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At least 1,351 records · Page 75Linked to original sources
PubMed1999

An arginine-faced amphipathic alpha helix is required for adenovirus type 5 e4orf6 protein function.

A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic alpha helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this alpha helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic alpha helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.

Adenovirus E1B Proteins↗
PubMed1996

Epstein-Barr virus latent and replicative gene expression in gastric carcinoma.

Recent reports demonstrated the presence of Epstein-Barr virus (EBV) in about 10% of gastric carcinoma cases, particularly in Asian populations. We carried out a retrospective assessment of the detection rate of EBV gene products in 59 cases of gastric carcinoma of various histological subtypes. In situ hybridization using non-isotopic EBER and BHLF1 oligoprobes, and immunohistochemistry using antibodies to latent membrane protein 1 (LMP-1) were applied to paraffin-embedded sections. Tumour cells in five out of 59 cases (8.5%) were found to be EBER positive by in situ hybridization, but no staining was observed with LMP-1 antibodies. Four EBER positive cases were lymphoepithelioma-like carcinomas and one case was a well differentiated adenocarcinoma, suggesting a stronger association with the former subtype. Among the four EBER positive lymphoepithelioma-like carcinomas, BHLF1 transcripts were expressed in one case in a few tumour cells, indicating the possible activation of a lytic cycle. In nine cases (including three EBER positive cases) a few scattered EBV-infected lymphocytes were seen in the normal mucosa but we were unable to detect any EBER positive normal epithelial cells. Our results show that, in a French population, the incidence of EBV-associated gastric carcinoma is similar to that in other geographic areas. The clinical implications of these findings, however, remain unclear.

Adult↗
PubMed2003

T' proteins influence JC virus biology.

The JC virus early mRNA is alternatively spliced to yield five transcripts that encode large T antigen, small t antigen, T'(135), T'(136), and T'(165). The splicing process is regulated differentially in transformed versus lytically infected cells and temporally during the course of a productive infection. The authors have identified a potential exonic splicing enhancer near the 3' end of the early viral mRNA that, when mutated, results in altered splice site usage. The authors have only recently begun investigating the function of the three T' proteins using genetic and biochemical approaches. These studies indicate that the T' proteins enhance viral DNA replication and bind differentially to the pRB family of cellular tumor suppressor proteins in vitro. Using a G418 selection scheme, the authors have created cell lines that express either T antigen or each of the T' proteins individually. Preliminary analyses of these lines suggest that T antigen may induce apoptosis in rodent cells, an activity that may be blocked by other JC virus early proteins. Furthermore, examination of protein-protein interactions within the G418-selected cells reveal differences in binding of the viral proteins to the pRB family members relative to that seen in vitro.

Alternative Splicing↗
PubMed2006

Natural killer cell function is well preserved in asymptomatic human immunodeficiency virus type 2 (HIV-2) infection but similar to that of HIV-1 infection when CD4 T-cell counts fall.

Natural killer (NK) cells are potent effectors of natural immunity and their activity prevents human immunodeficiency virus type 1 (HIV-1) viral entry and viral replication. We sought to determine whether NK immune responses are associated with different clinical course of HIV-1 and HIV-2 infections. A cross-sectional analysis of NK cell responses was undertaken in 30 HIV-1 and 30 HIV-2 subjects in each of three categories of CD4(+)-T-cell counts (>500, 200 to 500, and <200 cells/microl) and in 50 HIV-uninfected control subjects. Lytic activity and gamma interferon (IFN-gamma) secretion were measured by chromium release and enzyme-linked immunospot assays, respectively. Flow cytometry was used to assess intracellular cytokines and chemokines. Levels of NK cytotoxicity were significantly higher in HIV-2 than in HIV-1 infections in subjects with high CD4(+)-T-cell counts and were similar to that of the healthy controls. In these HIV-2 subjects, cytolytic activity was positively correlated to NK cell count and inversely related to plasma viremia. Levels of intracellular MIP-1beta, RANTES, tumor necrosis factor alpha, and IFN-gamma produced by NK CD56(bright) cells were significantly higher in HIV-2- than HIV-1-infected subjects with high CD4(+)-T-cell counts but fell to similar levels as CD4 counts dropped. The data suggest efficient cytolytic and chemokine-suppressive activity of NK cells early in HIV-2 infection, which is associated with high CD4(+) T-cell counts. Enhancement of these functions may be important in immune-based therapy to control HIV disease.

CD4 Lymphocyte Count↗
PubMed1989

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus.

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.

Cell Line↗
PubMed2006

Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions.

