bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal Structure of the RC-LH1 Core Complex from
Rhodopseudomonas palustris
Authors: Roszak Aleksander W. , Howard Tina D. , Southall June , Gardiner Alastair T. , Law Christopher J. , Isaacs Neil W. , Cogdell Richard J.
Published: December 2003

The crystal structure at 4.8 angstrom resolution of the reaction center–light harvesting 1 (RC–LH1) core complex from
Rhodopseudomonas palustris
shows the reaction center surrounded by an oval LH1 complex that consists of 15 pairs of transmembrane helical α- and β-apoproteins and their coordinated bacteriochlorophylls. Complete closure of the RC by the LH1 is prevented by a single transmembrane helix, out of register with the array of inner LH1 α-apoproteins. This break, located next to the binding site in the reaction center for the secondary electron acceptor ubiquinone (UQ
B
), may provide a portal through which UQ
B
can transfer electrons to cytochrome b/c
1
.
Anterior-Posterior Guidance of Commissural Axons by Wnt-Frizzled Signaling
Authors: Lyuksyutova Anna I. , Lu Chin-Chun , Milanesio Nancy , King Leslie A. , Guo Nini , Wang Yanshu , Nathans Jeremy , Tessier-Lavigne Marc , Zou Yimin
Published: December 2003

Commissural neurons in the mammalian dorsal spinal cord send axons ventrally toward the floor plate, where they cross the midline and turn anteriorly toward the brain; a gradient of chemoattractant(s) inside the spinal cord controls this turning. In rodents, several Wnt proteins stimulate the extension of commissural axons after midline crossing (postcrossing). We found that
Wnt4
messenger RNA is expressed in a decreasing anterior-to-posterior gradient in the floor plate, and that a directed source of Wnt4 protein attracted postcrossing commissural axons. Commissural axons in mice lacking the Wnt receptor Frizzled3 displayed anterior-posterior guidance defects after midline crossing. Thus, Wnt-Frizzled signaling guides commissural axons along the anterior-posterior axis of the spinal cord.
New Visions for Addressing Sustainability
Authors: McMichael A. J. , Butler C. D. , Folke Carl
Published: December 2003

Attaining sustainability will require concerted interactive efforts among disciplines, many of which have not yet recognized, and internalized, the relevance of environmental issues to their main intellectual discourse. The inability of key scientific disciplines to engage interactively is an obstacle to the actual attainment of sustainability. For example, in the list of Millennium Development Goals from the United Nations World Summit on Sustainable Development, Johannesburg, 2002, the seventh of the eight goals, to “ensure environmental sustainability,” is presented separately from the parallel goals of reducing fertility and poverty, improving gains in equity, improving material conditions, and enhancing population health. A more integrated and consilient approach to sustainability is urgently needed.
A Gene Expression Map of the
Arabidopsis
Root
Authors: Birnbaum Kenneth , Shasha Dennis E. , Wang Jean Y. , Jung Jee W. , Lambert Georgina M. , Galbraith David W. , Benfey Philip N.
Published: December 2003

A global map of gene expression within an organ can identify genes with coordinated expression in localized domains, thereby relating gene activity to cell fate and tissue specialization. Here, we present localization of expression of more than 22,000 genes in the
Arabidopsis
root. Gene expression was mapped to 15 different zones of the root that correspond to cell types and tissues at progressive developmental stages. Patterns of gene expression traverse traditional anatomical boundaries and show cassettes of hormonal response. Chromosomal clustering defined some coregulated genes. This expression map correlates groups of genes to specific cell fates and should serve to guide reverse genetics.
Subkelvin Cooling NO Molecules via "Billiard-like" Collisions with Argon
Authors: Elioff Michael S. , Valentini James J. , Chandler David W.
Published: December 2003

We report the cooling of nitric oxide using a single collision between an argon atom and a molecule of NO. We have produced significant numbers (10
8
to 10
9
molecules per cubic centimeter per quantum state) of translationally cold NO molecules in a specific quantum state with an upper-limit root mean square laboratory velocity of 15 plus or minus 1 meters per second, corresponding to a 406 plus or minus 23 millikelvin upper limit of temperature, in a crossed molecular beam apparatus. The technique, which relies on a kinematic collapse of the velocity distributions of the molecular beams for the scattering events that produce cold molecules, is general and independent of the energy of the colliding partner.
Fault Interactions and Large Complex Earthquakes in the Los Angeles Area
Authors: Anderson Greg , Aagaard Brad , Hudnut Ken
Published: December 2003

Faults in complex tectonic environments interact in various ways, including triggered rupture of one fault by another, that may increase seismic hazard in the surrounding region. We model static and dynamic fault interactions between the strike-slip and thrust fault systems in southern California. We find that rupture of the Sierra Madre–Cucamonga thrust fault system is unlikely to trigger rupture of the San Andreas or San Jacinto strike-slip faults. However, a large northern San Jacinto fault earthquake could trigger a cascading rupture of the Sierra Madre–Cucamonga system, potentially causing a moment magnitude 7.5 to 7.8 earthquake on the edge of the Los Angeles metropolitan region.
Importance of Surface Morphology in Interstellar H
2
Formation
Authors: Hornekær L. , Baurichter A. , Petrunin V. V. , Field D. , Luntz A. C.
Published: December 2003

