bluuebunny/crossref_metadata_2025 huggingface dataset:
Managing Environmental Impacts of Recreation and Tourism in Rainforests of the Wet Tropics of Queensland World Heritage Area
Authors: Turton Stephen M.
Published: June 2005

This paper describes environmental impacts of tourism and recreation activities in the world heritage listed rainforests of northeast Australia and presents management strategies for sustainable visitor use of the protected area. Tropical rainforests are characterised by their low resistance and moderate to high resilience to impacts associated with human visitation. Visitor use in the World Heritage Area is mostly associated with walking tracks, camping areas, day use areas and off‐road vehicle use of old forestry roads and tracks. Adverse environmental impacts range from vegetation trampling, soil compaction, water contamination and soil erosion at the local scale through to spread of weeds, feral animals and soil pathogens along extensive networks of old forestry roads and tracks at the regional scale. Concentration of visitor use is the most desirable management strategy for controlling adverse impacts at most World Heritage Area visitor nodes and sites, and includes methods such as site hardening and shielding to contain impacts. For dispersed visitor activities, such as off‐road vehicle driving and long‐distance walking, application of best practice methods by the tourist industry and recreational users such as removal of mud and soils from vehicle tyres and hiking boots before entering pathogen‐free catchments, together with seasonal closure of roads and tracks, are the preferred management strategies. Retention of canopy cover at camping areas and day use areas, as well as along walking tracks and forestry roads is a simple, yet effective, management strategy for reduction of a range of adverse impacts, including dispersal of weeds and feral animals, edge effects, soil erosion and nutrient loss, road kill and linear barrier effects on rainforest fauna.
Estimates of Historical Tree Densities in the North Lachlan River Catchment, New South Wales, Australia
Authors: Martin Warren K.
Published: June 2005

Vegetation structure and composition during the pre‐ and early‐European period in Australia is often proposed as a benchmark against which to measure the effects of modern landscape management practices. However, little quantifiable information exists for that historical period. One potential source of information is the land survey record, used in this study to provide estimates of tree densities that existed during the period 1870 to 1900 in part of central New South Wales. Estimates were obtained for 23 parishes in the north Lachlan River valley, between the towns of Condobolin and Tottenham. Densities were calculated from portion corner to nearest tree data obtained from historical survey plans, using the plotless, closest individual method. Densities ranged from 6.0 to 31.4 trees per hectare for parishes with enough data points for reliable calculation. These figures would account for trees of a diameter large enough to be blazed with a survey mark. Any smaller trees would be additional and, although no quantitative data existed for this, qualitative evidence suggested that the figures obtained were more likely to be underestimates due to the presence of such small trees. The study area was predominantly covered by woodlands which varied in density across the landscape, in comparison to the modern landscape where much of the native vegetation has been cleared for agriculture. Although the tree density figures should be considered to be indicative rather than precise, they provide quantifiable historical figures for past conditions in an area where the historical record is otherwise sparse. Such information is valuable for interpreting ecological changes brought about by modern land management.
Constructing Sustainable Rural Landscapes: Oil Mallees and the Western Australian Wheatbelt
Authors: Bell Sarah
Published: June 2005

This paper considers the landscapes of the Western Australian wheatbelt and the possibilities for sustainability signified by the Oil Mallee Project. The Oil Mallee Project aims to develop commercially viable tree crops for the low rainfall wheatbelt region as a means of profitably managing dryland salinity. Interviews with farmers and other stakeholders in the Oil Mallee Project and agriculture are analysed to reveal important elements of landscape construction in the wheatbelt and stages in the emergence of the Project. These elements include historical land clearing, international chemical and machinery companies and the technologies they supply, land of marginal economic productivity, global food markets and alley farming systems. The paper uses material semiotics and actor‐network theory in describing the networks of relationships that shape wheatbelt landscapes. Breakdowns in the dominant networks of industrial agriculture provide spaces for the Oil Mallee Project to build relationships that reconnect industrial systems to the specific ecology of the wheatbelt landscape. However, the networks of industrial agriculture remain powerful and the Project has worked strategically to become integrated with existing agricultural systems, rather than aiming to directly resist or entirely displace dominant patterns of production in the wheatbelt.
The Urban Emergence of a New Information Industry: Sydney's Multimedia Firms
Authors: Searle Glen , De Valence Gerard
Published: June 2005

