bluuebunny/crossref_metadata_2025_split huggingface dataset:
Up to 100-Fold Increase of Apparent Gene Expression in the Presence of Epstein-Barr Virus
oriP
Sequences and EBNA1: Implications of the Nuclear Import of Plasmids
Authors: Längle-Rouault Françoise , Patzel Volker , Benavente Annie , Taillez Martine , Silvestre Nathalie , Bompard Albine , Sczakiel Georg , Jacobs Eric , Rittner Karola 
Published: December 2019

ABSTRACT

A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV)
oriP
sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no
oriP
sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (
DS
) element within
oriP
. Luciferase production was not increased in the presence of only the
DS
element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without
oriP
sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying
oriP
from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of
oriP
sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression.
oriP
-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.
CXCR4 and CCR5 Genetic Polymorphisms in Long-Term Nonprogressive Human Immunodeficiency Virus Infection: Lack of Association with Mutations other than CCR5-Δ32
Authors: Cohen Oren J. , Paolucci Stefania , Bende Steven M. , Daucher MaryBeth , Moriuchi Hiroyuki , Moriuchi Masako , Cicala Claudia , Davey Richard T. , Baird Barbara , Fauci Anthony S. 
Published: December 2019

ABSTRACT
Polymorphisms in the coding sequences of CCR5 and CXCR4 were studied in a group of human immunodeficiency virus (HIV)-infected long-term nonprogressors. Two different point mutations were found in the CXCR4 coding sequence. One of these CXCR4 mutations was silent, and each was unique to two nonprogressors. The well-described 32-bp deletion within the CCR5 coding sequence (CCR5-Δ32) was found in 4 of 13 nonprogressors, and 12 different point mutations were found scattered over the CCR5 coding sequence from 8 nonprogressors. Most of the mutations created either silent or conservative changes in the predicted amino acid sequence: only one of these mutations was found in more than a single nonprogressor. All nonsilent mutations were tested in an HIV envelope-dependent fusion assay, and all functioned comparably to wild-type controls. Polymorphisms in the CXCR4 and CCR5 coding sequences other than CCR5-Δ32 do not appear to play a dominant mechanistic role in nonprogression among HIV-infected individuals.
Coreceptor Usage of Human Immunodeficiency Virus Type 2 Primary Isolates and Biological Clones Is Broad and Does Not Correlate with Their Syncytium-Inducing Capacities
Authors: Guillon Christophe , van der Ende Marchina E. , Boers Patrick H. M. , Gruters Rob A. , Schutten Martin , Osterhaus Albert D. M. E. 
Published: December 2019

ABSTRACT

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by binding of the surface envelope glycoprotein to the CD4 molecule. Interaction of the resulting CD4-glycoprotein complex with α- or β-chemokine receptors, depending on the biological phenotype of the virus, then initiates the fusion process. Here, we show that primary HIV-2 isolates and biological clones, in contrast to those of HIV-1, may use a broad range of coreceptors, including CCR-1, CCR-3, CCR-5, and CXCR-4. The syncytium-inducing capacity of these viruses did not correlate with the ability to infect via CXCR-4 or any other coreceptor. One cell-free passage of the intermediate isolates in mitogen-stimulated, CD8
+
cell-depleted peripheral blood mononuclear cells resulted in the outgrowth of variants with CCR-5 only, whereas the coreceptor usage of late and early isolates did not change. Since HIV-2 is less pathogenic in vivo than HIV-1, these data suggest that HIV pathogenicity in vivo is not directly related to the spectrum of coreceptors used in in vitro systems.
A Subset of Porcine Reproductive and Respiratory Syndrome Virus GP
3
Glycoprotein Is Released into the Culture Medium of Cells as a Non-Virion-Associated and Membrane-Free (Soluble) Form
Authors: Mardassi Helmi , Gonin Patrick , Gagnon Carl A. , Massie Bernard , Dea Serge 
Published: December 2019

