bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tissue- and Tumor-Specific Targeting of Murine Leukemia Virus-Based Replication-Competent Retroviral Vectors
Authors: Metzl Christian , Mischek Daniela , Salmons Brian , Günzburg Walter H. , Renner Matthias , Portsmouth Daniel 
Published: June 2006

ABSTRACT
Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human α1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver- and β-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.
Preferential Infection Shortens the Life Span of Human ImmunodeficiencyVirus-Specific CD4+T Cells In Vivo
Authors: Brenchley Jason M. , Ruff Laura E. , Casazza Joseph P. , Koup Richard A. , Price David A. , Douek Daniel C. 
Published: June 2006

ABSTRACTCD4+T-cell help is essential for effective immune responses to viruses. In human immunodeficiency virus (HIV) infection, CD4+T cells specific for HIV are infected by the virus at higher frequencies than other memory CD4+T cells. Here, we demonstrate that HIV-specific CD4+T cells are barely detectable in most infected individuals and that the corresponding CD4+T cells exhibit an immature phenotype compared to both cytomegalovirus (CMV)-specific CD4+T cells and other memory CD4+T cells. However, in two individuals, we observed a rare and diametrically opposed pattern in which HIV-specific CD4+T-cell populations of large magnitude exhibited a terminally differentiated immunophenotype; these cells were not preferentially infected in vivo. Clonotypic analysis revealed that the HIV-specific CD4+T cells from these individuals were cross-reactive with CMV. Thus, preferential infection can be circumvented in the presence of cross-reactive CD4+T cells driven to maturity by coinfecting viral antigens, and this physical proximity rather than activation status per se is an important determinant of preferential infection based on antigen specificity. These data demonstrate that preferential infection reduces the life span of HIV-specific CD4+T cells in vivo and thereby compromises the generation of effective immune responses to the virus itself; further, this central feature in the pathophysiology of HIV infection can be influenced by the cross-reactivity of responding CD4+T cells.
Consistent Effects of
TSG101
Genetic Variability on Multiple Outcomes of Exposure to Human Immunodeficiency Virus Type 1
Authors: Bashirova Arman A. , Bleiber Gabriela , Qi Ying , Hutcheson Holli , Yamashita Traci , Johnson Randall C. , Cheng Jie , Alter Galit , Goedert James J. , Buchbinder Susan , Hoots Keith , Vlahov David , May Margaret , Maldarelli Frank , Jacobson Lisa , O'Brien Stephen J. , Telenti Amalio , Carrington Mary 
Published: June 2006

ABSTRACT

Tumor susceptibility gene 101 (
TSG101
) encodes a host cellular protein that is appropriated by human immunodeficiency virus type 1 (HIV-1) in the budding process of viral particles from infected cells. Variation in the coding or noncoding regions of the gene could potentially affect the degree of TSG101-mediated release of viral particles. While the coding regions of the gene were found to lack nonsynonymous variants, two polymorphic sites in the
TSG101
5′ area were identified that were associated with the rate of AIDS progression among Caucasians. These single-nucleotide polymorphisms (SNPs), located at positions −183 and +181 relative to the translation start, specify three haplotypes termed A, B, and C, which occur at frequencies of 67%, 21%, and 12%, respectively. Haplotype C is associated with relatively rapid AIDS progression, while haplotype B is associated with slower disease progression. Both effects were dominant over the intermediate haplotype A. The haplotypes also demonstrated parallel effects on the rate of CD4 T-cell depletion and viral load increase over time, as well as a possible influence on HIV-1 infection. The data raise the hypothesis that noncoding variation in
TSG101
affects the efficiency of TSG101-mediated release of viral particles from infected cells, thereby altering levels of plasma viral load and subsequent disease progression.
The L4 22-Kilodalton Protein Plays a Role in Packaging of the Adenovirus Genome
Authors: Ostapchuk Philomena , Anderson Mary E. , Chandrasekhar Sharanya , Hearing Patrick 
Published: June 2006

ABSTRACT

Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence of a
cis
-acting region located at the left end of the genome referred to as the packaging domain. The functionally significant sequences within this domain consist of at least seven similar repeats, referred to as the A repeats, which have the consensus sequence 5′ TTTG-N
8
-CG 3′. In vitro and in vivo binding studies have demonstrated that the adenovirus protein IVa2 binds to the CG motif of the packaging sequences. In conjunction with IVa2, another virus-specific protein binds to the TTTG motifs in vitro. The efficient formation of these protein-DNA complexes in vitro was precisely correlated with efficient packaging activity in vivo. We demonstrate that the binding activity to the TTTG packaging sequence motif is the product of the L4 22-kDa open reading frame. Previously, no function had been ascribed to this protein. Truncation of the L4 22-kDa protein in the context of the viral genome did not reduce viral gene expression or viral DNA replication but eliminated the production of infectious virus. We suggest that the L4 22-kDa protein, in conjunction with IVa2, plays a critical role in the recognition of the packaging domain of the Ad genome that leads to viral DNA encapsidation. The L4 22-kDa protein is also involved in recognition of transcription elements of the Ad major late promoter.
Experimental Confirmation of Global Murine Cytomegalovirus Open Reading Frames by Transcriptional Detection and Partial Characterization of Newly Described Gene Products
Authors: Tang Qiyi , Murphy Eain A. , Maul Gerd G. 
Published: June 2006

