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1.
J Virol ; 75(7): 3250-8, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11238851

ABSTRACT

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. In latently infected cells, KSHV DNA is maintained as circularized, extrachromosomal episomes. To persist in proliferating cells, KSHV episomes must replicate and efficiently segregate to progeny nuclei. In uninfected B-lymphoblastoid cells, KSHV latency-associated nuclear antigen (LANA1) is necessary and sufficient for persistence of artificial episomes containing specific KSHV DNA. In previous work, the cis-acting sequence required for episome persistence contained KSHV terminal-repeat (TR) DNA and unique KSHV sequence. We now show that cis-acting KSHV TR DNA is necessary and sufficient for LANA1-mediated episome persistence. Furthermore, LANA1 binds TR DNA in mobility shift assays and a 20-nucleotide LANA1 binding sequence has been identified. Since LANA1 colocalizes with KSHV episomes along metaphase chromosomes, these results are consistent with a model in which LANA1 may bridge TR DNA to chromosomes during mitosis to efficiently segregate KSHV episomes to progeny nuclei.


Subject(s)
Antigens, Viral/physiology , DNA, Viral/metabolism , Herpesvirus 8, Human/genetics , Nuclear Proteins/physiology , Plasmids , Terminal Repeat Sequences , Base Sequence , DNA Replication , Herpesvirus 8, Human/immunology , Herpesvirus 8, Human/physiology , Molecular Sequence Data , Precipitin Tests
2.
Viral Immunol ; 14(4): 311-7, 2001.
Article in English | MEDLINE | ID: mdl-11792061

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (6,7,10). While the vast majority of tumor cells from these malignancies are latently infected, only a small subset of viral genes are actually expressed (5,40,47). Of these genes, the latency-associated nuclear antigen (LANA-1, LNA, or LNA1) is the only protein consistently shown to be highly expressed by in situ hybridization and immunohistochemistry (11,20,21,32). Moreover, within the past few years LANA-1 has proven to be a quite versatile protein, playing not only a pivotal role in KSHV episome persistence, but also in interacting with and influencing several cellular genes.


Subject(s)
Herpesvirus 8, Human/physiology , Nuclear Proteins/biosynthesis , Sarcoma, Kaposi/virology , Animals , Antigens, Viral , Herpesvirus 8, Human/immunology , Humans , Virus Latency
3.
Infect Dis Clin North Am ; 14(2): 475-87, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10829267

ABSTRACT

Improved understanding of the pharmacodynamics and toxicity of aminoglycoside antibiotics has resulted in the study of once-daily dosing regimens. Although studies have suggested a therapeutic advantage and possibly a decrease in toxicity with once-daily administration, these effects have been modest. The cost savings associated with once-daily aminoglycoside administration, however, makes this approach appealing. Although a syndrome of fever, tachycardia, hypotension, and rigors has been associated with once-daily dosing of gentamicin, this appears to have been the result of impurities in the antibiotic from a single offshore supplier. This syndrome has not been associated with other aminoglycoside antibiotics, and the FDA has now withdrawn its recommendation that once-daily aminoglycoside use be avoided. As with any medical regimen, the decision to use once-daily dosing of aminoglycoside agents must take into account special patient characteristics and the disease state being treated. Although once-daily dosing appears effective in limited studies in children, in individuals with neutropenia, and in individuals with cystic fibrosis, its role in gram-positive coccal endocarditis and in individuals with altered volumes of distribution remains uncertain. Further data are needed to clarify the role of once-daily dosing in these situations.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Bacterial Infections/drug therapy , Adult , Aminoglycosides , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/pharmacokinetics , Child , Drug Administration Schedule , Humans , Infant , Treatment Outcome
4.
J Virol ; 73(12): 10525-30, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10559372

ABSTRACT

An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675-683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-kappaB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.