ABSTRACT

The GP
3
protein of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in 293 cells by a recombinant human type 5 adenovirus carrying the open reading frame 3 gene. The protein exhibited a molecular mass of 42 kDa and comigrated with GP
3
expressed in PRRSV-infected MARC-145 cells. Removal of N-linked glycans from GP
3
resulted in a 27-kDa protein (P3), confirming its highly glycosylated nature. Pulse-chase experiments carried out either in the context of PRRSV infection or upon individual expression of GP
3
in 293 cells showed that the protein remains completely endo-β-
N
-acetylglucosaminidase H-sensitive even after 4 h of synthesis. Thus, the transport of GP
3
was restricted to the premedial Golgi compartment, presumably the endoplasmic reticulum (ER). However, a minor fraction of GP
3
was found to be secreted in the culture medium as a soluble membrane-free form. This released protein (sGP
3
) was readily identified upon individual expression of GP
3
in 293 cells as well as in the context of PRRSV infection, albeit at lower levels. The sGP
3
migrated as a smear and displayed a molecular mass ranging from 43 to 53 kDa. The unglycosylated form of sGP
3
comigrated with its intracellular deglycosylated counterpart, suggesting that the release from the cell of a subset of GP
3
did not result from cleavage of a putative membrane-anchor sequence. Strikingly, unlike GP
3
, the sGP
3
acquired Golgi-specific modifications of its carbohydrate side chains and folded into a disulfide-linked homodimer. Brefeldin A treatment completely abolished the release of sGP
3
, suggesting that the ER-to-Golgi compartment is an obligatory step in cellular secretion of sGP
3
. In contrast, 10 mM monensin did not prevent sGP
3
release but inhibited the terminal glycosylation that confers on the protein its diffuse pattern. Since GP
3
was found to be nonstructural in the case of the North American strain, secretion of a minor fraction of GP
3
might be an explanation for its high degree of immunogenicity in infected pigs. Furthermore, this secreted protein might be relevant as a model for further studies on the cellular subcompartments involved in the sorting of proteins to the extracellular milieu.
Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts
Authors: Ying Baoling , Smith Kimberley , Spindler Katherine R. 
Published: December 2019

ABSTRACT

Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from
Rb
+/+,
Rb
+/−, and
Rb
−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant,
pm
E109, or from infections with similar MAV-1 E1A null mutants,
pm
E112 and
pm
E113. The implications for the lack of a requirement of E1A in cell culture are discussed.
In Vitro Cell-Free Conversion of Noninfectious Moloney Retrovirus Particles to an Infectious Form by the Addition of the Vesicular Stomatitis Virus Surrogate Envelope G Protein
Authors: Abe Akihiro , Chen Shin-Tai , Miyanohara Atsushi , Friedmann Theodore 
Published: December 2019

ABSTRACT

In the absence of envelope gene expression, retrovirus packaging cell lines expressing Moloney murine leukemia virus (MLV)
gag
and
pol
genes produce large amounts of noninfectious virus-like particles that contain reverse transcriptase, processed Gag protein, and viral RNA (
gag-pol
RNA particles). We demonstrate that these particles can be made infectious in an in vitro, cell-free system by the addition of a surrogate envelope protein, the G spike glycoprotein of vesicular stomatitis virus (VSV-G). The appearance of infectivity is accompanied by physical association of the G protein with the immature, noninfectious virus particles. Similarly, exposure in vitro of wild-type VSV-G to a fusion-defective pseudotyped virus containing a mutant VSV-G markedly increases the infectivity of the virus to titers similar to those of conventional VSV-G pseudotyped viruses. Furthermore, similar treatment of an amphotropic murine leukemia virus significantly allows infection of BHK cells not otherwise susceptible to infection with native amphotropic virus. The partially cell-free virus maturation system reported here should be useful for studies aimed at the preparation of tissue-targeted retrovirus vectors and will also aid in studies of nucleocapsid-envelope interactions during budding and of virus assembly and virus-receptor interactions during virus uptake into infected cells. It may also represent a potentially useful step toward the eventual development of a completely cell-free retrovirus assembly system.
Multiple Virulence Determinants of Foot-and-Mouth Disease Virus in Cell Culture
Authors: Baranowski Eric , Sevilla Noemi , Verdaguer Nuria , Ruiz-Jarabo Carmen M. , Beck Ewald , Domingo Esteban 
Published: December 2019