ABSTRACT
Murine cytomegalovirus (MCMV) and human CMV (HCMV) share many features making the mouse system a potential small-animal model for HCMV. Although the genomic DNA sequence and the predicted open reading frames (ORFs) of MCMV have been determined, experimental evidence that the ORFs are actually transcribed has been lacking. We developed an MCMV global-DNA microarray that includes all previously predicted ORFs and 14 potential ones. A total of 172 ORFs were confirmed to be transcribed, including 7 newly discovered ORFs not previously predicted. No gene products from 10 previously predicted ORFs were detected by either DNA microarray analysis or reverse transcriptase PCR in MCMV-infected mouse fibroblasts, although 2 of those were expressed in a macrophage cell line, suggesting that potential gene products from these open reading fames are silenced in fibroblasts and required in macrophages. Immunohistochemical localization of the six newly described ORF products and three recently identified ones in cells transfected with the respective construct revealed four of the products in the nucleus and five in mitochondria. Analysis of two ORFs using site-directed mutagenesis showed that deletion of one of the mitochondrion-localized gene products led to significantly decreased replication in fibroblasts.
Dihydroxythiophenes Are Novel Potent Inhibitors of Human Immunodeficiency Virus Integrase with a Diketo Acid-Like Pharmacophore
Authors: Kehlenbeck S. , Betz U. , Birkmann A. , Fast B. , Göller A. H. , Henninger K. , Lowinger T. , Marrero D. , Paessens A. , Paulsen D. , Pevzner V. , Schohe-Loop R. , Tsujishita H. , Welker R. , Kreuter J. , Rübsamen-Waigmann H. , Dittmer F. 
Published: June 2006

ABSTRACTWe have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.
Hypersusceptibility to Substrate Analogs Conferred by Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Smith Robert A. , Anderson Donovan J. , Preston Bradley D. 
Published: June 2006

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed “random scanning mutagenesis” of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.
ICP0 Gene Expression Is a Herpes Simplex Virus Type 1 Apoptotic Trigger
Authors: Sanfilippo Christine M. , Blaho John A. 
Published: June 2006

ABSTRACT
Apoptosis is a highly regulated programmed cell death process which is activated during normal development and by various stimuli, such as viral infection, which disturb cellular metabolism and physiology. That herpes simplex virus type 1 (HSV-1) induces apoptosis but then prevents its killing of infected cells is well-established. However, little is known about the viral factor/event which triggers the apoptotic process. We previously reported that infections with either (i) a temperature-sensitive virus at its nonpermissive temperature which does not inject viral DNA into nuclei or (ii) various UV-inactivated wild-type viruses do not result in the induction of apoptosis (C. M. Sanfilippo, F. N. W. Chirimuuta, and J. A. Blaho, J. Virol. 78:224-239, 2004). This indicates that virus receptor binding/attachment to cells, membrane fusion, virion disassembly/tegument dispersal, virion RNAs, and capsid translocation to nuclei are not responsible for induction and implicates viral immediate-early (IE) gene expression in the process. Here, we systematically evaluated the contribution of each IE gene to the stimulation of apoptosis. Using a series of viruses individually deleted for α27, α4, and α22, we determined that these genes are not required for apoptosis induction but rather that their products play roles in its prevention, likely through regulatory effects. Sole expression of α0 acted as an “apoptoxin” that was necessary and sufficient to trigger the cell death cascade. Importantly, results using a recombinant virus which contains a stop codon in α0 showed that it was not the ICP0 protein which acted as the apoptotic inducer. Based on these findings, we propose that α0 gene expression acts as an initial inducer of apoptosis during HSV-1 infection. This represents the first description of apoptosis induction in infected cells triggered as a result of expression of a single viral gene. Expression of apoptotic viral genes is a unique mechanism through which human pathogens may modulate interactions with their host cells.
Evaluating Replication-Defective Vesicular Stomatitis Virus as a Vaccine Vehicle
Authors: Majid Ayaz M. , Ezelle Heather , Shah Sangeeta , Barber Glen N. 
Published: June 2006

ABSTRACT

We have generated replication-competent (VSV-C/E1/E2) and nonpropagating (VSVΔG-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVΔG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVΔG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-γ)-producing CD8
+
T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVΔG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN-γ-producing and E2-specific CD8
+
T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVΔG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease.
The 2.6-Angstrom Structure of Infectious Bursal Disease Virus-Derived T=1 Particles Reveals New Stabilizing Elements of the Virus Capsid
Authors: Garriga Damià , Querol-Audí Jordi , Abaitua Fernando , Saugar Irene , Pous Joan , Verdaguer Núria , Castón José R. , Rodriguez José F. 
Published: June 2006

