bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of HLA-DR Assembly, Transport, and Loading by Human Cytomegalovirus Glycoprotein US3: a Novel Mechanism for Evading Major Histocompatibility Complex Class II Antigen Presentation
Authors: Hegde Nagendra R. , Tomazin Roman A. , Wisner Todd W. , Dunn Claire , Boname Jessica M. , Lewinsohn David M. , Johnson David C.
Published: October 2002

Human cytomegalovirus (HCMV) establishes persistent lifelong infections and replicates slowly. To withstand robust immunity, HCMV utilizes numerous immune evasion strategies. The HCMV gene cassette encoding US2 to US11 encodes four homologous glycoproteins, US2, US3, US6, and US11, that inhibit the major histocompatibility complex class I (MHC-I) antigen presentation pathway, probably inhibiting recognition by CD8+T lymphocytes. US2 also inhibits the MHC-II antigen presentation pathway, causing degradation of human leukocyte antigen (HLA)-DR-α and -DM-α and preventing recognition by CD4+T cells. We investigated the effects of seven of the US2 to US11 glycoproteins on the MHC-II pathway. Each of the glycoproteins was expressed by using replication-defective adenovirus vectors. In addition to US2, US3 inhibited recognition of antigen by CD4+T cells by a novel mechanism. US3 bound to class II α/β complexes in the endoplasmic reticulum (ER), reducing their association with Ii. Class II molecules moved normally from the ER to the Golgi apparatus in US3-expressing cells but were not sorted efficiently to the class II loading compartment. As a consequence, formation of peptide-loaded class II complexes was reduced. We concluded that US3 and US2 can collaborate to inhibit class II-mediated presentation of endogenous HCMV antigens to CD4+T cells, allowing virus-infected cells to resist recognition by CD4+T cells.
A Sensitive, Quantitative Assay for Human Immunodeficiency Virus Type 1 Integration
Authors: O'Doherty Una , Swiggard William J. , Jeyakumar Deepa , McGain David , Malim Michael H.
Published: October 2002

Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomicAluelements and HIV-1gagsequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitativeAluPCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.
Use of Adenovirus Vectors Expressing Epstein-Barr Virus (EBV) Immediate-Early Protein BZLF1 or BRLF1 To Treat EBV-Positive Tumors
Authors: Feng Wen-hai , Westphal Eva , Mauser Amy , Raab-Traub Nancy , Gulley Margaret L. , Busson Pierre , Kenney Shannon C.
Published: October 2002

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.
Recombination of Poliovirus RNA Proceeds in Mixed Replication Complexes Originating from Distinct Replication Start Sites
Authors: Egger Denise , Bienz Kurt
Published: October 2002

Genetic recombination occurs frequently during replication of picornaviruses. To explore the intracellular site and structures involved in recombination, HeLa cells were infected with poliovirus type 1 Mahoney and type 2 Sabin. The two genomes were located by fluorescent in situ hybridization and confocal microscopy. For hybridization, type-specific fluorescent riboprobes were used to visualize the same genomic region where, in parallel, recombination was demonstrated with type-specific reverse transcription-PCR and sequencing. The hybridization analysis indicated that >85% of the replication complexes contained both type 1 and type 2 RNA sequences aligned at a lateral distance of 50 nm or less. Sequential infection of cells ruled out the possibility that the high percentage of mixed replication complexes was due to aggregation of input virus. Visualization of input genomic RNA over time showed that the viral genomes migrated to relatively few distinct, and thus presumably specific, perinuclear sites where replication started. The first recombinant RNA strands could be detected concomitantly with the onset of RNA replication. The limited number of start sites for replication may be the reason for the observed preferential formation of mixed replication complexes, each accommodating several parental RNA strands and thus allowing recombination.
Multiple Functions of Immunoglobulin A in Mucosal Defense against Viruses: an In Vitro Measles Virus Model
Authors: Yan Huimin , Lamm Michael E. , Björling Ewa , Huang Yung T.
Published: October 2002

Three defense functions of immunoglobulin A (IgA), immune exclusion, intracellular neutralization, and virus excretion, were assessed in a measles virus model using polarized epithelial cells expressing the polymeric immunoglobulin receptor and monoclonal antibodies against the viral H and F envelope proteins and the internal N protein. Anti-H IgA was the most effective antibody at preventing infection via the apical surface, i.e., immune exclusion. This IgA was also the most effective at intraepithelial cell neutralization after infection at the apical surface and endocytosis of IgA at the basolateral surface, although an antibody against the internal N protein was also effective. In the intracellular neutralization experiments, confocal immunofluorescence microscopy showed prominent colocalization of anti-H IgA and H protein inside virus-infected cells, whereas colocalization of anti-F and F protein and of anti-N and N protein was much less, in agreement with the neutralization results. Combinations of IgA anti-H, anti-F, and anti-N showed no synergistic effects in intracellular neutralization. In the immune excretion experiments, virus immune complexes with either anti-H or anti-F IgA placed beneath polarized epithelial cells could be transported to the apical supernatant. Anti-F IgA, which was relatively poor at immune exclusion and intracellular neutralization, was the most robust at virus excretion. Thus, the studies collectively demonstrated three different antiviral functions of IgA in relation to epithelium and also suggested that the particular viral component with which a given IgA antibody reacts is an important determinant of the magnitude of the antiviral effect.
Phosphorylation by the Varicella-Zoster Virus ORF47 Protein Serine Kinase Determines whether Endocytosed Viral gE Traffics to the
trans-
Golgi Network or Recycles to the Cell Membrane
Authors: Kenyon T. K. , Cohen Jeffrey I. , Grose Charles
Published: October 2002

Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the
trans
-Golgi network (TGN), while in cell-cell spread, surface glycoproteins fuse cells into syncytia. In this report, we delineate a potential role for serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoprotein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA
83:
8967-8971, 1986), although respective roles of viral and cellular protein kinases have never been delineated. VZV ORF47 is a viral serine protein kinase that recognized a consensus sequence similar to that of casein kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as “team players” in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.
A New Type of Adenovirus Vector That Utilizes Homologous Recombination To Achieve Tumor-Specific Replication
Authors: Bernt Kathrin , Liang Min , Ye Xun , Ni Shaoheng , Li Zong-Yi , Ye Sheng Long , Hu Fang , Lieber André
Published: October 2002

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.
Interaction between Long-Distance Transport Factor and Hsp70-Related Movement Protein of
Beet Yellows Virus
Authors: Prokhnevsky Alexey I. , Peremyslov Valera V. , Napuli Alberto J. , Dolja Valerian V.
Published: October 2002

Systemic spread of viruses in plants involves local movement from cell to cell and long-distance transport through the vascular system. The cell-to-cell movement of the
Beet yellows virus
(BYV) is mediated by a movement protein that is an Hsp70 homolog (Hsp70h). This protein is required for the assembly of movement-competent virions that incorporate Hsp70h. By using the yeast two-hybrid system, in vitro coimmunoprecipitation, and in planta coexpression approaches, we show here that the Hsp70h interacts with a 20-kDa BYV protein (p20). We further demonstrate that p20 is associated with the virions presumably via binding to Hsp70h. Genetic and immunochemical analyses indicate that p20 is dispensable for assembly and cell-to-cell movement of BYV but is required for the long-distance transport of virus through the phloem. These results reveal a novel activity for the Hsp70h that provides a molecular link between the local and systemic spread of a plant virus by docking a long-distance transport factor to virions.
Tissue Specificity of the Kaposi's Sarcoma-Associated Herpesvirus Latent Nuclear Antigen (LANA/orf73) Promoter in Transgenic Mice
Authors: Jeong Joseph H. , Hines-Boykin Rebecca , Ash John D. , Dittmer Dirk P.
Published: October 2002

Kaposi's sarcoma-associated herpesvirus
(KSHV/HHV-8) is a human-oncogenic herpesvirus. Cells from KSHV-associated tumors, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), are of endothelial and B-cell origin, respectively. KSHV persists indefinitely in these cell lineages during latent infection. Indeed, cellular latency is a hallmark of all herpesviruses that is intimately linked to their pathogenesis. We previously characterized the promoter for the KSHV latency-associated nuclear antigen LANA/orf73. LANA is required for latent episome maintenance and has also been implicated in oncogenesis. Hence, regulation of LANA expression is critical to KSHV persistence. We find that a region extending to bp −1299 upstream of the LANA transcription start site is able to drive
lacZ
-reporter gene expression in several lines of transgenic mice. In agreement with KSHV's natural tropism, we detected reporter gene expression in CD19-positive B cells but not in CD3-positive T cells. We also detected expression in the kidney and, at a lower level, in the liver. In contrast to KS tumors, transgene expression was localized to kidney tubular epithelium rather than vascular endothelial cells. This suggests that our promoter fragment contains all
cis
-regulatory elements sufficient for B-cell specificity but not those required for endothelial specificity. Alternatively, while the
trans
-acting factors required for LANA expression in B cells are evolutionarily conserved, those that regulate endothelial cell-specific expression are unique to humans. Our in vivo studies address a conundrum in KSHV biology: in culture, KSHV is able to infect a variety of cell types indiscriminately, while in healthy latent carriers KSHV is found in B lymphocytes. The transgenic-mouse experiments reported here suggest that tissue-restricted LANA gene expression could explain B-cell-specific viral persistence.
Productive Infection of Plasmacytoid Dendritic Cells with Human Immunodeficiency Virus Type 1 Is Triggered by CD40 Ligation
Authors: Fong Lawrence , Mengozzi Manuela , Abbey Nancy W. , Herndier Brian G. , Engleman Edgar G.
Published: October 2002

