bluuebunny/crossref_metadata_2025 huggingface dataset:
Some novel space propulsion systems
Authors: Bishop Forrest
Published: April 2003

Several spacecraft beam‐propulsion concepts are introduced. “Mesoparticle beam propulsion” uses a collimated beam of mesoscopic particles, very roughly on the order of a nanogram mass each. Molecular nanotechnologies may permit the inclusion of entire guidance systems in each particle. “Micro Lightsails for beam propulsion” proposes matter‐beams composed of small, thin film lightsails with nanoscale components. Pushing a spacecraft with small, high velocity lightsails may be currently viable. “Ultracold matter beam generators” are proposed as a new type of space‐based particle‐beam. Design‐variants include a laser‐cooled thermal jet and a laser‐cooled, neutralized‐ion beam. Possible uses include the shipment of condensed, ultracold matter through space, the formation of an “artificial aerobraking corridor”, and beam‐propulsion for micro and nanospacecraft.
Design, construction and testing of a low‐cost hybrid rocket motor
Authors: Mungas Greg S. , Das Debendra K. , Kulkarni Devdatta
Published: April 2003

Small hybrid rocket motors using solid propellant and gaseous oxidizer are becoming increasingly popular as a propulsion device. This paper describes the development of a one‐dimensional flow model for the design of a small rocket motor. Combustion of polyethylene as solid propellant with oxygen is used as a candidate hybrid fuel to test and evaluate the performance of this hybrid system. To assess the performance under different operating conditions, a computer program has been developed, which facilitates inputs to be varied and effects assessed. A system of governing equations is summarized in the main body of this paper and is numerically solved by the computer program. The results of the modelling are then used to design and build a small low‐cost rocket motor for experimental verification. Therefore, the materials presented herein could be used in the future design of hybrid rocket motors.
New forms of work organization: issues and challenges
Authors: Cartwright Susan
Published: April 2003

The changing nature of work and the emergence of new forms of work organization present particular challenges to leadership and management. This special issue examines some of the challenges and issues in the relationship between technology, stress and satisfaction within call centre environments, the problems of remote leadership and the rise in contingent workforce.
Temporary but seeking permanence: a study of New Zealand temps
Authors: Hardy Derrylea J. , Walker Robyn J.
Published: April 2003

Temporary employment, colloquially referred to as temping, is relatively new to New Zealand. Research is divided as to where the benefits of temporary employment lie – with the agencies, the employers, or the “temps” themselves. We review the literature on temporary employment, with particular reference to New Zealand. We also present some findings of a New Zealand survey of agency‐employed temps. We present the demographic profile of this population group, their reasons for engaging in temporary employment, and their preferred type of employment. Most temporary employees in this study preferred permanent work, and undertook temporary work as a step towards more permanent employment. We discuss the implications of the research findings for organisations that are increasingly being faced with issues surrounding the management of temporary employees.
Leadership in a global virtual team: an action learning approach
Authors: Pauleen David J.
Published: April 2003

This paper presents an action learning‐based case study investigating how one virtual team leader coped with a number of issues associated with the completion of a critical organizational task in a virtual environment. Exploring this one virtual team leader’s experiences and strategies opens a window for both practitioners and researchers. The insights shared by the team leader may lead to practical virtual team leadership strategies as well as avenues to research into virtual team leadership. The paper concludes with a discussion highlighting the important practical lessons learned about virtual team leadership and implications for practitioners and researchers.
A comparison between needed competencies of academic librarians and LIS curricula in Pakistan
Authors: Mahmood Khalid
Published: April 2003

Rapidly growing academic libraries are the major consumers of the product of seven LIS schools in Pakistan. The changing environment of academic life demands new competencies in academic librarians. This paper reviews the literature on the competencies needed for academic librarians in the Asia/Pacific region and discusses the changing environment of academic librarianship in Pakistan. It provides a list of competencies needed for entry‐level academic librarians, first prepared on the basis of a literature review and, then, validated by 70 chief librarians of universities and postgraduate level colleges from the public and private sector. The validated list of competencies is compared with the curricula of LIS programs. The paper highlights the deficiencies in the curricula and their implementation and recommendations are given to improve the situation.
Monitoring scientific publications over the WWW
Authors: Vu Ho Le , Cheung Hui Siu , Fong A.C.M.
Published: April 2003

