bluuebunny/crossref_metadata_2025 huggingface dataset:
Bordetella bronchisepticaFlagellin Is a Proinflammatory Determinant for Airway Epithelial Cells
Authors: López-Boado Yolanda S. , Cobb Laura M. , Deora Rajendar
Published: October 2005

Motility is an important virulence phenotype for many bacteria, and flagellin, the monomeric component of flagella, is a potent proinflammatory factor. Of the threeBordetellaspecies,Bordetella pertussisandBordetella parapertussisare nonmotile human pathogens, whileBordetella bronchisepticaexpresses flagellin and causes disease in animals and immunocompromised human hosts. The BvgAS two-component signal transduction system regulates phenotypic-phase transition (Bvg+, Bvg−, and Bvgi) in bordetellae. The Bvg−phase ofB. bronchisepticais characterized by the expression of flagellin and the repression of adhesins and toxins necessary for the colonization of the respiratory tract.B. bronchisepticanaturally infects a variety of animal hosts and constitutes an excellent model to studyBordetellapathogenesis. Using in vitro coculture models of bacteria and human lung epithelial cells, we studied the effects ofB. bronchisepticaflagellin on host defense responses. Our results show thatB. bronchisepticaflagellin is a potent proinflammatory factor that induces chemokine, cytokine, and host defense gene expression. Furthermore, we investigated receptor specificity in the response toB. bronchisepticaflagellin. Our results show thatB. bronchisepticaflagellin is able to signal effectively through both human and mouse Toll-like receptor 5.
Coordinate Expression of Fimbriae in Uropathogenic
Escherichia coli
Authors: Snyder Jennifer A. , Haugen Brian J. , Lockatell C. Virginia , Maroncle Nathalie , Hagan Erin C. , Johnson David E. , Welch Rodney A. , Mobley Harry L. T.
Published: October 2005

Uropathogenic
Escherichia coli
is the most common etiological agent of urinary tract infections. Bacteria can often express multiple adhesins during infection in order to favor attachment to specific niches within the urinary tract. We have recently demonstrated that type 1 fimbria, a phase-variable virulence factor involved in adherence, was the most highly expressed adhesin during urinary tract infection. Here, we examine whether the expression of type 1 fimbriae can affect the expression of other adhesins. Type 1 fimbrial phase-locked mutants of
E. coli
strain CFT073, which harbors genes for numerous adhesins, were employed in this study. CFT073-specific DNA microarray analysis of these strains demonstrates that the expression of type 1 fimbriae coordinately affects the expression of P fimbriae in an inverse manner. This represents evidence for direct communication between genes relating to pathogenesis, perhaps to aid the sequential occupation of different urinary tract tissues. While the role of type 1 fimbriae during infection has been clear, the role of P fimbriae must be further defined to assert the relevance of coordinated regulation in vivo. Therefore, we examined the ability of P fimbrial isogenic mutants, constructed in a type 1 fimbrial-negative background, to compete in the murine urinary tract over a period of 168 h. No differences in the colonization of these mutants were observed. However, comparison of these results with previous studies suggests that inversely coordinated expression of adhesin gene clusters does occur in vivo. Interestingly, the mutant that was incapable of expressing either type 1 or P fimbriae compensated by synthesizing F1C fimbriae.
Regulation of RANTES Promoter Activation in Gastric Epithelial Cells Infected withHelicobacter pylori
Authors: Kudo Takahiko , Lu Hong , Wu Jeng Yih , Graham David Y. , Casola Antonella , Yamaoka Yoshio
Published: October 2005