Subject(s)
B-Lymphocytes/virology , Cell Transformation, Neoplastic , Cell Transformation, Viral , Herpesvirus 4, Human/physiology , Viral Matrix Proteins/physiology , Binding Sites , Cells, Cultured , Enzyme Activation , Fibroblasts/cytology , Gene Expression , Genes, Viral , Herpesvirus 4, Human/genetics , Humans , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Phenotype , Recombination, Genetic , Viral Matrix Proteins/genetics , Virus Latency
5.
Science ; 284(5414): 641-4, 1999 Apr 23.
Article in English | MEDLINE | ID: mdl-10213686

ABSTRACT

Primary effusion lymphoma (PEL) cells harbor Kaposi's sarcoma-associated herpesvirus (KSHV) episomes and express a KSHV-encoded latency-associated nuclear antigen (LANA). In PEL cells, LANA and KSHV DNA colocalized in dots in interphase nuclei and along mitotic chromosomes. In the absence of KSHV DNA, LANA was diffusely distributed in the nucleus or on mitotic chromosomes. In lymphoblasts, LANA was necessary and sufficient for the persistence of episomes containing a specific KSHV DNA fragment. Furthermore, LANA colocalized with the artificial KSHV DNA episomes in nuclei and along mitotic chromosomes. These results support a model in which LANA tethers KSHV DNA to chromosomes during mitosis to enable the efficient segregation of KSHV episomes to progeny cells.


Subject(s)
Chromosomes/metabolism , DNA, Viral/metabolism , Herpesvirus 8, Human/genetics , Mitosis , Nuclear Proteins/metabolism , Plasmids , Antigens, Viral/analysis , Antigens, Viral/genetics , Antigens, Viral/metabolism , Cell Nucleus/chemistry , Chromosomes/chemistry , Cosmids , DNA, Viral/analysis , DNA, Viral/genetics , Herpesvirus 8, Human/physiology , Humans , Interphase , Lymphocytes/chemistry , Microscopy, Confocal , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Transfection , Tumor Cells, Cultured
7.
Oncogene ; 14(24): 2899-916, 1997 Jun 19.
Article in English | MEDLINE | ID: mdl-9205097

ABSTRACT

Expression of the Epstein-Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kappaB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187-231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-kappaB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an effect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-kappaB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-kappaB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-kappaB activation and IL6 secretion. The importance of TRAF interactions in mediating these effects was confirmed using dominant negative TRAF2 and TRAF3 mutants which also identified differences in the signalling events mediated by the two NF-kappaB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kappaB activity, also blocked NF-kappaB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.


Subject(s)
CD40 Antigens/physiology , Herpesvirus 4, Human/genetics , Interleukin-6/biosynthesis , NF-kappa B/physiology , Proteins/physiology , Receptors, Tumor Necrosis Factor/physiology , Viral Matrix Proteins/physiology , Amino Acid Sequence , Binding Sites , CD40 Antigens/genetics , Cell Division/physiology , Cell Transformation, Viral/physiology , Epithelium/embryology , Epithelium/physiology , Female , HeLa Cells/metabolism , HeLa Cells/physiology , Herpesvirus 4, Human/physiology , Humans , Interleukin-6/metabolism , Molecular Sequence Data , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Protein Structure, Tertiary , Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , TNF Receptor-Associated Factor 1 , TNF Receptor-Associated Factor 2 , TNF Receptor-Associated Factor 3 , Transfection , Tumor Cells, Cultured , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Viral Matrix Proteins/genetics
8.
Proc Natl Acad Sci U S A ; 94(4): 1447-52, 1997 Feb 18.
Article in English | MEDLINE | ID: mdl-9037073

ABSTRACT

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs). We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185-211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1 delta185-211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1 delta185-211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.


Subject(s)
B-Lymphocytes/virology , Cell Transformation, Neoplastic , Herpesvirus 4, Human/genetics , Viral Matrix Proteins/metabolism , Cell Transformation, Viral , Epitopes , Hematopoietic Stem Cells/virology , Lymphoma/etiology , Protein Binding , Proteins/metabolism , Sequence Deletion , TNF Receptor-Associated Factor 1 , TNF Receptor-Associated Factor 2 , TNF Receptor-Associated Factor 3 , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology
9.
Mol Cell Biol ; 16(12): 7098-108, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8943365

ABSTRACT

The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The negative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1(1-231)-mediated NF-kappaB activation. NF-kappaB activation by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-231)-mediated NF-kappaB activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF3 is a negative modulator of LMP1(1-231)-mediated NF-kappaB activation. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling.