The World Wide Web has become an important medium for disseminating scientific publications. To make their research works accessible to other researchers, most research institutions list their publications in an index page that sometimes includes links to online versions of the publications. As the index page is usually updated whenever new research papers are published, researchers need to check these index pages frequently in order to know of any new publications published in the targeted Web site or page. This manual publication monitoring process is tedious and time‐consuming. In this paper, a publication monitoring system, known as PubWatcher, is proposed to automatically track Web publications from user‐specified Web sites or pages. A publication extraction technique has been developed to extract publication information listed in the index pages of the monitored Web sites and pages.
Academic librarians’ perception of Lexis‐Nexis
Authors: Oulanov Alexei , Pajarillo Edmund J.Y.
Published: April 2003

This research is an examination of the perception by academic librarians of the Web version of Lexis‐Nexis academic datafile. Specifically, the investigation utilizes three parameters, namely: user‐effort, measures of effectiveness and retrieval techniques. These criteria were deemed essential when analyzing the performance of this information data source in the perspective of academic information specialists, results of which may be useful in its future redesigns and in the scrutiny of the nature and quality of reference services in a particular setting.
Journal publishing is ripe for change
Authors: Falk Howard
Published: April 2003

Focuses on the changes taking place in the world of scholarly publishing. Outlines the activities and future plans of a selection of new open distribution publishing sources.
A research and skills training framework for values‐driven leadership
Authors: Mussig Dennis James
Published: April 2003

Presents a framework for research into the exercise of values through organisational leadership. Leadership as a relationship and as a behaviour has values as a core dimension. Values underpin trust and credibility upon which relationships are built and sustained and inform the exercise of logical reasoning, communication, strategic thinking, and use of power. The proposed framework draws on work on emotional intelligence, spiritual intelligence and management behavioural competencies. It does not seek to identify a particular value set as optimal for leadership, but rather to identify the skills base required for the exercise of values through leadership. Whilst the contribution to leadership of senior executive coaching is increasingly recognised, extending this to line managers will, in practice, require tools and programs built around a testable research framework. Such a framework will assist HR managers to argue the strategic case for investment in values leadership training skills for supervisory and middle management personnel.
Tourism, linkages, and economic development in Jamaica
Authors: Karagiannis Nikolaos
Published: April 2003

This article offers, briefly, a production‐oriented development framework for Jamaica, based on growth‐promoting linkages between tourism, commodity production sectors, and complementary and related service industries. These linkages can boost the Jamaican endogenous competency and industrial competitiveness, while improving the country’s macroeconomic performance. Alternative development policy considerations are also within the scope of this article.
Terrorism and tourism: Bahamas and Jamaica fight back
Authors: Pratt Godfrey
Published: April 2003

This study highlights the fragility of the tourism industry in developing countries. The overdependence of the Bahamas on the tourism industry, for economic development, is brought into focus here. The adroit efforts of the directors of tourism for both the Bahamas and Jamaica are highlighted. Both governments saw the need to immediately fund a revised marketing and advertising campaign, in the aftermath of 9/11. An important factor in the recovery of both destinations is the extent to which a coalition of public and private sector tourism stakeholders in both countries committed themselves to resolving the crisis quickly at hand. The commitment of the public and private sector in these countries, to safeguarding and promoting this industry probably positively correlated to the importance of the industry to the economy of the countries, as evidenced by tourism’s contribution to their GDP.
Environmental management systems for internal corporate environmental benchmarking
Authors: Matthews Deanna H.
Published: April 2003