Detailed laboratory experiments on the formation of HD from atom recombination on amorphous solid water films show that this process is extremely efficient in a temperature range of 8 to 20 kelvin, temperatures relevant for H
2
formation on dust grain surfaces in the interstellar medium (ISM). The fate of the 4.5 electron volt recombination energy is highly dependent on film morphology. These results suggest that grain morphology, rather than the detailed chemical nature of the grain surface, is most important in determining the energy content of the H
2
as it is released from the grain into the ISM.
Social Capital and the Collective Management of Resources
Authors: Pretty Jules
Published: December 2003

The proposition that natural resources need protection from the destructive actions of people is widely accepted. Yet communities have shown in the past and increasingly today that they can collaborate for long-term resource management. The term social capital captures the idea that social bonds and norms are critical for sustainability. Where social capital is high in formalized groups, people have the confidence to invest in collective activities, knowing that others will do so too. Some 0.4 to 0.5 million groups have been established since the early 1990s for watershed, forest, irrigation, pest, wildlife, fishery, and microfinance management. These offer a route to sustainable management and governance of common resources.
The Challenge of Long-Term Climate Change
Authors: Hasselmann K. , Latif M. , Hooss G. , Azar C. , Edenhofer O. , Jaeger C. C. , Johannessen O. M. , Kemfert C. , Welp M. , Wokaun A.
Published: December 2003

Climate policy needs to address the multidecadal to centennial time scale of climate change. Although the realization of short-term targets is an important first step, to be effective climate policies need to be conceived as long-term programs that will achieve a gradual transition to an essentially emission-free economy on the time scale of a century. This requires a considerably broader spectrum of policy measures than the primarily market-based instruments invoked for shorter term mitigation policies. A successful climate policy must consist of a dual approach focusing on both short-term targets and long-term goals.
The Struggle to Govern the Commons
Authors: Dietz Thomas , Ostrom Elinor , Stern Paul C.
Published: December 2003

Human institutions—ways of organizing activities—affect the resilience of the environment. Locally evolved institutional arrangements governed by stable communities and buffered from outside forces have sustained resources successfully for centuries, although they often fail when rapid change occurs. Ideal conditions for governance are increasingly rare. Critical problems, such as transboundary pollution, tropical deforestation, and climate change, are at larger scales and involve nonlocal influences. Promising strategies for addressing these problems include dialogue among interested parties, officials, and scientists; complex, redundant, and layered institutions; a mix of institutional types; and designs that facilitate experimentation, learning, and change.
The Proteasome of
Mycobacterium tuberculosis
Is Required for Resistance to Nitric Oxide
Authors: Darwin K. Heran , Ehrt Sabine , Gutierrez-Ramos José-Carlos , Weich Nadine , Nathan Carl F.
Published: December 2003

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by
Mycobacterium tuberculosis
(
Mtb
). However, the protection is imperfect and infection persists. To identify genes that
Mtb
requires to resist RNI, we screened 10,100
Mtb
transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (
uvrB
) and the synthesis of a flavin cofactor (
fbiC
). Five mutants had insertions in proteasome-associated genes. An
Mtb
mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type
Mtb
to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.
Mono- Versus Polyubiquitination: Differential Control of p53 Fate by Mdm2
Authors: Li Muyang , Brooks Christopher L. , Wu-Baer Foon , Chen Delin , Baer Richard , Gu Wei
Published: December 2003

Although Mdm2-mediated ubiquitination is essential for both degradation and nuclear export of p53, the molecular basis for the differential effects of Mdm2 remains unknown. Here we show that low levels of Mdm2 activity induce monoubiquitination and nuclear export of p53, whereas high levels promote p53's polyubiquitination and nuclear degradation. A p53-ubiquitin fusion protein that mimics monoubiquitinated p53 was found to accumulate in the cytoplasm in an Mdm2-independent manner, indicating that monoubiquitination is critical for p53 trafficking. These results clarify the nature of ubiquitination-mediated p53 regulation and suggest that distinct mechanisms regulate p53 function in accordance with the levels of Mdm2 activity.
The Burden of Chronic Disease
Authors: Mascie-Taylor C. G. Nicholas , Karim Enamul
Published: December 2003

The shift from acute infectious and deficiency diseases to chronic noncommunicable diseases is not a simple transition but a complex and dynamic epidemiological process, with some diseases disappearing and others appearing or reemerging. The unabated pandemic of childhood and adulthood obesity and concomitant comorbidities are affecting both rich and poor nations, while infectious diseases remain an important public health problem, particularly in developing countries. More attention should be given to the high burden of disease associated with soil-transmitted helminths and schistosomiasis, which until recently was not considered a priority even though regular drug treatment is obtainable at relatively little cost. In developing countries, the pressing requirement is to provide an accessible and good quality health-care system, whereas industrialized countries have a major need for greater public health education and the promotion of healthy life-styles.
A Century of Oil, a Future of Options
Authors: Kammen Daniel M.
Published: December 2003

Energy at the Crossroads
Global Perspectives and Uncertainties.
By Vaclav Smil
. MIT Press, Cambridge, MA, 2003. 443 pp. $34.95, £22.95. ISBN 0-262-19492-9.

