bluuebunny/crossref_metadata_2025 huggingface dataset:
Respiratory Viruses Augment the Adhesion of Bacterial Pathogens to Respiratory Epithelium in a Viral Species- and Cell Type-Dependent Manner
Authors: Avadhanula Vasanthi , Rodriguez Carina A. , DeVincenzo John P. , Wang Yan , Webby Richard J. , Ulett Glen C. , Adderson Elisabeth E.
Published: January 2006

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable
Haemophilus influenzae
and S
treptococcus pneumoniae
to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.
Human Immunodeficiency Virus Type 1envEvolves toward Ancestral States upon Transmission to a New Host
Authors: Herbeck Joshua T. , Nickle David C. , Learn Gerald H. , Gottlieb Geoffrey S. , Curlin Marcel E. , Heath Laura , Mullins James I.
Published: January 2006

Selecting human immunodeficiency virus (HIV) sequences for inclusion within vaccines has been a difficult problem, as circulating HIV strains evolve relentlessly and become increasingly divergent over time. We report an assessment of this divergence from three perspectives: (i) across different hosts as a function of time of infection, (ii) between donors and recipients in known transmission pairs, and (iii) within individual hosts over time in relation to the initially replicating virus and to the deduced ancestral sequence of the intrahost viral population. Surprisingly, we consistently found less divergence between viruses from different individuals sampled in primary infection than in individuals sampled at more advanced stages of illness. Furthermore, longitudinal analysis of intrahost divergence revealed a 2- to 3-year period of evolution toward a common ancestral sequence at the start of infection, indicating that HIV recovers certain ancestral features when infecting a new host. These results have important implications for the study of HIV population genetics and rational vaccine design, including favoring the inclusion of viral gene sequences taken early in infection.
Generation of CD8+T-Cell Responses by a Recombinant NonpathogenicMycobacterium smegmatisVaccine Vector Expressing Human Immunodeficiency Virus Type 1 Env
Authors: Cayabyab Mark J. , Hovav Avi-Hai , Hsu Tsungda , Krivulka Georgia R. , Lifton Michelle A. , Gorgone Darci A. , Fennelly Glenn J. , Haynes Barton F. , Jacobs William R. , Letvin Norman L.
Published: January 2006

Because the vaccine vectors currently being evaluated in human populations all have significant limitations in their immunogenicity, novel vaccine strategies are needed for the elicitation of cell-mediated immunity. The nonpathogenic, rapidly growing mycobacteriumMycobacterium smegmatiswas engineered as a vector expressing full-length human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope protein. Immunization of mice with recombinantM. smegmatisled to the expansion of major histocompatibility complex class I-restricted HIV-1 epitope-specific CD8+T cells that were cytolytic and secreted gamma interferon. Effector and memory T lymphocytes were elicited, and repeated immunization generated a stable central memory pool of virus-specific cells. Importantly, preexisting immunity toMycobacterium bovisBCG had only a marginal effect on the immunogenicity of recombinantM. smegmatis. This mycobacterium may therefore be a useful vaccine vector.
Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle
Authors: Posthuma Clara C. , Nedialkova Danny D. , Zevenhoven-Dobbe Jessika C. , Blokhuis Jeroen H. , Gorbalenya Alexander E. , Snijder Eric J.
Published: January 2006

The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a
Xenopus laevis
endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.
Poliovirus Protein 3AB Displays Nucleic Acid Chaperone and Helix-Destabilizing Activities
Authors: DeStefano Jeffrey J. , Titilope Oduyebo
Published: January 2006

Poliovirus protein 3AB displayed nucleic acid chaperone activity in promoting the hybridization of complementary nucleic acids and destabilizing secondary structure. Hybridization reactions at 30°C between 20- and 40-nucleotide RNA oligonucleotides and 179- or 765-nucleotide RNAs that contained a complementary region were greatly enhanced in the presence of 3AB. The effect was nonspecific as reactions between DNA oligonucleotides and RNA or DNA templates were also enhanced. Reactions were optimal with 1 mM MgCl
2
and 20 mM KCl. Analysis of the reactions with various 3AB and template concentrations indicated that enhancement required a critical amount of 3AB that increased as the concentration of nucleic acid increased. This was consistent with a requirement for 3AB to “coat” the nucleic acids for enhancement. The helix-destabilizing activity of 3AB was tested in an assay with two 42-nucleotide completely complementary DNAs. Each complement formed a strong stem-loop (Δ
G
= −7.2 kcal/mol) that required unwinding for hybridization to occur. DNAs were modified at the 3′ or 5′ end with fluorescent probes such that hybridization resulted in quenching of the fluorescent signal. Under optimal conditions at 30°C, 3AB stimulated hybridization in a concentration-dependent manner, as did human immunodeficiency virus nucleocapsid protein, an established chaperone. The results are discussed with respect to the role of 3AB in viral replication and recombination.
Efficient Site-Specific Integration of Large Transgenes by an Enhanced Herpes Simplex Virus/Adeno-Associated Virus Hybrid Amplicon Vector
Authors: Liu Qiang , Perez Claudio F. , Wang Yaming
Published: January 2006

