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At least 919 records · Page 51Linked to original sources
PubMed1987

Persistent infection of Vero cells by paramyxoviruses. A morphological and immunoelectron microscopic investigation.

Two Vero cell lines persistently infected with canine distemper virus (CDV) or with both CDV and canine parainfluenza (CPI) viruses were investigated. Cells in the CPI-CDV cell line were 90-100% positive for CPI antigen and exhibited 10-80% CPI hemadsorption. Cytoplasmic CDV antigen expressed in both singly and dually infected monolayers varied weekly from 1 to 100%. Numerous cytolytic crises were observed in both cell lines. Cell replication was severely depressed in both cell lines when compared with uninfected Vero cells. Infrequent interfering activity against lytic CPI virus was present in the CPI-CDV cell line but not between lytic CDV and progeny virus from the CDV or the CPI-CDV cell line. Ultrastructurally, Vero cells persistently infected with both paramyxoviruses contained two types of viral nucleocapsids (NC). By immunoelectron microscopy, smooth NC were identified as CPI virus and rough NC were of CDV origin. The viral NC never intermingled but rather were restricted to discrete cytoplasmic areas containing either one type of NC or the other.

Animals↗
PubMed1989

Interactions of minute virus of mice and adenovirus with host nucleoli.

Biochemical evidence is presented that both minute virus of mice (MVM) and adenovirus interact with the nucleolus during lytic growth and that MVM can also target specific changes involving nucleolar components in adenovirus-infected cells. These virus-nucleolus interactions were studied by analysis of intranuclear compartmentalization of both viral DNAs and host nucleolar proteins: (i) MVM in mouse cells (its normal host) replicates its DNA in the host nucleoli; (ii) specific nucleolar proteins as well as small nuclear ribonucleoprotein antigens are recompartmentalized to multiple intranuclear foci in adenovirus-infected HeLa cells; and (iii) when adenovirus helps MVM DNA replication in a nonpermissive human cell (HeLa), the MVM DNA is also recompartmentalized for synthesis. The data suggest mechanisms for disruption of nucleolar function common to oncogenic or oncolytic virus lytic growth and cell transformation.

Adenoviridae↗
PubMed2001

Properties of two EBV Mta nuclear export signal sequences.

The Epstein-Barr virus (EBV) Mta protein is a posttranscriptional regulator of EBV lytic gene expression that affects RNA splicing and transport. Mta mediates cytoplasmic accumulation of unspliced EBV replication gene transcripts and shuttles between the nucleus and cytoplasm. Mta contains a recognized leucine-rich, putative nuclear export signal (NES) between aa 227 and 236. Deletion of this signal sequence eliminated shuttling, while mutation of the core LXL motif in the putative NES diminished but did not abolish the ability of Mta to shuttle from donor to recipient cells in a heterokaryon assay. A double mutation of the LXL motif plus an upstream VTL motif eliminated shuttling, suggesting that Mta may have two NES motifs. In confirmation of this, transfer of either the sequence encoding the leucine-rich aa 227-236 motif or that encoding the adjacent hydrophobic aa 218-227 sequence to a GFP-NLS-pyruvate kinase reporter protein conferred the property of cytoplasmic accumulation onto the heterologous protein. Cytoplasmic accumulation of both the aa 225-237 and 218-227 containing reporters was minimal in the presence of the inhibitor leptomycin B, indicating that both motifs mediated Crm-1-dependent export. Mutations in the NES signal sequences abolished the ability of Mta to mediate cytoplasmic accumulation of BALF2 replication gene transcripts. This included mutation of the LXL motif which still showed cytoplasmic shuttling, suggesting that the NES mutations might have additional effects on Mta function. Wild-type Mta co-immunoprecipitated with the splicing factor SC35 and colocalized with SC35 in transfected cells, modifying endogenous SC35 distribution within the nucleus to give more intense, rounded spots. Interestingly, the NES mutant proteins appeared to have altered interactions with the splicing complex, binding more tightly to SC35 in co-immunoprecipitation assays. These observations suggest a linkage between the splicing and export functions of Mta.

Amino Acid Sequence↗
PubMed1994

EBV-immortalized isogenic human B-cell clones exhibit differences in DNA-protein complex formation on the BZLF1 and BRLF1 promoter regions among latent, lytic and TPA-activated cell lines.

