ABSTRACT
Temperate grasses, such as wheat, become compact plants with small thick leaves after exposure to low temperature. These responses are associated with cold hardiness, but their underlying mechanisms remain largely unknown. Here we analyse the effects of low temperature on leaf morpho-anatomical structure, cell wall composition and activity of extracellular peroxidases, which play key roles in cell elongation and cell wall thickening, in two wheat cultivars with contrasting cold-hardening ability. A combined microscopy and biochemical approach was applied to study actively growing leaves of winter (ProINTA-Pincén) and spring (Buck-Patacón) wheat developed under constant warm (25 °C) or cool (5 °C) temperature. Cold-grown plants had shorter leaves but longer inter-stomatal epidermal cells than warm-grown plants. They had thicker walls in metaxylem vessels and mestome sheath cells, paralleled with accumulation of wall components, predominantly hemicellulose. These effects were more pronounced in the winter cultivar (Pincén). Cold also induced a sharp decrease in apoplastic peroxidase activity within the leaf elongating zone of Pincén, and a three-fold increase in the distal mature zone of the leaf. This was consistent with the enhanced cell length and thicker cell walls in this cultivar at 5 °C. The different response to low temperature of apoplastic peroxidase activity and hemicellulose between leaf zones and cultivar types suggests they might play a central role in the development of cold-induced compact morphology and cold hardening. New insights are presented on the potential temperature-driven role of peroxidases and hemicellulose in cell wall dynamics of grasses.
Subject(s)
Cell Wall/metabolism , Cold Temperature , Peroxidase/metabolism , Plant Leaves/anatomy & histology , Plant Leaves/physiology , Triticum/anatomy & histology , Triticum/physiology , Plant Proteins/metabolism , Polysaccharides/metabolism , SeasonsABSTRACT
Cardioviruses (e.g., Theiler's murine encephalomyelitis virus [TMEV]) are members of the Picornaviridae family that cause myocarditis and encephalitis in rodents. Recently, several studies have identified human cardioviruses, including Saffold virus (SAFV) and a related virus named human TMEV-like cardiovirus (HTCV). At least eight cardiovirus genotypes are now recognized, with SAFV and most strains of HTCV belonging to genotypes 1 and 2, respectively; genotype 2 strains are the most common in the population. Although a genotype 3 cardiovirus has recently been cultured (SAFV-3), the genotype 1 and 2 cardioviruses have been difficult to propagate in vitro, hindering efforts to understand their seroprevalence and pathogenicity. Here we present the isolation and characterization of a genotype 2 human cardiovirus (HTCV-UC6). Notably, successful cultivation of HTCV-UC6 from stool required the addition of cytokine-blocking antibodies to interrupt downstream antiviral pathways. Unlike SAFV-3, HTCV-UC6 exhibited slow replication kinetics and demonstrated only a moderate cytopathic effect. Serologic assays revealed that 91% of U.S. adults carry antibodies to the genotype 2 cardioviruses, of which 80% generate neutralizing antibodies, in agreement with previous data showing that cardiovirus infection is widespread in humans. We also demonstrate an acute cardiovirus seroconversion event in a child with diarrhea and vomiting, thus reporting for the first time evidence linking cardiovirus infection to diarrheal disease in humans.
Subject(s)
Cardiovirus Infections/epidemiology , Cardiovirus/isolation & purification , Cardiovirus/physiology , Diarrhea/virology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Cardiovirus/genetics , Cardiovirus/growth & development , Cardiovirus Infections/virology , Cell Line , Cytopathogenic Effect, Viral , Feces/virology , Female , Genotype , Humans , Male , Middle Aged , Molecular Sequence Data , RNA, Viral/genetics , Sequence Analysis, DNA , Seroepidemiologic Studies , United States/epidemiology , Virus Replication , Young AdultABSTRACT
Since they employ host gene expression machinery to execute their genetic programs, it is no surprise that DNA viruses also encode miRNAs. The small size of viral genomes, and the high degree of understanding of the functions of their gene products, make them particularly favorable systems for the examination of miRNA biogenesis and function. Here we review our computational and array-based approaches for viral miRNA discovery, and we discuss the structure and function of miRNAs identified by these approaches in polyomaviruses and herpesviruses.