We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (
rep
) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in ∼22% of transduced cells; of those cells, ∼70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.
Phosphorylation of the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein IE62 by the VZV Open Reading Frame 66 Protein Kinase
Authors: Eisfeld Amie J. , Turse Stephanie E. , Jackson Sara A. , Lerner Edwina C. , Kinchington Paul R.
Published: January 2006

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.
Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator
Authors: Gammoh Noor , Grm Helena Sterlinko , Massimi Paola , Banks Lawrence
Published: January 2006

In order to ensure a productive life cycle, human papillomaviruses (HPVs) require fine regulation of their gene products. Uncontrolled activity of the viral oncoproteins E6 and E7 results in the immortalization of the infected epithelial cells and thus prevents the production of mature virions. Ectopically expressed E2 has been shown to suppress transcription of the HPV E6 and E7 region in cell lines where the viral DNA is integrated into the host genome, resulting in growth inhibition. However, it has been demonstrated that growth control of these cell lines can also occur independently of HPV E2 transcriptional activity in high-risk HPV types. In addition, E2 is unable to suppress transcription of the same region in cell lines derived from cervical tumors that harbor only episomal copies of the viral DNA. Here we show that HPV type 16 (HPV-16) E2 is capable of inhibiting HPV-16 E7 cooperation with an activated
ras
oncogene in the transformation of primary rodent cells. Furthermore, we demonstrate a direct interaction between the E2 and E7 proteins which requires the hinge region of E2 and the zinc-binding domain of E7. These viral proteins interact in vivo, and E2 has a marked effect upon both the stability of E7 and its cellular location, where it is responsible for recruiting E7 onto mitotic chromosomes at the later stages of mitosis. These results demonstrate a direct role for E2 in regulating the function of E7 and suggest an important role for E2 in directing E7 localization during mitosis.
Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid
Authors: Butterfield-Gerson Kristin L. , Scheifele Lisa Z. , Ryan Eileen P. , Hopper Anita K. , Parent Leslie J.
Published: January 2006

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify
cis-
and
trans-
acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in
Saccharomyces cerevisiae
. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-β family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in
S. cerevisiae
, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-α/β (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.
Functional Analysis of Hepatitis C Virus Envelope Proteins, Using a Cell-Cell Fusion Assay
Authors: Kobayashi Mariko , Bennett Michael C. , Bercot Theodore , Singh Ila R.
Published: January 2006

Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.
Modulation of Cellular Protein Trafficking by Human Immunodeficiency Virus Type 1 Nef: Role of the Acidic Residue in the ExxxLL Motif
Authors: Coleman Scott H. , Madrid Ricardo , Van Damme Nanette , Mitchell Richard S. , Bouchet Jerome , Servant Cecile , Pillai Satish , Benichou Serge , Guatelli John C.
Published: January 2006