Considering what is possible and what is desirable in our energy future, Smil argues that human dependence on fossil fuels must be reduced not because of impending resource shortages but because of the environmental, economic, and political problems caused by our current consumption.
Global Food Security: Challenges and Policies
Authors: Rosegrant Mark W. , Cline Sarah A.
Published: December 2003

Global food security will remain a worldwide concern for the next 50 years and beyond. Recently, crop yield has fallen in many areas because of declining investments in research and infrastructure, as well as increasing water scarcity. Climate change and HIV/AIDS are also crucial factors affecting food security in many regions. Although agroecological approaches offer some promise for improving yields, food security in developing countries could be substantially improved by increased investment and policy reforms.
Tales from a Troubled Marriage: Science and Law in Environmental Policy
Authors: Houck Oliver
Published: December 2003

Early environmental policy depended on science, with mixed results. Newer approaches continue to rely on science to identify problems and solve them, but use other mechanisms to set standards and legal obligations. Given the important role that science continues to play, however, several cautionary tales are in order concerning “scientific management,” “good science,” the lure of money, and the tension between objectivity and involvement in important issues of our time.
Nota Bene: Steps into the Sky
Authors: Suter Sherman J.
Published: December 2003

The Wright Brothers and the Invention of the Aerial Age
.
By Tom D. Crouchand Peter L. Jakab
. National Geographic Society, Washington, DC, 2003. 240 pp. $35, C$55, ú25.50. ISBN 0-7922-6985-3.

Taking Flight
Inventing the Aerial Age From Antiquity Through the First World War.
By Richard P. Hallion
. Oxford University Press, New York, 2003. 553 pp. $35, £20. ISBN 0-19-516035-5.

Crouch and Jakab provide a lavishly illustrated account of the Wright brothers' lives and accomplishments. Hallion's comprehensive history places the stories of the Wrights, their predecessors, and competitors in the context of cultural, technological, scientific, political, and military developments.
Generation and Characterization of Closely Related Epizootic and Enzootic Infectious cDNA Clones for Studying Interferon Sensitivity and Emergence Mechanisms of Venezuelan Equine Encephalitis Virus
Authors: Anishchenko Michael , Paessler Slobodan , Greene Ivorlyne P. , Aguilar Patricia V. , Carrara Anne-Sophie , Weaver Scott C.
Published: December 2003

Venezuelan equine encephalitis virus (VEEV) is a reemerging pathogen and a continuing threat to humans and equines in the Americas. Identification of the genetic determinants that enable epizootic VEEV strains to arise and exploit equines as amplification hosts to cause widespread human disease is pivotal to understanding VEE emergence. The sensitivity to murine alpha/beta interferon-mediated antiviral activity was previously correlated to the epizootic phenotype of several VEEV strains. Infectious cDNA clones were generated from an epizootic subtype IC VEEV strain (SH3) isolated during the 1992 Venezuelan outbreak and a closely related enzootic, sympatric subtype ID strain (ZPC738). These VEEV strains had low-cell-culture-passage histories and differed by only 12 amino acids in the nonstructural and structural proteins. Rescued viruses showed similar growth kinetics to their parent viruses in several cell lines, and murine infections resulted in comparable viremia and disease. Unlike what was found in other studies of epizootic and enzootic VEEV strains, the sensitivities to murine alpha/beta interferon did not differ appreciably between these epizootic versus enzootic strains, calling into question the reliability of interferon sensitivity as a marker of epizootic potential.
Inhibition of S-Phase Cyclin-Dependent Kinase Activity Blocks Expression of Epstein-Barr Virus Immediate-Early and Early Genes, Preventing Viral Lytic Replication
Authors: Kudoh Ayumi , Daikoku Tohru , Sugaya Yutaka , Isomura Hiroki , Fujita Masatoshi , Kiyono Tohru , Nishiyama Yukihiro , Tsurumi Tatsuya
Published: December 2003