Subject(s)
Herpesvirus 4, Human , Proteins/genetics , Viral Matrix Proteins/genetics , B-Lymphocytes/metabolism , B-Lymphocytes/virology , Cell Transformation, Viral/genetics , Gene Expression Regulation , Humans , NF-kappa B/genetics , Proteins/metabolism , TNF Receptor-Associated Factor 1 , TNF Receptor-Associated Factor 2 , TNF Receptor-Associated Factor 3 , Tumor Cells, Cultured , Viral Matrix Proteins/metabolism
10.
Proc Natl Acad Sci U S A ; 93(20): 11085-90, 1996 Oct 01.
Article in English | MEDLINE | ID: mdl-8855313

ABSTRACT

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.


Subject(s)
Adaptor Proteins, Signal Transducing , B-Lymphocytes/metabolism , Cell Transformation, Viral , Gene Expression Regulation, Viral , Herpesvirus 4, Human/genetics , NF-kappa B/physiology , Proteins/metabolism , Proteins/physiology , Viral Matrix Proteins/physiology , Animals , Base Sequence , Binding, Competitive , Humans , Mice , Molecular Sequence Data , Protein Binding , TNF Receptor-Associated Factor 2
11.
Infect Dis Clin North Am ; 9(3): 547-59, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7490432

ABSTRACT

Topical antibacterial agents offer a useful alternative to systemic agents in certain circumstances. Uses include prophylaxis of infection for burns, traumatic wounds, and intravascular catheters, as well as eradication of S. aureus nasal carriage and treatment of primary and secondary pyodermas. Evidence supporting the use of topical agents for prophylaxis and treatment of skin and superficial wound infections and the indications for use of specific antimicrobials have been reviewed. Although topical agents are widely used, in many instances, data supporting their efficacy are only beginning to emerge.


Subject(s)
Anti-Infective Agents, Local/therapeutic use , Acne Vulgaris/drug therapy , Bacterial Infections/prevention & control , Burns/complications , Catheterization/adverse effects , Humans , Pyoderma/drug therapy , Wound Infection/prevention & control
12.
Infect Dis Clin North Am ; 9(3): 731-45, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7490441

ABSTRACT

Azithromycin and clarithromycin are structural analogues of erythromycin that have similar mechanisms of action. The newer macrolides have several distinct advantages over erythromycin, including improved oral bioavailability; longer half-life, allowing once or twice daily administration; higher tissue concentrations; and fewer gastrointestinal adverse effects. Clarithromycin and azithromycin also have enhanced antimicrobial activity. The clinical efficacy of the newer macrolides has been similar to erythromycin for the treatment of upper and lower respiratory tract and skin and soft tissue infections. New therapeutic roles include the use of azithromycin for C. trachomatis infections and the inclusion of clarithromycin or azithromycin as part of therapeutic regimens for disseminated MAC infections in HIV-infected patients. Further clinical trials are needed to determine the optimal roles for and uses of these new macrolides.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Azithromycin/therapeutic use , Clarithromycin/therapeutic use , Azithromycin/adverse effects , Azithromycin/pharmacology , Clarithromycin/adverse effects , Clarithromycin/pharmacology , Drug Resistance, Microbial , Humans , Respiratory Tract Infections/drug therapy
13.
J Virol ; 69(2): 675-83, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7815530