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. phiYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of phiYS40, most of which have no similarity to sequences in public databases, were identified by mass spectrometric analysis of purified virions. Various phiYS40 proteins have different phylogenetic neighbors, including myovirus, podovirus, and siphovirus gene products, bacterial genes and, in one case, a dUTPase from a eukaryotic virus. phiYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.

Amino Acid Sequence↗
PubMed1992

Use of second-site homologous recombination to demonstrate that Epstein-Barr virus nuclear protein 3B is not important for lymphocyte infection or growth transformation in vitro.

Recombinant Epstein-Barr viruses with a stop codon inserted into the nuclear protein 3B (EBNA 3B) open reading frame were generated by second-site homologous recombination. These mutant viruses infected and growth transformed primary B lymphocytes, resulting in the establishment of lymphoblastoid cell lines (LCLs). Polymerase chain reaction analysis and Southern hybridizations with infected cell DNA demonstrated the presence of the mutant EBNA 3B and the absence of wild-type EBNA 3B. Immunoblot analysis of the LCLs with affinity-purified EBNA 3B antibodies confirmed the absence of EBNA 3B cross-reactive protein. Virus was reactivated from two of these infected LCLs and serially passaged through primary B lymphocytes. The newly infected cells contained only the mutant recombinant virus. No difference was noted between mutant and wild-type recombinants, derived in parallel, in latent (other than EBNA 3B) or lytic cycle-infected cell virus protein expression or in the growth of the latently infected transformed cell lines. These data indicate that the EBNA 3B protein is not critical for primary B-lymphocyte infection, growth transformation, or lytic virus infection in vitro.

Antigens, Viral↗
PubMed1988

Identification of a protein encoded by the vpu gene of HIV-1.

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture. The complexity of virus-host cell interaction is reflected in the complex organization of the viral genome. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3' orf proteins, as well as a protein of unknown function from the open reading frame called R. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV) that do not have the capacity to encode a similar protein.

Acquired Immunodeficiency Syndrome↗
PubMed1982

Fine structure of virus chromatin in thin sections of SV40-infected cells: a cytochemical and autoradiographical study.

The ultrastructural organization of virus chromatin was studied within nuclei prepared from CV1 cells (cultured monkey kidney cells) lytically infected with SV40 virus (simian virus 40) by a procedure which allows a mild loosening of nucleoproteins. In addition to dispersed host components, DNA-containing nucleoplasmic structures could be identified as virus chromatin. Both standard staining for structure and specific staining for DNA clearly revealed in thin sections the nucleosomal structure of well-extended virus chromatin as well as alignments of virions on host chromatin. In addition, replicating and transcribing virus chromatins were abundant as revealed by high-resolution autoradiography. Therefore, the procedure of loosening of nucleoproteins used in this report also preserves the active SV40 chromatin and allows the in situ visualization in Epon sections of transcription and replication of the virus genome.

Animals↗
PubMed1991

Establishment of latency in vitro by the herpes simplex virus type 1 mutant in1814.

The herpes simplex virus type 1 (HSV-1) mutant in1814 possesses an insertion mutation that abolishes trans-activation of immediate early (IE) transcription by the virion protein Vmw65. Interactions between in1814 and the host cell were examined by use of an in vitro latency system which relies on infection of human foetal lung (HFL) cells at 42 degrees C to prevent lytic growth of virus. Mutant in1814 was retained in HFL cells after infection at low m.o.i. and incubation at 42 degrees C, and was reactivated by superinfection of monolayers with viruses that express the HSV-1 IE protein Vmw110. Moreover, latency was established by in1814 in an analogous manner at 37 degrees C. The low cytotoxicity of in1814 enabled an investigation of latency after infection at high m.o.i. (five particles per cell) to be undertaken. At 42 degrees C, or at 37 degrees C in the presence of an inhibitor of DNA synthesis, in1814 DNA was maintained at low abundance (one to eight copies per infected cell) in a non-linear configuration. The absence of trans-activation by Vmw65 therefore predisposes HSV to latency, as opposed to lytic growth, in HFL cells, resulting in the retention of the genome in a form resembling that found in vivo.

Cell Line↗
PubMed1988

Generation and characterization of a temperature-sensitive mutation in the NS-1 gene of the autonomous parvovirus minute virus of mice.

In-phase single-codon insertion mutations were constructed in the open reading frames of the NS-1 and NS-2 genes of the autonomous parvovirus minute virus of mice. A viral mutant containing an isoleucine insertion exclusively within NS-1 between residues 229 and 230 was isolated that produced approximately 3 orders of magnitude fewer plaques at 39 degrees C than at 32 degrees C. Preliminary characterization of the mutant demonstrated that the NS-1 gene product is independently required for both genome amplification and the regulation of the temporal expression between the two viral transcription units during lytic infection.