Immature plasmacytoid dendritic cells are the principal alpha interferon-producing cells (IPC), responsible for primary antiviral immunity. IPC express surface molecules CD4, CCR5, and CXCR4, which are known coreceptors required for human immunodeficiency virus (HIV) infection. Here we show that IPC are susceptible to and replicate HIV type 1 (HIV-1). Importantly, viral replication is triggered upon activation of IPC with CD40 ligand, a signal physiologically delivered by CD4 T cells. Immunohistochemical staining of tonsil from HIV-infected individuals reveals HIV p24+IPC, consistent with in vivo infection of these cells. IPC exposed in vitro to HIV produce alpha interferon, which partially inhibits viral replication. Nevertheless, IPC efficiently transmit HIV-1 to CD4 T-cells, and such transmission is also augmented by CD40 ligand activation. IPC produce RANTES/CCL5 and MIP-1α/CCL3 when exposed to HIV in vitro. IPC also induce naïve CD4 T cells to proliferate and would therefore preferentially infect these cells. These results indicate that IPC may play an important role in the dissemination of HIV.
Cooperative Activation of Human Papillomavirus Type 8 Gene Expression by the E2 Protein and the Cellular Coactivator p300
Authors: Müller Andreas , Ritzkowsky Andreas , Steger Gertrud
Published: October 2002

The E2 proteins of papillomaviruses (PV) bind to the coactivator CBP/p300 as do many other transcription factors, but the precise role of CBP/p300 in E2-specific functions is not yet understood. We show that the E2 protein of human PV type 8 (HPV8) directly binds to p300. Activation of HPV8 gene expression by low amounts of HPV8 E2 was stimulated up to sevenfold by coexpression of p300. The interaction between E2 and p300 may play a role in differentiation-dependent activation of PV gene expression, since we can show that the expression level of p300 increases during keratinocyte differentiation. Surprisingly, sequence-specific binding of E2 to its recognition sites within the regulatory region of HPV8 is not necessary for this cooperation, indicating that E2 can be recruited to the promoter via protein-protein interaction. HPV8 E2 binds via its N-terminal activation domain (AD), its C-terminal DNA binding domain (DBD), and its internal hinge region to p300 in vitro
.
Transient-transfection assays revealed that the AD is necessary and sufficient for cooperative activation with p300. However, we provide evidence that the interaction of the hinge and the DBD of HPV8 E2 with p300 may contribute. Our data suggest an important role of p300 in regulation of HPV8 gene expression and reveal a new mechanism by which E2 may be recruited to a promoter to activate transcription without sequence specific DNA binding.
Inhibition of PACT-Mediated Activation of PKR by the Herpes Simplex Virus Type 1 Us11 Protein
Authors: Peters Gregory A. , Khoo David , Mohr Ian , Sen Ganes C.
Published: October 2002

PACT, a protein activator of PKR, can cause inhibition of cellular protein synthesis and apoptosis. Here, we report that the Us11 protein of herpes simplex virus type 1 can block PKR activation by PACT both in vitro and in vivo. Although Us11 can bind to both PKR and PACT, mutational analyses revealed that the binding of Us11 to PKR, and not to PACT, was essential for its inhibitory action. Similar analyses also revealed that the inhibitory effect was mediated by an interaction between the C-terminal half of Us11 and the N-terminal domain of PKR. The binding of Us11 to PKR did not block the binding of PKR to PACT but prevented its activation. Us11 is the first example of a viral protein that can inhibit the action of PACT on PKR.
Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59
Authors: Yount Boyd , Denison Mark R. , Weiss Susan R. , Baric Ralph S.
Published: October 2002

A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ∼31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes. RNA transcripts derived from the full-length MHV-A59 construct were infectious, although transfection frequencies were enhanced 10- to 15-fold in the presence of transcripts encoding the nucleocapsid protein N. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar growth kinetics, plaque morphology, and cytopathology in murine cells as did wild-type MHV-A59. Molecularly cloned viruses recognized the MHV receptor (MHVR) for docking and entry, and pretreatment of cells with monoclonal antibodies against MHVR blocked virus entry and replication. Cells infected with molecularly cloned MHV-A59 virus expressed replicase (gene 1) proteins identical to those of laboratory MHV-A59. Importantly, the molecularly cloned viruses contained three marker mutations that had been derived from the engineered component clones. Full-length infectious constructs of MHV-A59 will permit genetic modifications of the entire coronavirus genome, particularly in the replicase gene. The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.
Synergistic Activation of Human Immunodeficiency Virus Type 1 Promoter Activity by NF-κB and Inhibitors of Deacetylases: Potential Perspectives for the Development of Therapeutic Strategies
Authors: Quivy Vincent , Adam Emmanuelle , Collette Yves , Demonte Dominique , Chariot Alain , Vanhulle Caroline , Berkhout Ben , Castellano Rémy , de Launoit Yvan , Burny Arsène , Piette Jacques , Bours Vincent , Van Lint Carine
Published: October 2002