RANTES, a CC chemokine, plays an important role in the inflammatory response associated withHelicobacter pyloriinfection. However, the mechanism by whichH. pyloriinduces RANTES expression in the gastric mucosa is unknown. We cocultured gastric epithelial cells with wild-typeH. pylori, isogenicoipAmutants,cagpathogenicity island (PAI) mutants, or double knockout mutants. Reverse transcriptase PCR showed that RANTES mRNA was induced byH. pyloriand that the expression was both OipA andcagPAI dependent. Luciferase reporter gene assays and electrophoretic mobility shift assays showed that maximalH. pylori-inducedRANTESgene transcription required the presence of the interferon-stimulated responsive element (ISRE), the cyclic AMP-responsive element (CRE), nuclear factor-interleukin 6 (NF-IL-6), and two NF-κB sites. OipA- andcagPAI-dependent pathways included NF-κB→NF-κB/NF-IL-6/ISRE pathways, andcagPAI-dependent pathways additionally included Jun N-terminal kinase→CRE/NF-κB pathways. The OipA-dependent pathways additionally included p38→CRE/ISRE pathways. We confirmed the in vitro effects in vivo by examining RANTES mRNA levels in biopsy specimens from human gastric antral mucosa. RANTES mRNA levels in the antral mucosa were significantly higher for patients infected withcagPAI/OipA-positiveH. pylorithan for those infected withcagPAI/OipA-negativeH. pylorior uninfected patients. The mucosal inflammatory response toH. pyloriinfection involves different signaling pathways for activation of the RANTES promoter, with both OipA and thecagPAI being required for full activation of the RANTES promoter.
Interleukin-4 (IL-4) and IL-10 Collude in Vaccine Failure for Novel Exacerbatory Antigens in Murine
Leishmania major
Infection
Authors: Roberts Mark T. M. , Stober Carmel B. , McKenzie Andrew N , Blackwell Jenefer M.
Published: October 2005

Leishmaniasis affects 12 million people but there are no vaccines in routine use. Recently, we used DNA vaccination in a susceptible BALB/c high-dose model of infection to screen 100 novel
Leishmania major
genes as vaccine candidates. In addition to finding novel protective antigens, we identified several antigens that reproducibly exacerbated disease. Here we examined the immune response to two of these antigens, lmd29 and 584C, that were originally identified in an expressed sequence tag cDNA sequencing project. We show that, in addition to exacerbating disease in susceptible BALB/c mice, these antigens retain a propensity to exacerbate disease in resistant C57BL/6 mice. This ability to exacerbate disease was lost when susceptible BALB/c mice were rendered resistant by disruption of the genes encoding interleukin-4 (IL-4) alone, IL-4/IL-13, or IL-4, IL-5, IL-9, and IL-13. Failure to exacerbate disease was associated with reduced IL-5 and IL-10 production in IL-4 knockout mice. Treatment of lmd29-vaccinated mice with anti-IL-10 receptor antibody prior to challenge infection converted exacerbation in wild-type BALB/c mice into highly significant antigen-specific protection. These studies demonstrate that some highly immunogenic antigens of
L. major
, while having an intrinsic capacity to exacerbate disease in the context of otherwise T helper 1-promoting DNA vaccine delivery, can be rendered protective by the removal of functional IL-10.
Effects of
Anaplasma phagocytophilum
on Host Cell Ferritin mRNA and Protein Levels
Authors: Carlyon Jason A. , Ryan Dara , Archer Kristina , Fikrig Erol
Published: October 2005

Ferritin is a major intracellular iron storage protein and also functions as a cytoprotectant by sequestering iron to minimize the formation of reactive oxygen species.
Anaplasma phagocytophilum
, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils. We have previously reported that human promyelocytic HL-60 cells infected with
A. phagocytophilum
demonstrate increased transcription of ferritin heavy chain and also that the bacterium stimulates neutrophil NADPH oxidase assembly and degranulation during the initial hours of infection (J. A. Carlyon, W. T. Chan, J. Galan, D. Roos, and E. Fikrig, J. Immunol.
169:
7009-7018, 2002, and J. A. Carlyon, D. Abdel-Latif, M. Pypaert, P. Lacy, and E. Fikrig, Infect. Immun. 72:4772-4783, 2004). In this study, we assessed ferritin mRNA and protein levels during
A. phagocytophilum
infection in vitro using HL-60 cells and neutrophils and in vivo using neutrophils from infected mice. The addition of
A. phagocytophilum
, as well as
Escherichia coli
and serum-opsonized zymosan, to neutrophils results in a pronounced increase in ferritin light-chain transcription and a concomitant rise in ferritin protein levels. Neutrophils from
A. phagocytophilum
-infected mice demonstrate elevated ferritin heavy-chain mRNA expression, a phenomenon consistent with infections by intracellular pathogens. Notably, ferritin protein levels of infected HL-60 cells were markedly diminished in a dose- and time-dependent manner. These studies provide insight into the effects
A. phagocytophilum
has on the ferritin levels of its host cell.
Apical Spore Phagocytosis Is Not a Significant Route of Infection of Differentiated Enterocytes by
Encephalitozoon intestinalis
Authors: Leitch Gordon J. , Ward Tarsha L. , Shaw Andrew P. , Newman Gale
Published: October 2005

