin flanked by CA-rich sequence that may be involved in enhancing systemic infection (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003). In this study, sequence and structural requirements of the M1H were further assayed by replacing the 28-base M1H with 10 random bases and then subjecting the pool of satellite RNA to functional selection in plants. Unlike previous results with 28-base replacement sequences (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003), only a few of the 10-base SELEX (systematic evolution of ligands by exponential enrichment) assay winners contained short motifs in their minus-sense orientation that were similar to TCV replication elements. However, all second- and third-round winning replacement sequences folded into hairpins flanked by CA-rich sequence predicted to be more stable on plus strands than minus strands. Plus strands of several of the most fit satellite RNAs contained insertions of CA-rich sequence at the base of their hairpins whose presence correlated with enhanced replication and reduced detection of virions. Deletion of the M1H resulted in no detectable virions despite very low satellite accumulation. These results support the hypothesis that a sequence-nonspecific plus-strand hairpin brings together flanking CA-rich sequences in the M1H region that confers fitness to satC by reducing the accumulation of stable virions.
Transmissible Gastroenteritis Coronavirus Packaging Signal Is Located at the 5′ End of the Virus Genome
Authors: Escors David , Izeta Ander , Capiscol Carmen , Enjuanes Luis
Published: June 2003

To locate the transmissible gastroenteritis coronavirus (TGEV) packaging signal, the incorporation of TGEV subgenomic mRNAs (sgmRNAs) into virions was first addressed. TGEV virions were purified by three different techniques, including an immunopurification using an M protein-specific monoclonal antibody. Detection of sgmRNAs in virions by specific reverse transcription-PCRs (RT-PCRs) was related to the purity of virus preparations. Interestingly, virus mRNAs were detected in partially purified virus but not in virus immunopurified using stringent conditions. Analyses by quantitative RT-PCR confirmed that virus mRNAs were not present in highly purified preparations. Lack of sgmRNA encapsidation was probably due to the absence of a packaging signal (Ψ) within these mRNAs. This information plus that from the encapsidation of a collection of TGEV-derived minigenomes suggested that Ψ is located at the 5′ end of the genome. To confirm that this was the case, a set of minigenomes was expressed that included an expression cassette for an mRNA including the β-glucuronidase gene (GUS) plus variable sequence fragments from the 5′ end of the virus genome potentially including Ψ. Insertion of the first 649 nucleotides (nt) of the TGEV genome led to the specific encapsidation of the mRNA, indicating that a Ψ was located within this region which was absent from all of the other virus mRNAs. The presence of this packaging signal was further confirmed by showing the expression and rescue of the mRNA including the first 649 nt of the TGEV genome under control of the cytomegalovirus promoter in TGEV-infected cells. This mRNA was successfully amplified and encapsidated, indicating that the first 649 nt of TGEV genome also contained the 5′
cis
-acting replication signals. The encapsidation efficiency of this mRNA was about 30-fold higher than the genome encapsidation efficiency, as estimated by quantitative RT-PCR. In contrast, viral mRNAs presented significantly lower encapsidation efficiencies (about 100-fold) than those of the virus genome, strongly suggesting that TGEV mRNAs in fact lacked an alternative TGEV Ψ.
Sendai Virus Targets Inflammatory Responses, as Well as the Interferon-Induced Antiviral State, in a Multifaceted Manner
Authors: Strähle Laura , Garcin Dominique , Le Mercier Philippe , Schlaak Joerg F. , Kolakofsky Daniel
Published: June 2003

We have used cDNA arrays to compare the activation of various cellular genes in response to infection with Sendai viruses (SeV) that contain specific mutations. Three groups of cellular genes activated by mutant SeV infection, but not by wild-type SeV, were identified in this way. While some of these genes are well known interferon (IFN)-stimulated genes, others, such as those for interleukin-6 (IL-6) and IL-8, are not directly induced by IFN. The gene for beta IFN (IFN-β), which is critical for initiating an antiviral response, was also specifically activated in mutant SeV infections. The SeV-induced activation of IFN-β was found to depend on IFN regulatory factor 3, and the activation of all three cellular genes was independent of IFN signaling. Mutations that disrupt four distinct elements in the SeV genome (the leader RNA, two regions of the C protein, and the V protein) all lead to enhanced levels of IFN-β mRNA, and at least three of these viral genes also appear to be involved in preventing activation of IL-8. Our results suggest that SeV targets the inflammatory and adaptive immune responses as well as the IFN-induced intracellular antiviral state by using a multifaceted approach.
Frequent Recovery and Broad Genotype 2 Diversity Characterize Hepatitis C Virus Infection in Ghana, West Africa
Authors: Candotti Daniel , Temple Jillian , Sarkodie Francis , Allain Jean-Pierre
Published: June 2003

Hepatitis C virus (HCV) infection is thought to mostly become chronic and rarely resolves. HCV infection was serologically screened in 4,984 samples from Ghanaian blood donors, and 1.3% prevalence was found. At least 53% of confirmed anti-HCV carriers had no detectable viral RNA and were considered to have cleared the virus and recovered from the infection. Confirmation was authenticated by the presence of antibodies specific to at least two viral antigens, mostly NS3 and E2. Reactivity to HCV core antigens was lower in Ghanaian than United Kingdom blood donors. The minority of chronically infected donors carried a viral load significantly lower than an unselected comparative group of United Kingdom blood donors (2.5 × 105versus 2.9 × 106IU/ml;P= 0.004). HCV genotype 2 was largely predominant (87%). Sequence clustering was similarly broad in the E1/E2 and NS5 regions. The phylogenetic diversity and the incapacity to distinguish subtypes within genotype 2 in our and others' West African strains suggested that West Africa may be the origin of HCV genotype 2. The genetic diversity extended to the identification of strains clearly separated from known subtypes of genotype 2 and genotype 1. One strain appears to be part of a new HCV genotype. HCV infection in Ghana is characterized by a high rate of recovery and the predominance of broadly divergent genotype 2 strains.