The transcription factor NF-κB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment with deacetylase inhibitors such as trichostatin A (TSA) or sodium butyrate (NaBut) markedly induces HIV-1 transcriptional activity of the long terminal repeat (LTR) promoter. Here, we demonstrate that TSA (NaBut) synergized with both ectopically expressed p50/p65 and tumor necrosis factor alpha/SF2 (TNF)-induced NF-κB to activate the LTR. This was confirmed for LTRs from subtypes A through G of the HIV-1 major group, with a positive correlation between the number of κB sites present in the LTRs and the amplitude of the TNF-TSA synergism. Mechanistically, TSA (NaBut) delayed the cytoplasmic recovery of the inhibitory protein IκBα. This coincided with a prolonged intranuclear presence and DNA binding activity of NF-κB. The physiological relevance of the TNF-TSA (NaBut) synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Therefore, our results open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression.
Evolution of Phenotypic Drug Susceptibility and Viral Replication Capacity during Long-Term Virologic Failure of Protease Inhibitor Therapy in Human Immunodeficiency Virus-Infected Adults
Authors: Barbour Jason D. , Wrin Terri , Grant Robert M. , Martin Jeffrey N. , Segal Mark R. , Petropoulos Christos J. , Deeks Steven G.
Published: October 2002

Continued use of antiretroviral therapy despite the emergence of drug-resistant human immunodeficiency virus (HIV) has been associated with the durable maintenance of plasma HIV RNA levels below pretherapy levels. The factors that may account for this partial control of viral replication were assessed in a longitudinal observational study of 20 HIV-infected adults who remained on a stable protease inhibitor-based regimen despite ongoing viral replication (plasma HIV RNA levels consistently >500 copies/ml). Longitudinal plasma samples (
n
= 248) were assayed for drug susceptibility and viral replication capacity (measured by using a single-cycle recombinant-virus assay). The initial treatment-mediated decrease in plasma viremia was directly proportional to the reduction in replicative capacity (
P
= 0.01). Early virologic rebound was associated the emergence of a virus population exhibiting increased protease inhibitor phenotypic resistance, while replicative capacity remained low. During long-term virologic failure, plasma HIV RNA levels often remained stable or increased slowly, while phenotypic resistance continued to increase and replicative capacity decreased slowly. The emergence of primary genotypic mutations within protease (particularly V82A, I84V, and L90M) was temporally associated with increasing phenotypic resistance and decreasing replicative capacity, while the emergence of secondary mutations within protease was associated with more-gradual changes in both phenotypic resistance and replicative capacity. We conclude that HIV may be constrained in its ability to become both highly resistant and highly fit and that this may contribute to the continued partial suppression of plasma HIV RNA levels that is observed in some patients with drug-resistant viremia.
Differential Requirements for COPI Coats in Formation of Replication Complexes among Three Genera of
Picornaviridae
Authors: Gazina Elena V. , Mackenzie Jason M. , Gorrell Rebecca J. , Anderson David A.
Published: October 2002

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, β-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain β-COP. In contrast, β-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells β-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.
Baculovirus Infection Raises the Level of TATA-Binding Protein That Colocalizes with Viral DNA Replication Sites
Authors: Quadt Ilja , Mainz Daniela , Mans Ruud , Kremer Andreas , Knebel-Mörsdorf Dagmar
Published: October 2002

During the infection cycle of
Autographa californica
multicapsid nuclear polyhedrosis virus, the TATA-binding protein (TBP) of the insect host cell likely participates in early viral transcription, which is mediated by the host RNA polymerase II. However, the role of TBP in late and very late viral transcription, which is accomplished by an alpha-amanitin-resistant RNA polymerase, is unclear. We observed a dramatic increase of TBP protein during the late phases of infection. TBP mRNA levels, however, were not coordinately increased. Indirect-immunofluorescence studies revealed a nuclear redistribution of TBP during infection. After labeling of viral replication centers with bromodeoxyuridine (BrdU), costaining of TBP and BrdU showed that TBP localized to viral DNA replication centers. These results suggest a putative role of TBP during late viral transcription, which may occur in close proximity to viral DNA replication.
In Vitro-Assembled Alphavirus Core-Like Particles Maintain a Structure Similar to That of Nucleocapsid Cores in Mature Virus
Authors: Mukhopadhyay Suchetana , Chipman Paul R. , Hong Eunmee M. , Kuhn Richard J. , Rossmann Michael G.
Published: October 2002

In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 Å with 240 copies of the capsid protein arranged in a T=4 icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.
Implication of the Lymphocyte-Specific Nuclear Body Protein Sp140 in an Innate Response to Human Immunodeficiency Virus Type 1
Authors: Madani Navid , Millette Robert , Platt Emily J. , Marin Mariana , Kozak Susan L. , Bloch Donald B. , Kabat David
Published: October 2002