ABSTRACT
Hypervirulent variants of foot-and-mouth disease virus (FMDV) of serotype C arise upon serial cytolytic or persistent infections in cell culture. A specific mutation in the internal ribosome entry site of persistent FMDV was previously associated with enhanced translation initiation activity that could contribute to the hypervirulent phenotype for BHK-21 cells. Here we report that several hypervirulent FMDV variants arising upon serial cytolytic passage show an invariant internal ribosome entry site but have a number of mutations affecting structural and nonstructural viral proteins. The construction of chimeric type O-type C infectious transcripts has allowed the mapping of a major determinant of hypervirulence to the viral capsid. Tissue culture-adapted FMDV displayed enhanced affinity for heparin, but binding to cell surface heparan sulfate moieties was not required for expression of the hypervirulent phenotype in Chinese hamster ovary (CHO) cells. Virulence was identical or even higher for glycosaminoglycan-deficient CHO cells than for wild-type CHO cells. FMDV variants with decreased affinity for heparin were selected from a high-binding parental population and analyzed. Substitutions associated with decreased heparin binding were located at positions 173 of capsid protein VP3 and 144 of capsid protein VP1. These substitutions had a moderate effect on virulence for BHK-21 cells but completely abrogated infection of CHO cells. The comparative results with several FMDV isolates show that (i) increased affinity for heparin and alterations in cell tropism may be mediated by a number of independent sites on the viral capsid and (ii) the same capsid modifications may have different effects on different cell types.
Determinants for Sensitivity of Human Immunodeficiency Virus Coreceptor CXCR4 to the Bicyclam AMD3100
Authors: Labrosse Béatrice , Brelot Anne , Heveker Nikolaus , Sol Nathalie , Schols Dominique , De Clercq Erik , Alizon Marc 
Published: December 2019

ABSTRACT
The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.
Intestinal Intraepithelial Lymphocytes Are Primed for Gamma Interferon and MIP-1β Expression and Display Antiviral Cytotoxic Activity despite Severe CD4+T-Cell Depletion in Primary Simian Immunodeficiency Virus Infection
Authors: Mattapallil Joseph J. , Smit-McBride Zeljka , McChesney Michael , Dandekar Satya 
Published: December 2019

ABSTRACTIntraepithelial lymphocytes (IEL) are a critical effector component of the gut-associated lymphoid tissue (GALT) and play an important role in mucosal immunity as well as in the maintenance of the epithelial cell integrity and barrier function. The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of rhesus macaques would cause alterations in the immunophenotypic profiles of IEL and their mitogen-specific cytokine (gamma interferon [IFN-γ] and MIP-1β) responses (by flow cytometry) and virus-specific cytotoxic T-cell (CTL) activity (by the chromium release assay). Virally infected IEL were detected through the entire course of SIV infection by in situ hybridization. Severe depletion of CD4+single-positive and CD4+CD8+double-positive T cells occurred early in primary SIV infection, which was coincident with an increased prevalence of CD8+T cells. This was in contrast to a gradual depletion of CD4+T cells in peripheral blood. The CD8+IEL were the primary producers of IFN-γ and MIP-1β and were found to retain their potential to produce both IFN-γ and MIP-1β through the entire course of SIV infection. SIV-specific CTL activity was detected in primary IEL at 1, 2, and 4 weeks post-SIV infection. These results demonstrated that IEL may be involved in generating antiviral immune responses early in SIV infection and in suppressing viral infection thereafter. Alterations in homeostasis in epithelia due to severe CD4+T-cell depletion accompanied by changes in the cytokine and chemokine production by IEL may play a role in the enteropathogenesis of SIV infection.
Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells
Authors: Egorov Andrej , Brandt Sabine , Sereinig Sabine , Romanova Julia , Ferko Boris , Katinger Dietmar , Grassauer Andreas , Alexandrova Galina , Katinger Hermann , Muster Thomas 
Published: December 2019

ABSTRACT

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (
ts
) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the
ts
phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40°C selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.
The Second Extracellular Loop of CXCR4 Determines Its Function as a Receptor for Feline Immunodeficiency Virus
Authors: Willett Brian J. , Adema Karen , Heveker Nikolaus , Brelot Anne , Picard Laurent , Alizon Marc , Turner Julie D. , Hoxie James A. , Peiper Stephen , Neil James C. , Hosie Margaret J. 
Published: December 2019