The reactivation of Epstein-Barr virus from latency requires the transcriptional induction of the viral encoded lytic cycle initiator gene, BZLF1, and a concomitant switch from OriP to OriLyt directed viral DNA replication. To investigate the role of host cell factors in these events, a series of EBV-immortalized clonal lymphoblastoid cell lines (LCL) were derived from the spontaneous outgrowth of peripheral blood lymphocytes from a single EBV-seropositive individual. We show that the state of virus activation among this family of isogenic clonal LCL differs, suggesting that each B-cell clone expresses a different complement of cellular factors that influence the state of viral activation. As a first step in the identification of factors involved in EBV reactivation, nuclear extracts were prepared from tightly latent, spontaneously replicating and latent LCL treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and sodium butyrate. The extracts were used in gel mobility shift analyses to compare DNA-protein complex formation among a series of target DNA sequences, including OriLyt and promoter sequences from BZLF1 and BRLF1. The DNA-protein complex patterns were reproducible and indistinguishable among extracts obtained from the latent LCL, but differed from those observed in extracts obtained from the spontaneously replicating LCL, particularly in regard to the binding of a CREB protein to the BZLF1 promoter. Moreover, extracts prepared from LCL treated with TPA to induce virus reactivation resulted in the formation of complexes that differed from those prepared from the spontaneously replicating LCL. Taken together, these data suggest that B-cell factors govern the state of viral activation and that EBV may be reactivated by more than one pathway.

B-Lymphocytes↗
PubMed1996

Development of an expression strategy using a lytic phage to trigger explosive plasmid amplification and gene expression.

A novel plasmid-based expression strategy, exploiting two features of lytic bacteriophages, was developed in Lactococcus lactis. Components of this system include a phage origin of replication and phage expression signals, which were induced to high efficiency upon phage infection of the host. Phage-specific expression signals were cloned from phi 31 in a promoter-screening strategy using the lacZ gene from Streptococcus thermophilus. One clone exhibited a significant induction in beta-galactosidase production and concomitant increase in lacZ mRNA during the phi 31 infection cycle of the host. Molecular characterization of the cloned insert revealed 888 bp positioned near the phi 31 cos site. Primer extension analysis showed that transcription was induced approximately 20 min following phi 31 infection at four points, apparently organized in two sets of tandem promoters on the cloned phage insert. One of these middle phage promoters also showed a basal level of activity prior to phage infection. The phi 31 promoter lacZ cassette was cloned into a low-copy-number vector plasmid containing the phi 31 origin of replication (ori31) and the resulting low-copy-number plasmid exhibited negligible beta-galactosidase production in L. lactis. However, > 2,000 units were detected following a deliberate infection with phi 31. A control expression plasmid without ori31 could only be induced to 85 units. The combination of these phage-inducible expression signals together with ori31 functioned synergistically to drive rapid and high efficiency expression of a heterologous gene in L. lactis.

Amino Acid Sequence↗
PubMed2002

K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus.

The murine gamma-herpesvirus-68 (MHV-68) K3 protein, like that of the Kaposi's sarcoma associated herpesvirus, down-regulates major histocompatibility complex (MHC) class I expression. However, how this contributes to viral replication in vivo is unclear. After intranasal MHV-68 infection, K3 was transcribed both during acute lytic infection in the lung and during latency establishment in lymphoid tissue. K3-deficient viruses were not cleared more rapidly from the lung, but the number of latently infected spleen cells was reduced and the frequency of virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) was increased. CTL depletion reversed the viral latency deficit. Thus, a major function of K3 appears to be CTL evasion during viral latency expansion.

3T3 Cells↗
PubMed1998

The Epstein-Barr virus Rta protein activates lytic cycle genes and can disrupt latency in B lymphocytes.

