bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo
Authors: Qing Keyun , Khuntirat Benjawan , Mah Cathryn , Kube Dagmar M. , Wang Xu-Shan , Ponnazhagan Selvarangan , Zhou Shangzhen , Dwarki Varavani J. , Yoder Mervin C. , Srivastava Arun 
Published: December 2019

ABSTRACT

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34
+
primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1
+
lin
−
primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.
Essential Roles of NF-κB and C/EBP in the Regulation of Intercellular Adhesion Molecule-1 after Respiratory Syncytial Virus Infection of Human Respiratory Epithelial Cell Cultures
Authors: Chini B. A. , Fiedler M. A. , Milligan L. , Hopkins T. , Stark J. M. 
Published: December 2019

ABSTRACTTo determine the molecular mechanism(s) of respiratory syncytial virus (RSV)-induced intercellular adhesion molecule-1 (ICAM-1) upregulation in respiratory epithelial cells (REC; A549 cell cultures), we investigated the roles of the transcription factors NF-κB and C/EBP. Increases in ICAM-1 message required de novo mRNA synthesis. ICAM-1 promoter constructs (luciferase reporter gene) transfected into A549 monolayers demonstrated promoter activation following RSV infection. Activation was abolished by site-specific mutation of the NF-κB (−228) or C/EBP (−239) sites. These data support the critical role of the activation of NF-κB and C/EBP in RSV-induced ICAM-1 expression by REC.
Herpesvirus Saimiri-Transformed Human CD4+T-Cell Lines: an Efficient Target Cell System for the Analysis of Human Immunodeficiency Virus-Specific Cytotoxic CD8+T-Lymphocyte Activity
Authors: Bauer Monika , Lucchiari-Hartz Maria , Fickenscher Helmut , Eichmann Klaus , McKeating Jane , Meyerhans Andreas 
Published: December 2019

ABSTRACTHerpesvirus saimiri growth-transformed human CD4+T lymphocytes were examined for their suitability as a target cell system for investigating human immunodeficiency virus (HIV)-specific HLA class I-restricted cytotoxic T-cell activity. Besides CD4, they express the chemokine receptors CCR5 and CXCR4, the common coreceptors of HIV. They are infectible by a range of HIV strains, including primary isolates, becoming efficient targets for CD8-positive HIV-specific cytotoxic T lymphocytes.
Coadministration of DNA Encoding Interleukin-6 and Hemagglutinin Confers Protection from Influenza Virus Challenge in Mice
Authors: Larsen Diane L. , Dybdahl-Sissoko Naomi , McGregor Martha W. , Drape Robert , Neumann Veronica , Swain William F. , Lunn D. Paul , Olsen Christopher W. 
Published: December 2019

ABSTRACTThis study was conducted to investigate whether Accell gene gun coadministration of DNA encoding human interleukin-6 (IL-6) would enhance protective immune responses in mice to an equine influenza A virus hemagglutinin (HA) DNA vaccine. Mice that received HA DNA alone exhibited accelerated clearance of homologous challenge virus but were not protected from infection. In contrast, mice that received both HA and IL-6 DNA had no detectable virus in their lungs after challenge. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination.
Proteolytic Processing at the Amino Terminus of Human Coronavirus 229E Gene 1-Encoded Polyproteins: Identification of a Papain-Like Proteinase and Its Substrate
Authors: Herold Jens , Gorbalenya Alexander E. , Thiel Volker , Schelle Barbara , Siddell Stuart G. 
Published: December 2019

ABSTRACT

Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitro
trans
-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus pp1a and pp1ab polyproteins. By combining the
trans
-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861–Glu-975 and Asn-1209–Gln-1285. Finally, codon mutagenesis was used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity, suggesting that these amino acids most likely have a catalytic function.
Kinetic Analysis of the Specific Host Response to a Murine Gammaherpesvirus
Authors: Stevenson Philip G. , Doherty Peter C. 
Published: December 2019