Subject(s)
MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Viruses/genetics , Viruses/metabolism , Animals , Genome, Viral , Herpesviridae/genetics , Herpesviridae/metabolism , Humans , Mutation , Oligonucleotide Array Sequence Analysis , Polyomavirus/genetics , Polyomavirus/metabolism , Simian virus 40/genetics , Simian virus 40/metabolism , SoftwareABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus associated with Kaposi's sarcoma (KS) and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease. Studies on the biology and pathogenesis of KSHV have been limited by lack of efficient cell culture systems and lack of a suitable animal model for KS. Here we report on the experimental inoculation of SIV-positive and SIV-negative rhesus macaques with KSHV-infected PEL cells or KSHV preparations derived from PEL cells. Low levels of viral DNA could be detected in cultivated peripheral blood mononuclear cell of all animals, as well as in the bone marrow of one monkey that died from SAIDS. However, we were not able to detect KSHV-specific antibodies or transcripts, nor did we observe any symptoms clearly related to KSHV infection (e.g. KS or lympho-proliferative disease). Hence, although KSHV replicates in rhesus macaques at very low levels, this non-human primate host is unlikely to provide a useful animal model for disease.
Subject(s)
Disease Models, Animal , Herpesviridae Infections/transmission , Herpesvirus 8, Human/growth & development , Herpesvirus 8, Human/genetics , Macaca mulatta/virology , Animals , Antibodies, Viral/blood , DNA Primers , DNA, Viral/blood , Enzyme-Linked Immunosorbent Assay , Reverse Transcriptase Polymerase Chain Reaction , Simian Immunodeficiency Virus/immunologyABSTRACT
There may be drugs and a useful vaccine available to combat the hepatitis B virus, but one aspect of this pathogen remains a puzzle: the function of its HBx protein. As Ganem reveals in his Perspective, new findings (Bouchard et al.) show that this versatile viral protein is an activator of calcium-dependent Ras signaling, an observation that may explain many of its biological effects.
Subject(s)
Hepatitis B virus/genetics , Hepatitis B virus/physiology , Trans-Activators/metabolism , Animals , Calcium/metabolism , Calcium Signaling , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA Replication , DNA, Viral/biosynthesis , Focal Adhesion Kinase 2 , Gene Expression Regulation, Viral , Genes, Viral , Liver/virology , Mutation , Open Reading Frames , Phosphorylation , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Trans-Activators/genetics , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism , Viral Regulatory and Accessory Proteins , Virus Replication , ras Proteins/metabolism , src-Family Kinases/metabolismABSTRACT
Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses.
Subject(s)
Hepatitis Delta Virus/physiology , RNA, Viral/physiology , Virus Replication/genetics , Animals , Birds , Humans , Mammals , Species SpecificityABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types. The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency. Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP). Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation. The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3' to it. Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required. The 3' flanking sequences also play a critical role in DNA binding and transactivation. The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element. Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide.
Subject(s)
Carrier Proteins/genetics , DNA/metabolism , Gene Expression Regulation, Viral , Genes, Viral , Herpesvirus 8, Human/metabolism , Immediate-Early Proteins/metabolism , Promoter Regions, Genetic , Repressor Proteins/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Transcriptional Activation , Viral Proteins/genetics , Animals , Baculoviridae/genetics , Basic-Leucine Zipper Transcription Factors , Cell Line , Chlorocebus aethiops , Enhancer Elements, Genetic , Escherichia coli , Gene Expression , Humans , Immediate-Early Proteins/genetics , Recombination, Genetic , TATA BoxABSTRACT
Kaposi's sarcoma-associated (KS-associated) herpesvirus (KSHV) is a B-lymphotropic agent linked to AIDS-related lymphoproliferative disorders and KS. We and others have earlier identified two viral genes, K3 and K5, that encode endoplasmic reticulum proteins that downregulate surface MHC-I chains by enhancing their endocytosis. Here we have examined the ability of these proteins to influence the disposition of other host surface proteins implicated in immune recognition and activation. We report that K5, but not K3, expression in BJAB cells dramatically reduces ICAM-1 and B7-2 surface expression; B7-1 expression is unaffected. This K5-induced reduction can be reversed by coexpression of a dominant negative mutant of dynamin, indicating that the loss of ICAM and B7-2 surface expression is due to their enhanced endocytosis. This downregulation is functionally significant, because K5-transfected B cells show substantial impairment in their ability to induce T cell activation. K5 is thus the first example of a viral modulator of immunological synapse formation and T cell costimulation. We propose that its expression reduces T cell responses to KSHV-infected B cells early in infection, thereby diminishing antiviral cytokine release and the production of stimulatory signals for CTL generation.