ABSTRACT

Recombinant Epstein-Barr viruses (EBVs) were made with mutated latent membrane protein 1 (LMP1) genes that express only the LMP1 amino-terminal cytoplasmic and six transmembrane domains (MS187) or these domains and the first 44 amino acids of the 200-residue LMP1 carboxy-terminal domain (MS231). After infection of primary B lymphocytes with virus stocks having small numbers of recombinant virus and large numbers of P3HR-1 EBV which is transformation defective but wild type (WT) for LMP1, all lymphoblastoid cell lines (LCLs) that had MS187 or MS231 LMP1 also had WT LMP1 provided by the coinfecting P3HR-1 EBV. Lytic virus infection was induced in these coinfected LCLs, and primary B lymphocytes were infected. In over 200 second-generation LCLs, MS187 LMP1 was never present without WT LMP1. Screening of over 600 LCLs infected with virus from MS231 recombinant virus-infected LCLs identified two LCLs which were infected with an MS231 recombinant without WT LMP1. The MS231 recombinant virus could growth transform primary B lymphocytes when cells were grown on fibroblast feeders. Even after 6 months on fibroblast feeder layers, cells transformed by the MS231 recombinant virus died when transferred to medium without fibroblast feeder cells. These data indicate that the LMP1 carboxy terminus is essential for WT growth-transforming activity. The first 44 amino acids of the carboxy-terminal cytoplasmic domain probably include an essential effector of cell growth transformation, while a deletion of the rest of LMP1 can be complemented by growth on fibroblast feeder layers. LMP1 residues 232 to 386 therefore provide a growth factor-like effect for the transformation of B lymphocytes. This effect may be indicative of the broader role of LMP1 in cell growth transformation.


Subject(s)
Antigens, Viral/physiology , B-Lymphocytes/physiology , Cell Transformation, Viral , Herpesvirus 4, Human/genetics , Viral Matrix Proteins/physiology , Antigens, Viral/genetics , Base Sequence , Cell Line , Codon , Cytoplasm , DNA, Recombinant , Fibroblasts/virology , Humans , Molecular Sequence Data , Viral Matrix Proteins/genetics
14.
J Virol ; 68(7): 4369-76, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8207810

ABSTRACT

Previous recombinant Epstein-Barr virus molecular genetic experiments with specifically mutated LMP1 genes indicate that LMP1 is essential for primary B-lymphocyte growth transformation and that the amino-terminal cytoplasmic and first transmembrane domains are together an important mediator of transformation. EBV recombinants with specific deletions in the amino-terminal cytoplasmic domain have now been constructed and tested for the ability to growth transform primary B lymphocytes into lymphoblastoid cell lines. Surprisingly, deletion of DNA encoding EHDLER or GPPLSSS from the full LMP1 amino-terminal cytoplasmic domain (MEHDLERGPPGPRRPPRGPPLSSS) had no discernible effect on primary B-lymphocyte transformation. These two motifs distinguish the LMP1 amino-terminal cytoplasmic domain from other arginine-rich membrane proximal sequences that anchor hydrophobic transmembrane domains. Two deletions which included the ERGPPGPRRPPR motif adversely affected but did not prevent transformation. This arginine- and proline-rich sequence is probably important in anchoring the first transmembrane domain in the plasma membrane, since these mutated LMP1s had altered stability and cell membrane localization. The finding that overlapping deletions of the entire amino-terminal cytoplasmic domain do not ablate transformation is most consistent with a model postulating that the transmembrane and carboxyl-terminal cytoplasmic domains are the likely biochemical effectors of transformation.


Subject(s)
Antigens, Viral/physiology , B-Lymphocytes , Cell Transformation, Viral/physiology , Herpesvirus 4, Human/physiology , Viral Matrix Proteins/physiology , Amino Acid Sequence , Animals , Antigens, Viral/chemistry , Antigens, Viral/genetics , B-Lymphocytes/microbiology , Base Sequence , Callithrix , Cell Division/genetics , Cell Line , Cell Transformation, Neoplastic/genetics , DNA Primers , DNA, Viral , Herpesvirus 4, Human/genetics , Humans , Molecular Sequence Data , Mutation , Recombination, Genetic , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics
15.
Proc Natl Acad Sci U S A ; 90(19): 9150-4, 1993 Oct 01.
Article in English | MEDLINE | ID: mdl-8415670