Thenefgene contributes to the replication of primate lentiviruses by altering the trafficking of cellular proteins involved in adaptive immunity (class I and II major histocompatibility complex [MHC]) and viral transmission (CD4 and DC-SIGN). A conserved acidic leucine-based sequence (E160xxxLL) within human immunodeficiency virus type 1 (HIV-1) Nef binds to the cellular adaptor protein (AP) complexes, which mediate protein sorting into endosomal vesicles. The leucine residues in this motif are required for the down-regulation of CD4 and for the up-regulation of DC-SIGN and the invariant chain of MHC class II, but the role of the acidic residue is unclear. Here, substitution of E160 with uncharged residues impaired the ability of Nef to up-regulate the expression of the invariant chain and DC-SIGN at the cell surface, whereas substitution with a basic residue was required for a similar effect on the down-regulation of CD4. All substitutions of E160 relieved the Nef-mediated block to transferrin uptake. E160 was required for the efficient interaction of Nef with AP-1 and AP-3 and for the stabilization of these complexes on endosomal membranes in living cells. Systematic mutation of the ExxxLL sequence together with correlation of binding and functional data leads to the hypotheses that AP-1 and AP-3 are major cofactors for the effect of Nef on the trafficking of transferrin, are less important but contribute to the modulation of the invariant chain and DC-SIGN, and are least critical for the modulation of CD4. The data suggest that the E160 residue plays a differential role in the modulation of leucine-dependent Nef-targets and support a model in which distinct AP complexes are used by Nef to modulate different cellular proteins.
CD4-Specific Transgenic Expression of Human Cyclin T1 Markedly Increases Human Immunodeficiency Virus Type 1 (HIV-1) Production by CD4+T Lymphocytes and Myeloid Cells in Mice Transgenic for a Provirus Encoding a Monocyte-Tropic HIV-1 Isolate
Authors: Sun Jinglin , Soos Timothy , KewalRamani Vineet N. , Osiecki Kristin , Zheng Jian Hua , Falkin Laurie , Santambrogio Laura , Littman Dan R. , Goldstein Harris
Published: January 2006

Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical for efficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate the nascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specific structural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1 replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under the control of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4+T lymphocytes and monocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mouse cells and selectively increase HIV-1 production in this cellular population, which is infected primarily in HIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying the full-length HIV-1JR-CSFprovirus under the control of the endogenous HIV-1 long terminal repeat and demonstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primary mouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 production by these stimulated cells. Increased HIV-1 production by CD4+T lymphocytes was paralleled with their specific depletion in the peripheral blood of the JR-CSF/hu-cycT1 mice, which increased over time. In addition, increased HIV-1 transgene expression due to human cyclin T1 expression was associated with increased lipopolysaccharide-stimulated monocyte chemoattractant protein 1 production by JR-CSF mouse monocytes/macrophages in vitro. Therefore, the JR-CSF/hu-cycT1 mice should provide an improved mouse system for investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeutic interventions.
Transient and Stable Knockdown of the Integrase Cofactor LEDGF/p75 Reveals Its Role in the Replication Cycle of Human Immunodeficiency Virus
Authors: Vandekerckhove Linos , Christ Frauke , Van Maele Bénédicte , De Rijck Jan , Gijsbers Rik , Van den Haute Chris , Witvrouw Myriam , Debyser Zeger
Published: January 2006

After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three- to fivefold inhibition of HIV-1 (strain NL4.3) replication. Quantitative PCR was used to pinpoint the replication block to the integration step. Next, polyclonal and monoclonal HeLaP4-derived cell lines were selected with a stable knockdown of LEDGF/p75 mediated by a lentiviral vector (lentivector) encoding a short hairpin RNA (shRNA) targeting this protein. Cell lines stably transduced with a lentivector encoding an unrelated hairpin or a double-mismatch hairpin served as controls. Again, a two- to fourfold reduction of HIV-1 replication was observed. The extent of LEDGF/p75 knockdown closely correlated with the reduction of HIV-1 replication. After the back-complementation of LEDGF/p75 in the poly- and monoclonal knockdown cell lines using an shRNA-resistant expression plasmid, viral replication was restored to nearly wild-type levels. The Q168A mutation in integrase has been shown to interfere with the interaction with LEDGF/p75 without reducing the enzymatic activity. Transduction by HIV-1-derived lentivectors carrying the Q168A IN mutant was severely hampered, pointing again to a requirement for LEDGF/p75. Altogether, our data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.
The Leader Proteinase of Foot-and-Mouth Disease Virus Inhibits the Induction of Beta Interferon mRNA and Blocks the Host Innate Immune Response
Authors: de los Santos Teresa , de Avila Botton Sonia , Weiblen Rudi , Grubman Marvin J.
Published: January 2006