The viral infectivity factor (Vif) of human immunodeficiency virus type 1 (HIV-1) neutralizes an unidentified antiviral pathway that occurs only in nonpermissive (NP) cells. Using a yeast two-hybrid screen of a human lymphocyte cDNA library, we identified several potential Vif partners. One, the nuclear body protein Sp140, was found specifically in all NP cells (
n
= 12 cell lines tested;
P
≤ 0.001), and HIV-1 infection induced its partial dispersal from nuclear bodies into cytosolic colocalization with Vif. Our results implicate Sp140 in a response to HIV-1 that may be related to or coordinated with the pathway that inactivates HIV-1 lacking
vif
.
Evidence for Early Infection of Nonneoplastic Natural Killer Cells by Epstein-Barr Virus
Authors: Trempat Pascal , Tabiasco Julie , Andre Pascale , Faumont Nathalie , Meggetto Fabienne , Delsol Georges , Gascoyne Randy D. , Fournie Jean-Jacques , Vivier Eric , Brousset Pierre
Published: October 2002

We examined lymph nodes and tonsils from patients with infectious mononucleosis by combined detection of EBV-encoded RNA and a specific marker of natural killer (NK) cells, PEN5. A small number of Epstein-Barr virus (EBV) latently infected nonneoplastic NK cells were detected. Our data demonstrate that NK cells are natural targets of EBV and that infection of these cells is an early event observed during primary EBV infection.
Identification of the Hepatitis C Virus E2 Glycoprotein Binding Site on the Large Extracellular Loop of CD81
Authors: Drummer Heidi E. , Wilson Kirilee A. , Poumbourios Pantelis
Published: October 2002

The binding of hepatitis C virus glycoprotein E2 to the large extracellular loop (LEL) of CD81 has been shown to modulate human T-cell and NK cell activity in vitro. Using random mutagenesis of a chimera of maltose-binding protein and LEL residues 113 to 201, we have determined that the E2-binding site on CD81 comprises residues Ile
182
, Phe
186
, Asn
184
, and Leu
162
. These findings reveal an E2-binding surface of approximately 806 Å
2
and potential target sites for the development of small-molecule inhibitors of E2 binding.
Comparative Analysis of Anti-Hepatitis C Virus Activity and Gene Expression Mediated by Alpha, Beta, and Gamma Interferons
Authors: Cheney I. Wayne , Lai Vicky C. H. , Zhong Weidong , Brodhag Tessa , Dempsey Shannon , Lim Charmaine , Hong Zhi , Lau Johnson Y. N. , Tam Robert C.
Published: October 2002

A direct comparison of the inhibitory effects of alpha, beta, and gamma interferons (IFNs) on replication of a hepatitis C virus subgenomic replicon in a hepatoma cell line revealed similarities in antiviral potency. However, alternate IFN-induced antiviral mechanisms were suggested following observations of striking differences between IFN-γ and IFN-α/β with respect to strength and durability of the antiviral response and the magnitude and pattern of IFN-mediated gene expression.
Identification of a Hepatitis B Virus Genome in Wild Chimpanzees (Pan troglodytes schweinfurthi) from East Africa Indicates a Wide Geographical Dispersion among Equatorial African Primates
Authors: Vartanian Jean-Pierre , Pineau Pascal , Henry Michel , Hamilton William D. , Muller Martin N. , Wrangham Richard W. , Wain-Hobson Simon
Published: October 2002

DNAs from four wild chimpanzees (Pan troglodytes schweinfurthi) from eastern Africa were screened for 14 DNA viruses and retroviruses. Between two and three viruses were found in each animal. An entire hepatitis B virus (HBV) genome was amplified and sequenced from samples taken from one animal. This indicates that HBV is distributed across the entire range of chimpanzee habitats.
Evidence for Regulation of Long Terminal Repeat Transcription by Wnt Transcription Factor TCF-4 in Human Astrocytic Cells
Authors: Wortman Bethany , Darbinian Nune , Sawaya Bassel E. , Khalili Kamel , Amini Shohreh
Published: October 2002