ABSTRACT

The gene encoding latent-infection membrane protein 1 (LMP1) was specifically mutated in Epstein-Barr virus (EBV) recombinants by inserting a nonsense linker after codon 9 or codon 84 or into an intron 186 bp 3' to the latter insertion site. EBV recombinants with the LMP1 intron mutation were wild type for LMP1 expression and for growth transformation of primary B lymphocytes. In contrast, EBV recombinants with the mutations in the LMP1 open reading frame expressed N-terminally truncated crossreactive proteins and could initiate or maintain primary B-lymphocyte transformation only when wild-type LMP1 was provided in trans by a coinfecting, transformation-defective EBV, P3HR-1. These data indicate that LMP1 is essential for EBV-mediated transformation of primary B lymphocytes, that the first 43 amino acids are critical for LMP1's function, and that codon 44-initiated LMP1 does not have a dominant negative effect on transformation.


Subject(s)
Antigens, Viral/metabolism , Herpesvirus 4, Human/genetics , Lymphocyte Activation , Viral Matrix Proteins/metabolism , Antigens, Viral/analysis , Antigens, Viral/biosynthesis , B-Lymphocytes , Base Sequence , Blotting, Southern , Cell Division , Cell Line, Transformed , Cell Membrane/immunology , Cell Membrane/metabolism , Herpesvirus 4, Human/immunology , Humans , Immunoblotting , Molecular Sequence Data , Oligonucleotides, Antisense , Open Reading Frames , Polymerase Chain Reaction , Protein Structure, Secondary , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Recombination, Genetic , Tumor Cells, Cultured , Viral Matrix Proteins/analysis , Viral Matrix Proteins/biosynthesis
16.
Clin Infect Dis ; 14(5): 1010-4, 1992 May.
Article in English | MEDLINE | ID: mdl-1600002

ABSTRACT

Three cases of catheter infection due to Methylobacterium extorquens are reported. Each patient had a history of acute leukemia and was immunocompromised; two had undergone bone marrow transplantation, and the third was receiving consolidation chemotherapy. All three patients survived after removal of the central venous catheter and antibiotic treatment. The clinical features of these cases are compared with those of the 12 previously reported cases of infection due to Methylobacterium species.


Subject(s)
Catheterization, Central Venous/adverse effects , Gram-Negative Aerobic Bacteria/isolation & purification , Gram-Negative Bacterial Infections/microbiology , Immunocompromised Host , Adult , Child , Child, Preschool , Female , Humans , Male , Treatment Outcome
18.
J Virol ; 59(1): 90-7, 1986 Jul.
Article in English | MEDLINE | ID: mdl-3012122

ABSTRACT

In this paper we provide a step by step comparison of the pathogenesis of murine infection caused by reovirus type 3 (Dearing) and an antigenic variant (K) selected by its resistance to neutralization with a monoclonal antibody (G5) directed against the T3 hemagglutinin. To show that specific changes in the biologic properties of variant K were due to mutation in the S1 double-stranded RNA segment (gene), which encodes the viral hemagglutinin, we generated a reassortant virus ("1 HA K") containing the variant K S1 gene and compared its properties to variant K and to a reassortant ("1 HA 3") containing the T3 (Dearing) S1 gene. These studies, in conjunction with our previous nucleotide sequence analysis of the S1 genes of variant K and T3 (Dearing) [R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizel, Jr., and B. N. Fields, Nature (London) 315:421-423, 1985; R. Bassel-Duby, D. R. Spriggs, K. L. Tyler, and B. N. Fields, submitted for publication], indicate that a single amino acid change in the T3 hemagglutinin can alter viral growth and tropism within the central nervous system without affecting either its primary replication in the intestine or its pattern of spread to or within the central nervous system.


Subject(s)
Brain/microbiology , Hemagglutinins, Viral/genetics , Mammalian orthoreovirus 3/genetics , Reoviridae/genetics , Animals , Genes, Viral , Hemagglutinins, Viral/immunology , Intestines/microbiology , Mammalian orthoreovirus 3/growth & development , Mammalian orthoreovirus 3/immunology , Mammalian orthoreovirus 3/pathogenicity , Mice , Neutralization Tests , Reoviridae Infections/microbiology , Spleen/microbiology , Time Factors , Viral Proteins/genetics , Viral Proteins/immunology , Virus Replication
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