The transition of Epstein-Barr virus (EBV) from latency into the lytic cycle is associated with the expression of two immediate-early viral genes, BZLF1 and BRLF1. Overexpression of ZEBRA, the product of BZLF1, is sufficient to disrupt latency in B lymphocytes and epithelial cells by stimulating expression of lytic cycle genes, including BRLF1. The BRLF1 product Rta functions as a transcriptional activator in both B lymphocytes and epithelial cells. However, Rta has recently been reported to disrupt latency in an epithelial specific manner (S. Zalani, E. Holley-Guthrie, and S. Kenney, Proc. Natl. Acad. Sci. USA 93:9194-9199, 1996). Here we demonstrate that expression of Rta is also sufficient for disruption of latency in a permissive B-cell line. In HH514-16 cells, transfection of Rta leads to synthesis of ZEBRA, viral DNA replication, and late gene expression. However, Rta by itself is less potent than ZEBRA in the ability to activate most early and late lytic cycle genes. In light of previous work implicating ZEBRA in the activation of Rta, we suggest a cooperative model for EBV entry into the lytic cycle. Expression of either BZLF1 or BRLF1 triggers expression of the other immediate-early factor, and together these activators act individually or in synergy on downstream targets to activate the viral lytic cycle.

B-Lymphocytes↗
PubMed2005

Genome replication and progeny virion production of herpes simplex virus type 1 mutants with temperature-sensitive lesions in the origin-binding protein.

Genome replication of herpes simplex viruses (HSV) in cultured cells is thought to be started by the action of the virus-encoded origin-binding protein (OBP). In experiments using two HSV-1 mutants with temperature-sensitive lesions in the helicase domain of OBP, we demonstrated that this function is essential during the first 6 hours of the lytic cycle. Once DNA synthesis has started, this function is no longer required, suggesting that origin-driven initiation of viral DNA replication is a single event rather than a continuous process.

Animals↗
PubMed1987

A colorimetric assay based on cell viability for the indirect detection of intracellular replication and killing of Trypanosoma cruzi.

We have developed an 'in vitro' colorimetric assay for the detection of replication and/or destruction of the intracellular amastigote form of Trypanosoma cruzi. The assay can be applied to other intracellular parasites having lytic effects on the infected cells. The assay is based on the relationship between the content of the lysosomal enzyme hexosaminidase and the number of viable cells. The level of this enzyme can be detected by a simple and sensitive procedure in microtiter wells using a p-nitrophenol derivative as enzyme substrate and scanning the absorbance at 405 nm. Macrophages or other suitable host cells which support T. cruzi intracellular replication have detectable levels of this enzyme whereas the protozoan parasites do not. The assay exhibited a good inverse correlation between the number of amastigotes released and the amount of enzyme in the infected cultures. Furthermore, the method was used for the detection of macrophage-activating factors and gave results similar to those obtained by microscopical examination of the cells. The advantages of the procedure are objectivity, sensitivity and simplicity.

Animals↗
PubMedDate not supplied

A second post-transcriptional trans-activator gene required for HTLV-III replication.

Evidence is provided for the existence of a seventh gene in the genome of human T-lymphotropic virus type III/lymphadenopathy-associated virus. The gene is necessary for replication and acts post-transcriptionally to relieve negative regulation of the messenger RNA for the virion capsid and envelope proteins. These observations suggest mechanisms for regulating both the latent and lytic phases of the virus life cycle.

Cell Line↗
PubMed2006

Differences in the frequency and function of HHV8-specific CD8 T cells between asymptomatic HHV8 infection and Kaposi sarcoma.

It is unclear how the immune response controls human herpesvirus 8 (HHV8; also known as Kaposi sarcoma-associated herpesvirus [KSHV]) replication and thereby prevents Kaposi sarcoma (KS). We compared CD8 T-cell responses to HHV8 latent (K12) and lytic (glycoprotein B, ORF6, ORF61, and ORF65) antigens in patients who spontaneously controlled the infection and in patients with posttransplantation, AIDS-related, or classical KS. We found that anti-HHV8 responses were frequent, diverse, and strongly differentiated toward an effector phenotype in patients who controlled the infection. Conversely, HHV8-specific CD8 cells were very rare in patients who progressed to KS, and were not recruited to the tumoral tissue, as visualized by in situ tetramer staining of KS biopsies. Last, HHV8-specific CD8 T cells were observed in a seronegative recipient of an HHV8infected graft who remained persistently aviremic and antibody negative, suggesting that specific cytotoxic T lymphocytes (CTLs) may provide protection from persistent HHV8 infection. These results support the crucial role of cellular immune responses in controlling HHV8 replication, in preventing malignancies in latently infected subjects, and in conferring genuine resistance to persistent infection. They may also have important implications for the design of prophylactic and therapeutic HHV8 vaccines, and for adoptive immunotherapy of KS.