This paper analyses the multimedia industry in Sydney. It seeks to understand whether the emergence of the industry has involved dense horizontal and vertical inter‐firm linkages characteristic of an industrial cluster, or whether the industry has grown because of general urbanisation economies related to such factors as skilled labour supply and market demand. The industry is highly concentrated in and around central Sydney. This provides the best access to firms’ main clients, notably advertising companies and other multimedia firms. It also reflects the centralised location of skilled labour such as web designers. While there is evidence of an emerging inner Sydney multimedia cluster which includes graphic design and advertising and related media, the main driver of the industry's development appears to have been general demand from advertising and related media companies, in association with the pool of computer graphics talent generated by these companies.
Physical Limits and Design Principles for Plant and Fungal Movements
Authors: Skotheim Jan M. , Mahadevan L.
Published: May 2005

The typical scales for plant and fungal movements vary over many orders of magnitude in time and length, but they are ultimately based on hydraulics and mechanics. We show that quantification of the length and time scales involved in plant and fungal motions leads to a natural classification, whose physical basis can be understood through an analysis of the mechanics of water transport through an elastic tissue. Our study also suggests a design principle for nonmuscular hydraulically actuated structures: Rapid actuation requires either small size or the enhancement of motion on large scales via elastic instabilities.
Identifying Vibrations That Destabilize Crystals and Characterize the Glassy State
Authors: Greaves G. N. , Meneau F. , Majérus O. , Jones D. G. , Taylor J.
Published: May 2005

High-resolution inelastic neutron scattering was used to identify major sources of low-frequency vibrations in zeolite crystals. Dispersed and nondispersed modes were found, both of which are prominent in the early stages of compressive amorphization but decline dramatically in strength once a glass of conventional density is created. By identifying the dispersed modes with the characteristic vibrations of the various secondary building units of zeolitic structures, the Boson peak, a characteristic of the glassy state, can be attributed to vibrations within connected rings of many different sizes. The nondispersed phonon features in zeolites, retained in the amorphized glass, were also replicated in silica. These modes are librational in origin and are responsible for destabilizing the microporous crystalline structure, for converting the resulting glass from a low- to a high-density phase, and for the associated changes in network topology that affect the Boson peak.
A Microbial Arsenic Cycle in a Salt-Saturated, Extreme Environment
Authors: Oremland Ronald S. , Kulp Thomas R. , Blum Jodi Switzer , Hoeft Shelley E. , Baesman Shaun , Miller Laurence G. , Stolz John F.
Published: May 2005

Searles Lake is a salt-saturated, alkaline brine unusually rich in the toxic element arsenic. Arsenic speciation changed from arsenate [As(V)] to arsenite [As(III)] with sediment depth. Incubated anoxic sediment slurries displayed dissimilatory As(V)-reductase activity that was markedly stimulated by H
2
or sulfide, whereas aerobic slurries had rapid As(III)-oxidase activity. An anaerobic, extremely haloalkaliphilic bacterium was isolated from the sediment that grew via As(V) respiration, using either lactate or sulfide as its electron donor. Hence, a full biogeochemical cycle of arsenic occurs in Searles Lake, driven in part by inorganic electron donors.
An Asymmetric Energetic Type Ic Supernova Viewed Off-Axis, and a Link to Gamma Ray Bursts
Authors: Mazzali Paolo A. , Kawabata Koji S. , Maeda Keiichi , Nomoto Ken'ichi , Filippenko Alexei V. , Ramirez-Ruiz Enrico , Benetti Stefano , Pian Elena , Deng Jinsong , Tominaga Nozomu , Ohyama Youichi , Iye Masanori , Foley Ryan J. , Matheson Thomas , Wang Lifan , Gal-Yam Avishay
Published: May 2005