Subject(s)
Antigens, CD/metabolism , Endocytosis , Immediate-Early Proteins/pharmacology , Intercellular Adhesion Molecule-1/metabolism , Membrane Glycoproteins/metabolism , T-Lymphocytes/immunology , Antigen Presentation , B-Lymphocytes/immunology , B-Lymphocytes/virology , B7-2 Antigen , Cell Line , Cytotoxicity Tests, Immunologic , Down-Regulation , Dynamins , GTP Phosphohydrolases/genetics , Humans , Jurkat Cells , Lymphocyte Activation , Transfection , Viral Proteins/pharmacologyABSTRACT
Cytotoxic T lymphocytes (CTL) recognize and kill virus-infected cells and contribute to immunologic control of viral replication. For many herpesviruses (e.g., Epstein-Barr and cytomegalovirus), virus-specific CTL responses can be readily detected in infected persons, but CTL responses against Kaposi's sarcoma-associated herpesvirus (KSHV) appear to be weak and remain poorly characterized. Using a human leukocyte antigen (HLA) binding motif-based epitope prediction algorithm, we identified 37 HLA-A*0201 binding peptides from 8 KSHV open-reading frames (ORFs). After in vitro stimulation of peripheral blood mononuclear cells from KSHV-infected persons, CTL responses against 1 peptide in the KSHV kaposin protein (ORF K12) were detected in 2 HLA-A*0201-positive subjects. The optimal CTL epitope was identified by HLA restriction analysis and peptide titration assays. These data describe a latent phase viral gene product targeted by CTL that may be relevant for KSHV immunopathogenesis.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Epitopes , HLA-A Antigens/immunology , Herpesvirus 8, Human/immunology , T-Lymphocytes, Cytotoxic/immunology , Viral Proteins/immunology , Gene Expression Regulation, Viral , HumansABSTRACT
The Kaposi's sarcoma-associated herpesvirus (KSHV) K1 gene encodes a polypeptide bearing an immunoreceptor tyrosine-based activation motif (ITAM) that is constitutively active for ITAM-based signal transduction. Although ectopic overexpression of K1 in cultured fibroblasts can lead to growth transformation, in vivo this gene is primarily expressed in lymphoid cells undergoing lytic infection. Here we have examined function of K1 in the setting of lytic replication, through the study of K1 mutants lacking functional ITAMs. Expression of such mutants in BJAB cells cotransfected with wild-type K1 results in dramatic inhibition of K1 signal transduction, as judged by impaired activation of Syk kinase and phospholipase C-gamma2 as well as by diminished expression of a luciferase reporter gene dependent upon K1-induced calcium and Ras signaling. Thus, the mutants behave as dominantly acting inhibitors of K1 function. To assess the role of K1 in lytic replication, we introduced these K1 mutants into BCBL-1 cells, a B-cell lymphoma line latently infected with KSHV, and induced lytic replication by ectopic expression of the KSHV ORF50 transactivator. Expression of lytic cycle genes was diminished up to 80% in the presence of a K1 dominant negative mutant. These inhibitory effects could be overridden by tetradecanoyl phorbol acetate treatment, indicating that inhibition was not due to irreversible cell injury and suggesting that other signaling events could bypass the block. We conclude that ITAM-dependent signaling by K1 is not absolutely required for lytic reactivation but functions to modestly augment lytic replication in B cells, the natural reservoir of KSHV.