The Wnt signaling pathway plays an important role in neural cell development and function. The key components of this pathway, β-catenin and its partner TCF-4/LEF-1, exert their effects on transcription by entering the nuclei, where they associate with the TCF-4/LEF-1 DNA motif positioned in the promoters of several important genes. Here we examined the role of TCF-4 upon transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in human astrocytic cells. Our results showed that expression of TCF-4 in human astrocytic cells (U-87MG cells) decreased the basal and Tat-mediated transcription of the HIV-1 long terminal repeat (LTR). Results from promoter deletion studies revealed that the promoter sequence of the LTR with no classical binding motif for TCF-4/LEF-1, which spans positions −80 to +80 of the LTR, remained responsive to down-regulation by TCF-4. Noticeably, removal of the sequences between positions −80 and −68 decreased the negative effect of TCF-4 on viral gene transcription. A mutant variant of TCF-4 with no binding site for β-catenin was able to down-regulate LTR transcription, suggesting that β-catenin may not be directly involved in the observed regulatory events. Results from the glutathione
S
-transferase pull-down assay as well as the combined immunoprecipitation and Western blot analysis of protein extract from U-87MG cells revealed an interaction of Tat with TCF-4. Subcellular examination of TCF-4 and Tat in cells expressing either protein alone showed a predominantly nuclear accumulation of these proteins. However, in cells which coexpressed both TCF-4 and Tat, significant levels of these proteins were found in the cytoplasm. All together, these observations provide evidence for the cooperative interaction of TCF-4, the important transcription factor of the Wnt pathway, with Tat; this interaction may determine the level of viral gene transcription in human astrocytic cells.
The Influenza A Virus NS1 Protein Inhibits Activation of Jun N-Terminal Kinase and AP-1 Transcription Factors
Authors: Ludwig Stephan , Wang Xiuyan , Ehrhardt Christina , Zheng Hongyong , Donelan Nicola , Planz Oliver , Pleschka Stephan , García-Sastre Adolfo , Heins Gudrun , Wolff Thorsten
Published: October 2002

The influenza A virus nonstructural NS1 protein is known to modulate host cell gene expression and to inhibit double-stranded RNA (dsRNA)-mediated antiviral responses. Here we identify NS1 as the first viral protein that antagonizes virus- and dsRNA-induced activation of the stress response-signaling pathway mediated through Jun N-terminal kinase.
Regions of Mouse Mammary Tumor Virus Superantigen Involved in Interaction with the Major Histocompatibility Complex Class II I-A Molecule
Authors: Wirth Susanne , Vessaz Annelyse , Krummenacher Claude , Baribaud Frédéric , Acha-Orbea Hans , Diggelmann Heidi
Published: October 2002

To study the major histocompatibility complex class II I-E dependence of mouse mammary tumor virus (MMTV) superantigens, we constructed hybrids between the I-E-dependent MMTV(GR) and the I-E-independent
mtv
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7
superantigens and tested them in vivo. Our results suggest that, although the C-terminal third mediates I-A interaction, additional binding sites are located elsewhere in the superantigen.
The Stereoselective Preparation of Fluorinated Dienes via Stille-Liebeskind Cross-Coupling Reactions
Authors: Zhang Xin , Lu Long , Burton Donald J.
Published: October 2002

Readily prepared fluorinated vinylstannane reagents stereoselectively undergo Stille-Liebeskind cross-coupling reactions with vinyl halides (including fluorine-containing vinyl halides) with Pd(PPh3)4/CuI catalysis.
A Mild and Practical Preparation of Trifluoromethaneselenenyl Chloride
Authors: Magnier Emmanuel , Wakselman Claude
Published: October 2002

Trifluoromethaneselenenyl chloride is prepared by a two-step procedure with an 85% overall yield. Benzyl trifluoromethyl selenide is formed from dibenzyl diselenide and trifluoromethyl iodide in the presence of sodium hydroxymethanesulfinate. The benzyl-selenium bond is then cleaved with sulfuryl chloride to give the corresponding selenenyl chloride.
Synthesis of 2',3'-Dideoxy-2'-trifluoromethyl-N-azanucleosides
Authors: Qing Feng-Ling , Yu Jiang , Fu Xiang-Kai
Published: October 2002

The trifluoromethylation of (Z)/(E)-tert-butyl (4S)-4-(2'-ethoxycarbonyl-2'-bromoprop-1'-enyl)-2,2-dimethyloxazolidine-3-carboxylate (4), which is derived from chiral serine aldehyde, yields α-trifluoromethyl-α,β-unsaturated ester 1. Compound 1 is used as a key intermediate for the synthesis of a number of 2',3'-dideoxy-2'-trifluoromethyl-N-azanucleosides.
Polyhalogenated Heterocyclic Compounds. Part 48. Multisubstituted Bis-pyridyl Derivatives from Pentafluoropyridine
Authors: Chambers Richard D. , Hoskin Philip R. , Sandford Graham
Published: October 2002

Various polyfunctional and ring-fused bis-pyridine systems have been synthesised from pentafluoropyridine by nucleophilic substitutions.
Radical Reaction Using an Organocopper Reagent Derived from Ethyl Bromodifluoroacetate
Authors: Sato Kazuyuki , Ogawa Yuko , Tamura Misato , Harada Mika , Ohara Terumasa , Omote Masaaki , Ando Akira , Kumadaki Itsumaro
Published: October 2002

In our previous work, reaction of ethyl bromodifluoroacetate (1) in the presence of Cu powder with olefins activated by an electron-withdrawing group gave the Michael-type adducts through an anionic intermediate. The same reaction with non-activated olefins was found to give addition products by a radical mechanism. The radical intermediate from α-methylstyrene was stabilized by a benzene ring and gave novel dimerization products. This reaction with 1-decene provided a convenient route to 2,2-difluorododecanoic acid.
Fluoride-Catalyzed Nucleophilic Perfluoroalkylations of Acetylenic Ketones or Aldehydes
Authors: Manandhar Sudha , Singh Rajendra P. , Shreeve Jean'ne M.
Published: October 2002