ABSTRACTRespiratory infection of BALB/c mice with the murine gammaherpesvirus 68 (MHV-68) induces the clonal expansion of virus-specific cytotoxic T-lymphocyte (CTL) precursors (CTLp) in the regional, mediastinal lymph nodes (MLN). Some of these CTLps differentiate to become fully functional CTL effectors, which can be detected in both the lymphoid tissue and in the site of pathology in the lung. Though the lymph nodes and spleen harbor substantial populations of latently infected B cells for life, the level of virus-specific CTL activity decreases rapidly in all sites. The CD8+CTLp numbers fall to background levels in the MLN within several months of the termination of the productive phase of MHV-68 infection in the respiratory epithelium but are maintained at relatively low frequency in the spleen. The continued presence of a gamma interferon-producing, MHV-68-specific CD4+set can also be demonstrated in cultured spleen cells. The virus-specific immunoglobulin G (IgG) response is slow to develop, with serum neutralizing antibody and enzyme-linked immunosorbent assay titers continuing to rise for several months. The level of total serum IgG increases dramatically within 2 weeks of infection, probably as a consequence of polyclonal B-cell activation, and remains high. The immune response profile is clearly influenced by the persistence of this DNA virus.
Multiple Residues Contribute to the Inability of Murine CCR-5 To Function as a Coreceptor for Macrophage-Tropic Human Immunodeficiency Virus Type 1 Isolates
Authors: Ross Ted M. , Bieniasz Paul D. , Cullen Bryan R. 
Published: December 2019

ABSTRACT
Infection of CD4-positive cells by human immunodeficiency virus type 1 (HIV-1) requires functional interaction of the viral envelope protein with a coreceptor belonging to the chemokine receptor family of seven-membrane-spanning receptors. For the majority of macrophage-tropic HIV-1 isolates, the physiologically relevant coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection, chimeric hCCR-5/mCCR-5 molecules containing single extracellular domains derived from hCCR-5 are effective coreceptors for certain macrophage-tropic HIV-1 isolates. Here, we have sought to identify residues in hCCR-5 critical for HIV-1 infection by substitution of mCCR-5-derived residues into the context of functional chimeric hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate that residues 7, 13, and 15 in the first extracellular domain and residue 180 in the third extracellular domain of CCR-5 are important for HIV-1 envelope-mediated membrane fusion. Of interest, certain substitutions, for example, at residues 184 and 185 in the third extracellular domain, have no phenotype when introduced individually but strongly inhibit hCCR-5 coreceptor function when present together. We hypothesize that these changes, which do not preclude chemokine receptor function, may inhibit a conformational transition in hCCR-5 that contributes to HIV-1 infection. Finally, we report that substitution of glycine for valine at residue 5 in CCR-5 can significantly enhance the level of envelope-dependent cell fusion by expressing cells. The diversity of the mutant phenotypes observed in this mutational analysis, combined with their wide distribution across the extracellular regions of CCR-5, emphasizes the complexity of the interaction between HIV-1 envelope and coreceptor.
Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication
Authors: Poon Betty , Chen Irvin S. Y. 
Published: December 2019

ABSTRACTIn vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication.
The Genome of Salmonid Herpesvirus 1
Authors: Davison Andrew J. 
Published: December 2019

ABSTRACT

Salmonid herpesvirus 1 (SalHV-1) is a pathogen of the rainbow trout (
Oncorhynchus mykiss
). Restriction endonuclease mapping, cosmid cloning, DNA hybridization, and targeted DNA sequencing experiments showed that the genome is 174.4 kbp in size, consisting of a long unique region (U
L
; 133.4 kbp) linked to a short unique region (U
S
; 25.6 kbp) which is flanked by an inverted repeat (R
S
; 7.7 kbp). U
S
is present in virion DNA in either orientation, but U
L
is present in a single orientation. This structure is characteristic of the
Varicellovirus
genus of the subfamily
Alphaherpesvirinae
but has evidently evolved independently, since an analysis of randomly sampled DNA sequence data showed that SalHV-1 shares at least 18 genes with channel catfish virus (CCV), a fish herpesvirus whose complete sequence is known and which is unrelated to mammalian herpesviruses. The use of oligonucleotide probes demonstrated that in comparison with CCV, the conserved SalHV-1 genes are located in U
L
in at least five rearranged blocks. Large-scale gene rearrangements of this type are also characteristic of the three mammalian herpesvirus subfamilies. The junction between two SalHV-1 gene blocks was confirmed by sequencing a 4,245-bp region which contains the dUTPase gene, part of a putative spliced DNA polymerase gene, and one other complete gene. The implications of these findings in herpesvirus taxonomy are discussed.
Nucleocapsid and Matrix Protein Contributions to Selective Human Immunodeficiency Virus Type 1 Genomic RNA Packaging
Authors: Poon Dexter T. K. , Li Guangde , Aldovini Anna 
Published: December 2019