Encephalitozoon intestinalis
is a microsporidian species that infects the intestinal mucosal epithelium, primarily in immunodeficient individuals. The present study employed undifferentiated and differentiated human colonic carcinoma cell lines to determine if this parasite species infected polarized epithelial cells by spore phagocytosis or by impalement with the deployed spore polar tube. Apical surface spore attachment differed between cell lines such that SW480>HT-29>Caco-2>HCT-8, with attachment being greater to undifferentiated Caco-2 cells than differentiated cells and greater to partially differentiated HCT-8 cells than differentiated HCT-8 cells. Attachment was inhibited by chondroitin sulfate A, suggesting that it was mediated by host cell sulfated glycoaminoglycans. Infection rates 3 days postinfection paralleled spore attachment in the various cell lines. The undifferentiated cell line SW480 and undifferentiated Caco-2 and HCT-8 cells exhibited modest spore phagocytosis while the more differentiated cell line HT29 and differentiated Caco-2 and HCT-8 cells did not. All cell lines were impaled by the polar tubes of germinating spores. When normalized to the number of spores attached to the apical membrane, such impalement was greatest in the more differentiated Caco-2 and HCT-8 cells. The host cell apical surface influenced parasite spore germination, as in populations of large undifferentiated Caco-2 cells to which >3 spores had attached, the frequency distribution of the percentages of spores germinated per cell was bimodal, indicating that the surface of some cells favored germination, while others did not. This study suggests that phagocytosis is not a biologically significant mode of infection in differentiated intestinal epithelial cells.
Host and Bacterial Factors Contributing to the Clearance of Colonization byStreptococcus pneumoniaein a Murine Model
Authors: van Rossum Annemarie M. C. , Lysenko Elena S. , Weiser Jeffrey N.
Published: October 2005

Nasopharyngeal colonization is the first step in the interaction betweenStreptococcus pneumoniae(the pneumococcus) and its human host. Factors that contribute to clearance of colonization are likely to affect the spread of the pneumococcus and the rate of pneumococcal disease in the population. To identify host and bacterial factors contributing to this process, we examined the time course of colonization using genetically modified mice and pneumococci. Severe combined immunodeficient mice remained persistently colonized (>6 weeks). Major histocompatibility complex II-deficient mice, but not μMT mice, were unable to clear colonization and showed a diminished T helper 1 response. Thus, CD4+T cells, rather than the generation of specific antibody, appear to be required for effective Th1-mediated clearance. In addition, the microbial pattern recognition receptor toll-like receptor 2 (TLR2), but not TLR4, was necessary for efficient clearance of colonization. In contrast, no role of complement component 3, inducible nitric oxide synthetase, interleukin 12 (IL-12), or IL-4 could be demonstrated. Expression of the pneumococcal toxin pneumolysin enhanced acute localized inflammatory responses and promoted clearance of colonization in a TLR4-independent manner. We conclude that both innate and CD4+T-cell-mediated immunity and proinflammatory bacterial factors, rather than a humoral adaptive immune response, are important for clearance ofS. pneumoniaefrom the murine nasopharynx.
Redundant Roles for Nucleocapsid and Matrix RNA-Binding Sequences in Human Immunodeficiency Virus Type 1 Assembly
Authors: Ott David E. , Coren Lori V. , Gagliardi Tracy D.
Published: October 2005

RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in
trans
by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.
Assembly of Severe Acute Respiratory Syndrome Coronavirus RNA Packaging Signal into Virus-Like Particles Is Nucleocapsid Dependent
Authors: Hsieh Ping-Kun , Chang Shin C. , Huang Chu-Chun , Lee Ting-Ting , Hsiao Ching-Wen , Kou Yi-Hen , Chen I-Yin , Chang Chung-Ke , Huang Tai-Huang , Chang Ming-Fu
Published: October 2005

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm
3
. Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3′ noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.
Kinetic Analyses of the Surface-Transmembrane Disulfide Bond Isomerization-Controlled Fusion Activation Pathway in Moloney Murine Leukemia Virus
Authors: Wallin Michael , Löving Robin , Ekström Maria , Li Kejun , Garoff Henrik
Published: October 2005

