pinecone/core-2020-05-10-deduplication huggingface dataset:
Epstein–Barr Virus Latent Membrane Protein-1 Induction by Histone Deacetylase Inhibitors Mediates Induction of Intercellular Adhesion Molecule-1 Expression and Homotypic Aggregation

AbstractEpstein–Barr virus (EBV) latent membrane protein (LMP)-1 induces B lymphocyte immortalization and activates constitutive signal transduction, including NF-κB, JNK/p38, and JAK/STAT pathways. During EBV latency, LMP-1 expression induces several B lymphocyte activation markers, including intercellular adhesion molecule (ICAM)-1. We found that various structurally distinct histone deacetylase inhibitors (HDACI), as well as phorbol ester treatment, induced homotypic aggregation in EBV-positive Burkitt's lymphoma lines. Cell-surface expression of ICAM-1 was concurrently strongly up-regulated by both HDACI and phorbol ester treatments. Cell-surface expression of ICAM-1 was concurrently strongly induced by both HDACI and phorbol ester treatment. Among several ICAM family members, only ICAM-1 was up-regulated by both HDACI and phorbol ester treatments, suggesting that up-regulated ICAM-1 expression might mediate the observed increase in homotypic aggregation. HDACI-induced homotypic aggregation was blocked by exposure to a monoclonal antibody specific for the β-chain (CD18) of an ICAM-1 ligand, LFA-1. Unexpectedly, HDAC inhibition, but not phorbol ester treatment, induced LMP-1 expression in EBV-positive cell lines, and this LMP-1 species was identified by RT-PCR and immunoblot analyses as the latent form of LMP-1. Control of EBV LMP-1 gene expression by HDACI inhibition occurs at the transcriptional level, as indicated by nuclear runoff studies and analysis of steady-state mRNA levels. Dominant-negative LMP-1 efficiently blocked HDACI-induced ICAM-1 up-regulation, and ectopic expression of LMP-1 activated expression of an ICAM-1 promoter-driven reporter gene. The HDACI-induced up-regulation of ICAM-1, and consequent homotypic aggregation, were efficiently blocked by the addition of N-acetyl-l-cysteine and by ectopic expression of a super-repressor IκBα, while LPM-1 induction was unaffected, suggesting that these effects are mediated by NF-κB. We demonstrate, therefore, that the latent isoform of LMP-1 is induced by HDAC inhibition, and that HDACI-induced latent LMP-1 expression, through NF-κB activation, is responsible for ICAM-1 expression up-regulation and homotypic adhesion
Apoptosis Modulation of Epstein–Barr Virus-Encoded Latent Membrane Protein 1 in the Epithelial Cell Line HeLa Is Stimulus-Dependent

AbstractEpstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is required for viral transformation and has been shown to protect lymphocytes from apoptosis. However, the effect of LMP1 on cells of epithelial origin remains poorly understood. Using the epithelial cell line HeLa in which the expression of LMP1 is inducibly regulated by tetracycline, we demonstrate that apoptosis triggered by ligation of the death receptor, Fas, or by the chemotherapeutic agent, etoposide, is potentiated by LMP1. Apoptosis was assessed by nuclear condensation and activation of caspase-3-like enzymes with concomitant proteolysis of the nuclear caspase substrate, poly(ADP-ribose) polymerase. However, the effect of LMP1 in HeLa cells appeared to be stimulus-dependent since apoptosis induced by tumor necrosis factor (TNF) was inhibited. Moreover, we observed an upregulation of the zinc finger protein A20 and a decrease in expression of Bcl-2 upon induction of LMP1 in HeLa cells. Taken together, these data further our understanding of the function of LMP1 in epithelial cells and suggest that LMP1, similar to its mammalian homolog CD40, can exert opposing effects on cell survival depending on the nature of the apoptosis trigger
A Glycosylation-Defective Variant of the Ecotropic Murine Retrovirus Receptor Is Expressed in Rat XC Cells

