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1.
Virology ; 287(1): 62-70, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11504542

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) encodes a G protein-coupled receptor (vGPCR) in open reading frame (ORF) 74, which is homologous to human chemokine receptors. KSHV vGPCR is constitutively active and induces VEGF-mediated angiogenesis. Previous studies have shown that ORF 74 is transcribed as part of a bicistronic message containing ORF K14 upstream of ORF 74, with an early lytic pattern of expression. We have now extended these studies by analyzing three different KSHV-positive primary effusion lymphoma (PEL) cell lines and three PEL clinical samples. In addition, we have identified another less abundant monocistronic transcript containing only ORF 74. Both transcripts were identified at low but similar levels in two PEL clinical samples. We evaluated the degree of sequence and functional conservation of ORF74 in three additional PELs and two KS clinical specimens, demonstrating complete identity at the amino acid level among all isolates. While it is expressed as an early lytic transcript in PEL cell lines, in primary clinical PEL samples transcription of KSHV vGPCR can be readily detected.


Subject(s)
Herpesvirus 8, Human/genetics , Lymphoma/virology , Receptors, Chemokine/biosynthesis , Amino Acid Sequence , Base Sequence , Castleman Disease/genetics , Castleman Disease/virology , Humans , Lymphoma/genetics , Molecular Sequence Data , Open Reading Frames , Receptors, Chemokine/genetics , Sarcoma, Kaposi/genetics , Sarcoma, Kaposi/virology , Structure-Activity Relationship , Tumor Cells, Cultured
2.
J Virol ; 75(5): 2435-43, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11160746

ABSTRACT

Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8. At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR. The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR. Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission.


Subject(s)
Herpesviridae Infections/virology , Herpesvirus 8, Human/physiology , Herpesvirus 8, Human/pathogenicity , Keratinocytes/virology , Phosphoproteins , Antigens, Viral/genetics , Antigens, Viral/metabolism , Cells, Cultured , Cytokines/biosynthesis , Endothelial Growth Factors/genetics , Fibroblast Growth Factor 2/genetics , Herpesvirus 8, Human/genetics , Humans , Immunohistochemistry , Keratinocytes/physiology , Lymphokines/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Virus Replication
3.
J Infect Dis ; 180(5): 1466-76, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10515805

ABSTRACT

Molecular polymorphism was found in Kaposi's sarcoma-associated herpesvirus (KSHV) latent nuclear antigen (LNA), mapped to the internal repeat domain of the encoding orf73 gene, and used to develop a novel genotyping technique, KSHV LNA genotyping (KVNAtyping). KVNAtype was stable during latent and lytic viral replication in cell culture and in humans. Diverse KVNAtypes were identified in 43 specimens: 6 KSHV cell lines and 6 Kaposi's sarcoma (KS) and 4 primary effusion lymphoma (PEL) tumor samples from the United States, 15 KS tumor samples from Italy, and 12 KS tumor samples from Zambia. A single KVNAtype was detected in each of 41 specimens, and 2 KVNAtypes were detected in each of 2 KS specimens. Multifocal KS from 3 patients showed the same single KVNAtype at all sites in each patient. These results demonstrate a large repertoire of KSHV genotypes and suggest that the development of most KSs and PELs is associated with a single viral genotype.


Subject(s)
Antigens, Viral/genetics , Herpesvirus 8, Human/genetics , Nuclear Proteins/genetics , Polymorphism, Genetic , Sarcoma, Kaposi/virology , Cell Line , Chromosome Mapping , DNA, Viral/analysis , DNA, Viral/genetics , Genotype , Herpesvirus 8, Human/classification , Herpesvirus 8, Human/immunology , Humans , Polymerase Chain Reaction/methods
4.
Nature ; 394(6693): 588-92, 1998 Aug 06.
Article in English | MEDLINE | ID: mdl-9707121