Corporate environmental benchmarking is difficult with the range and inconsistency of environmental information available, even from facilities within the same firm. Environmental management systems can assist firms in organizing internal corporate benchmarking efforts. They attempt to capture environmental impacts from activities throughout a facility under a single system and generally follow traditional benchmarking cycles of plan, do, check, and act. However, the systems lack important features that enable benchmarking. Based on a critical analysis of environmental management systems, the article recommends minor changes to extend environmental management systems for corporate environmental benchmarking. Consistent goals should be encouraged at all facilities to produce common metrics. Procedures should require data collection and reporting to a central office. Management review should monitor performance and determine where leading facilities can transfer better processes to lagging facilities.
Theory and practice of environmental benchmarking in a major consumer electronics company
Authors: Boks Casper , Stevels Ab
Published: April 2003

Environmental benchmarking has, since 1997, been the basis of many ecodesign‐related activities at both Delft University of Technology and Philips Consumer Electronics in Eindhoven, The Netherlands. Cooperative efforts have led to a robust, reproducible and practical environmental benchmark method. The method is based on the assessment of the five focal areas: energy, material and weight, packaging, potentially toxic substances, and recyclability. The generation and prioritisation of green improvement options is done by addressing consumer and societal feasibility as well as technical and financial feasibility. Ongoing research continuously stimulates the methodology and practical implementation. This has created a tremendous awareness in the Philips Consumer Electronics organisation regarding product‐related environmental matters, because the method is embedded in an overall strategy that considers the interests of all internal and external stakeholders.
Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages
Authors: Ménard Carine , Wagner Markus , Ruzsics Zsolt , Holak Karina , Brune Wolfram , Campbell Ann E. , Koszinowski Ulrich H.
Published: April 2003

The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.
Intranasal Vaccination Using Interleukin-12 and Cholera Toxin Subunit B as Adjuvants To Enhance Mucosal and Systemic Immunity to Human Immunodeficiency Virus Type 1 Glycoproteins
Authors: Albu Diana I. , Jones-Trower Agnes , Woron Amy M. , Stellrecht Kathleen , Broder Christopher C. , Metzger Dennis W.
Published: April 2003

We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants. It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n. immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes. Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120. However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6. Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.
L-Particle Production during Primary Replication of Pseudorabies Virus in the Nasal Mucosa of Swine
Authors: Alemañ Nuria , Quiroga María Isabel , López-Peña Mónica , Vázquez Sonia , Guerrero Florentina H. , Nieto José M.
Published: April 2003

Different tissue culture cell lines infected with a number of alphaherpesviruses produce, in addition to virions, light particles (L particles). L particles are composed of the envelope and tegument components of the virion but totally lack the proteins of the capsid and the virus genome; therefore, they are noninfectious. In this electron microscopy report, we show that L particles are produced during primary replication of the alphaherpesvirus pseudorabies virus (PRV) in the nasal mucosa of experimentally infected swine, its natural host. Although PRV infected different types of cells of the respiratory and olfactory mucosae, PRV L particles were found to be produced exclusively by epithelial cells and fibroblasts. We observed that formation of noninfectious particles occurred by budding of condensed tegument at the inner nuclear membrane and at membranes of cytoplasmic vesicles, resulting in intracisternal and intravesicular L particles, respectively. Both forms of capsidless particles were clearly distinguishable by the presence of prominent surface projections on the envelope and the higher electron density of the tegument, morphological features which were only observed in intravesicular L particles. Moreover, intravesicular but not intracisternal L particles were found to be released by exocytosis and were also identified extracellularly. Comparative analysis between PRV virion and L-particle morphogenesis indicates that both types of virus particles share a common intracellular pathway of assembly and egress but that they show different production patterns during the replication cycle of PRV.
A Novel Genetic Pathway of Human Immunodeficiency Virus Type 1 Resistance to Stavudine Mediated by the K65R Mutation
Authors: García-Lerma J. Gerardo , MacInnes Hamish , Bennett Diane , Reid Patrick , Nidtha Soumya , Weinstock Hillard , Kaplan Jonathan E. , Heneine Walid
Published: April 2003