We have previously shown that the leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) blocks cap-dependent mRNA translation and that a genetically engineered FMDV lacking the leader proteinase coding region (A12-LLV2) is attenuated in cell culture and susceptible animals. The attenuated phenotype apparently is a consequence of the inability of A12-LLV2 to block the expression of type I interferon (IFN-α/β) protein, resulting in IFN-induced inhibition of FMDV replication. Here we show that in addition to preventing IFN-α/β protein synthesis, Lproreduces the level of immediate-early induction of IFN-β mRNA and IFN-stimulated gene products such as double-stranded RNA-dependent protein kinase R (PKR), 2′,5′-oligoadenylate synthetase, and Mx1 mRNAs in swine cells. Down-regulation of cellular PKR by RNA interference did not affect wild-type virus yield but resulted in a higher yield of A12-LLV2, indicating a direct role of PKR in controlling FMDV replication in the natural host. The observation that Lprocontrols the transcription of genes involved in innate immunity reveals a novel role of this protein in antagonizing the cellular response to viral infection.
Insights into Gene Expression Changes Impacting B-Cell Transformation: Cross-Species Microarray Analysis of Bovine Leukemia Virus Tax-Responsive Genes in Ovine B Cells
Authors: Klener Pavel , Szynal Maud , Cleuter Yvette , Merimi Makram , Duvillier Hugues , Lallemand Françoise , Bagnis Claude , Griebel Philip , Sotiriou Christos , Burny Arsène , Martiat Philippe , Van den Broeke Anne
Published: January 2006

Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor incidence, experimental infection of sheep leads to overt leukemia in the majority of infected animals, providing a model for studying the pathogenesis associated with BLV and HTLV-1. Tax
BLV
, the major oncoprotein, initiates a cascade of events leading toward malignancy, although the basis of transformation is not fully understood. We have taken a cross-species ovine-to-human microarray approach to identify Tax
BLV
-responsive transcriptional changes in two sets of cultured ovine B cells following retroviral vector-mediated delivery of Tax
BLV
. Using cDNA-spotted microarrays comprising 10,336 human genes/expressed sequence tags, we identified a cohort of differentially expressed genes, including genes related to apoptosis, DNA transcription, and repair; proto-oncogenes; cell cycle regulators; transcription factors; small Rho GTPases/GTPase-binding proteins; and previously reported Tax
HTLV-1
-responsive genes. Interestingly, genes known to be associated with human neoplasia, especially B-cell malignancies, were extensively represented. Others were novel or unexpected. The results suggest that Tax
BLV
deregulates a broad network of interrelated pathways rather than a single B-lineage-specific regulatory process. Although cross-species approaches do not permit a comprehensive analysis of gene expression patterns, they can provide initial clues for the functional roles of genes that participate in B-cell transformation and pinpoint molecular targets not identified using other methods in animal models.
Nuclear Import of Epstein-Barr Virus Nuclear Antigen 1 Mediated by NPI-1 (Importin α5) Is Up- and Down-Regulated by Phosphorylation of the Nuclear Localization Signal for Which Lys379 and Arg380 Are Essential
Authors: Kitamura Ryo , Sekimoto Toshihiro , Ito Sayuri , Harada Shizuko , Yamagata Hideo , Masai Hisao , Yoneda Yoshihiro , Yanagi Kazuo
Published: January 2006

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin α NPI-1 (importin α5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin α1) bound only weakly and Qip1 (importin α3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.
Human Immunodeficiency Virus Fusion to Dendritic Cells Declines as Cells Mature
Authors: Cavrois Marielle , Neidleman Jason , Kreisberg Jason F. , Fenard David , Callebaut Christian , Greene Warner C.
Published: January 2006

The maturation of dendritic cells (DCs) is associated with a diminished ability to support human immunodeficiency virus (HIV) replication; however, the precise step in the HIV life cycle impaired by DC maturation remains uncertain. Using an HIV virion-based fusion assay, we now show that HIV fusion to monocyte-derived DCs (MDDCs) both decreases and kinetically slows when DCs are induced to mature with poly(I:C) and tumor necrosis factor alpha. Specifically, laboratory-adapted CCR5-tropic 81A virions fused with markedly lower efficiency to mature MDDCs than immature DCs. In contrast, fusion of NL4-3, the isogenic CXCR4-tropic counterpart of 81A, was low in both immature and mature MDDCs. Fusion mediated by primary HIV envelopes, including seven CCR5- and four CXCR4-tropic envelopes, also decreased with DC maturation. The kinetics of virion fusion were also altered by both the state of DC maturation and the coreceptor utilized. Fusion of 81A and NL4-3 virions was delayed in mature compared to immature MDDCs, and NL4-3 fused more slowly than 81A in both mature and immature MDDCs. Surprisingly, primary envelopes with CXCR4 tropism mediated fusion to immature MDDCs with efficiencies similar to those of primary CCR5-tropic envelopes. This result contrasted with the marked preferential fusion of the laboratory-adapted 81A over NL4-3 in immature MDDCs and in ex vivo Langerhans cells, indicating that these laboratory-adapted HIV strains do not fully recapitulate all of the properties of primary HIV isolates. In conclusion, our results demonstrate that the defect in HIV replication observed in mature MDDCs stems at least in part from a decline in viral fusion.
Cytotoxic T-Lymphocyte Epitope Vaccination Protects against Human Metapneumovirus Infection and Disease in Mice
Authors: Herd Karen A. , Mahalingam Suresh , Mackay Ian M. , Nissen Michael , Sloots Theo P. , Tindle Robert W.
Published: January 2006