Subject(s)
Herpesvirus 8, Human/physiology , Viral Proteins/physiology , Virus Replication , B-Lymphocytes/virology , Cell Line , Humans , Phosphorylation , Tetradecanoylphorbol Acetate/pharmacology , Tyrosine/metabolismABSTRACT
The K8 locus in Kaposi's sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three alternatively spliced transcripts. The fully spliced transcript encodes K-bZIP, the KSHV homologue of the EBV immediate-early transcriptional transactivator Z. Here we show that despite the presence of alternatively spliced transcripts, the protein from the fully spliced RNA, K-bZIP, is the principal product detectable in KSHV-infected B cells. The protein is detected only in lytically infected cells and is localized to the nucleus. We further characterized K-bZIP by determining its phosphorylation status. Phosphoamino acid analysis revealed phosphorylation on serine and threonine. Analysis of the sites of K-bZIP phosphorylation by tandem mass spectrometry revealed that K-bZIP was phosphorylated on Thr 111 and Ser 167. These phosphorylation sites are contained within cyclin-dependent kinase (CDK) recognition sites with the consensus sequence (S/T)PXR, suggesting that K-bZIP could be phosphorylated by CDKs. We tested this hypothesis using an in vitro kinase reaction performed in whole-cell extracts that resemble in vivo conditions more closely than standard in vitro kinase reactions. We found that the three CDK-cyclin complexes we tested phosphorylated K-bZIP but not the control ORF 73 protein, which contains four (S/T)PXR sites. Ectopic expression of K-bZIP cannot reactivate KSHV from latency, and single and double mutants of K-bZIP in which alanines replaced the phosphorylated serine and/or threonine also failed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the protein.
Subject(s)
Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/metabolism , Herpesvirus 8, Human/physiology , Transcription Factors/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Basic-Leucine Zipper Transcription Factors , COS Cells , G-Box Binding Factors , Molecular Sequence Data , Phosphorylation , Protein Isoforms/analysis , Rabbits , Serine/metabolism , Threonine/metabolism , Virus Activation , Virus LatencyABSTRACT
Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.
Subject(s)
Cell Division , Hepatitis Delta Virus/physiology , Hepatitis Delta Virus/pathogenicity , Virus Replication , Animals , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line , Dactinomycin/pharmacology , Flow Cytometry , Gene Expression/drug effects , HeLa Cells , Hepatitis Delta Virus/drug effects , Humans , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Time Factors , Transfection , Tumor Necrosis Factor-alpha/pharmacology , Viral Proteins/biosynthesis , Virus Replication/drug effectsSubject(s)
Herpesvirus 8, Human/genetics , Sarcoma, Kaposi/virology , Skin Neoplasms/virology , Acquired Immunodeficiency Syndrome/complications , Acquired Immunodeficiency Syndrome/pathology , DNA, Viral/genetics , Disease Progression , Foot , Humans , Liver Neoplasms/pathology , Lung Neoplasms/pathology , Male , Melanoma/pathology , Neoplasm Metastasis , Open Reading Frames , Photomicrography , Sarcoma, Kaposi/pathology , Skin Neoplasms/pathology , Time Factors , Virus LatencyABSTRACT
Open reading frame 71 (ORF 71) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a death effector domain-containing protein that is homologous to cellular FLIPs (FLICE-inhibitory proteins) and is proposed to inhibit Fas-mediated apoptosis. Transcripts bearing ORF 71 (v-FLIP) sequences are present in all latently infected cells. However, mapping studies reveal these to be bi- or tricistronic mRNAs with ORF 71 located 3' to ORFs 72 (v-cyclin) and 73 (latency-associated nuclear antigen), raising the question of how efficient expression of v-FLIP is achieved. We explored this question by examining the expression of model bicistronic (v-cyclin/LUC) transcripts in which a luciferase (LUC) reporter replaced v-FLIP coding sequences. SLK spindle cells transfected with such constructs efficiently expressed luciferase from the 3' position, and this expression was independent of the expression of the 5' v-cyclin gene. Surprisingly, transcript mapping showed that in these cultures, efficient splicing occurred to remove v-cyclin sequences and generate monocistronic LUC transcripts. Similar splicing events produced monocistronic v-FLIP transcripts in KSHV-infected primary effusion lymphoma cells. However, these RNAs were of low abundance and were inducible by treatment with 12-O-tetradecanoylphorbol-13-acetate. Examination of the more abundant bicistronic latent RNAs revealed the presence of an efficient internal ribosome entry site (IRES) overlapping ORF 72 coding sequences. Thus, two potential mechanisms exist for v-FLIP expression, but the evidence suggests that IRES-mediated internal translational initiation on latent polycistronic mRNAs is the principal source of v-FLIP in latency.