Capsid↗
PubMed2004

Phosphorylation of Epstein-Barr virus ZEBRA protein at its casein kinase 2 sites mediates its ability to repress activation of a viral lytic cycle late gene by Rta.

ZEBRA, a member of the bZIP family, serves as a master switch between latent and lytic cycle Epstein-Barr virus (EBV) gene expression. ZEBRA influences the activity of another viral transactivator, Rta, in a gene-specific manner. Some early lytic cycle genes, such as BMRF1, are activated in synergy by ZEBRA and Rta. However, ZEBRA suppresses Rta's ability to activate a late gene, BLRF2. Here we show that this repressive activity is dependent on the phosphorylation state of ZEBRA. We find that two residues of ZEBRA, S167 and S173, that are phosphorylated by casein kinase 2 (CK2) in vitro are also phosphorylated in vivo. Inhibition of ZEBRA phosphorylation at the CK2 substrate motif, either by serine-to-alanine substitutions or by use of a specific inhibitor of CK2, abolished ZEBRA's capacity to repress Rta activation of the BLRF2 gene, but did not alter its ability to initiate the lytic cycle or to synergize with Rta in activation of the BMRF1 early-lytic-cycle gene. These studies illustrate how the phosphorylation state of a transcriptional activator can modulate its behavior as an activator or repressor of gene expression. Phosphorylation of ZEBRA at its CK2 sites is likely to play an essential role in proper temporal control of the EBV lytic life cycle.

Casein Kinase II↗
PubMed1994

Differential expression of viral and human interleukin-10 (IL-10) by primary B cell tumors and B cell lines.

Human and viral interleukin-10 (IL-10) possess growth factor activity for human B cells and may act as autocrine growth factors in B cell malignancies. To study this possibility we have measured viral (v) and human (h) IL-10 expression in EBV-positive and negative B lineage tumors and tumor cell lines. Previous studies demonstrated IL-10 expression in cell lines and now we describe the pattern of IL-10 expression in primary Burkitt's lymphoma (BL) tumor biopsies and in BL lines of defined phenotypes. vIL-10 was expressed during the lytic (productive) phase of EBV infection, but not during virus latency. Although hIL-10 was expressed in the majority of B cell lines, it was not expressed in two BL biopsy specimens. Expression of hIL-10 did not correlate with the presence of EBV, but was associated with the differentiation state of the B cell line. Thus, vIL-10 may enhance the persistence of B cells infected at sites of virus replication, and while hIL-10 may be a factor in the growth both in vivo and in vitro of some BLs and EBV-transformed B cells, it is not an absolute requirement.

B-Lymphocytes↗
PubMed2002

Transcription mapping as a tool in phage genomics: the case of the temperate Streptococcus thermophilus phage Sfi21.

For the lytic growth cycle of the temperate cos-site Streptococcus thermophilus phage Sfi21 a transcription map was developed on the basis of systematic Northern blot hybridizations. All deduced 5' ends were confirmed by primer extension analysis. Three time classes of transcripts were observed. Early transcripts were identified in four different genome regions. One prominent early mRNA of 4.8 kb length covered a group of 12 genes located between the origin of replication and the cos-site. Two short early mRNAs represented a single gene from the direct vicinity of the cos-site and the superinfection immunity gene from the lysogeny module, respectively. A fourth early transcript covered a group of four genes located between the lysin and the integrase gene. Middle transcripts of 2.1 and 5.8 kb length covered cro-like and ant-like repressor genes and the DNA replication module, respectively. Four types of late transcripts were identified. The transcripts covered the likely DNA packaging genes, the head morphogenesis module plus the major tail gene, the remainder of the tail genes, and the putative tail fiber plus lysis genes, respectively. Only the transcript from the head morphogenesis genes yielded defined late mRNA species. The transcription map concurred with most of the in silico predictions for the genome organization of phage Sfi21 except for the separation of the DNA replication module from a possible transcription regulation module. Most 5' ends of the transcripts determined in primer-extension experiments were not preceded by a consensus promoter sequence. The involvement of phage-encoded regulators for middle and late transcription was suggested by chloramphenicol-inhibition experiments.

5' Untranslated Regions↗
PubMed1987

Coronavirus mouse hepatitis virus (MHV)-A59 causes a persistent, productive infection in primary glial cell cultures.