AbstractThe XC rat sarcoma cell line undergoes extensive cell-to-cell fusion (syncytium formation) after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope (Env) protein in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R+ Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To assess whether this property of the XC cell line arises from differences in its ecotropic MLV receptor, CAT1, we isolated CAT1 cDNA clones from XC cells. A variant CAT1 (xcCAT1) was found together with the wild-type rat CAT1 (rCAT1). xcCAT1 cDNA encodes a protein with a single amino acid change that destroys a conserved N-linked glycosylation site proximal to the Env-binding motif. We found that xcCAT1 expressed in Chinese hamster ovary (CHO) cells undergoes less glycosylation than rCAT1 and that the expression of xcCAT1 rendered the CHO cells more susceptible to infection with Moloney MLV. Thus, N-glycosylation negatively regulates the receptor activity of rCAT1. This is supported by the observation that treatment of rat F10 cells with the N-glycosylation inhibitor tunicamycin enhanced their susceptibility to Mo-MLV. However, xcCAT1-expressing CHO cells did not fuse with 293T cells expressing R+ Env, indicating that xcCAT1 expression is not sufficient to induce the XC cell-specific characteristic of forming syncytia in response to R+ Env
The HTLV-1 Tax Oncoprotein Attenuates DNA Damage Induced G1 Arrest and Enhances Apoptosis in p53 Null Cells

AbstractTransformation of cells by the human T cell leukemia virus type 1 occurs via mechanisms unique among oncogenic retroviruses. A prevailing hypothesis for HTLV-1-mediated cellular transformation is that expression of the viral transactivator, Tax, induces genomic instability. Tax-mediated failure in the cellular repair response is one possible mechanism for loss in genomic integrity. Here we have examined the in vivo repair response of Tax-expressing cells to determine the underlying defects that contribute to loss of genomic integrity. In these studies we examined the effects of de novo Tax-expression in naive “pre-neoplastic” REF52 cells. DNA-damage-induced p53 stabilization and concomitant transient stabilization of p21 were clearly evident in Tax-expressing cells. Likewise, the damage-induced apoptotic response of Tax-expressing cells was normal. However, the damage-induced G1 checkpoint was abrogated in either p53+ or p53− cellular backgrounds. Although nucleotide excision repair (NER) of asynchronous Tax-expressing cells was impaired, cell-cycle-independent assessment of NER in the global excision repair assay demonstrated comparable NER activity in Tax-expressing cells, suggesting that the failure of G1 checkpoint contributes to NER deficiency. Interestingly, we observed a dramatic increase in apoptosis and UV sensitivity of Tax-expressing p53−/− cells when compared to Tax-expressing p53+/+ cells. These data demonstrate that Tax-mediated cellular genomic instability arises from attenuation of cell-cycle checkpoint and imply a clonal dependence on p53 status separate from genomic integrity
Replication of HIV-1 Deleted Nef Mutants in Chronically Immune Activated Human T Cells

AbstractLymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4+ T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication
Different Substitutions at Conserved Amino Acids in Domains II and III in the Sendai L RNA Polymerase Protein Inactivate Viral RNA Synthesis

AbstractThe Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I–VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis
A Pregenomic RNA Sequence Adjacent to DR1 and Complementary to Epsilon Influences Hepatitis B Virus Replication Efficiency

AbstractHepatitis B virus (HBV) possesses a 3.2-kb partially double-stranded DNA genome that is generated inside the nucleocapsid by the reverse transcription of the 3.5-kb pregenomic viral transcript. The initial steps in viral replication involve the recognition of an encapsidation signal termed epsilon (ϵ) at the 5′-end of the pregenomic RNA by the HBV polymerase. The polymerase-bound pregenomic RNA is subsequently incorporated into an immature nucleocapsid particle and minus-strand HBV DNA synthesis is initiated utilizing the bulge region of ϵ as a template and a tyrosine residue in the amino-terminal region of the polymerase as a primer. Three nucleotides complementary to the 3′-end of the bulge region of ϵ are synthesized and subsequently translocated with the polymerase molecule to the acceptor site located in the DR1 sequence present at the 3′-end of the pregenomic RNA. Using mutagenesis analysis, a sequence element designated phi (φ) located upstream of the 3′ DR1 sequence has been identified that is complementary to ϵ and is important for efficient viral replication. This element may bring the 3′ DR1 sequence into proximity with the three nucleotide primer synthesized at the bulge of ϵ and facilitate primer translocation to the 3′ DR1 acceptor sequence. Sequence elements with similar proximity to the 3′ DR1 sequences and complementarity to ϵ are present in the woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV), suggesting the φ regulatory element may be phylogenetically conserved due to its functional importance in hepadnavirus minus-strand DNA synthesis
Expression of IL-18 by SIV Does Not Modify the Outcome of the Antiviral Immune Response