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is invariably present in Kaposi's sarcoma lesions. KSHV contains several viral oncogenes and serological evidence suggests that KSHV infection is necessary for the development of Kaposi's sarcoma, but cellular transformation by this virus has not so far been demonstrated. KSHV is found in the microvascular endothelial cells in Kaposi's sarcoma lesions and in the spindle 'tumour' cells, which are also thought to be of endothelial origin. Here we investigate the biological consequences of infecting human primary endothelial cells with purified KSHV particles. We find that infection causes long-term proliferation and survival of these cells, which are associated with the acquisition of telomerase activity and anchorage-independent growth. KSHV was present in only a subset of cells, and paracrine mechanisms were found to be responsible for the survival of uninfected cells. Their survival may have been mediated by upregulation of a receptor for vascular endothelial growth factor. Our results indicate that transformation of endothelial cells by KSHV, as well as paracrine mechanisms that are induced by this virus, may be critical in the pathogenesis of Kaposi's sarcoma.


Subject(s)
Cell Transformation, Neoplastic , Cell Transformation, Viral , Endothelium, Vascular/virology , Herpesvirus 8, Human/physiology , Sarcoma, Kaposi/virology , Antigens, Viral/biosynthesis , Cell Adhesion , Cell Division , Cell Survival , Cells, Cultured , DNA, Viral/analysis , Endothelial Growth Factors/physiology , Endothelium, Vascular/pathology , Humans , Lymphokines/physiology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Telomerase/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Virus Replication
5.
J Virol ; 72(2): 1005-12, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9444993

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) gene transcription in the BC-1 cell line (KSHV and Epstein-Barr virus coinfected) was examined by using Northern analysis with DNA probes extending across the viral genome except for a 3-kb unclonable rightmost region. Three broad classes of viral gene transcription have been identified. Class I genes, such as those encoding the v-cyclin, latency-associated nuclear antigen, and v-FLIP, are constitutively transcribed under standard growth conditions, are unaffected by tetradecanoylphorbol acetate (TPA) induction, and presumably represent latent viral transcripts. Class II genes are primarily clustered in nonconserved regions of the genome and include small polyadenylated RNAs (T0.7 and T1.1) as well as most of the virus-encoded cytokines and signal transduction genes. Class II genes are transcribed without TPA treatment but are induced to higher transcription levels by TPA treatment. Class III genes are primarily structural and replication genes that are transcribed only following TPA treatment and are presumably responsible for lytic virion production. These results indicate that BC-1 cells have detectable transcription of a number of KSHV genes, particularly nonconserved genes involved in cellular signal transduction and regulation, during noninduced (latent) virus culture.


Subject(s)
Genes, Viral , Genome, Viral , Herpesvirus 8, Human/genetics , Lymphoma/virology , Humans , Signal Transduction , Transcription, Genetic , Tumor Cells, Cultured
6.
AIDS Res Hum Retroviruses ; 13(14): 1229-33, 1997 Sep 20.
Article in English | MEDLINE | ID: mdl-9310290

ABSTRACT

Epidemiological studies strongly suggest that a newly discovered herpesvirus, Kaposi's sarcoma associated-herpesvirus (KSHV/HHV8), is the likely infectious cause of Kaposi's sarcoma (KS). Identification of this agent suggests the possibility that existing anti-herpesvirus chemotherapeutics have activity against KSHV. Using KSHV/Epstein-Barr virus (EBV)-coinfected cell line BC-1 and KSHV-infected/EBV-negative cell line BC-3, the effect of DNA polymerase inhibitors in the presence of virus-inducing agents was examined. The phorbol ester TPA induced 8.2-fold EBV replication in BC-1 cells with only minimal concurrent KSHV genome replication in BC-1, but induced fourfold KSHV in BC-3 cells. TPA, however, induced transcripts encoded by the lytic cycle major capsid protein gene that were inhibited by both phosphonoacetic acid and phosphonoformic acid either in the KSHV/EBV-infected cell line or in the EBV-negative/KSHV-infected cell line. Transcripts hybridizing to a KSHV-encoded cyclin gene were unaffected by either TPA or DNA polymerase inhibitors in both cell lines. These results show in vitro activity of DNA polymerase inhibitors on KSHV lytic transcript accumulation and may provide a simple assay for evaluating the efficacy of potential anti-KSHV chemotherapeutics.