ABSTRACT

Infectious
bursal disease virus (IBDV), a member of the
Birnaviridae
family, is a double-stranded RNA virus that causes a highly contagious
disease in young chickens leading to significant economic losses in the
poultry industry. The VP2 protein, the only structural component of the
IBDV icosahedral capsid, spontaneously assembles into T=1 subviral
particles (SVP) when individually expressed as a chimeric gene. We have
determined the crystal structure of the T=1 SVP to 2.60 Å
resolution. Our results show that the 20 trimeric VP2 clusters forming
the T=1 shell are further stabilized by calcium ions located at the
threefold icosahedral axes. The structure also reveals a new unexpected
domain swapping that mediates interactions between adjacent trimers: a
short helical segment located close to the end of the long C-terminal
arm of VP2 is projected toward the threefold axis of a neighboring VP2
trimer, leading to a complex network of interactions that increases the
stability of the T=1 particles. Analysis of crystal packing shows that
the exposed capsid residues, His253 and Thr284, determinants of IBDV
virulence and the adaptation of the virus to grow in cell culture, are
involved in particle-particle
interactions.
Both Spike and Background Genes Contribute to Murine Coronavirus Neurovirulence
Authors: Iacono Kathryn T. , Kazi Lubna , Weiss Susan R. 
Published: June 2006

ABSTRACTVarious strains of mouse hepatitis virus (MHV) exhibit different pathogenic phenotypes. Infection with the A59 strain of MHV induces both encephalitis and hepatitis, while the highly neurovirulent JHM strain induces a fatal encephalitis with little, if any, hepatitis. The pathogenic phenotype for each strain is determined by the genetic composition of the viral genome, as well as the host immune response. Using isogenic recombinant viruses with A59 background genes differing only in the spike gene, we have previously shown that high neurovirulence is associated with the JHM spike protein, the protein responsible for attachment to the host cell receptor (J. J. Phillips, M. M. Chua, G. F. Rall, and S. R. Weiss, Virology301:109-120, 2002). Using another set of isogenic recombinant viruses with JHM background genes expressing either the JHM or A59 spike, we have further investigated the roles of viral genes in pathogenesis. Here, we demonstrate that the high neurovirulence of JHM is associated with accelerated spread through the brain and a heightened innate immune response that is characterized by high numbers of infiltrating neutrophils and macrophages, suggesting an immunopathogenic component to neurovirulence. While expression of the JHM spike is sufficient to confer a neurovirulent phenotype, as well as increased macrophage infiltration, background genes contribute to virulence as well, at least in part, by dictating the extent of the T-cell immune response.
Epstein-Barr Virus LMP2A Enhances B-Cell Responses In Vivo and In Vitro
Authors: Swanson-Mungerson Michelle , Bultema Rebecca , Longnecker Richard 
Published: June 2006

ABSTRACT
Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.
Vaccinia Virus Activation of CCR5 Invokes Tyrosine Phosphorylation Signaling Events That Support Virus Replication
Authors: Rahbar Ramtin , Murooka Thomas T. , Hinek Anna A. , Galligan Carole L. , Sassano Antonella , Yu Celeste , Srivastava Kishore , Platanias Leonidas C. , Fish Eleanor N. 
Published: June 2006

ABSTRACTVaccinia virus, a poxvirus, produces structurally distinct forms of virions for which the immediate events following cell entry are ill-defined. We provide evidence that intracellular mature virus (IMV) enters both permissive and nonpermissive T-cell lines and that introduction of CCR5 into nonpermissive mouse fibroblasts or human primary T cells renders the cells permissive for vaccinia replication. Notably, T cells expressing CCR5 in which tyrosine 339 in the intracellular region is replaced by phenylalanine no longer support virus replication or virus-inducible activation of specific host cell signaling effectors IRS-2, Grb2, and Erk1/2. We show that following IMV entry into the cell, the intact but not the tyrosine-deficient CCR5 is rapidly internalized and colocalizes with virus. This colocalization precedes virus-inducible signaling and replication.
Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus
Authors: Throsby Mark , Geuijen Cecile , Goudsmit Jaap , Bakker Arjen Q. , Korimbocus Jehanara , Kramer R. Arjen , Clijsters-van der Horst Marieke , de Jong Maureen , Jongeneelen Mandy , Thijsse Sandra , Smit Renate , Visser Therese J. , Bijl Nora , Marissen Wilfred E. , Loeb Mark , Kelvin David J. , Preiser Wolfgang , ter Meulen Jan , de Kruif John 
Published: June 2006

ABSTRACTMonoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 μg/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.