Subject(s)
Gene Expression Regulation, Viral , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/physiology , Open Reading Frames , Base Sequence , Cell Line , Cyclins/genetics , Cyclins/metabolism , Genes, Reporter , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , Plasmids/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomes/metabolism , Transcription, Genetic , Transfection , Viral Proteins/genetics , Viral Proteins/metabolism , Virus LatencyABSTRACT
Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.
Subject(s)
Endocytosis/physiology , HLA-B7 Antigen/immunology , Herpesviridae/immunology , Ligases/metabolism , Mitochondrial Proteins , Phosphate Transport Proteins , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chimera , Flow Cytometry , Gene Expression Regulation , Glutathione Transferase/metabolism , HLA-B7 Antigen/genetics , HeLa Cells , Humans , Intracellular Membranes/metabolism , Isoenzymes/metabolism , Major Histocompatibility Complex/genetics , Molecular Sequence Data , Recombinant Proteins/metabolism , Sequence Alignment , Transfection , Ubiquitin-Protein LigasesABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)-and from NF-kappaB-dependent reporter genes-was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection.
Subject(s)
Antigens, Viral/physiology , Gene Expression Regulation , Herpesvirus 8, Human/physiology , Nuclear Proteins/physiology , Animals , Blotting, Northern , COS Cells , DNA-Binding Proteins/genetics , Humans , Minor Histocompatibility Antigens , Promoter Regions, Genetic , RNA, Messenger/analysis , Repressor Proteins/genetics , STAT1 Transcription Factor , Terminal Repeat Sequences , Trans-Activators/genetics , Transcription Factors/genetics , Tripartite Motif ProteinsABSTRACT
BACKGROUND: The genome of human herpesvirus 8 (HHV-8) contains at least 84 open reading frames, including the highly immunogenic K8.1. Other studies have determined that K8.1 gene generates at least two spliced transcripts in the HHV-8 infected BCBL-1 cells, termed as glycoprotein (gp)K8.1A and gpK8.1B. OBJECTIVE: To analyze the expression, post-translational modification and localization of HHV-8 gpK8.1 by monoclonal antibody (mAb). STUDY DESIGN: Mabs to HHV-8 produced by conventional hybridization and several clones identified. A mAb was used by various immunological assays to analyze HHV-8 K8.1 proteins in BCBL-1- and Sf9 insect cells. RESULTS: MAb clone 19B4 identified a 0.75-kb insert from the lambdaZAP cDNA expression library of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced BCBL-1 cells. Sequence analysis revealed that the cDNA insert corresponds to the published spliced ORF K8.1 mRNA of HHV-8. By immunofluorescence assay, the mAb stained the cell membrane, cytoplasm and perinuclear region of TPA induced BCBL-1 cells and showed no cross-reactivity with other herpesviruses. By immunoblotting assay, mAb 19B4 reacted with two species polypeptides giving a diffuse band with rMW from 42 to 64 kDa (gpK8.1A) and two closely migrating polypeptides of rMW 35/37 kDa (gpK8.1B). Both species were labeled by [14C]glucosamine, indicating that they are glycosylated and only gpK8.1A was detected in the virions. Expression of the full length K8.1 derived from cDNA in baculovirus system confirmed that these two glycoproteins are encoded by K8.1 gene. Enzymatic deglycosylation with endoF/peptide-N-glycosidase F, led to the reduction of rMW of both polypeptides whereas deglycosylation with O-glycosidase led only the reduction of rMW of K8.1A. CONCLUSION: The mAb 19B4 reacts specifically with BCBL-1 and Sf9 cells infected with recombinant baculovirus containing HHV-8 K8.1 gene. In several assays the mAb reacts with gpK8.1A and gpK8.1B. Only the mature spliced gpK8.1A is incorporated into virion. Enzymatic deglacosylation determined that gpK8.1A is N- and O-glycosylated, whereas gpK8.1B may lack O-glycosylation.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Cell Membrane/virology , Cell Nucleus/virology , Cytoplasm/virology , Glycoproteins/metabolism , Herpesvirus 8, Human/metabolism , Protein Processing, Post-Translational , Alternative Splicing , Animals , Baculoviridae/genetics , Cell Line , Glycoproteins/genetics , Glycoproteins/immunology , Glycoside Hydrolases/chemistry , Glycosylation , Herpesvirus 8, Human/drug effects , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/immunology , Humans , Mice , Microscopy, Fluorescence , Molecular Weight , Open Reading Frames , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , RNA, Viral/analysis , Spodoptera/virology , Tetradecanoylphorbol Acetate/pharmacology , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism , Virion/metabolismABSTRACT