MHV-A59 causes a chronic demyelinating disease in mice which is accompanied by persistence of viral genome in white matter. As part of the investigation into the mechanism of viral persistence, infection of glial cells, probable targets for chronic infection, was studied by the use of mixed glial, enriched oligodendrocyte and enriched astrocyte cultures. Following MHV-A59 infection in vitro, approximately 10% of oligodendrocytes and 30% of astrocytes expressed viral antigens in the absence of overt cytopathic effect. All cultures released infectious virus for the lifetime of the cultures, for at least 45 days in the case of mixed glial cultures. Cultures derived from previously infected mice were similar to those infected in vitro with respect to percentage of cells expressing viral antigen and levels of infectious virus produced. These results show (1) that glial cells are early sites of infection in vivo as well as sites of infection in vitro cultures, and (2) that glial cells support a non-lytic but productive infection in vitro and thus may contribute to viral persistence in vivo.

Animals↗
PubMed2001

Epstein-Barr virus detection in non-Hodgkin's lymphoma of the oral cavity: an immunocytochemical and in situ hybridization study.

OBJECTIVE: The purpose of this study was to histologically characterize a series of oral non-Hodgkin's lymphomas (NHLs) and to investigate latent and lytic Epstein-Barr virus (EBV) infection in these. STUDY DESIGN: The revised European-American Lymphoma classification system (41) was used to categorize 58 cases of oral NHL, which included 9 immunosuppression-related NHLs. EBV infection was determined by in situ hybridization for Epstein-Barr virus-encoded RNA and by immunohistochemistry for the EBV antigens latency membrane protein, Epstein-Barr nuclear antigen-2 (EBNA2) and Z EBV replication activator protein. RESULTS: Most tumors were B-cell lymphomas (78%), but the proportion of T-cell lymphomas was surprisingly high (22%). The most common histologic subtypes were diffuse large B-cell lymphomas (45%), peripheral T-cell lymphomas (19%), and follicle center lymphomas (14%). Two thirds of the known immunosuppression-related NHLs were T-cell lymphomas. All of the immunosuppression-related tumors were EBV-infected, whereas the EBV infection rate in the NHLs of the remaining patients presumed to be immunocompetent was only 9%. Most EBV-positive tumors expressed neither of the latent antigens (ie, latency membrane protein and Epstein-Barr nuclear antigen-2), and coexpression of the 2 was observed only in immunosuppressed patients. Z EBV replication activator protein expression, which is indicative of replicative infection, occurred only in immunosuppressed individuals. CONCLUSIONS: Diffuse large B-cell lymphomas were the most common histologic subtype of oral NHLs, but T-cell lymphomas were relatively common and frequently occurred in states of immunosuppression. EBV may play a limited role in the initiation of lymphoma in the immunocompetent patient, but the virus may be of importance in progression of the disease in those patients with more aggressive tumors, as immunosuppression occurs.

Adult↗
PubMed1984

Construction and properties of a ribosome-binding site mutation in gene E of phi X174 bacteriophage.

Oligodeoxyribonucleotide mutagenesis has been used to produce a G----A mutation at nucleotide 557 of the phi X174 genome. This changes the ribosome-binding sequence GAGG of gene E to GAAG without affecting the amino acid, glutamine, encoded by the overlapping gene D. The phi X174rb(E)557 mutant does not lyse infected Escherichia coli C and therefore results in the accumulation of a large number intracellular mature phage particles. Thus, the mutation inactivates production of the gene E lytic product, presumably by blocking translation of gene E, without affecting other phage functions.

Bacteriophage phi X 174↗
PubMed2000

Efficient lytic infection of human arterial endothelial cells by human cytomegalovirus strains.

Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) in vivo during acute disease. However, studies of HCMV-EC interactions in vitro have generated discordant results. While lytic infection of cultured venous EC has been well established, Fish et al. (K. N. Fish, C. Soderberg Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson, J. Virol. 72:5661-5668) have reported noncytopathic persistence of the virus in cultured aortic EC. We propose that interstrain differences in viral host cell tropism rather than the vascular bed of origin of infected EC might account for these discrepancies. In the present investigation we compared the responses of EC derived from human adult iliac artery, placental microvasculature, and umbilical vein to infection with various HCMV strains. Regardless of the vascular bed of origin, infection with EC-propagated HCMV strains induced 100% efficient cytopathic change progressing to complete lysis of inoculated monolayers. While fibroblast-propagated strains persisted at low titer in infected arterial EC cultures, they were also cytolytic for individual infected cells. The finding of cytopathic lytic infection of arterial EC by HCMV implicates a mechanism of vascular injury in the pathogenesis of HCMV infection.

Adult↗