AbstractInterleukin 18 (IL-18) is a proinflammatory cytokine expressed by several cell types, including activated dendritic cells and macrophages, that acts in synergy with IL-12 as an important amplifying factor for IFN-γ production and Th1 development. To study the immunological and virological effects of IL-18 expression in the context of a lentiviral infection, we inoculated rhesus macaques with a high dose of replication-competent simian immunodeficiency virus (SIV) vectors carrying the rhesus IL-18 gene in the sense (SIVIL-18) or antisense (SIVFIGI) orientation. Both vectors behaved as attenuated viruses, resulting in low viral loads, induction of low and transient levels of inflammatory cytokines, no CD4+ T cell depletion, and mild activation of T lymphocytes. Although IL-18-expressing virus could be isolated from some SIVIL18-infected macaques for 12 weeks postinfection, the anti-SIV humoral and cellular immune responses of macaques inoculated with SIVIL18 and SIVFIGI were similar to each other, with the exception of an early IFN-γ response in animals infected with SIVIL18. In summary, expression of IL-18 during the acute phase of SIV infection does not increase viral replication or influence the outcome of the antiviral immune response
Virus-Derived Tubular Structure Displaying Foreign Sequences on the Surface Elicit CD4+ Th Cell and Protective Humoral Responses

AbstractParticulate vector systems for the presentation of immunogenic epitopes provide an alternate and powerful approach for the delivery of immunogens of interest. In this article, we have exploited a viral protein of unknown function, bluetongue virus (BTV) nonstructural protein NS1, which forms distinct tubular aggregates in infected cells, as an immunogen delivery system. Tubules are helical assemblies of NS1 protein that present the C-terminus of the protein to the outer edge effectively displaying appended residues in a regular and repeating array akin to the coat of a filamentous phage. To assess the breadth of response induced following tubule-based immunization, two different immunodominant foreign peptides were inserted at the C-terminus of NS1 and chimeric tubules generated following expression in the baculovirus expression system. Both constructs, one carrying a peptide of foot and mouth disease virus (FMDV) (aa 135–144 of VP1) and the other, a peptide of influenza A virus (aa 186–205 of HA), effectively assembled into tubules and were easily purified. Subsequently, using in vitro assay systems, we demonstrated that each purified chimeric particle was capable of eliciting strong immune responses. Further, NS1-FMDV chimeric tubules could induce a potent immune response that could protect against disease
Repeated Cycles of Alternate Administration of Fludarabine and Zidovudine Plus Didanosine Inhibits Murine AIDS and Reduces Proviral DNA Content in Lymph Nodes to Undetectable Levels

AbstractThe results of the combined use of Fludarabine, an anticancer agent that may be able to target latently infected cells, and conventional antiretroviral therapy (AZT+DDI) in a murine model of AIDS, i.e., LP-BM5 infection, are reported. Eighty percent of infected mice, treated with four cycles of alternate administration of Fludarabine and AZT+DDI, showed undetectable levels of proviral DNA in lymph nodes. After 8 weeks of treatment interruption, the infected/treated animals, although still alive at a time when all untreated animals had succumbed to the infection, showed disease progression and reappearance of proviral DNA in lymph nodes. The retrospective analysis of proviral DNA content in spleen and bone marrow at the end of the fourth cycle of treatment revealed a low but detectable amount of BM5d proviral DNA. We thus concluded that the spleen and bone marrow may be less sensitive to lympholitic drugs and therefore act as viral reservoirs in LP-BM5 infection. This study suggests that optimized protocols of alternate administration of cytolytic and antiretroviral drugs may represent a useful strategy to eradicate retroviral infections
Thymocyte and Peripheral Blood T Lymphocyte Subpopulation Changes in Piglets Following in Utero Infection with Porcine Reproductive and Respiratory Syndrome Virus

AbstractPiglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) are born severely immunocompromised. In this article we more closely examine the effects of in utero PRRSV infection on circulating and thymic T cell populations. Numbers of CD4+, CD8+, and dual-positive lymphocytes were quantitated in circulation and in the thymus during the 2 weeks following birth. At birth we found that the number of circulating lymphocytes was suppressed by 60%. Lymphocyte numbers were also suppressed by 42% at day 7, but by day 14 the number of lymphocytes had rebounded and was actually 47% greater than controls. At birth and day 7, a drop in the number of CD4+ cells could partially explain the suppression we observed, while the rebound in total lymphocyte numbers seen at day 14 was due to a nearly fourfold increase in the number of circulating CD8+ cells. As a result, the normal CD4+:CD8+ ratio of between 1.4 and 2.2 for neonatal pigs was reduced to 0.1–0.5. The thymuses of infected piglets were found to be 50% smaller than those of control pigs and were characterized by cortical involution and severe cortical depletion of thymocytes. Analysis of the population of thymocytes revealed that double-positive thymocytes were suppressed to a greater degree than either single positive subpopulation. In addition, we show that the number of thymocytes undergoing apoptosis was increased twofold in piglets infected with PRRSV. Taken together, these results help explain the dramatic immunosuppression observed in neonatal animals infected in utero with PRRSV
Reduced IL-2 and IL-4 mRNA Expression in CD4+ T Cells from Bovine Leukemia Virus-Infected Cows with Persistent Lymphocytosis