ABSTRACT
The nucleocapsid protein (NC) of retroviruses plays a major role in genomic RNA packaging, and some evidence has implicated the matrix protein (MA) of certain retroviruses in viral RNA binding. To further investigate the role of NC in the selective recognition of genomic viral RNA and to address the potential contribution of MA in this process, we constructed chimeric and deletion human immunodeficiency virus type 1 (HIV-1) mutants that alter the NC or MA protein. Both HIV and mouse mammary tumor virus (MMTV) NC proteins have two zinc-binding domains and similar basic amino acid compositions but differ substantially in total length, amino acid sequence, and spacing of the zinc-binding motifs. When the entire NC coding sequence of HIV was replaced with the MMTV NC coding sequence, we found that the HIV genome was incorporated into virions at 50% of wild-type levels. Viruses produced from chimeric HIV genomes with complete NC replacements, or with the two NC zinc-binding domains replaced with MMTV sequences, preferentially incorporated HIV genomes when both HIV and MMTV genomes were simultaneously present in the cell. Viruses produced from chimeric MMTV genomes in which the MMTV NC had been replaced with HIV NC preferentially incorporated MMTV genomes when both HIV and MMTV genomes were simultaneously present in the cell. In contrast, viruses produced from chimeric HIV genomes containing the Moloney NC, which contains a single zinc-binding motif, were previously shown to preferentially incorporate Moloney genomic RNA. Taken together, these results indicate that an NC protein with two zinc-binding motifs is required for specific HIV RNA packaging and that the amino acid context of these motifs, while contributing to the process, is less crucial for specificity. The data also suggest that HIV NC may not be the exclusive determinant of RNA selectivity. Analysis of an HIV MA mutant revealed that specific RNA packaging does not require MA protein.
Adenovirus Endocytosis via α
v
Integrins Requires Phosphoinositide-3-OH Kinase
Authors: Li Erguang , Stupack Dwayne , Klemke Richard , Cheresh David A. , Nemerow Glen R. 
Published: December 2019

ABSTRACT

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by α
v
integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas α
v
integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with α
v
integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.
Functional Characterization of Naturally Occurring Variants of Human Hepatitis B Virus Containing the Core Internal Deletion Mutation
Authors: Yuan Thomas Ta-Tung , Lin Min-Hui , Qiu Sui Min , Shih Chiaho 
Published: December 2019

ABSTRACTNaturally occurring variants of human hepatitis B virus (HBV) containing the core internal deletion (CID) mutation have been found frequently in HBV carriers worldwide. Despite numerous sequence analysis reports of CID variants in patients, in the past decade, CID variants have not been characterized functionally, and thus their biological significance to HBV infection remains unclear. We report here two different CID variants identified from two patients that are replication defective, most likely due to the absence of detectable core protein. In addition, we were unable to detect the presence of the precore protein and e antigen from CID variants. However, the production of polymerase appeared to be normal. The replication defect of the CID variants can be rescued intransby complementation with wild-type core protein. The rescued CID variant particles, which utilize the wild-type core protein, presumably are enveloped properly since they can be secreted into the medium and band at a position similar to that of mature wild-type Dane particles, as determined by gradient centrifugation analysis. Our results also provide an explanation for the association of CID variants with helper or wild-type HBV in nature. The significance of CID variants in HBV infection and pathogenesis is discussed.
Targeting a Polyepitope Protein Incorporating Multiple Class II-Restricted Viral Epitopes to the Secretory/Endocytic Pathway Facilitates Immune Recognition by CD4+Cytotoxic T Lymphocytes: a Novel Approach to Vaccine Design
Authors: Thomson Scott A. , Burrows Scott R. , Misko Ihor S. , Moss Denis J. , Coupar Barbara E. H. , Khanna Rajiv 
Published: December 2019

ABSTRACTThe role of CD4+and CD8+cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4+-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4+memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.
Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassette
Authors: Massie Bernard , Couture France , Lamoureux Linda , Mosser Dick D. , Guilbault Claire , Jolicoeur Pierre , Bélanger François , Langelier Yves 
Published: December 2019

ABSTRACT
We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(Δ2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce ΔR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5ΔR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.