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocirculate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HLA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathology in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytokines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.
Deacetylation of the Herpes Simplex Virus Type 1 Latency-Associated Transcript (LAT) Enhancer and a Decrease in LAT Abundance Precede an Increase in ICP0 Transcriptional Permissiveness at Early Times Postexplant
Authors: Amelio Antonio L. , Giordani Nicole V. , Kubat Nicole J. , O'Neil Jerome E. , Bloom David C.
Published: January 2006

Only the latency-associated transcript (LAT) of the herpes simplex virus type 1 (HSV-1) genome is transcribed during latency, while the lytic genes are suppressed, possibly by LAT antisense mechanisms and/or chromatin modifications. In the present study, latently infected dorsal root ganglia were explanted to assess both relative levels of LAT and histone H3 (K9, K14) acetylation of the LAT locus and ICP0 promoter at early times postexplant. We observed that a decrease in both LAT enhancer histone H3 (K9, K14) acetylation and LAT RNA abundance occurs prior to an increase in acetylation, or transcriptional permissiveness, at the ICP0 promoter.
APOBEC3F and APOBEC3G mRNA Levels Do Not Correlate with Human Immunodeficiency Virus Type 1 Plasma Viremia or CD4
+
T-Cell Count
Authors: Cho Soo-Jin , Drechsler Henning , Burke Ryan C. , Arens Max Q. , Powderly William , Davidson Nicholas O.
Published: January 2006

APOBEC3F and APOBEC3G (hA3F and hA3G) are part of an innate mechanism of antiretroviral defense. The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif targets both proteins for proteasomal degradation. Using mRNA from peripheral blood mononuclear cells of 92 HIV-infected subjects not taking antiretroviral therapy and 19 HIV-uninfected controls, we found that hA3F (
P
< 0.001) and hA3G (
P
= 0.016) mRNA levels were lower in HIV-infected subjects and were positively correlated with one another (
P
= 0.003). However, we found no correlation in the abundance of either hA3F or hA3G mRNA with either viral load or CD4 counts in HIV-infected subjects.
Lymphatic vessel density in the neoplastic progression of Barrett’s oesophagus to adenocarcinoma
Authors: Brundler M-A , Harrison J A , de Saussure B , de Perrot M , Pepper M S
Published: January 2006

Background:Oesophageal adenocarcinoma is an aggressive neoplasm with poor prognosis as a result of early lymph node metastasis.

Aims:To measure lymphatic vessel density (LVD) in the neoplastic progression from Barrett’s metaplasia to adenocarcinoma and determine whether LVD can predict the risk of cancer. In addition, to correlate LVD with lymph node metastasis and assess whether LVD could be used as a prognostic indicator for outcome or survival.

Methods:LVD and microvascular density (MVD) were assessed after immunohistochemical staining of vessels in Barrett’s metaplasia, dysplasia, and adenocarcinoma tissues and were correlated with clinicopathological features.

Results:LVD was significantly reduced in adenocarcinoma, being half that seen in normal stomach/oesophagus or metaplasia/dysplasia. LVD did not correlate with tumour grade, stage, or clinical outcome; however, patients who had either lymph node metastasis or invasion of tumour cells into peritumorous lymphatic vessels had a significantly worse overall survival. MVD was also assessed as a prognostic marker; its increase appeared to be linked more with the development of Barrett’s metaplasia than adenocarcinoma.