The induction of lytic replication of the Epstein-Barr virus (EBV) completely arrests cell cycle progression, in spite of elevation of S-phase cyclin-dependent kinase (CDK) activity, thereby causing accumulation of hyperphosphorylated forms of retinoblastoma (Rb) protein (A. Kudoh, M. Fujita, T. Kiyono, K. Kuzushima, Y. Sugaya, S. Izuta, Y. Nishiyama, and T. Tsurumi, J. Virol.77:851-861, 2003). Thus, the EBV lytic program appears to promote specific cell cycle-associated activity involved in the progression from G1to S phase. We have proposed that this provides a cellular environment that is advantageous for EBV productive infection. Purvalanol A and roscovitine, inhibitors of S-phase CDKs, blocked the viral lytic replication when cells were treated at the early stage of lytic infection, while well-characterized inhibitors of enzymes, such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C, known to be involved in BZLF1 gene expression did not. Inhibition of CDK activity resulted in the accumulation of the hypophosphorylated form of Rb protein and inhibition of expression of EBV immediate-early and early proteins. Cycloheximide block-and-release experiments clearly demonstrated that even in the presence of enough amounts of the BZLF1 protein, purvalanol A blocked expression of lytic viral proteins at transcription level. Furthermore, reporter gene experiments confirmed that BZLF1-induced activation of early EBV promoters was impaired in the presence of the CDK inhibitor. We conclude here that the EBV lytic program promotes specific cell cycle-associated activity involved in the progression from G1to S phase because the S-phase-like cellular environment is essential for the expression of immediate-early and early genes supplying the viral replication proteins and hence for lytic viral replication.
Vaccination of Rabbits with an Adenovirus Vector Expressing the Papillomavirus E2 Protein Leads to Clearance of Papillomas and Infection
Authors: Brandsma Janet L. , Shlyankevich Mark , Zhang Lixin , Slade Martin D. , Goodwin Edward C. , Peh Woei , Deisseroth Albert B.
Published: December 2003

Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV). Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papilloma-forming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.
Role for Bovine Viral Diarrhea Virus E
rns
Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA
Authors: Iqbal Munir , Poole Emma , Goodbourn Stephen , McCauley John W.
Published: December 2003

Production of alpha/beta interferon in response to viral double-stranded RNA (dsRNA) produced during viral replication is a first line of defense against viral infections. Here we demonstrate that the E
rns
glycoprotein of the pestivirus bovine viral diarrhea virus can act as an inhibitor of dsRNA-induced responses of cells. This effect is seen whether E
rns
is constitutively expressed in cells or exogenously added to the culture medium. The E
rns
effect is specific to dsRNA since activation of NF-κB in cells infected with Semliki Forest virus or treated with tumor necrosis factor alpha was not affected. We also show that E
rns
contains a dsRNA-binding activity, and its RNase is active against dsRNA at a low pH. Both the dsRNA binding and RNase activities are required for the inhibition of dsRNA signaling, and we discuss here a model to account for these observations.
A Single Injection of Recombinant Measles Virus Vaccines Expressing Human Immunodeficiency Virus (HIV) Type 1 Clade B Envelope Glycoproteins Induces Neutralizing Antibodies and Cellular Immune Responses to HIV
Authors: Lorin Clarisse , Mollet Lucile , Delebecque Frédéric , Combredet Chantal , Hurtrel Bruno , Charneau Pierre , Brahic Michel , Tangy Frédéric
Published: December 2003

The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8
+
and CD4
+
cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.
Residues of Human Cytomegalovirus DNA Polymerase Catalytic Subunit UL54 That Are Necessary and Sufficient for Interaction with the Accessory Protein UL44
Authors: Loregian Arianna , Appleton Brent A. , Hogle James M. , Coen Donald M.
Published: December 2003

The human cytomegalovirus DNA polymerase contains a catalytic subunit, UL54, and an accessory protein, UL44. Recent studies suggested that UL54 might interact via its extreme C terminus with UL44 (A. Loregian, R. Rigatti, M. Murphy, E. Schievano, G. Palu', and H. S. Marsden, J. Virol. 77:8336-8344, 2003). To address this hypothesis, we quantitatively measured the binding of peptides corresponding to the extreme C terminus of UL54 to UL44 by using isothermal titration calorimetry. A peptide corresponding to the last 22 residues of UL54 was sufficient to bind specifically to UL44 in a 1:1 complex with a dissociation constant of ca. 0.7 μM. To define individual residues in this segment that are crucial for interacting with UL44, we engineered a series of mutations in the C-terminal region of
UL54
. The UL54 mutants were tested for their ability to interact with UL44 by glutathione
S
-transferase pulldown assays, for basal DNA polymerase activity, and for long-chain DNA synthesis in the presence of UL44. We observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction. Thus, like the herpes simplex virus UL30-UL42 interaction, a few specific side chains in the C terminus of UL54 are crucial for UL54-UL44 interaction. However, the UL54 residues important for interaction with UL44 are hydrophobic and not basic. This information might aid in the rational design of new drugs for the treatment of human cytomegalovirus infection.
Genomes of the Parapoxviruses Orf Virus and Bovine Papular Stomatitis Virus
Authors: Delhon G. , Tulman E. R. , Afonso C. L. , Lu Z. , de la Concha-Bermejillo A. , Lehmkuhl H. D. , Piccone M. E. , Kutish G. F. , Rock D. L.
Published: December 2003