Type Ic supernovae, the explosions after the core collapse of massive stars that have previously lost their hydrogen and helium envelopes, are particularly interesting because of their link with long-duration gamma ray bursts. Although indications exist that these explosions are aspherical, direct evidence has been missing. Late-time observations of supernova SN 2003jd, a luminous type Ic supernova, provide such evidence. Recent Subaru and Keck spectra reveal double-peaked profiles in the nebular lines of neutral oxygen and magnesium. These profiles are different from those of known type Ic supernovae, with or without a gamma ray burst, and they can be understood if SN 2003jd was an aspherical axisymmetric explosion viewed from near the equatorial plane. If SN 2003jd was associated with a gamma ray burst, we missed the burst because it was pointing away from us.
Peptide Antagonists That Inhibit Sin Nombre Virus and Hantaan Virus Entry through the β
3
-Integrin Receptor
Authors: Larson Richard S. , Brown David C. , Ye Chunyan , Hjelle Brian
Published: May 2005

Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the α
v
β
3
integrin, and transfection of human β
3
integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-β
3
antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified α
v
β
3
, we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 μg/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use β
3
integrins and PHV uses a different receptor, β1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via β
3
integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.
A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation
Authors: Grdzelishvili Valery Z. , Smallwood Sherin , Tower Dallas , Hall Richard L. , Hunt D. Margaret , Moyer Sue A.
Published: May 2005

The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5′-cap structures methylated at the guanine-
N
7 and 2′-
O
-adenosine positions (7mGpppA
m
). Previously, our laboratory showed that a VSV host range (
hr
) and temperature-sensitive (
ts
) mutant,
hr1
, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the
hr1
defect in vitro. Here, we sequenced the L, P, and N genes of mutant
hr1
and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate
hr1
from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for
hr1
. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the
hr
and
ts
phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5′-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved
S
-adenosyl-
l
-methionine-binding domain of the L protein.
A New Class of Receptor for Herpes Simplex Virus Has Heptad Repeat Motifs That Are Common to Membrane Fusion Proteins
Authors: Perez Aleida , Li Qing-Xue , Perez-Romero Pilar , DeLassus Gregory , Lopez Santiago R. , Sutter Sarah , McLaren Ning , Fuller A. Oveta
Published: May 2005

We isolated a human cDNA by expression cloning and characterized its gene product as a new human protein that enables entry and infection of herpes simplex virus (HSV). The gene, designated
hfl-
B5, encodes a type II cell surface membrane protein, B5, that is broadly expressed in human primary tissue and cell lines. It contains a high-scoring heptad repeat at the extracellular C terminus that is predicted to form an α-helix for coiled coils like those in cellular SNAREs or in some viral fusion proteins. A synthetic 30-mer peptide that has the same sequence as the heptad repeat α-helix blocks HSV infection of B5-expressing porcine cells and human HEp-2 cells. Transient expression of human B5 in HEp-2 cells results in increased polykarocyte formation even in the absence of viral proteins. The B5 protein fulfills all criteria as a receptor or coreceptor for HSV entry. Use by HSV of a human cellular receptor, such as B5, that contains putative membrane fusion domains provides an example where a pathogenic virus with broad tropism has usurped a widely expressed cellular protein to function in infection at events that lead to membrane fusion.
Exon 3 of the Human Cytomegalovirus Major Immediate-Early Region Is Required for Efficient Viral Gene Expression and for Cellular Cyclin Modulation
Authors: White Elizabeth A. , Spector Deborah H.
Published: May 2005

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Δ30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Δ30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Δ30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Δ30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Δ30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.
Induction of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen by the Lytic Transactivator RTA: a Novel Mechanism for Establishment of Latency
Authors: Lan Ke , Kuppers Daniel A. , Verma Subhash C. , Sharma Nikhil , Murakami Masanao , Robertson Erle S.
Published: May 2005

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jκ (RBP-Jκ), as a RBP-Jκ-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jκ. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.
Complement Activation Is Required for Induction of a Protective Antibody Response against West Nile Virus Infection
Authors: Mehlhop Erin , Whitby Kevin , Oliphant Theodore , Marri Anantha , Engle Michael , Diamond Michael S.
Published: May 2005