Down-regulation of the cell surface display of class I MHC proteins is an important mechanism of immune evasion by human and animal viruses. Herpesviruses in particular encode a variety of proteins that function to lower MHC I display by several mechanisms. These include binding and retention of MHC I chains in the endoplasmic reticulum, dislocation of class I chains from the ER, inhibition of the peptide transporter (TAP) involved in antigen presentation, and shunting of newly assembled chains to lysosomes. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human herpesvirus strongly linked to the development of KS and to certain AIDS-associated lymphoproliferative disorders. Here we show that KSHV encodes two distinctive gene products that function to dramatically reduce cell surface MHC I expression. These viral proteins are localized predominantly to the ER. However, unlike previously described MHC I inhibitors, they do not interfere with the synthesis, translocation, or assembly of class I chains, nor do they retain them in the ER. Rather, they act to enhance endocytosis of MHC I from the cell surface; internalized class I chains are delivered to endolysosomal vesicles, where they undergo degradation. These KSHV proteins define a mechanism of class I down-regulation distinct from the mechanisms of other herpesviruses and are likely to contribute importantly to immune evasion during viral infection.
Subject(s)
Endocytosis , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/immunology , Histocompatibility Antigens Class I/metabolism , Viral Proteins/immunology , Amino Acid Sequence , Biological Transport , Fluorescent Antibody Technique, Indirect , Genes, Viral , HeLa Cells , Humans , Microscopy, Confocal , Molecular Sequence DataABSTRACT
Hepatitis delta virus (HDV), a single-stranded RNA virus, bears a single coding region whose product, the hepatitis delta antigen (HDAg), is expressed in two isoforms, small (S-HDAg) and large (L-HDAg). S-HDAg is required for replication of HDV, while L-HDAg inhibits viral replication and is required for the envelopment of the HDV genomic RNA by hepatitis B virus proteins. Here we have examined the spatial distribution of HDV RNA and proteins in infected nuclei, with particular reference to specific nuclear domains. We found that L-HDAg was aggregated in specific nuclear domains and that over half of these domains were localized beside nuclear domain 10 (ND10). At later times, ND10-associated proteins like PML were found in larger HDAg complexes that had developed into apparently hollow spheres. In these larger complexes, PML was found chiefly in the rims of the spheres, while the known ND10 components Sp100, Daxx, and NDP55 were found in the centers of the spheres. Thus, ND10 proteins that normally are closely linked separate within HDAg-associated complexes. Viral RNA of antigenomic polarity, whether expressed from genomic RNA or directly from introduced plasmids, colocalizes with L-HDAg and the transcriptional repressor PML. In contrast, HDV genomic RNA was distributed more uniformly throughout the nucleus. These results suggest that different host protein complexes may assemble on viral RNA strands of different polarities, and they also suggest that this RNA virus, like DNA viruses, can alter the distribution of ND10-associated proteins. The fact that viral components specifically linked to repression of replication can associate with one of the ND10-associated proteins (PML) raises the possibility that this host protein may play a role in the regulation of HDV RNA synthesis.
Subject(s)
Antigens, Nuclear , Cell Nucleus/virology , Hepatitis Antigens/metabolism , Hepatitis Delta Virus , Intracellular Signaling Peptides and Proteins , RNA, Viral/metabolism , Virus Replication , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Autoantigens/metabolism , Binding Sites , Carrier Proteins/metabolism , Cell Nucleus/metabolism , Co-Repressor Proteins , Hepatitis Delta Virus/genetics , Hepatitis Delta Virus/immunology , Hepatitis Delta Virus/physiology , Hepatitis delta Antigens , Humans , Macromolecular Substances , Molecular Chaperones , Molecular Sequence Data , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , SUMO-1 Protein , Transcription Factors/metabolism , Tumor Cells, Cultured , Tumor Suppressor Proteins , Ubiquitins/metabolismABSTRACT
Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.