Conclusions:The reduction in lymphatic vessel numbers was not useful for determining disease outcome in the patient group studied. It is the entry of tumour cells into pre-existing peritumorous lymphatic vessels that confers a significantly worse overall survival.
BACK REACTION IN SCHWARZSCHILD–de SITTER SPACETIME WITH A MASSLESS QUANTUM FIELD
Authors: KURIAKOSE P. I. , KURIAKOSE V. C.
Published: January 2006

Back reaction in the Schwarzschild–de Sitter black hole in thermal equilibrium with conformal massless quantum field is discussed using the method of York. The presence of quantum field and back reaction ensures the entropy of dressed black hole. In the perturbed spacetime geometry, the nature of the effective potential and the orbits of massless and massive particles are also investigated.
ON SERIES OF MULTIQUBIT BELL'S INEQUALITIES
Authors: PATEREK TOMASZ , LASKOWSKI WIESŁAW , ŻUKOWSKI MAREK
Published: January 2006

We review series of multiqubit Bell's inequalities which apply to correlation functions and present conditions that quantum states must satisfy to violate such inequalities.
SINGLE Si AD-DIMER DEPOSITION BEHAVIOR ON P(2×2)Si SURFACE
Authors: HUANG YAN , ZHU XIAOYAN , CHEN XIAOSHUANG
Published: January 2006

The deposition of single Si ad-dimer on the p(2×2) reconstructed Si (100) surface has been simulated by an empirical tight-binding (ETB) method. Using the clean and defective Si surfaces as the deposition substrates, the deposition energies are mapped out around the clean surface and dimer vacancies. From the calculated energy plots, the binding sites and several possible diffusion paths are achieved. It is easy for the ad-dimer to form stable state A1 and metal stable state A2 on the clean surface. The change from A1 to A2 state appears as the rotation of the ad-dimer, which has been observed experimentally. The ad-dimers are found to prefer filling into the missing dimer vacancies. Finally, the diffusion traces of the ad-dimers on the surfaces have been simulated.
LATTICE EQUILIBRIUM THEORY AND SIZE EFFECTS FOR BOSONS IN A BOUNDED HARMONIC POTENTIAL
Authors: LUMB S. , MUTHU S. K. , SINGH K. K.
Published: January 2006

Effects of finite spatial size of boson assemblies in traps are studied in a self-consistent lattice theory by modeling the trap as a bounded harmonic potential of size R0. The thermodynamic quantities exhibit scaling and crossover from ideal gas behaviour at small (R0/a0) to that appropriate to an unbounded harmonic potential at large (R0/a0) with a crossover parameter [Formula: see text], a0 being the harmonic oscillator length, and τ denoting the dimensionless thermal energy. The numerical results obtained earlier by computing the energy levels of the bounded harmonic oscillator fit the general structure predicted by the theory very well. For a1>10, the spatial size effects are negligible but for a1<10 they become appreciable and experimentally measurable in suitably designed traps. At low temperatures the self consistent cell size is found to be about 2.5a0 implying that the condensate is essentially a single coherent state contained in the central cell.
A NEW PHOSPHOR MADE BY A HIGHLY EMITTED EUROPIUM COMPLEX ADSORBED ON NANOSTRUCTURED TiO2 FILMS
Authors: STATHATOS ELIAS
Published: January 2006

Transparent mesoporous TiO 2 films consisting of uniform anatase particles have been deposited on glass slides by using sol-gel technique. A Europium complex Eu(TTA) 3 (bpy) , where the TTA ligand is β-diketonate thenoyltrifluoroacetonate and the bpy is 2,2-bipyridine, was formed on TiO 2 surface by physical adsorption of europium ions and successive formation of stable chelate complexes with TTA and bpy ligands. In this way a well dispersion of the complex has been achieved giving significant emission intensity. The excitation of the material in the UV region (374 nm) results in the emission of a nearly monochromatic light at 613 nm (5D0→7F2) via energy transfer from the excited organic ligands to europium emissive state.
DEVELOPMENT AND EVALUATION OF AN ALTERNATIVE METHOD FOR PROCESSING ELASTIC-LIDAR RETURN SIGNALS
Authors: CASTREJÓN GARCÍA R. , VARELA J. R. , CASTREJÓN PITA A. A. , CASTREJÓN PITA J. R.
Published: January 2006