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.
Adenovirus Binding to Blood Factors Results in Liver Cell Infection and Hepatotoxicity
Authors: Shayakhmetov Dmitry M. , Gaggar Anuj , Ni Shaoheng , Li Zong-Yi , Lieber André
Published: May 2005

Adenoviruses (Ad) are efficient vehicles for gene delivery in vitro and in vivo. Therefore, they are a promising tool in gene therapy, particularly in the treatment of cancer and cardiovascular diseases. However, preclinical and clinical studies undertaken during the last decade have revealed a series of problems that limit both the safety and efficacy of Ad vectors, specifically after intravenous application. Major obstacles to clinical use include innate toxicity and Ad sequestration by nontarget tissues. The factors and mechanisms underlying these processes are poorly understood. The majority of intravenously injected Ad particles are sequestered by the liver, which in turn causes an inflammatory response characterized by acute transaminitis and vascular damage. Here, we describe a novel pathway that is used by Ad for infection of hepatocytes and Kupffer cells upon intravenous virus application in mice. We found that blood factors play a major role in targeting Ad vectors to hepatic cells. We demonstrated that coagulation factor IX and complement component C4-binding protein can bind the Ad fiber knob domain and provide a bridge for virus uptake through cell surface heparan sulfate proteoglycans and low-density lipoprotein receptor-related protein. An Ad vector, Ad5
mut
, which contained mutations in the fiber knob domain ablating blood factor binding, demonstrated significantly reduced infection of liver cells and liver toxicity in vivo. This study contributes to a better understanding of adenovirus-host interactions for intravenously applied vectors. It also provides a rationale for novel strategies to target adenovirus vector to specific tissues and to reduce virus-associated toxicity after systemic application.
Intestinal Dysplasia Induced by Simian Virus 40 T Antigen Is Independent of p53
Authors: Markovics Jennifer A. , Carroll Patrick A. , Robles M. Teresa Sáenz , Pope Hannah , Coopersmith Craig M. , Pipas James M.
Published: May 2005

Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.
Differential Regulation of Interferon Regulatory Factor (IRF)-7 and IRF-9 Gene Expression in the Central Nervous System during Viral Infection
Authors: Ousman Shalina S. , Wang Jianping , Campbell Iain L.
Published: May 2005

Interferon regulatory factors (IRFs) are a family of transcription factors involved in the regulation of the interferons (IFNs) and other genes that may have an essential role in antiviral defense in the central nervous system, although this is currently not well defined. Therefore, we examined the regulation of IRF gene expression in the brain during viral infection. Several IRF genes (IRF-2, -3, -5, -7, and -9) were expressed at low levels in the brain of uninfected mice. Following intracranial infection with lymphocytic choriomeningitis virus (LCMV), expression of the IRF-7 and IRF-9 genes increased significantly by day 2. IRF-7 and IRF-9 gene expression in the brain was widespread at sites of LCMV infection, with the highest levels in infiltrating mononuclear cells, microglia/macrophages, and neurons. IRF-7 and IRF-9 gene expression was increased in LCMV-infected brain from IFN-γ knockout (KO) but not IFN-α/βR KO animals. In the brain, spleen, and liver or cultured glial and spleen cells, IRF-7 but not IRF-9 gene expression increased with delayed kinetics in the absence of STAT1 but not STAT2 following LCMV infection or IFN-α treatment, respectively. The stimulation of IRF-7 gene expression by IFN-α in glial cell culture was prevented by cycloheximide. Thus, (i) many of the IRF genes were expressed constitutively in the mouse brain; (ii) the IRF-7 and IRF-9 genes were upregulated during viral infection, a process dependent on IFN-α/β but not IFN-γ; and (iii) IRF-7 but not IRF-9 gene expression can be stimulated in a STAT1-independent but STAT2-dependent fashion via unidentified indirect pathways coupled to the activation of the IFN-α/β receptor.
Proteome Analysis of Liver Cells Expressing a Full-Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients
Authors: Jacobs Jon M. , Diamond Deborah L. , Chan Eric Y. , Gritsenko Marina A. , Qian Weijun , Stastna Miroslava , Baas Tracey , Camp David G. , Carithers Robert L. , Smith Richard D. , Katze Michael G.
Published: May 2005