The surface (SU) and transmembrane (TM) subunits of Moloney murine leukemia virus (Mo-MLV) Env are disulfide linked. The linking cysteine in SU is part of a conserved CXXC motif in which the other cysteine carries a free thiol. Recently, we showed that receptor binding activates its free thiol to isomerize the intersubunit disulfide bond into a disulfide within the motif instead (M. Wallin, M. Ekström and H. Garoff, EMBO J.
23:
54-65, 2004). This facilitated SU dissociation and activation of TM for membrane fusion. The evidence was mainly based on the finding that alkylation of the CXXC-thiol prevented isomerization. This arrested membrane fusion, but the activity could be rescued by cleaving the intersubunit disulfide bond with dithiothreitol (DTT). Here, we demonstrate directly that receptor binding causes SU-TM disulfide bond isomerization in a subfraction of the viral Envs. The kinetics of the isomerization followed that of virus-cell membrane fusion. Arresting the fusion with lysophosphatidylcholine did not arrest isomerization, suggesting that isomerization precedes the hemifusion stage of fusion. Our earlier finding that native Env was not possible to alkylate but required isomerization induction by receptor binding intimated that alkylation trapped an intermediate form of Env. To further clarify this possibility, we analyzed the kinetics by which the alkylation-sensitive Env was generated during fusion. We found that it followed the fusion kinetics. In contrast, the release of fusion from alkylated, isomerization-blocked virus by DTT reduction of the SU-TM disulfide bond was much faster. These results suggest that the alkylation-sensitive form of Env is a true intermediate in the fusion activation pathway of Env.
Transcriptional Regulation of Early Transposon Elements, an Active Family of Mouse Long Terminal Repeat Retrotransposons
Authors: Maksakova Irina A. , Mager Dixie L.
Published: October 2005

While early transposon (ETn) endogenous retrovirus (ERV)-like elements are known to be active insertional mutagens in the mouse, little is known about their transcriptional regulation. ETns are transcribed during early mouse embryogenesis in embryonic stem (ES) and embryonic carcinoma (EC) cell lines. Despite their lack of coding potential, some ETns remain transposition competent through their use of reverse transcriptase encoded by a related group of ERVs—MusD elements. In this study, we have confirmed high expression levels of ETn and MusD elements in ES and EC cells and have demonstrated an increase in the copy number of ETnII elements in the EC P19 cell line. Using transient transfections, we have shown that ETnII and MusD LTRs are much more active as promoters in P19 cells than in NIH 3T3 cells, indicating that genomic context and methylation are not the only factors determining endogenous transcriptional activity of ETns. Three sites in the 5′ part of the long terminal repeat (LTR) were demonstrated to bind Sp1 and Sp3 transcription factors and were found to be important for high LTR promoter activity in P19 cells, suggesting that as yet unidentified Sp binding partners are involved in the regulation of ETn activity in undifferentiated cells. Finally, we found multiple transcription start sites within the ETn LTR and have shown that the LTR retains significant promoter activity in the absence of its noncanonical TATA box. These findings lend insight into the transcriptional regulation of this family of mobile mouse retrotransposons.
Stress-Activated Protein Kinases Are Involved in Coxsackievirus B3 Viral Progeny Release
Authors: Si Xiaoning , Luo Honglin , Morgan Andrew , Zhang Jingchun , Wong Jerry , Yuan Ji , Esfandiarei Mitra , Gao Guang , Cheung Caroline , McManus Bruce M.
Published: October 2005

Stress-activated protein kinases (SAPKs), consisting of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), are activated upon various environmental stimuli, including viral infections. Cellular survival and death signaling events following coxsackievirus B3 (CVB3) infection have been studied in relationship to viral replication, but the role of SAPKs has not been scrutinized. In this study, we found that the phosphorylation of JNK1/2 and p38 MAPK was increased during active replication of CVB3 and that their phosphorylation was independent of CVB3-induced caspase activation or production of reactive oxygen species. The roles of these kinases in CVB3 infection were further evaluated using specific inhibitors: SP600125 for JNK1/2 and SB203580 for p38 MAPK. JNK1/2 inhibitors reduced CVB3-induced phosphorylation of activating transcription factor 2, and the p38 MAPK inhibitor reduced CVB3-induced phosphorylation of heat shock protein 27. Although inhibition of these kinases by specific inhibitors did not affect CVB3 viral protein synthesis, inhibition of p38 MAPK but not of JNK1/2 resulted in significant reduction of viral progeny release, suppression of CVB3-induced cell death, and blockage of CVB3-induced caspase-3 activation in infected cells. We conclude that SAPK pathways play critical roles in the life cycle of CVB3, particularly in viral progeny release.
Viral Protein VP4 Is a Target of Human Antibodies Enhancing Coxsackievirus B4- and B3-Induced Synthesis of Alpha Interferon
Authors: Chehadeh Wassim , Lobert Pierre-Emmanuel , Sauter Pierre , Goffard Anne , Lucas Bernadette , Weill Jacques , Vantyghem Marie-Christine , Alm Gunnar , Pigny Pascal , Hober Didier
Published: October 2005

Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-α) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-α were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56°C from CVB4E2 (VP4CVB4) and CVB3 (VP4CVB3) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis. There was no cross-reaction between VP4CVB4and VP4CVB3in the inhibiting effect. IFN-α levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4CVB4). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-α levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis by PBMC.
Inhibition of Polyprotein Processing and RNA Replication of Human Rhinovirus by Pyrrolidine Dithiocarbamate Involves Metal Ions
Authors: Krenn B. M. , Holzer B. , Gaudernak E. , Triendl A. , van Kuppeveld F. J. , Seipelt J.
Published: October 2005

Pyrrolidine dithiocarbamate (PDTC) is an antiviral compound that was shown to inhibit the replication of human rhinoviruses (HRVs), poliovirus, and influenza virus. To elucidate the mechanism of PDTC, the effects on the individual steps of the infection cycle of HRV were investigated. PDTC did not interfere with receptor binding or internalization by receptor mediated endocytosis of HRV2 particles into HeLa cells. But we demonstrate that the processing of the viral polyprotein was prevented by PDTC treatment in HeLa cells infected with HRV2. Furthermore, PDTC inhibited the replication of the viral RNA, even when added four hours post infection. As PDTC is described as a metal ion binding agent, we investigated the effect of other metal chelators on the multiplication of HRV2. We show that EDTA,ο-phenanthroline, and bathocuproine disulfonic acid do not exhibit any antiviral properties. Surprisingly, these substances, coadministered with PDTC, abolished the antiviral effect of PDTC, suggesting that metal ions play a pivotal role in the inhibition of virus multiplication. These results suggest that PDTC inhibits the activity of the viral proteases in a metal ion dependent way.
Modulation of the Immune Response to the Severe Acute Respiratory Syndrome Spike Glycoprotein by Gene-Based and Inactivated Virus Immunization
Authors: Kong Wing-pui , Xu Ling , Stadler Konrad , Ulmer Jeffrey B. , Abrignani Sergio , Rappuoli Rino , Nabel Gary J.
Published: October 2005

Although the initial isolates of the severe acute respiratory syndrome (SARS) coronavirus (CoV) are sensitive to neutralization by antibodies through their spike (S) glycoprotein, variants of S have since been identified that are resistant to such inhibition. Optimal vaccine strategies would therefore make use of additional determinants of immune recognition, either through cellular or expanded, cross-reactive humoral immunity. Here, the cellular and humoral immune responses elicited by different combinations of gene-based and inactivated viral particles with various adjuvants have been assessed. The T-cell response was altered by different prime-boost immunizations, with the optimal CD8 immunity induced by DNA priming and replication-defective adenoviral vector boosting. The humoral immune response was enhanced most effectively through the use of inactivated virus with adjuvants, either MF59 or alum, and was associated with stimulation of the CD4 but not the CD8 response. The use of inactivated SARS virus with MF59 enhanced the CD4 and antibody response even after gene-based vaccination. Because both cellular and humoral immune responses are generated by gene-based vaccination and inactivated viral boosting, this strategy may prove useful in the generation of SARS-CoV vaccines.
Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells
Authors: Cai Zhaohui , Zhang Chen , Chang Kyung-Soo , Jiang Jieyun , Ahn Byung-Chul , Wakita Takaji , Liang T. Jake , Luo Guangxiang
Published: October 2005

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5′ end and a hepatitis delta virus ribozyme at the 3′ end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10
8
copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.
Identification of Interferon-Stimulated Gene 15 as an Antiviral Molecule during Sindbis Virus Infection In Vivo
Authors: Lenschow Deborah J. , Giannakopoulos Nadia V. , Gunn Lacey J. , Johnston Christine , O'Guin Andy K. , Schmidt Robert E. , Levine Beth , Virgin Herbert W.
Published: October 2005