Stavudine (d4T) and zidovudine (AZT) are thymidine analogs widely used in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected persons. Resistance to d4T is not fully understood, although the selection of AZT resistance mutations in patients treated with d4T suggests that both drugs have similar pathways of resistance. Through the analysis of genotypic changes in nine recombinant viruses cultured with d4T, we identified a new pathway for d4T resistance mediated by K65R, a mutation not selected by AZT. Passaged viruses were derived from treatment-naïve persons or HIV-1HXB2and had wild-type reverse transcriptase (RT) or T215C/D mutations. K65R was selected in seven viruses and was associated with a high level of enzymatic resistance to d4T-triphosphate (median, 16-fold; range, 5- to 48-fold). The role of K65R in d4T resistance was confirmed in site-directed mutants generated in three different RT backgrounds. Phenotypic assays based on recombinant single-cycle replication or a whole-virus multiple replication cycle were unable to detect d4T resistance in d4T-selected mutants with K65R but detected cross-resistance to other nucleoside RT inhibitors. Four of the six viruses that had 215C/D mutations at baseline acquired the 215Y mutation alone or in association with K65R. Mutants having K65R and T215Y replicated less efficiently than viruses that had T215Y only, suggesting that selection of T215Y in patients treated with d4T may be favored. Our results demonstrate that K65R plays a role in d4T resistance and indicate that resistance pathways for d4T and AZT may not be identical. Biochemical analysis and improved replication assays are both required for a full phenotypic characterization of resistance to d4T. These findings highlight the complexity of the genetic pathways of d4T resistance and its phenotypic expression.
In Vitro and In Ovo Expression of Chicken Gamma Interferon by a Defective RNA of Avian Coronavirus Infectious Bronchitis Virus
Authors: Hackney Karen , Cavanagh Dave , Kaiser Pete , Britton Paul
Published: April 2003

Coronavirus defective RNAs (D-RNAs) have been used for site-directed mutagenesis of coronavirus genomes and for expression of heterologous genes. D-RNA CD-61 derived from the avian coronavirus infectious bronchitis virus (IBV) was used as an RNA vector for the expression of chicken gamma interferon (chIFN-γ). D-RNAs expressing chIFN-γ were shown to be capable of rescue, replication, and packaging into virions in a helper virus-dependent system following electroporation of in vitro-derived T7 RNA transcripts into IBV-infected cells. Secreted chIFN-γ, under the control of an IBV transcription-associated sequence derived from gene 5 of the Beaudette strain, was expressed from two different positions within CD-61 and shown to be biologically active. In addition, following infection of 10-day-old chicken embryos with IBV containing D-RNAs expressing chIFN-γ, the allantoic fluid was shown to contain biologically active chIFN-γ, demonstrating that IBV D-RNAs can express heterologous genes in vivo.
Disruption of Gammaherpesvirus 68 Gene 50 Demonstrates that Rta Is Essential for Virus Replication
Authors: Pavlova Iglika V. , Virgin Herbert W. , Speck Samuel H.
Published: April 2003