The development of a reproducible model system for the study of hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large-scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full-length HCV replicon. We detected >4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Consistent with the literature, a comparison of HCV replicon-positive and -negative Huh-7.5 cells identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where a total of >1,500 proteins were detected from only 2 μg of liver biopsy protein digest using the Huh-7.5 protein database and the accurate mass and time tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting in the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.
Noncytopathic Replication of Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus Replicons in Mammalian Cells
Authors: Petrakova Olga , Volkova Eugenia , Gorchakov Rodion , Paessler Slobodan , Kinney Richard M. , Frolov Ilya
Published: May 2005

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5′ untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.
Defective Infectious Particles and Rare Packaged Genomes Produced by Cells Carrying Terminal-Repeat-Negative Epstein-Barr Virus
Authors: Feederle R. , Shannon-Lowe C. , Baldwin G. , Delecluse H. J.
Published: May 2005

The Epstein-Barr virus (EBV) lytic program includes lytic viral DNA replication and the production of a viral particle into which the replicated viral DNA is packaged. The terminal repeats (TRs) located at the end of the linear viral DNA have been identified as the packaging signals. A TR-negative (TR
−
) mutant therefore provides an appropriate tool to analyze the relationships between EBV DNA packaging and virus production. Here, we show that supernatants from lytically induced 293 cells carrying TR mutant EBV genomes (293/TR
−
) contain large amounts of viral particles devoid of viral DNA which are nevertheless able to bind to EBV target cells. This shows that viral DNA packaging is not a prerequisite for virion formation and egress. Rather surprisingly, supernatants from lytically induced 293/TR
−
cells also contained rare infectious viruses carrying the viral mutant DNA. This observation indicates that the TRs are important but not absolutely essential for virus encapsidation.
Hepatitis C Virus Core Protein Suppresses NF-κB Activation and Cyclooxygenase-2 Expression by Direct Interaction with IκB Kinase β
Authors: Joo Myungsoo , Hahn Young S. , Kwon Minjae , Sadikot Ruxana T. , Blackwell Timothy S. , Christman John W.
Published: May 2005

In addition to hepatocytes, hepatitis C virus (HCV) infects immune cells, including macrophages. However, little is known concerning the impact of HCV infection on cellular functions of these immune effector cells. Lipopolysaccharide (LPS) activates IκB kinase (IKK) signalsome and NF-κB, which leads to the expression of cyclooxygenase-2 (COX-2), which catalyzes production of prostaglandins, potent effectors on inflammation and possibly hepatitis. Here, we examined whether expression of HCV core interferes with IKK signalsome activity and COX-2 expression in activated macrophages. In reporter assays, HCV core inhibited NF-κB activation in RAW 264.7 and MH-S murine macrophage cell lines treated with bacterial LPS. HCV core inhibited IKK signalsome and IKKβ kinase activities induced by tumor necrosis factor alpha in HeLa cells and coexpressed IKKγ in 293 cells, respectively. HCV core was coprecipitated with IΚΚβ and prevented nuclear translocation of IKKβ. NF-κB activation by either LPS or overexpression of IKKβ was sufficient to induce robust expression of COX-2, which was markedly suppressed by ectopic expression of HCV core. Together, these data indicate that HCV core suppresses IKK signalsome activity, which blunts COX-2 expression in macrophages. Additional studies are necessary to determine whether interrupted COX-2 expression by HCV core contributes to HCV pathogenesis.
Role of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Immune Response to Influenza Virus Infection in Mice
Authors: Ishikawa Eri , Nakazawa Masatoshi , Yoshinari Masahiro , Minami Mutsuhiko
Published: May 2005