ABSTRACT
The feline homolog of the α-chemokine receptor CXCR4 has recently been shown to support cell-cell fusion mediated by CXCR4-dependent strains of human immunodeficiency virus (HIV) and strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney (CrFK) cell line. In this report we demonstrate that expression of CXCR4 alone is sufficient to render cells from diverse species permissive for fusion with FIV-infected cells, suggesting that CXCR4 is the sole receptor for CrFK-tropic strains of FIV, analogous to CD4-independent strains of HIV-2. To identify the regions of CXCR4 involved in fusion mediated by FIV, we screened panels of chimeric CXCR4 molecules for the ability to support fusion with FIV-infected cells. Human CXCR4 supported fusion more efficiently than feline CXCR4 and feline/human CXCR4 chimeras, suggesting that the second and third extracellular loops of human CXCR4 contain a critical determinant for receptor function. Rat/human CXCR4 chimeras suggested that the second extracellular loop contained the principal determinant for receptor function; however, chimeras constructed between human CXCR2 and CXCR4 revealed that the first and third loops of CXCR4 contribute to the FIV Env binding site, as replacement of these domains with the corresponding domains of CXCR2 rendered the molecule nonfunctional in fusion assays. Mutation of the DRY motif and the C-terminal cytoplasmic tail of CXCR4 did not affect the ability of the molecule to support fusion, suggesting that neither signalling via G proteins nor receptor internalization was required for fusion mediated by FIV; similarly, truncation of the N terminus of CXCR4 did not affect the function of the molecule as a receptor for FIV. CXCR4-transfected feline cells were rendered permissive for infection with both the CrFK-tropic PET isolate of FIV and the CXCR4-dependent RF strain of HIV-1, and susceptibility to infection correlated well with ability to support fusion. The data suggest that the second extracellular loop of CXCR4 is the major determinant of CXCR4 usage by FIV.
Critical Role for CD4+T Cells in Controlling Retrovirus Replication and Spread in Persistently Infected Mice
Authors: Hasenkrug Kim J. , Brooks Diane M. , Dittmer Ulf 
Published: December 2019

ABSTRACTReactivations of persistent viral infections pose a significant medical problem in immunocompromised cancer, transplant, and AIDS patients, yet little is known about how persistent viral infections are immunologically controlled. Here we describe a mouse model for investigating the role of the immune response in controlling a persistent retroviral infection. We demonstrate that, following recovery from acute Friend virus infection, a small number of B cells evade immunological destruction and harbor persistent virus. In vivo depletions of T-cell subsets in persistently infected mice revealed a critical role for CD4+T cells in controlling virus replication, spread to the erythroid lineage, and induction of erythroleukemia. The CD4+T-cell effect was independent of CD8+T cells and in some cases was also independent of virus-neutralizing antibody responses. Thus, the CD4+T cells may have had a direct antiviral effect. These results may have relevance for human immunodeficiency virus (HIV) infections where loss of CD4+T cells is associated with an increase in HIV replication, reactivation of persistent viruses, and a high incidence of virus-associated cancers.
Apoptosis Induction by the Binding of the Carboxyl Terminus of Human Immunodeficiency Virus Type 1 gp160 to Calmodulin
Authors: Ishikawa Hiroki , Sasaki Masafumi , Noda Satoshi , Koga Yasuhiro 
Published: December 2019

ABSTRACTThe role of calmodulin (CaM) in apoptosis induced by gp160 of human immunodeficiency virus type 1 was investigated with cells undergoing single-cell killing. These cells were found to express, under the control of an inducible promoter, wild-type gp160 or mutant gp160 devoid of various lengths of the carboxyl terminus. Immunoprecipitation accompanied by immunoblotting revealed binding of CaM to wild-type gp160 but not to mutant gp160 bearing a carboxyl terminus with a deletion spanning more than five amino acid residues. A significant coenzyme activity was detected in the CaM bound to gp160 even in the presence of a Ca2+chelater, EGTA. The cells forming this gp160-CaM complex exhibited an elevated intracellular Ca2+level followed by DNA fragmentation, which is a hallmark of apoptosis, and finally cell killing, while the cells not forming this complex did not show any significant elevation in Ca2+level or DNA fragmentation. These results thus indicated that CaM plays a key role in gp160-induced apoptosis.