Subject(s)
Capsid/drug effects , Capsid/genetics , Cyclins/drug effects , Herpesvirus 8, Human/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Blotting, Northern , Carcinogens/pharmacology , Foscarnet/pharmacology , Gene Expression/drug effects , Humans , Phosphonoacetic Acid/pharmacology , RNA, Messenger/analysis , RNA, Messenger/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/drug effects , Tumor Cells, Cultured
7.
Protein Sci ; 3(10): 1651-69, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7849583

ABSTRACT

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 A resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VP0 to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N-terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N-terminal extension of VP1, as well as portions corresponding to VP4 and the N-terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP0 scissile bond is located some 20 A away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoil-shaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA-dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N-terminal network and in generating stable virions.


Subject(s)
Capsid/chemistry , Poliovirus/chemistry , Crystallization , Crystallography, X-Ray , Models, Molecular , Poliovirus/physiology , Protein Folding , RNA, Viral/chemistry
8.
Int J Syst Bacteriol ; 42(1): 37-43, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1736970

ABSTRACT

The bacteriolytic activities of different group D streptococcal species on various media and substrates were studied. Our results showed that all of the enterococcal species which we tested had bacteriolytic activity on at least one of the media used, while the group D nonenterococcal species had no such activity. In addition, using culture media containing different additives and different pH values, we defined seven major groups of bacteriolytic activity (lyogroups), each of which overlapped with one species (four lyogroups), two species (two lyogroups), or four species (one lyogroup). The detection of enterococcal lyogroups proved to be as reliable for species identification as the conventional methods presently in use.


Subject(s)
Bacteriolysis , Streptococcus/classification , Streptococcus/physiology
9.
Clin Exp Obstet Gynecol ; 16(1): 16-20, 1989.
Article in English | MEDLINE | ID: mdl-2713989

ABSTRACT

A new pyrimidine analog, 5-(2-bromoethyl)-2'-deoxyuridine (BEUdR), was tested in vitro for antiviral activity on Herpes simplex virus types 1 and 2. As reference compounds, ACG, BVUdR and PAA were used. Compared to ACG and BVUdR, BEUdR resulted less potent on both HSV-1 and HSV-2. However, a 50% inhibition of the multiplication of uninfected cells could be obtained only at very high BEUdR concentration (ID50 = 8500 microM). This makes BEUdR the least toxic analog known and gives it a selective index comparable to, if not better, than of ACG and BVUdR.


Subject(s)
Antiviral Agents/pharmacology , Bromodeoxyuridine/analogs & derivatives , Herpes Simplex/drug therapy , Bromodeoxyuridine/pharmacology , Humans
10.
Cytogenet Cell Genet ; 48(1): 60-2, 1988.
Article in English | MEDLINE | ID: mdl-2846239

ABSTRACT

When fixed chromosome preparations were allowed to age for 1-72 h, they became progressively more susceptible to digestion by exonuclease III and by S1 nuclease. Analysis of DNA from these aged preparations on agarose gels showed that the molecular weight of the DNA decreased as ageing progressed. We conclude that DNA in fixed chromosome preparations becomes progressively degraded as the preparations age.