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of various tumor cells but not normal cells. However, various cytokines and virus infection differentially regulate TRAIL and TRAIL receptor expression. It has been demonstrated that virus infection changes the pattern of human TRAIL-receptor expression on normal cells, which were resistant to TRAIL-mediated apoptosis, and makes them susceptible to TRAIL-mediated apoptosis. Since previous studies on the function of TRAIL have been performed mainly in vitro, its physiological role in the immune response to virus infection remains unknown. In the present study, we investigated the expression of TRAIL in the lungs of influenza virus-infected mice and the function of TRAIL in the immune response to infection. Influenza virus infection increased TRAIL mRNA expression in the lung. TRAIL protein expression was induced on NK cells in the lung 4 days after infection. At 7 days after infection, TRAIL protein expression was also detected on CD4+and CD8+T cells. However, NK cells and T cells in the lungs of uninfected mice did not express a detectable level of TRAIL on their cell surfaces. DR5, which is a mouse TRAIL receptor, was also induced to express after virus infection. Expression of both TRAIL and DR5 mRNAs was reduced to normal level at 6 weeks after virus infection. Administration of anti-TRAIL monoclonal antibody, which blocks TRAIL without killing TRAIL-expressing cells, to mice during influenza virus infection significantly delayed virus clearance in the lung. These results suggest that TRAIL plays an important role in the immune response to virus infection.
Single Amino Acid Insertions at the Junction of the Sindbis Virus E2 Transmembrane Domain and Endodomain Disrupt Virus Envelopment and Alter Infectivity
Authors: Hernandez Raquel , Ferreira Davis , Sinodis Christine , Litton Katherine , Brown Dennis T.
Published: May 2005

The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 ΔK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 ΔK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K→F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.
Cytokine Responses in Severe Acute Respiratory Syndrome Coronavirus-Infected Macrophages In Vitro: Possible Relevance to Pathogenesis
Authors: Cheung Chung Y. , Poon Leo L. M. , Ng Iris H. Y. , Luk Winsie , Sia Sin-Fun , Wu Mavis H. S. , Chan Kwok-Hung , Yuen Kwok-Yung , Gordon Siamon , Guan Yi , Peiris Joseph S. M.
Published: May 2005

The pathogenesis of severe acute respiratory syndrome (SARS) remains unclear. Macrophages are key sentinel cells in the respiratory system, and it is therefore relevant to compare the responses of human macrophages to infections with the SARS coronavirus (SARS-CoV) and other respiratory viruses. Primary human monocyte-derived macrophages were infected with SARS-CoV in vitro. Virus replication was monitored by measuring the levels of positive- and negative-strand RNA, by immunofluorescence detection of the SARS-CoV nucleoprotein, and by titration of the infectious virus. The gene expression profiles of macrophages infected with SARS-CoV, human coronavirus 229E, and influenza A (H1N1) virus were compared by using microarrays and real-time quantitative reverse transcriptase PCR. Secreted cytokines were measured with an enzyme-linked immunosorbent assay. SARS-CoV initiated viral gene transcription and protein synthesis in macrophages, but replication was abortive and no infectious virus was produced. In contrast to the case with human coronavirus 229E and influenza A virus, there was little or no induction of beta interferon (IFN-β) in SARS-CoV-infected macrophages. Furthermore, SARS-CoV induced the expression of chemokines such as CXCL10/IFN-γ-inducible protein 10 and CCL2/monocyte chemotactic protein 1. The poor induction of IFN-β, a key component of innate immunity, and the ability of the virus to induce chemokines could explain aspects of the pathogenesis of SARS.
Characterization of Human Cytomegalovirus Glycoprotein-Induced Cell-Cell Fusion
Authors: Kinzler Eric R. , Compton Teresa
Published: May 2005