Reactions of 4-phenylbut-3-yn-2-one (1a) and phenylpropynal (1b) with trimethyl(perfluoroalkyl)silanes (Me3SiRf) (Rf = CF3, C2F5, n-C6F13, n-C7F15, n-C8F17) in the presence of catalytic amounts of cesium fluoride (CsF) have been studied. Compounds 1a, 1b were reacted with 0.5 equivalent excess of Me3SiRf in ethylene glycol dimethyl ether (monoglyme) at 25 °C for Rf = CF3, C2F5 and at 50 °C for Rf = n-C6F13, n-C7F15, n-C8F17 to give the corresponding perfluoroalkylated alcohols in good yields after acid hydrolysis. The new compounds were characterized by IR, NMR (1H, 19F, 13C), MS and elemental analysis. In these reactions, tetrabutylammonium fluoride (TBAF) is also effective as the fluoride catalyst. The alcohols with CF3 or C2F5 are viscous liquids whereas those with n-C6F13, n-C7F15 or n-C8F17 are solids. They are soluble in common organic solvents and stable to air and moisture.
Parallel Solid-Phase Synthesis of Partially Modified Retro and Retro-Inverso ψ[NHCH(CF3)]-Gly Peptides
Authors: Sani Monica , Bravo Pierfrancesco , Volonterio Alessandro , Zanda Matteo
Published: October 2002

The parallel solid-phase synthesis of small libraries of molecules belonging to a novel class of retro and retro-inverso peptides having a ψ[NHCH(CF3)] surrogate of the conventional retro-peptide bond (NH-CO) has been accomplished. Key step for the synthesis of the -NHCH(CF3)- unit is a Michael-type N-addition of resin bound α-amino acid esters H-AA1-OWang (1), dipeptide H-Val-Gly-OWang (8), and tripeptide H-Val-Val-Ala-OWang (12) to (S)-3-(E-enoyl)-1,3-oxazolidin-2-one (3), which took place very effectively under mild condition. Chemoselective exocyclic oxazolidinone cleavage, followed by parallel couplings of the resulting polymer-bound pseudopeptides WangO-AA1-ψ[NHCH(CF3)]-Gly-OH (5), WangO-Gly-Val-ψ[NHCH(CF3)]-Gly-OH (10), and WangO-Ala-Val-Val-ψ[NHCH(CF3)]-Gly-OH (14) with different α-amino acid esters afforded, after release from the resin, a representative set of ψ[NHCH(CF3)] retro and retro-inverso tripeptides HO-AA1-ψ[NHCH(CF3)]-Gly-AA2-OX1 (7), tetrapeptides HO-Gly-Val-ψ[NHCH(CF3)]-Gly-AA3-OX2 (11), and pentapeptides HO-Ala-Val-Val-ψ[NHCH(CF3)]-Gly-AA4-OX3 (15), respectively, with good to excellent purity in all cases.
A Simple Method for Preparing Difluorodiiodomethane from Difluoro(fluorosulfonyl)acetyl Fluoride
Authors: Xiao Ji-Chang , Duan Jian-Xin , Li An-Rong , Guo Yong , Chen Qing-Yun
Published: October 2002

Difluorodiiodomethane is prepared in 60% yield by the reaction of difluoro(fluorosulfonyl)acetyl fluoride with I2/KI in the presence of a catalytic amount of SiO2in acetonitrile at 60-65 °C.
Synthesis of α-Fluoro-β-lactones and Their Thermal Conversion to 1-Fluoroalkenes
Authors: Ocampo Rogelio , Dolbier William R. , Zuluaga Fabio
Published: October 2002

α-Fluoro-β-lactones have been synthesized and isolated for the first time from α-fluoro-β-hydroxy acids by using the couple TsCl/DMAP as lactonization agent. A detailed description of the synthesis and spectroscopic properties of α-fluoro-β-lactones is presented. Preliminary results indicate that thermolysis of these new β-lactones produces 1-fluoroalkenes.
Preparation of (Fluoromethyl)- and (Difluoromethyl)imidazoles
Authors: Dolensky Bohumil , Kirk Kenneth L.
Published: October 2002

2-(Fluoromethyl)- and 2-(difluoromethyl)imidazoles, and 4-(fluoromethyl)- and 4-(difluoromethyl)imidazoles have been prepared by deoxyfluorination of (hydroxymethyl)imidazole or formylimidazole precursors.
Synthesis of (Polyfluoroalkyl)cyclopentenes as Model Compounds for Fluorophilic Cyclopentadienes
Authors: Kvíčala Jaroslav , Bříza Tomáš , Paleta Oldřich , Čermák Jan
Published: October 2002