Bovine papular stomatitis virus
(BPSV) and
orf virus
(ORFV), members of the genus
Parapoxvirus
of the
Poxviridae
, are etiologic agents of worldwide diseases affecting cattle and small ruminants, respectively. Here we report the genomic sequences and comparative analysis of BPSV strain BV-AR02 and ORFV strains OV-SA00, isolated from a goat, and OV-IA82, isolated from a sheep. Parapoxvirus (PPV) BV-AR02, OV-SA00, and OV-IA82 genomes range in size from 134 to 139 kbp, with an average nucleotide composition of 64% G+C. BPSV and ORFV genomes contain 131 and 130 putative genes, respectively, and share colinearity over 127 genes, 88 of which are conserved in all characterized chordopoxviruses. BPSV and ORFV contain 15 and 16 open reading frames (ORFs), respectively, which lack similarity to other poxvirus or cellular proteins. All genes with putative roles in pathogenesis, including a vascular endothelial growth factor (VEGF)-like gene, are present in both viruses; however, BPSV contains two extra ankyrin repeat genes absent in ORFV. Interspecies sequence variability is observed in all functional classes of genes but is highest in putative virulence/host range genes, including genes unique to PPV. At the amino acid level, OV-SA00 is 94% identical to OV-IA82 and 71% identical to BV-AR02. Notably, ORFV 006/132, 103, 109, 110, and 116 genes (VEGF, homologues of vaccinia virus A26L, A33R, and A34R, and a novel PPV ORF) show an unusual degree of intraspecies variability. These genomic differences are consistent with the classification of BPSV and ORFV as two PPV species. Compared to other mammalian chordopoxviruses, PPV shares unique genomic features with molluscum contagiosum virus, including a G+C-rich nucleotide composition, three orthologous genes, and a paucity of nucleotide metabolism genes. Together, these data provide a comparative view of PPV genomics.
Inefficient Signalase Cleavage Promotes Efficient Nucleocapsid Incorporation into Budding Flavivirus Membranes
Authors: Lobigs Mario , Lee Eva
Published: December 2003

The mechanism for efficient nucleocapsid (NC) uptake into flavivirus particles which form by budding through the membranes of the endoplasmic reticulum (ER) was investigated by using Murray Valley encephalitis virus as a model. Budding of flavivirus membranes is driven by the viral transmembrane proteins prM and E independently of NC interaction. We show that control of signalase cleavage of the multimembrane-spanning flavivirus polyprotein by the catalytic function of the viral protease is critical for efficient virus morphogenesis. In wild-type virus, signalase cleavage of prM remains inefficient until cleavage of capsid at the cytosolic side of the signal sequence separating the two proteins has occurred. This obligatory sequence of cleavages was uncoupled in a mutant virus with the consequence of greatly reduced incorporation of NC into budding membranes and augmented release of NC-free virus-like particles. Efficient signalase cleavage of prM in the mutant virus resulted in partial inhibition of cleavage of capsid by the viral NS2B-3 protease. Our results support a model for flavivirus morphogenesis involving temporal and spatial coordination of NC assembly and envelopment by regulated cleavages of an ER membrane-spanning capsid-prM intermediate.
Control of Heterologous Hepatitis C Virus Infection in Chimpanzees Is Associated with the Quality of Vaccine-Induced Peripheral T-Helper Immune Response
Authors: Rollier C. , Depla E. , Drexhage J. A. R. , Verschoor E. J. , Verstrepen B. E. , Fatmi A. , Brinster C. , Fournillier A. , Whelan J. A. , Whelan M. , Jacobs D. , Maertens G. , Inchauspé G. , Heeney J. L.
Published: December 2003

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-γ), protein-specific lymphoproliferative responses, IFN-γ, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.
Evasion of Cellular Antiviral Responses by Human Cytomegalovirus
TRS1
and
IRS1
Authors: Child Stephanie J. , Hakki Morgan , De Niro Katherine L. , Geballe Adam P.
Published: December 2003

During infection with human cytomegalovirus (HCMV), cellular protein synthesis continues even as viral proteins are being synthesized in abundance. Thus, HCMV may have a mechanism for counteracting host cell antiviral pathways that act by shutting off translation. Consistent with this view, HCMV infection of human fibroblasts rescues the replication of a vaccinia virus mutant lacking the double-stranded RNA-binding protein gene E3L (VVΔE3L). HCMV also prevents the phosphorylation of the eukaryotic translation initiation factor eIF-2α, the activation of RNase L, and the shutoff of viral and cellular protein synthesis that otherwise result from VVΔE3L infection. To identify the HCMV gene(s) responsible for these effects, we prepared a library of VVΔE3L recombinants containing HCMV genomic fragments. By infecting nonpermissive cells with this library and screening for VV gene expression and replication, we isolated a virus containing a 2.8-kb HCMV fragment that rescues replication of VVΔE3L. The fragment comprises the 3′ end of the J1S open reading frame through the entire
TRS1
gene. Analyses of additional VVΔE3L recombinants revealed that the protein encoded by
TRS1
, pTRS1, as well as the closely related
IRS1
gene, rescues VVΔE3L replication and prevent the shutoff of protein synthesis, the phosphorylation of eIF-2α, and activation of RNase L. These results demonstrate that
TRS1
and
IRS1
are able to counteract critical host cell antiviral response pathways.
Subsets of Memory Cytotoxic T Lymphocytes Elicited by Vaccination Influence the Efficiency of Secondary Expansion In Vivo
Authors: Seaman Michael S. , Peyerl Fred W. , Jackson Shawn S. , Lifton Michelle A. , Gorgone Darci A. , Schmitz Jörn E. , Letvin Norman L.
Published: December 2003