Human cytomegalovirus (CMV) infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteins mediate the initial attachment and fusion events that occur between the viral envelope and a host cell membrane, as well as virion-independent cell-cell spread of the infection. Here we have utilized a cell-based fusion assay to identify the fusogenic glycoproteins of CMV. To deliver the glycoprotein genes to various cell lines, we constructed recombinant retroviruses encoding gB, gH, gL, and gO. Cells expressing individual CMV glycoproteins did not form multinucleated syncytia. Conversely, cells expressing gH/gL showed pronounced syncytium formation, although expression of gH or gL alone had no effect. Anti-gH neutralizing antibodies prevented syncytium formation. Coexpression of gB and/or gO with gH/gL did not yield detectably increased numbers of syncytia. For verification, these results were recapitulated in several cell lines. Additionally, we found that fusion was cell line dependent, as nonimmortalized fibroblast strains did not fuse under any conditions. Thus, the CMV gH/gL complex has inherent fusogenic activity that can be measured in certain cell lines; however, fusion in fibroblast strains may involve a more complex mechanism involving additional viral and/or cellular factors.
Regulatory T Cells Suppress In Vitro Proliferation of Virus-Specific CD8+T Cells during Persistent Hepatitis C Virus Infection
Authors: Rushbrook Simon M. , Ward Scott M. , Unitt Esther , Vowler Sarah L. , Lucas Michaela , Klenerman Paul , Alexander Graeme J. M.
Published: May 2005

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8+T cells. The role of CD4+CD25+T regulatory (Treg) cells in priming and expanding virus-specific CD8+T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8+T-cell proliferation and gamma interferon (IFN-γ) frequency were analyzed with/without depletion of Tregcells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4+CD25+Tregcells inhibited anti-CD3/CD28 CD8+T-cell proliferation and perforin expression. Depletion of CD4+CD25+Tregcells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P= 0.0005 andP= 0.002, respectively) and also the number of HCV- and EBV-specific IFN-γ-expressing CD8+T cells. Although stimulated CD8+T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4+CD25+regulatory T cells on CD8+T-cell proliferation. In conclusion, marked CD4+CD25+regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8+T-cell responses and viral persistence.
Disruption of Human TRIM5α Antiviral Activity by Nonhuman Primate Orthologues
Authors: Berthoux Lionel , Sebastian Sarah , Sayah David M. , Luban Jeremy
Published: May 2005

TRIM5 is a determinant of species-specific differences in susceptibility to infection by retroviruses bearing particular capsids. Human immunodeficiency virus type 1 (HIV-1) infection is blocked by the alpha isoform of macaque TRIM5 (TRIM5α
rh
) or by the product of the owl monkey TRIM5-cyclophilin A gene fusion (TRIMCyp). Human TRIM5α potently restricts specific strains of murine leukemia virus (N-MLV) but has only a modest effect on HIV-1. The amino termini of TRIM5 orthologues are highly conserved and possess a coiled-coil domain that promotes homomultimerization. Here we show that heterologous expression of TRIM5α
rh
or TRIMCyp in human cells interferes with the anti-N-MLV activity of endogenous human TRIM5α (TRIM5α
hu
). Deletion of the cyclophilin domain from TRIMCyp has no effect on heteromultimerization or colocalization with TRIM5α
hu
but prevents interference with anti-N-MLV activity. These data demonstrate that TRIM5 orthologues form heteromultimers and indicate that C-terminal extensions alter virus recognition by multimers of these proteins.
Investigation of the Multimerization Region of the Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Protein K-bZIP: the Proposed Leucine Zipper Region Encodes a Multimerization Domain with an Unusual Structure
Authors: Al Mehairi Salama , Cerasoli Eleanora , Sinclair Alison J.
Published: May 2005

The
K8
gene of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) shares many functional similarities with the
BZLF1
gene of Epstein-Barr virus. The protein products of
K8
and
BZLF1
, K-bZIP (RAP, K8) and Zta (BZLF1, ZEBRA, Z) have both been proposed to be members of the bZIP family of transcription factors, forming multimers via a coiled-coil motif termed a leucine zipper. Substantial evidence supporting this model for Zta is published. Here, we demonstrate that the proposed leucine zipper region of K-bZIP (amino acids 182 to 218) is required for multimer formation but that it does not fold as a coiled coil.
Determination of the Minimal Distance between the Matrix and Transmembrane Domains of the Large Hepatitis B Virus Envelope Protein
Authors: Kluge Britta , Schläger Michaela , Pairan Alexander , Bruss Volker
Published: May 2005