The innate immune response, and in particular the alpha/beta interferon (IFN-α/β) system, plays a critical role in the control of viral infections. Interferons α and β exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-α/β. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-α/β receptor (IFN-α/βR)−/−mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.
Epstein-Barr Virus Lytic Infection Contributes to Lymphoproliferative Disease in a SCID Mouse Model
Authors: Hong Gregory K. , Gulley Margaret L. , Feng Wen-Hai , Delecluse Henri-Jacques , Holley-Guthrie Elizabeth , Kenney Shannon C.
Published: October 2005

Most Epstein-Barr virus (EBV)-positive tumor cells contain one of the latent forms of viral infection. The role of lytic viral gene expression in EBV-associated malignancies is unknown. Here we show that EBV mutants that cannot undergo lytic viral replication are defective in promoting EBV-mediated lymphoproliferative disease (LPD). Early-passage lymphoblastoid cell lines (LCLs) derived from EBV mutants with a deletion of either viral immediate-early gene grew similarly to wild-type (WT) virus LCLs in vitro but were deficient in producing LPD when inoculated into SCID mice. Restoration of lytic EBV gene expression enhanced growth in SCID mice. Acyclovir, which prevents lytic viral replication but not expression of early lytic viral genes, did not inhibit the growth of WT LCLs in SCID mice. Early-passage LCLs derived from the lytic-defective viruses had substantially decreased expression of the cytokine interleukin-6 (IL-6), and restoration of lytic gene expression reversed this defect. Expression of cellular IL-10 and viral IL-10 was also diminished in lytic-defective LCLs. These results suggest that lytic EBV gene expression contributes to EBV-associated lymphoproliferative disease, potentially through induction of paracrine B-cell growth factors.
The Vaccinia Virus F1L Protein Interacts with the Proapoptotic Protein Bak and Inhibits Bak Activation
Authors: Wasilenko Shawn T. , Banadyga Logan , Bond David , Barry Michele
Published: October 2005

Many viruses have evolved strategies to counteract cellular immune responses, including apoptosis. Vaccinia virus, a member of the poxvirus family, encodes an antiapoptotic protein, F1L. F1L localizes to mitochondria and inhibits apoptosis by preventing the release of cytochrome
c
by an undetermined mechanism (S. T. Wasilenko, T. L. Stewart, A. F. Meyers, and M. Barry, Proc. Natl. Acad. Sci. USA
100:
14345-14350, 2003; T. L. Stewart, S. T. Wasilenko, and M. Barry, J. Virol.
79:
1084-1098, 2005). Here, we show that in the absence of an apoptotic stimulus, F1L associates with Bak, a proapoptotic member of the Bcl-2 family that plays a pivotal role in the release of cytochrome
c
. Cells infected with vaccinia virus were resistant to Bak oligomerization and the initial N-terminal exposure of Bak following the induction of apoptosis with staurosporine. A mutant vaccinia virus missing F1L was no longer able to inhibit apoptosis or Bak activation. In addition, the expression of F1L was essential to inhibit tBid-induced cytochrome
c
release in both wild-type murine embryonic fibroblasts (MEFs) and Bax-deficient MEFs, indicating that F1L could inhibit apoptosis in the presence and absence of Bax. tBid-induced Bak oligomerization and N-terminal exposure of Bak in Bax-deficient MEFs were inhibited during virus infection, as assessed by cross-linking and limited trypsin proteolysis. Infection with the F1L deletion virus no longer provided protection from tBid-induced Bak activation and apoptosis. Additionally, infection of Jurkat cells with the F1L deletion virus resulted in cellular apoptosis, as measured by loss of the inner mitochondrial membrane potential, caspase 3 activation, and cytochrome
c
release, indicating that the presence of F1L was pivotal for inhibiting vaccinia virus-induced apoptosis. Our data indicate that F1L expression during infection inhibits apoptosis and interferes with the activation of Bak.
Immunodeficiency in the Absence of High Viral Load in Pig-Tailed Macaques Infected with Simian Immunodeficiency Virus SIVsun or SIVlhoest
Authors: Beer Brigitte E. , Brown Charles R. , Whitted Sonya , Goldstein Simoy , Goeken Robert , Plishka Ronald , Buckler-White Alicia , Hirsch Vanessa M.
Published: October 2005