AbstractThe role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4+ T cells to the expression of mRNAs encoding interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-γ, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from cows seronegative to BLV (BLV−), seropositive without persistent lymphocytosis (BLV+PL−), and seropositive with PL (BLV+PL+) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-γ, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV+PL+ cows as compared to BLV− cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4+ T cells from BLV+PL+ cows. In contrast, Con A stimulated PBMCs and CD4+ T cells did not differ significantly in expression of IFN-γ, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4+ T cells and semiquantitative RT-PCR the frequencies of CD4+ T cells transcribing IFN-γ, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV−, BLV+PL−, and BLV+PL+ cows were determined. There were no significant differences in the frequencies of CD4+ T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4+ T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4+ T cells, thus suggesting aberrant Th regulation in subclinically infected animals
Subgenomic Hepatitis C Virus Replicons Inducing Expression of a Secreted Enzymatic Reporter Protein

AbstractWe constructed dicistronic, subgenomic hepatitis C virus (HCV) replicons in which the sequence encoding the human immunodeficiency virus (HIV) tat protein was placed in the upstream cistron, between the HCV 5′NTR and a picornaviral 2A proteinase sequence fused to the selectable marker Neo. Stably transformed Huh7 cells expressing secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV LTR promoter actively secreted SEAP following transfection with these replicon RNAs. Extracellular SEAP activity correlated closely with intracellular HCV RNA levels, as determined by Northern blotting and real-time RT-PCR analysis. These RNAs replicated efficiently despite the absence of core-protein-coding sequence downstream of the HCV IRES. The replication efficiency of replicons derived from the HCV-N strain of HCV was significantly greater than those derived from Con1 in transiently transfected cells. Using this reporter system, we have demonstrated significant differences in the response to interferon α-2b in cell lines containing replicons derived from these two strains of HCV
Species-Specific Effects of HIV-1 Nef-Mediated MHC-I Downmodulation

AbstractIn vitro studies have revealed that human immunodeficiency virus-1 (HIV-1) Nef functionally interacts with amino acid residues in the cytoplasmic tail of major histocompatibility complex class I (MHC-I) molecules, reducing their expression on the cell surface and protecting them from cytotoxic T lymphocyte (CTL) lysis. To obtain a better understanding of Nef's effects in vivo, it would be helpful to have a mouse model system. However, it is not known whether Nef will affect murine MHC-I proteins. We find that Nef downmodulates human MHC-I HLA-A2 more efficiently than murine MHC-I molecules in HeLa cells and that Nef does not function efficiently in murine endothelial cells. Studies with chimeric molecules indicate that the MHC-I cytoplasmic tail is primarily responsible for species-specific differences. However, there are also effects attributable to the extracellular domain
Bovine Herpesvirus 1 US ORF8 Protein Induces Apoptosis in Infected Cells and Facilitates Virus Egress

AbstractThe bovine herpesvirus 1 (BHV-1) US ORF8 protein with homology to the Us9 protein of other alphaherpesviruses induces apoptosis in rabbit kidney (RK13) cells without the presence of other BHV-1-encoded proteins. In this article, we have characterized the cytotoxicity and growth behavior of a BHV-1 recombinant, BHV-1/D8, which fails to express the US ORF8 protein in infected cells. BHV-1/D8 exhibited a reduced cytotoxicity to RK13 cells when compared to the cytotoxicity of control BHV-1 strains. In RK13 cells, the onset of apoptosis was not observed during the infection with BHV-1/D8, and the virus multiplication of BHV-1/D8 was markedly greater than that of control viruses. However, virus release of progeny viruses from the infected RK13 cells into culture supernatant was significantly decreased by the loss of the US ORF8 protein. These data demonstrate that the US ORF8 protein activates the apoptotic process and facilitates virus release from the BHV-1-infected cells