A method for interpreting elastic-lidar return signals in heavily-polluted atmospheres is presented. It is based on an equation derived directly from the classic lidar equation, which highlights gradients of the atmospheric backscattering properties along the laser optical path. The method is evaluated by comparing its results with those obtained with the differential absorption technique. The results were obtained from locating and ranging measurements in pollutant plumes and contaminated environments around central México.
Glutamine's protection against cellular injury is dependent on heat shock factor-1
Authors: Morrison Angela L. , Dinges Martin , Singleton Kristen D. , Odoms Kelli , Wong Hector R. , Wischmeyer Paul E.
Published: January 2006

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1+/+) and knockout (HSF-1−/−) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1+/+ cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1−/− cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1−/− cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation.
Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium
Authors: Sahai Atul , Pan Xiaomin , Paul Rachelle , Malladi Padmini , Kohli Rohit , Whitington Peter F.
Published: January 2006

Feeding mice a methionine and choline-deficient (MCD) diet serves as an experimental animal model for nonalcoholic steatohepatitis (NASH). In the present study we examined the effect of exposing AML-12 hepatocytes to MCD culture medium in regard to mechanisms of steatosis and alanine amino-transferase (ALT) release. Cells exposed to MCD medium developed significant and progressive steatosis from 6 to 24 h and also had significantly increased loss of ALT into the medium at 18 and 24 hours of incubation. No increased oxidative injury or cell death was observed. Osteopontin (OPN) mRNA in cells and protein expression in medium were significantly increased during 6–24 hours of incubation. MCD medium treatment also resulted in activation of PI3-kinase by 30 minutes and its downstream target p-Akt within 1hour of incubation. Steatosis was associated with increased expression of microsomal triglyceride transfer protein (MTTP) mRNA and increased ALT release with over expression of ALT mRNA, all of which were completely prevented by inhibition of PI3-kinase (LY294002). Blocking OPN signaling by treating with anti-OPN or anti-β3-integrin antibody prevented the increased ALT release while only partially prevented the increased ALT mRNA expression, but had no effect on either steatosis or MTTP expression. In conclusion, incubation of cultured hepatocytes with MCD medium results in cellular steatosis and OPN dependent ALT release. PI3-kinase plays a central role in signaling the MCD medium-induced steatosis and increased OPN expression, whereas OPN appears to play a role in signaling hepatocyte ALT release but not steatosis.
Common cavity pressure during gastroesophageal reflux: reassessment using simultaneous pressure, impedance, and ultrasound imaging
Authors: Tipnis Neelesh A. , Liu Jianmin , Puckett James L. , Mittal Ravinder K.
Published: January 2006

An increase in intraesophageal pressure during transient lower esophageal sphincter (LES) relaxation [referred to as common cavity (CC) pressure] is thought to be a marker of gastroesophageal reflux (GER). Multiluminal impedance (MII) measurement is a sensitive marker of reflux entry into the esophagus during GER. We recorded GER using esophageal pressure, pH, impedance, and intraluminal ultrasound (US) images to understand the genesis of the esophageal CC pressure. Nine normal subjects underwent simultaneous MII/pH/pressure and US image recording of the esophagus for 2 h following a standardized meal. MII and pressure transducers were located at 5 and 15 cm above the LES. The US transducer and pH sensors were also placed at 5 cm above the LES. Refluxate entry into the esophagus by MII criteria was determined relative to the onset of CC pressure wave. Esophageal lumen cross-sectional area (CSA) and muscle CSA during GER were determined from the US images. Eighty liquid GER episodes identified using MII criteria, of which 55 were clearly associated with CC pressure waves, were analyzed. The GER reached 15 cm above LES in 49 of 55 (89%) by MII criteria, but the CC pressure wave was observed at 5 and 15 cm during all episodes. The propagation of the CC pressure wave was simultaneous between 5 and 15 cm during 49 of 55 (89%) of the GER episodes, but reflux entry by MII criteria was retrograde during 53 of 55 (96%) of these episodes. During 5 air-reflux episodes, MII showed a simultaneous reflux entry between the 5- and 15-cm site, however, the CC pressure preceded reflux entry during all of these episodes. There was poor correlation between the luminal CSA and the magnitude of CC pressure ( R2 = 0.144). US images revealed a close temporal correlation between CC pressure and the increase in esophageal muscle thickness and muscle CSA (markers of longitudinal muscle contraction). Disassociation between CC pressure and MII-detected reflux suggests that the onset of CC pressure is not due to GER. We speculate that longitudinal muscle contraction plays an important role in the genesis of CC pressure.