Simian immunodeficiency virus (SIV) is known to result in an asymptomatic infection of its natural African monkey host. However, some SIV strains are capable of inducing AIDS-like symptoms and death upon experimental infection of Asian macaques. To further investigate the virulence of natural SIV isolates from African monkeys, pig-tailed (PT) macaques were inoculated intravenously with either of two recently discovered novel lentiviruses, SIVlhoest and SIVsun. Both viruses were apparently apathogenic in their natural hosts but caused immunodeficiency in PT macaques. Infection was characterized by a progressive loss of CD4
+
lymphocytes in the peripheral blood and lymph nodes, generalized lymphoid depletion, a wasting syndrome, and opportunistic infections, such as
Mycobacterium avium
or
Pneumocystis carinii
infections. However, unlike SIVsm/mac infection of macaques, SIVlhoest and SIVsun infections in PT macaques were not accompanied by high viral loads during the chronic disease stage. In addition, no significant correlation between the viral load at set point (12 weeks postinfection) and survival could be found. Five out of eight SIVlhoest-infected and three out of four SIVsun-infected macaques succumbed to AIDS during the first 5 years of infection. Thus, the survival of SIVsun- and SIVlhoest-infected animals was significantly longer than that of SIVagm- or SIVsm-infected macaques. All PT macaques maintained strong SIV antibody responses despite progression to SIV-induced AIDS. The development of immunodeficiency in the face of low viremia suggests that SIVlhoest and SIVsun infections of macaques may model unique aspects of the pathogenesis of human immunodeficiency virus infection in humans.
Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0
Authors: Potel Corinne , Elliott Gillian
Published: October 2005

Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Δ22 virus replicated poorly in epithelial MDBK cells. We have also previously shown that VP22 interacts with the tegument protein VP16 and the cellular microtubule network. While the majority of VP22 in infected cells is highly phosphorylated, the nonphosphorylated form of VP22 is the predominant species in the virion, suggesting a differential requirement for phosphorylation through virus replication. Hence, to study the significance of VP22 phosphorylation, we have now constructed two recombinant viruses expressing green fluorescent protein-VP22 (G22) in which the previously identified serine phosphorylation sites have been mutated either to alanine to abolish the phosphorylation status of VP22 (G22P−) or to glutamic acid to mimic permanent phosphorylation (G22P+). Localization studies indicated that the G22P− protein associated tightly with microtubules in some infected cells, suggesting that VP22 phosphorylation may control its interaction with the microtubule network. By contrast, VP22 phosphorylation had no effect on its ability to interact with VP16 and, importantly, had no effect on the relative packaging of VP22. Intriguingly, virion packaging of ICP0 was reduced in the G22P+ virus while ICP0 expression was reduced in the G22P− virus, suggesting that these two ICP0 defects, previously observed in the Δ22 virus, were attributable to different forms of VP22. Furthermore, the Δ22 virus replication defect in MDBK cells correlated with the expression of constitutively charged VP22 in the G22P+ virus. Taken together, these results suggest an important role for VP22 phosphorylation in its relationship with ICP0.
Crystal Structure of Enteric Adenovirus Serotype 41 Short Fiber Head
Authors: Seiradake Elena , Cusack Stephen
Published: October 2005

Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding.
Gene and Genome Duplication in
Acanthamoeba polyphaga Mimivirus
Authors: Suhre Karsten
Published: October 2005

Gene duplication is key to molecular evolution in all three domains of life and may be the first step in the emergence of new gene function. It is a well-recognized feature in large DNA viruses but has not been studied extensively in the largest known virus to date, the recently discovered
Acanthamoeba polyphaga Mimivirus
. Here, I present a systematic analysis of gene and genome duplication events in the mimivirus genome. I found that one-third of the mimivirus genes are related to at least one other gene in the mimivirus genome, either through a large segmental genome duplication event that occurred in the more remote past or through more recent gene duplication events, which often occur in tandem. This shows that gene and genome duplication played a major role in shaping the mimivirus genome. Using multiple alignments, together with remote-homology detection methods based on Hidden Markov Model comparison, I assign putative functions to some of the paralogous gene families. I suggest that a large part of the duplicated mimivirus gene families are likely to interfere with important host cell processes, such as transcription control, protein degradation, and cell regulatory processes. My findings support the view that large DNA viruses are complex evolving organisms, possibly deeply rooted within the tree of life, and oppose the paradigm that viral evolution is dominated by lateral gene acquisition, at least in regard to large DNA viruses.
Epstein-Barr Virus mRNA Export Factor EB2 Is Essential for Intranuclear Capsid Assembly and Production of gp350
Authors: Batisse Julien , Manet Evelyne , Middeldorp Jaap , Sergeant Alain , Gruffat Henri
Published: October 2005