Gammaherpesvirus pathogenesis is dependent on the ability of these viruses to establish a lifelong latent infection and the ability to reactivate from latency. Immediate-early genes of theses viruses are thought to be critical regulators of lytic replication and reactivation from latency. The gene 50-encoded Rta is the only immediate-early gene product that appears to be conserved among all characterized gammaherpesviruses. Previous studies have demonstrated that, in Epstein-Barr virus (EBV), Kaposi's sarcoma-associated virus, and gammaherpesvirus 68 (γHV68, also referred to as murine gammaherpesvirus 68), ectopic expression of Rta in latently infected cell lines can lead to induction of the viral cycle. Recently, studies employing null mutants of EBV have provided a formal demonstration that both Rta and the BZLF1 gene product, Zta, the two EBV immediate-early gene products, are essential for EBV replication. Here we generate and characterize a gene 50-null mutant γHV68 and demonstrate that the gene 50 product Rta is essential for virus replication. Providing γHV68 Rta in
trans
was sufficient to restore replication of the gene 50-null virus. Notably, Rta expressed from the spliced form of the gene 50 transcript was sufficient to complement growth of the gene 50-null virus. In addition, we provide evidence that loss of Rta expression leads to a complete defect in viral DNA replication and a significant defect in late antigen expression. This work lays the foundation for characterizing the role of Rta in γHV68 chronic infection of mice.
Human Herpesvirus 8-Encoded vGPCR Activates Nuclear Factor of Activated T Cells and Collaborates with Human Immunodeficiency Virus Type 1 Tat
Authors: Pati Shibani , Foulke James S. , Barabitskaya Oxana , Kim Jynho , Nair B. C. , Hone David , Smart Jennifer , Feldman Ricardo A. , Reitz Marvin
Published: April 2003

Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi's sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-κB, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidylinositol 3-kinase-Akt-glycogen synthetase kinase 3 (PI3-K/Akt/GSK-3) pathway and resulted in increased expression of NF-AT-dependent cell surface molecules (CD25, CD29, Fas ligand), proinflammatory cytokines (interleukin-2 [IL-2], IL-4), and proangiogenic factors (granulocyte-macrophage colony-stimulating factor GMCSF and TNFα). vGPCR expression also increased endothelial cell-T-cell adhesion. Although infection with HHV-8 is necessary to cause KS, coinfection with human immunodeficiency virus type 1 (HIV-1), in the absence of antiretroviral suppressive therapy, increases the risk of KS by many orders of magnitude. NF-AT and NF-κB activation by vGPCR was greatly increased by the HIV-1 Tat protein, although Tat alone had little effect on NF-AT. The enhancement of NF-AT by Tat appears to be mediated through collaborative stimulation of the PI3-K/Akt/GSK-3 pathway by vGPCR and Tat. Our data further support the idea that vGPCR contributes to the pathogenesis of KS by a paracrine mechanism and, in addition, provide the first evidence of collaboration between an HIV-1 protein and an HHV-8 protein.
Comparison of Adjuvant Efficacy of Herpes Simplex Virus Type 1 Recombinant Viruses Expressing TH1 and TH2 Cytokine Genes
Authors: Osorio Yanira , Ghiasi Homayon
Published: April 2003

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-γ) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-γ or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-γ or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-γ. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a TH1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4+and CD8+T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-γ production by CD4+and CD8+T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-γ-expressing virus. Taken together, our results suggest that, in contrast to IFN-γ which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy.
Infection-Triggered Regulatory Mechanisms Override the Role of STAT 4 in Control of the Immune Response to Influenza Virus Antigens
Authors: Bot A. , Rodrigo E. , Wolfe T. , Bot S. , Von Herrath M. G.
Published: April 2003

Accurate control of the balance of the T1 and T2 cells during antiviral immunity is essential for optimizing immune effector functions and for avoiding potentially severe immunopathology. We examined the in vivo role of the signal transducer and activator of transcription (STAT) 4 in regulating the T1/T2 balance during the response to live influenza virus and isolated viral proteins. We found that the differentiation of gamma interferon (IFN-γ)-producing Th1 and Tc1 cells after inoculation of live virus occurred independently of STAT 4 expression. Influenza virus-specific T2 and Tc2 responses were well controlled in such STAT 4-deficient mice unless IFN-γ was eliminated as well. In contrast, the STAT 4-dependent signaling pathway played a more essential role in regulating the T1/T2 balance after immunization with viral proteins and, in particular, inactivated nonreplicating virus. Pulmonary infection was cleared even in the absence of both functional STAT 4 genes and functional IFN-γ genes because virus-neutralizing antibodies were still generated, consistent with a substantial redundancy in different antiviral effector pathways. Thus, replicating agents such as live influenza virus can elicit IFN-γ and control T2 immunity independently of STAT 4, whereas the profile of immunity to isolated proteins is more reliant on an intact STAT 4 signaling pathway.
Comprehensive Investigation of the Molecular Defect in
vif
-Deficient Human Immunodeficiency Virus Type 1 Virions
Authors: Gaddis Nathan C. , Chertova Elena , Sheehy Ann M. , Henderson Louis E. , Malim Michael H.
Published: April 2003