Vaccine-elicited cytotoxic T lymphocytes (CTL) should be long-lived memory cells that can rapidly expand in number following re-exposure to antigen. The present studies were initiated to analyze the ability of plasmid interleukin-12 (IL-12) to augment CTL responses in mice when delivered during the peak phase of an immune response elicited by a plasmid human immunodeficiency virus type 1 gp120 DNA vaccine. Delivery of plasmid IL-12 on day 10 postimmunization resulted in a robust expansion of gp120-specific CD8+T cells, as measured by tetramer, gamma interferon ELISPOT, and functional-killing assays. Interestingly, this delayed administration of plasmid IL-12 had no significant effect on antigen-specific CD4+-T-cell and antibody responses. Phenotypic analyses suggested that administration of plasmid IL-12 near the time of the peak CTL response activated and expanded antigen-specific effector cells, preventing their loss through apoptosis. However, this IL-12-augmented population of gp120-specific CD8+T cells did not efficiently expand following gp120 boost immunization, suggesting that these effector cells would be of little utility in expanding to contain a viral infection. Analyses of the phenotypic profile and anatomic distribution of the plasmid IL-12-augmented CTL population indicated that these lymphocytes were primarily effector memory rather than central memory T cells. These observations suggest that CTL-based vaccines should elicit central memory rather than effector memory T cells and illustrate the importance of monitoring the phenotype and functionality of vaccine-induced, antigen-specific CTL.
N-Terminal Domain of the Murine Coronavirus Receptor CEACAM1 Is Responsible for Fusogenic Activation and Conformational Changes of the Spike Protein
Authors: Miura Hideka S. , Nakagaki Keiko , Taguchi Fumihiro
Published: December 2003

The mouse hepatitis virus (MHV) receptor (MHVR), CEACAM1, has two different functions for MHV entry into cells: binding to MHV spike protein (S protein) and activation of the S protein to execute virus-cell membrane fusion, the latter of which is accompanied by conformational changes of the S protein. The MHVR comprising the N-terminal and fourth domains [R1(1,4)] displays these two activities, and the N domain is thought to be critical for binding to MHV. In this study, we have addressed whether or not the N domain alone is sufficient for these activities. We examined three types of soluble form MHVR (soMHVR), one consisting of the N domain alone [soR1(1)], one with the N and second domains [soR1(1,2)], and one [soR1(1,4)] expressed by recombinant baculoviruses. We assessed the abilities of these three types of soMHVR to bind to MHV, activate fusogenicity, and induce conformational changes of the S protein. All three types of soMHVR similarly bound to MHV, as examined by a solid-phase binding assay and neutralized MHV infectivity. They also activated S protein fusogenicity and induced its conformational changes with similar levels of efficiency. However, R1(1) expressed on the BHK cell surface failed to serve as a receptor in spite of a sufficient level of expression. The inability of expressed R1(1) to work as a receptor was due to the inaccessibility of virions to R1(1); however, these were accessible using the MHVR-specific monoclonal antibody CC1. These results collectively indicated that the N domain retains all biological activities necessary for receptor function.
Herpes Simplex Virus Type 1 Immediate-Early Gene Expression Is Required for the Induction of Apoptosis in Human Epithelial HEp-2 Cells
Authors: Sanfilippo Christine M. , Chirimuuta Fungai N. W. , Blaho John A.
Published: December 2003

Wild-type herpes simplex virus type 1 (HSV-1) induces apoptosis in human epithelial HEp-2 cells, but infected cell proteins produced later in infection block the process from killing the cells. Thus, HSV-1 infection in the presence of the translational inhibitor cycloheximide (CHX) results in apoptosis. Our specific goal was to gain insight as to the viral feature(s) responsible for triggering apoptosis during HSV-1 infection. We now report the following. (i) No viral protein synthesis or death factor processing was detected after infection with HSV-1(HFEM
ts
B7) at 39.5°C; this mutant virus does not inject its virion DNA into the nucleus at this nonpermissive temperature. (ii) No death factor processing or apoptotic morphological changes were detected following infection with UV-irradiated, replication-defective viruses possessing transcriptionally active incoming VP16. (iii) Addition of the transcriptional inhibitor actinomycin D prevented death factor processing upon infection with the apoptotic, ICP27-deletion virus HSV-1(vBSΔ27). (iv) Apoptotic morphologies and death factor processing were not observed following infection with HSV-1(
d
109), a green fluorescent protein-expressing recombinant virus possessing deletions of all five immediate-early (IE) (or α) genes. (v) Finally, complete death factor processing was observed upon infection with the VP16 transactivation domain-mutant HSV-1(V422) in the presence of CHX. Based on these findings, we conclude that (vi) the expression of HSV-1 α/IE genes is required for the viral induction of apoptosis and (vii) the transactivation activity of VP16 is not necessary for this induction.
ICP27 Selectively Regulates the Cytoplasmic Localization of a Subset of Viral Transcripts in Herpes Simplex Virus Type 1-Infected Cells
Authors: Pearson Angela , Knipe David M. , Coen Donald M.
Published: December 2003