Nucleophilic opening of the epoxide ring in 6-oxabicyclo[3.1.0]hexane (1) with ethenylmagnesium bromide was employed for the preparation of 2-ethenylcyclopentan-1-ol (2). Radical addition of perfluoroalkyl iodides 3 to alcohol 2 afforded (polyfluoroiodoalkyl)cyclopentanols 4, which were deiodinated with tributylstannane to (polyfluoroalkyl)cyclopentanols 5, followed by dehydration with Nafion-H to the target (polyfluoroalkyl)cyclopentenes 6, which are potential intermediates for fluorous chemistry. Attempts to synthesize (polyfluoroalkyl)cyclopentadienes or bis(polyfluoroalkyl)cyclopentadienes failed due to the exclusive formation of unexpected side product, polyfluoroiodoalkanol 7, in the course of radical addition of perfluoroalkyl iodides 3 to ethenylcyclopentenol (8) or diethenylcyclopentanediol (9), respectively.
Synthesis of (Trifluoromethyl)aziridines in 1,1,1,3,3,3-Hexafluoropropan-2-ol: First Example of Coupling Reactions of Fluoral, an Amine and a Diazo Compound
Authors: Spanedda Maria Vittoria , Crousse Benoit , Narizuka Satoru , Bonnet-Delpon Danièle , Bégué Jean-Pierre
Published: October 2002

The influence of solvent on the Yb(OTf)3 or BF3·Et2O-catalyzed reaction of (trifluoromethyl)aldimines with ethyl diazoacetate has been investigated. The reaction of fluoral ethyl hemiacetal, an aromatic amine, and ethyl diazoacetate could be performed in 1,1,1,3,3,3-hexafluoropropan-2-ol to provide the corresponding (trifluoromethyl)aziridines.
2,2':6',2''-Terpyridine as Monodentate Ligand: Halogen Bonding Driven Formation of Discrete 2 : 1 Aggregates with 1,2,4,5-Tetrafluoro-3,6-diiodobenzene
Authors: Liantonio Rosalba , Logothetis Thomas A. , Messina Maria T. , Metrangolo Pierangelo , De Santis Alessandra , Pilati Tullio , Resnati Giuseppe
Published: October 2002

2,2':6',2''-Terpyridine (1) is a well-known electron donor module in metal coordination chemistry and typically works as a tridentate ligand. Here it is shown that 1 can also work as electron donor towards iodoperfluorocarbons both in solution and in the solid phase. Halogen-bonded supramolecular systems are thus obtained. Specifically, terpyridine 1 self-assembles with 1,2,4,5-tetrafluoro-3,6-diiodobenzene (2) and affords the trimeric adduct 3, which is stable and crystalline in the air at room temperature. Single crystal X-ray analysis shows how in adduct 3 both iodine atoms of one molecule of 2 are halogen-bonded to the nitrogen atoms of external pyridine rings of two molecules of 1 that act as monodentate electron donors.
New mannotriosides and trimannosides as potential ligands for mannose-specific binding proteins
Authors: Furneaux Richard H , Pakulski Zbigniew , Tyler Peter C
Published: October 2002

The α-D-mannopyranosyl and 3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl neoglycolipids 36 and the branched and cluster trimannosidic acids 38 and 41 have been made in connection with studies of liposomes as transporters of antigens to dendritic cells.Key words: mannotrioside, trimannoside, neoglycoconjugate, glycolipid, synthesis.
Synthesis, structure, and electrochemistry of cobalt(III) complexes with bis(benzoylacetone)ethylenediimine Schiff base
Authors: Amirnasr Mehdi , Vafazadeh Rasoul , Mahmoudkhani Amir H
Published: October 2002

A series of complexes of the type trans-[Co((BA)2en)(amine)2]ClO4  where (BA)2en is the bis(benzoylacetone)ethylenediimine dianion and the amines are morpholine (mrpln) 1, aniline (aniln) 2, benzylamine (bzlan) 3, piperidine (pprdn) 4, p-toluidine (p-toldn) 5, and pyrrolidine (prldn) 6  has been synthesized and characterized by elemental analysis, UVvis, 1H NMR, and 13C NMR spectroscopy. The crystal and molecular structure of trans-[Co((BA)2en)(mrpln)2]ClO4 (1) has been determined by X-ray crystallography. The electrochemical reduction of these complexes at a glassy-carbon electrode, in the potential range of 0.5 to 2.0 V (vs. Ag/AgCl), in acetonitrile solution, indicates that the first reduction potential of Co(III/II) is irreversible, which is accompanied by the dissociation of the axial aminecobalt bonds. This potential is dependent on the pKa of the axial amines. The second reduction potential of Co(II/I) shows reversible behavior and is also independent of the axial amine pKa. These results indicate the loss of the axial amines in the first redox process and the involvement of a four-coordinate species in the second redox step.Key words: Schiff base, cobalt(III), axial amine, electrochemical properties, crystal structure.