Most human herpesviruses, including Epstein-Barr virus (EBV), express a protein which functions primarily as an mRNA export factor. Previously, we deleted the gene for the Epstein-Barr virus mRNA export factor EB2 from the EBV genome and then introduced the mutated genome into 293 cells. Using a transcomplementation assay in which ectopic expression of the transcription factor EB1/ZEBRA was sufficient to induce the EBV productive cycle, we showed that Ori-Lyt-dependent replication of the EBV DNA occurs in the absence of EB2, indicating that EB2 is not essential for the expression and export of early mRNAs. However, in the absence of EB2, no infectious viral particles are produced (H. Gruffat, J. Batisse, D. Pich, B. Neuhierl, E. Manet, W. Hammerschmidt, and A. Sergeant, J. Virol.
76:
9635-9644, 2002). In this report, we now show that EB2 is essential for the nuclear export of most, but not all, late mRNAs produced from intronless genes that translate into proteins involved in intranuclear capsid assembly and maturation. As a consequence, we show that EB2 is essential for the proper assembly of intranuclear capsids. Interestingly, the late BLLF1 gene contains an intron, and both unspliced and spliced mRNAs must be exported to the cytoplasm to be translated into gp350 and gp220, respectively (M. Hummel, D. A. Thorley-Lawson, and E. Kieff, J. Virol.
49:
413-417, 1984). Our results also demonstrate that although BLLF1 spliced mRNAs are exported to the cytoplasm independently of EB2, EB2 is essential for the nuclear export of unspliced BLLF1 mRNA. In the same assay, herpes simplex virus 1 ICP27 completely inhibited the nuclear export of BLLF1 spliced mRNAs whereas unspliced BLLF1 mRNAs were exported, confirming that in a physiological assay, ICP27 inhibits splicing.
Association of Human Immunodeficiency Virus Type 1 Gag with Membrane Does Not Require Highly Basic Sequences in the Nucleocapsid: Use of a Novel Gag Multimerization Assay
Authors: Ono Akira , Waheed Abdul A. , Joshi Anjali , Freed Eric O.
Published: October 2005

Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterol-enriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.
A Single Amino Acid Change in Rabies Virus Glycoprotein Increases Virus Spread and Enhances Virus Pathogenicity
Authors: Faber Milosz , Faber Marie-Luise , Papaneri Amy , Bette Michael , Weihe Eberhard , Dietzschold Bernhard , Schnell Matthias J.
Published: October 2005

Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu
333
remained unchanged after five mouse passages, an Asn
194
→Lys
194
mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn
194
with Lys
194
in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn
194
→Lys
194
mutation does not arise when Asn
194
is exchanged with Ser
194
(SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.
Immunogenicity of Recombinant Fiber-Chimeric Adenovirus Serotype 35 Vector-Based Vaccines in Mice and Rhesus Monkeys
Authors: Nanda Anjali , Lynch Diana M. , Goudsmit Jaap , Lemckert Angelique A. C. , Ewald Bonnie A. , Sumida Shawn M. , Truitt Diana M. , Abbink Peter , Kishko Michael G. , Gorgone Darci A. , Lifton Michelle A. , Shen Ling , Carville Angela , Mansfield Keith G. , Havenga Menzo J. E. , Barouch Dan H.
Published: October 2005

Preexisting immunity to adenovirus serotype 5 (Ad5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. A potential solution to this problem is to utilize rAd vectors derived from rare Ad serotypes, such as Ad35. However, rAd35 vectors have appeared less immunogenic than rAd5 vectors in preclinical studies to date. In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vectors may be due in part to differences between the fiber proteins of these viruses. We constructed capsid chimeric rAd35 vectors containing the Ad5 fiber knob (rAd35k5) and compared the immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing simian immunodeficiency virus Gag and HIV-1 Env in mice and rhesus monkeys. In vitro studies demonstrated that rAd35k5 vectors utilized the Ad5 receptor CAR rather than the Ad35 receptor CD46. In vivo studies showed that rAd35k5 vectors were more immunogenic than rAd35 vectors in both mice and rhesus monkeys. These data suggest that the Ad5 fiber knob contributes substantially to the immunogenicity of rAd vectors. Moreover, these studies demonstrate that capsid chimeric rAd vectors can be constructed to combine beneficial immunologic and serologic properties of different Ad serotypes.