Evidence suggests that the herpes simplex virus regulatory protein ICP27 mediates the nuclear export of viral transcripts; however, the extent of this activity during infection is unclear. ICP27 is required for efficient expression of the long, leaky-late
UL24
transcripts, but not for that of the short, early
UL24
transcripts. We found that infection by an
ICP27
-null mutant resulted in undetectable UL24 protein expression, which represented at least a 70-fold decrease relative to that of wild-type virus. Because lack of ICP27 had a greater effect on levels of UL24 protein than on transcripts, we examined its effect on subcellular localization of
UL24
transcripts. In wild-type-infected cells, both short and long
UL24
transcripts fractionated predominantly with the cytoplasm. However, in the absence of ICP27, greater than 50% of long
UL24
transcripts were nuclear, while the percentage of short
UL24
transcripts that were cytoplasmic was not reduced. These results also imply that the short
UL24
transcripts are translated poorly. The effect of ICP27 on cytoplasmic localization of the long
UL24
transcripts did not extend to other transcripts with which it shared a common 3′ end or to other transcripts tested, including
gC
and
UL42
, whose overall expression is highly dependent on ICP27. Thus, the dual effects of ICP27 on mRNA accumulation and cytoplasmic localization are not always linked. These results identify viral transcripts that are dependent on ICP27 for efficient cytoplasmic localization during infection, but they also indicate the existence of ICP27-independent nuclear export pathways that are accessible to many viral transcripts during infection.
Structural Differences among Hemagglutinins of Influenza A Virus Subtypes Are Reflected in Their Antigenic Architecture: Analysis of H9 Escape Mutants
Authors: Kaverin Nikolai V. , Rudneva Irina A. , Ilyushina Natalia A. , Lipatov Aleksandr S. , Krauss Scott , Webster Robert G.
Published: December 2003

We used a panel of monoclonal antibodies to H9 hemagglutinin to select 18 escape mutants of mouse-adapted influenza A/Swine/Hong Kong/9/98 (H9N2) virus. Cross-reactions of the mutants with the antibodies and the sequencing of hemagglutinin genes revealed two minimally overlapping epitopes. We mapped the amino acid changes to two areas of the recently reported three-dimensional structure of A/Swine/Hong Kong/9/98 hemagglutinin. The grouping of the antigenically relevant amino acid positions in H9 hemagglutinin differs from the pattern observed in H3 and H5 hemagglutinins. Several positions in site B of H3 hemagglutinin are distributed in two sites of H9 hemagglutinin. Unlike any subtype analyzed so far, H9 hemagglutinin does not contain an antigenic site corresponding to site A in H3 hemagglutinin. Positions 145 and 193 (H3 numbering), which in H3 hemagglutinin belong to sites A and B, respectively, are within one site in H9 hemagglutinin. This finding is consistent with the peculiarity of the three-dimensional structure of the H9 molecule, that is, the absence from H9 hemagglutinin of the lateral loop that forms site A in H3 and the equivalent site in H5 hemagglutinins. The escape mutants analyzed displayed phenotypic variations, including decreased virulence for mice and changes in affinity for sialyl substrates. Our results demonstrate a correlation between intersubtype differences in three-dimensional structure and variations among subtypes in the distribution of antigenic areas. Our findings also suggest that covariation and pleiotropic effects of antibody-selected mutations may be important in the evolution of H9 influenza virus, a possible causative agent of a future pandemic.
A JC Virus-Induced Signal Is Required for Infection of Glial Cells by a Clathrin- and eps15-Dependent Pathway
Authors: Querbes W. , Benmerah A. , Tosoni D. , Di Fiore P. P. , Atwood Walter J.
Published: December 2003

Infectious entry of JC virus (JCV) into human glial cells occurs by receptor-mediated clathrin-dependent endocytosis. In this report we demonstrate that the tyrosine kinase inhibitor genistein blocks virus entry and inhibits infection. Transient expression of dominant-negative eps15 mutants, including a phosphorylation-defective mutant, inhibited both virus entry and infection. We also show that the JCV-induced signal activates the mitogen-activated protein kinases ERK1 and ERK2. These data demonstrate that JC virus binding to human glial cells induces an intracellular signal that is critical for entry and infection by a ligand-inducible clathrin-dependent mechanism.
Immune Failure in the Absence of Profound CD4+T-Lymphocyte Depletion in Simian Immunodeficiency Virus-Infected Rapid Progressor Macaques
Authors: Hirsch Vanessa M. , Santra Sampa , Goldstein Simoy , Plishka Ronald , Buckler-White Alicia , Seth Aruna , Ourmanov Ilnour , Brown Charles R. , Engle Ronald , Montefiori David , Glowczwskie Jennifer , Kunstman Kevin , Wolinsky Steven , Letvin Norman L.
Published: December 2003