The cytosolic matrix domain (MD) located between amino acids (aa) 103 and 124 of the large hepatitis B virus envelope protein L is essential for virion formation. We reduced the distance between MD and the transmembrane domain (TD; aa 254 to 272) by deletions starting at aa 132. Six mutants with deletions of up to aa 234 were wild type, and four mutants with slightly larger deletions were blocked with respect to virion morphogenesis. Thus, the minimal distance between MD and TD was around 26 aa. This spacer might be required by MD to reach contact sites on the capsid.
The Morphology and Composition of Influenza A Virus Particles Are Not Affected by Low Levels of M1 and M2 Proteins in Infected Cells
Authors: Bourmakina Svetlana V. , García-Sastre Adolfo
Published: May 2005

We generated a recombinant influenza A virus (Mmut) that produced low levels of matrix (M1) and M2 proteins in infected cells. Mmut virus propagated to significantly lower titers than did wild-type virus in cells infected at low multiplicity. By contrast, virion morphology and incorporation of viral proteins and vRNAs into virus particles were similar to those of wild-type virus. We propose that a threshold amount of M1 protein is needed for the assembly of viral components into an infectious particle and that budding is delayed in Mmut virus-infected cells until sufficient levels of M1 protein accumulate at the plasma membrane.
Enhanced Immunogenicity of gp120 Protein When Combined with Recombinant DNA Priming To Generate Antibodies That Neutralize the JR-FL Primary Isolate of Human Immunodeficiency Virus Type 1
Authors: Wang Shixia , Arthos James , Lawrence John M. , Van Ryk Donald , Mboudjeka Innocent , Shen Siyuan , Chou Te-hui W. , Montefiori David C. , Lu Shan
Published: May 2005

Strategies are needed for human immunodeficiency virus type 1 vaccine development that improves the neutralizing antibody response against primary isolates of the virus. Here we examined recombinant DNA priming followed by subunit protein boosting as a strategy to generate neutralizing antibodies. Both plasmid-based and recombinant protein envelope (Env) glycoprotein immunogens were derived from a primary viral isolate, JR-FL. Serum from rabbits immunized with either gp120 or gp140 DNA vaccines delivered by gene gun inoculation followed by recombinant gp120 protein boosting was capable of neutralizing JR-FL. Neither the DNA vaccines alone nor the gp120 protein alone generated a detectable neutralizing antibody response against this virus. Neutralizing antibody responses using gp120 DNA and gp140 DNA for priming were similar. The results suggest that Env DNA priming followed by gp120 protein boosting provides an advantage over either approach alone for generating a detectable neutralizing antibody response against primary isolates that are not easily neutralized.
Reversible Acid-Induced Inactivation of the Membrane Fusion Protein of Semliki Forest Virus
Authors: Waarts Barry-Lee , Smit Jolanda M. , Aneke Onwuchekwa J. C. , McInerney Gerald M. , Liljeström Peter , Bittman Robert , Wilschut Jan
Published: May 2005

Previously, it has been shown that the exposure of Semliki Forest virus (SFV) to a mildly acidic environment induces a rapid and complete loss of the ability of the virus to bind and fuse to target membranes added subsequently. In the present study, incubation of SFV at low pH followed by a specific reneutralization step resulted in a partial reversion of this loss of viral fusion capacity, as assessed in a liposomal model system. Also, the ability of the viral E1 fusion protein to undergo liposome-stimulated trimerization was restored. Furthermore, acid-treated and neutralized SFV largely retained infectivity. Exposure of SFV to low pH induced dissociation of the E1/E2 heterodimer, which was not reversed upon neutralization. It is concluded that the SFV E1 fusion protein, after acid-induced dissociation from E2, rapidly adopts an intermediate, nontrimeric conformation in which it is no longer able to interact with target membrane lipids. Neutralization restores the ability of E1 to interact with membranes. This interaction, however, remains strictly dependent on low pH.