Replication of human immunodeficiency virus type 1 (HIV-1) in primary blood lymphocytes, certain T-cell lines (nonpermissive cells), and most likely in vivo is highly dependent on the virally encoded Vif protein. Evidence suggests that Vif acts late in the viral life cycle during assembly, budding, and/or maturation to counteract the antiviral activity of the CEM15 protein and possibly other antiviral factors. Because HIV-1 virions produced in the absence of Vif are severely restricted at a postentry, preintegration step of infection, it is presumed that such virions differ from wild-type virions in some way. In the present study, we established a protocol for producing large quantities of
vif
-deficient HIV-1 (HIV-1/Δ
vif
) from an acute infection of nonpermissive T cells and performed a thorough examination of the defect in these virions. Aside from the expected lack of Vif, we observed no apparent abnormalities in the packaging, modification, processing, or function of proteins in Δ
vif
virions. In addition, we found no consistent defect in the ability of Δ
vif
virions to perform intravirion reverse transcription under a variety of assay conditions, suggesting that the reverse transcription complexes in these particles can behave normally under cell-free conditions. Consistent with this finding, neither the placement of the primer tRNA3Lys nor its ability to promote reverse transcription in an in vitro assay was affected by a lack of Vif. Based on the inability of this comprehensive analysis to uncover molecular defects in Δ
vif
virions, we speculate that such defects are likely to be subtle and/or rare.
In Vivo Evolution of Human Immunodeficiency Virus Type 1 toward Increased Pathogenicity through CXCR4-Mediated Killing of Uninfected CD4 T Cells
Authors: Jekle Andreas , Keppler Oliver T. , De Clercq Erik , Schols Dominique , Weinstein Mark , Goldsmith Mark A.
Published: April 2003

The destruction of the immune system by progressive loss of CD4 T cells is the hallmark of AIDS. CCR5-dependent (R5) human immunodeficiency virus type 1 (HIV-1) isolates predominate in the early, asymptomatic stages of HIV-1 infection, while CXCR4-dependent (X4) isolates typically emerge at later stages, frequently coinciding with a rapid decline in CD4 T cells. Lymphocyte killing in vivo primarily occurs through apoptosis, but the importance of apoptosis of HIV-1-infected cells relative to apoptosis of uninfected bystander cells is controversial. Here we show that in human lymphoid tissues ex vivo, apoptosis of uninfected bystander CD4 T cells is a major mechanism of lymphocyte depletion caused by X4 HIV-1 strains but is only a minor mechanism of depletion by R5 strains. Further, X4 HIV-1-induced bystander apoptosis requires the interaction of the viral envelope glycoprotein gp120 with the CXCR4 coreceptor on CD4 T cells. These results emphasize the contribution of bystander apoptosis to HIV-1 cytotoxicity and suggest that in association with a coreceptor switch in HIV disease, T-cell killing evolves from an infection-restricted stage to generalized toxicity that involves a high degree of bystander apoptosis.
Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human Immunodeficiency Virus Type 1 gp120
Authors: Zwick Michael B. , Parren Paul W. H. I. , Saphire Erica O. , Church Sarah , Wang Meng , Scott Jamie K. , Dawson Philip E. , Wilson Ian A. , Burton Dennis R.
Published: April 2003

IgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities.
Hyperglycosylated Mutants of Human Immunodeficiency Virus (HIV) Type 1 Monomeric gp120 as Novel Antigens for HIV Vaccine Design
Authors: Pantophlet Ralph , Wilson Ian A. , Burton Dennis R.
Published: April 2003

The ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gp120, has generally failed to elicit such antibodies. We postulated that gp120 might be a better immunogen if it could be engineered to preferentially bind known broadly neutralizing antibodies. In a first study, we found that four alanine substitutions on the perimeter of the so-called Phe-43 cavity of gp120 could reduce binding of weakly neutralizing CD4-binding site antibodies (R. Pantophlet, E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton, J. Virol. 77:642-658, 2003), while slightly enhancing binding of the potent, broadly neutralizing antibody b12. In the present study, we sought to reduce or abolish the binding of a wider range of nonneutralizing antibodies, by incorporating extra N-glycosylation motifs at select positions into the hypervariable loops and the gp120 core. A hyperglycosylated mutant containing seven extra glycosylation sequons (consensus sequences) and the four alanine substitutions described above did not bind an extensive panel of nonneutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HIVIG) of low neutralizing potency. Binding of b12, at lowered affinity, and of four antibodies to the C1 and C5 regions was maintained. Removal of N- and C-terminal residues in the C1 and C5 regions, respectively, reduced or abolished binding of the four antibodies, but this also adversely affected b12 binding. The hyperglycosylated mutant and its analogues described here are novel antigens that may provide a new approach to eliciting antibodies with b12-like neutralizing properties.
The Latency-Associated Nuclear Antigen Homolog of Herpesvirus Saimiri Inhibits Lytic Virus Replication
Authors: Schäfer Alexandra , Lengenfelder Doris , Grillhösl Christian , Wieser Carsten , Fleckenstein Bernhard , Ensser Armin
Published: April 2003

Herpesvirus saimiri (HVS), a T-lymphotropic tumor virus of neotropical primates, and the Kaposi's sarcoma-associated human herpesvirus 8 (KSHV) belong to the gamma-
2
-herpesvirus (
Rhadinovirus
) subfamily and share numerous features of genome structure and organization. The KSHV latency-associated nuclear antigen (LANA) protein appears to be relevant for viral persistence, latency, and transformation. It binds to DNA, colocalizes with viral episomal DNA, and presumably mediates efficient persistence of viral genomes. LANA further represses the transcriptional and proapoptotic activities of the p53 tumor suppressor protein. Here we report on the ORF73 gene of HVS strain C488, which is the positional and structural homolog of KSHV LANA. The ORF73 gene in OMK cells can encode a 62-kDa protein that localizes to the nucleus in a pattern similar to that of LANA. We show that the ORF73 gene product can regulate viral gene expression by acting as a transcriptional modulator of latent and lytic viral promoters. To define the HVS ORF73 function in the background of a replication-competent virus, we constructed a viral mutant that expresses ORF73 under the transcriptional control of a mifepristone (RU-486)-inducible promoter. The HVS ORF73 gene product efficiently suppresses lytic viral replication in permissive cells, indicating that it defines a critical control point between viral persistence and lytic replication.
Sodium-Dependent
myo
-Inositol Transporter 1 Is a Cellular Receptor for
Mus cervicolor
M813 Murine Leukemia Virus
Authors: Hein Sibyll , Prassolov Vladimir , Zhang Yuanming , Ivanov Dmitry , Löhler Jürgen , Ross Susan R. , Stocking Carol
Published: April 2003

Retrovirus infection is initiated by binding of the surface (SU) portion of the viral envelope glycoprotein (Env) to specific receptors on cells. This binding triggers conformational changes in the transmembrane portion of Env, leading to membrane fusion and cell entry, and is thus a major determinant of retrovirus tissue and species tropism. The M813 murine leukemia virus (MuLV) is a highly fusogenic gammaretrovirus, isolated from
Mus cervicolor
, whose host range is limited to mouse cells. To delineate the molecular mechanisms of its restricted host range and its high fusogenic potential, we initiated studies to characterize the cell surface protein that mediates M813 infection. Screening of the T31 mouse-hamster radiation hybrid panel for M813 infectivity localized the receptor gene to the distal end of mouse chromosome 16. Expression of one of the likely candidate genes (
slc5a3
) within this region in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity.
slc5a3
encodes sodium
myo
-inositol transporter 1 (SMIT1), thus adding another sodium-dependent transporter to the growing list of proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologues in different species identified several amino acid variations within two extracellular loops that may restrict susceptibility to M813 infection.
Reovirus σNS and μNS Proteins Form Cytoplasmic Inclusion Structures in the Absence of Viral Infection
Authors: Becker Michelle M. , Peters Timothy R. , Dermody Terence S.
Published: April 2003

Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, σNS and μNS, and structural protein σ3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed σNS, μNS, and σ3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, σNS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed σNS is functional as it complements the defect in temperature-sensitive, σNS-mutant virustsE320. In both transfected and infected cells, μNS is found in punctate cytoplasmic structures and σ3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of μNS and σ3 is not altered when the proteins are expressed together or with σNS. However, when expressed with μNS, σNS colocalizes with μNS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both σNS and μNS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, σNS interacts with μNS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that σNS and μNS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.
Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus
Authors: Flick Ramon , Flick Kirsten , Feldmann Heinz , Elgh Fredrik
Published: April 2003

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus
Nairovirus
, family
Bunyaviridae
. The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family
Bunyaviridae
. This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.
Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses
Authors: Huarte Maite , Falcón Ana , Nakaya Yuri , Ortín Juan , García-Sastre Adolfo , Nieto Amelia
Published: April 2003

Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo
.
In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.
Discordant Outcomes following Failure of Antiretroviral Therapy Are Associated with Substantial Differences in Human Immunodeficiency Virus-Specific Cellular Immunity
Authors: Price David A. , Scullard George , Oxenius Annette , Braganza Ruth , Beddows Simon A. , Kazmi Shamim , Clarke John R. , Johnson Gabriele E. , Weber Jonathan N. , Phillips Rodney E.
Published: April 2003

Many individuals chronically infected with human immunodeficiency virus type 1 (HIV-1) experience a recrudescence of plasma virus during continuous combination antiretroviral therapy (ART) due either to the emergence of drug-resistant viruses or to poor compliance. In most cases, virologic failure on ART is associated with a coincident decline in CD4
+
T lymphocyte levels. However, a proportion of discordant individuals retain a stable or even increasing CD4
+
T lymphocyte count despite virological failure. In order to address the nature of these different outcomes, we evaluated virologic and immunologic variables in a prospective, single-blinded, nonrandomized cohort of 53 subjects with chronic HIV-1 infection who had been treated with continuous ART and monitored intensively over a period of 19 months. In all individuals with detectable viremia on ART, multiple drug resistance mutations with similar impacts on viral growth kinetics were detected in the
pol
gene of circulating plasma virus. Further, C2V3
env
gene analysis demonstrated sequences indicative of CCR5 coreceptor usage in the majority of those with detectable plasma viremia. In contrast to this homogeneous virologic pattern, comprehensive screening with a range of antigens derived from HIV-1 revealed substantial immunologic differences. Discordant subjects with stable CD4
+
T lymphocyte counts in the presence of recrudescent virus demonstrated potent virus-specific CD4
+
and CD8
+
T lymphocyte responses. In contrast, subjects with virologic failure associated with declining CD4
+
T lymphocyte counts had substantially weaker HIV-specific CD4
+
T lymphocyte responses and exhibited a trend towards weaker HIV-specific CD8
+
T lymphocyte responses. Importantly the CD4
+
response was sustained over periods as long as 11 months, confirming the stability of the phenomenon. These correlative data lead to the testable hypothesis that the consequences of viral recrudescence during continuous ART are modulated by the HIV-specific cellular immune response.
The NB Protein of Influenza B Virus Is Not Necessary for Virus Replication In Vitro
Authors: Hatta Masato , Kawaoka Yoshihiro
Published: April 2003

The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.