A fraction of simian immunodeficiency virus (SIV)-infected macaques develop rapidly progressive disease in the apparent absence of detectable SIV-specific antibody responses. To characterize the immunopathogenesis of this syndrome, we studied viral load, CD4+T-lymphocyte numbers as well as cellular and humoral immune responses to SIV and other exogenous antigens in four SIVsm-infected rhesus macaques that progressed to AIDS 9 to 16 weeks postinoculation. Each of these animals exhibited high levels of viremia but showed relatively preserved CD4 T lymphocytes in blood and lymphoid tissues at the time of death. Transient SIV-specific antibody responses and cytotoxic T-lymphocyte responses were observed at 2 to 4 weeks postinoculation. Two of the macaques that were immunized sequentially with tetanus toxoid and hepatitis A virus failed to develop antibody to either antigen. These studies show that the SIV-infected rapid progressor macaques initially mounted an appropriate but transient cellular and humoral immune response. The subsequent immune defect in these animals appeared to be global, affecting both cellular and humoral immunity to SIV as well as immune responses against unrelated antigens. The lack of CD4 depletion and loss of humoral and cellular immune responses suggest that their immune defect may be due to an early loss in T helper function.
Phosphorylation of Human Cytomegalovirus Glycoprotein B (gB) at the Acidic Cluster Casein Kinase 2 Site (Ser
900
) Is Required for Localization of gB to the
trans-
Golgi Network and Efficient Virus Replication
Authors: Jarvis Michael A. , Jones Thomas R. , Drummond Derek D. , Smith Patsy P. , Britt William J. , Nelson Jay A. , Baldick Carl J.
Published: December 2003

Human cytomegalovirus (HCMV) glycoprotein B (gB), encoded by the UL55 open reading frame, is an essential envelope glycoprotein involved in cell attachment and entry. Previously, we identified residue serine 900 (Ser
900
) as a unique site of reversible casein kinase 2 phosphorylation in the cytoplasmic domain of HCMV gB. We have also recently shown that gB is localized to the
trans
-Golgi network (TGN) in HCMV-permissive cells, thereby identifying the TGN as a possible site of virus envelopment. The aim of the current study was to determine the role of Ser
900
phosphorylation in transport of gB to the TGN and in HCMV biogenesis. Recombinant HCMV strains were constructed that expressed gB molecules containing either an aspartic acid (gBAsp
900
) or alanine residue (gBAla
900
) substitution at Ser
900
to mimic the phosphorylated or nonphosphorylated form, respectively. Immunofluorescence analysis of the trafficking of gB mutant molecules in fibroblasts infected with the HCMV recombinants revealed that gBAsp
900
was localized to the TGN. In contrast, gBAla
900
was partially mislocalized from the TGN, indicating that phosphorylation of gB at Ser
900
was necessary for TGN localization. The increased TGN localization of gBAsp
900
was due to a decreased transport of the molecule to post-TGN compartments. Remarkably, the substitution of an aspartic acid residue for Ser
900
also resulted in an increase in levels of progeny virus production during HCMV infection of fibroblasts. Together, these results demonstrate that phosphorylation of gB at Ser
900
is necessary for gB localization to the TGN, as well as for efficient viral replication, and further support the TGN as a site of HCMV envelopment.
Selectively Receptor-Blind Measles Viruses: Identification of Residues Necessary for SLAM- or CD46-Induced Fusion and Their Localization on a New Hemagglutinin Structural Model
Authors: Vongpunsawad Sompong , Oezgun Numan , Braun Werner , Cattaneo Roberto
Published: December 2003

Measles virus (MV) enters cells either through the signaling lymphocyte activation molecule SLAM (CD150) expressed only in immune cells or through the ubiquitously expressed regulator of complement activation, CD46. To identify residues on the attachment protein hemagglutinin (H) essential for fusion support through either receptor, we devised a strategy based on analysis of morbillivirus H-protein sequences, iterative cycles of mutant protein production followed by receptor-based functional assays, and a novel MV H three-dimensional model. This model uses the Newcastle disease virus hemagglutinin-neuraminidase protein structure as a template. We identified seven amino acids important for SLAM- and nine for CD46 (Vero cell receptor)-induced fusion. The MV H three-dimensional model suggests (i) that SLAM- and CD46-relevant residues are located in contiguous areas in propeller β-sheets 5 and 4, respectively; (ii) that two clusters of SLAM-relevant residues exist and that they are accessible for receptor contact; and (iii) that several CD46-relevant amino acids may be shielded from direct receptor contacts. It appears likely that certain residues support receptor-specific H-protein conformational changes. To verify the importance of the H residues identified with the cell-cell fusion assays for virus entry into cells, we transferred the relevant mutations into genomic MV cDNAs. Indeed, we were able to recover recombinant viruses, and we showed that these replicate selectively in cells expressing SLAM or CD46. Selectively receptor-blind viruses will be used to study MV pathogenesis and may have applications for the production of novel vaccines and therapeutics.