Subject(s)
Cytological Techniques , DNA/metabolism , Cell Survival , Endonucleases , Exodeoxyribonucleases , Humans , Metaphase , Molecular Weight , Pharyngeal Neoplasms , Single-Strand Specific DNA and RNA Endonucleases , Time Factors , Tumor Cells, Cultured
11.
J Virol ; 61(2): 561-9, 1987 Feb.
Article in English | MEDLINE | ID: mdl-3027383

ABSTRACT

A previously undetected subviral particle, designated the 55S particle because of its position in sucrose density gradients, has been found in cytoplasmic extracts of poliovirus-infected cells. It contains no RNA, is composed of equimolar amounts of the structural polypeptides P1AB, P1C, and P1D, and is stable in vitro under a variety of conditions: presence or absence of EDTA, dilution in low- or high-ionic-strength buffers, suspension in buffers up to pH 10, incubation at 37 degrees C, and centrifugation to equilibrium in CsCl gradients (where it bands at a density of 1.285 g/cm3). Conventional pulse-chase experiments show that 55S particles are the products of the assembly of 14S subunits and the precursors of virions. These data led to the formulation of a model of poliovirus morphogenesis in which the conversion of capsomers into 73S empty capsids does not occur directly, but through the formation of an intermediate structure, the 55S particle.


Subject(s)
Morphogenesis , Poliovirus/isolation & purification , Cell Line , HeLa Cells/cytology , Humans , Hydrogen-Ion Concentration , Molecular Weight , Osmolar Concentration , Peptides/analysis , Poliovirus/ultrastructure , Viral Proteins/analysis
12.
Experientia ; 36(3): 304, 1980 Mar 15.
Article in English | MEDLINE | ID: mdl-6245914

ABSTRACT

Glycyrrhizic acid inhibits the growth of several DNA and RNA viruses in cell cultures and inactivates Herpes simplex 1 virus irreversibly.


Subject(s)
Glycyrrhetinic Acid/analogs & derivatives , Viruses/drug effects , Cells, Cultured , Glycyrrhetinic Acid/pharmacology , Glycyrrhizic Acid , Humans , Simplexvirus/drug effects
13.
Nature ; 281(5733): 689-90, 1979 Oct 25.
Article in English | MEDLINE | ID: mdl-233133

ABSTRACT

Screening investigations in antiviral action of plant extracts have revealed that a component of Glycyrrhiza glabra roots, found to be glycyrrhizie acid, is active against viruses. We report here that this drug inhibits growth and cytopathology of several unrelated DNA and RNA viruses, while not affecting cell activity and ability to replicate. In addition, glycyrrhizic acid inactivates herpes simplex virus particles irreversibly.


Subject(s)
Antiviral Agents , Glycyrrhetinic Acid/analogs & derivatives , Glycyrrhiza , Plants, Medicinal , Virus Replication/drug effects , Biological Transport/drug effects , Cells, Cultured , Glycyrrhetinic Acid/pharmacology , Glycyrrhizic Acid , Poliovirus/drug effects , Simplexvirus/drug effects , Vaccinia virus/drug effects , Vesicular stomatitis Indiana virus/drug effects
14.
Experientia ; 35(3): 321-2, 1979 Mar 15.
Article in English | MEDLINE | ID: mdl-446602

ABSTRACT

2-Amino-4,6-dichloropyrimidine prevents maturation of Vaccinia virus. Proteins synthesized in the presence of the drug are not assembled into virions.


Subject(s)
Pyrimidines/pharmacology , Vaccinia virus/drug effects , DNA, Viral/biosynthesis , Vaccinia virus/growth & development , Vaccinia virus/metabolism , Viral Proteins/metabolism
15.
Experientia ; 34(11): 1528-9, 1978 Nov 15.
Article in English | MEDLINE | ID: mdl-214330

ABSTRACT

The presence of hydrocortisone in virus-infected cell cultures leads to enhancement of the syncytia forming ability of Newcastle disease virus and to production of vescicular stomatitis virus particles which loose their infectivity upon storage below 0 degrees C.


Subject(s)
Hydrocortisone/pharmacology , Newcastle disease virus/drug effects , Cells, Cultured , Cytopathogenic Effect, Viral/drug effects , Freezing , Vesicular stomatitis Indiana virus/drug effects , Vesicular stomatitis Indiana virus/pathogenicity
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