Acquired Immunodeficiency Syndrome↗
PubMed1981

Phenotypic characterization of mice of thymus target cells susceptible to productive infection by the radiation leukemia virus.

The spread of virus replication was studied by electron microscopy in the thymuses of inbred C57BL/Ka mice after intrathymic inoculation of the radiation leukemia virus (RadLV). The first type C-budding virus particles appeared in scarce blast cells of the subcapsular zone. Most of these blast cells were "X-cells," i.e., the thymus lymphoid cells most actively engaged in DNA synthesis. Virus replication spread to the entire cortical blast cell population and, from day 7 on, to the small cortical lymphocytes. The first virus-producing cells were derived from a very few target cells (approximately 0.001-0.003% of thymocytes) susceptible to RadLV infection. For determination of the phenotypes of these target cells, various thymocyte subpopulations obtained through a battery of cell separation methods were tested for their ability to support the replication of RadLV/VL3 virus in short-term culture. Most of these target cells were sensitive to the lytic effect of hydrocortisone and migrated in the fastest fraction of a 1Xg sedimentation gradient, together with the majority of [3H] thymidine-incorporating blast cells. They exhibited an intermediate density and expressed H-2 and Thy 1,2 cell surface antigens, although they were not found preferentially among the high Thy 1,2 population to which most of the cortical blast cells belonged. The spread of RadLV within the thymus and the surface phenotype characteristics of target cells indicate that these cells correspond to the thymocyte subset at the earliest stage of thymic lymphopoiesis and may be transitional between the prothymocytes and the subcapsular blast cell population.

Animals↗
PubMed2001

A classification scheme for human polyomavirus JCV variants based on the nucleotide sequence of the noncoding regulatory region.

The human polyomavirus JCV is responsible for the central nervous system (CNS) demyelination observed in cases of progressive multifocal leukoencephalopathy (PML). Lytic infection of oligodendrocytes, the cells that constitute the basis of myelin in the CNS, is established by JCV in conjunction with immunosuppressive conditions. Beyond this, however, many questions related to JCV pathogenesis remain unanswered. The JCV regulatory region is a hypervariable noncoding sequence positioned between the early and late protein-coding regions. The particular nucleotide sequence of a JCV regulatory region affects levels of viral transcription and replication. Modifications to this promoter/enhancer structure can alter the cellular host range and may be responsible for switching JCV between states of lytic and latent infection. The regulatory region structure has, therefore, been used to distinguish JCV variants. Nucleotide sequencing studies have uncovered numerous variations of regulatory region structure. Until now, however, no inclusive nomenclature existed that linked variants by regulatory region structure and/or activity. We have arranged all known variant JCV regulatory regions into quadrants according to the integration of particular sequence sections and repetition of sequence section groups. This arrangement of regulatory regions results in an updated nomenclature that is well-suited for describing the relationships between JCV variants. Four distinct structural forms (I-S, I-R, II-S, and II-R) are defined along with tissue tropisms. This design provides logical connections between the variant regulatory regions and may be useful for elucidating crucial steps in JCV pathogenesis.

DNA, Viral↗
PubMed2005

Marked variation in response of consensus binding elements for the Rta protein of Epstein-Barr virus.

The R transactivator (Rta) protein activates Epstein-Barr virus (EBV) lytic-cycle genes by several distinct mechanisms that include direct binding to viral promoters, synergy with BamHI Z EBV replication activator (ZEBRA), and activation of cellular signaling pathways. In the direct and synergistic mechanisms of action, Rta binds to specific DNA sequences that are present in the promoters of responsive genes. It has been difficult to demonstrate the capacity of Rta expressed in mammalian cells to bind DNA in vitro in order to study the relative affinities of Rta binding elements. We discovered that a short C-terminal region of Rta inhibits the ability of Rta to bind DNA in vitro. C-terminally truncated versions of Rta bind DNA efficiently and thus facilitate a comparison of consensus Rta binding elements (CRBEs) found in promoters of five Rta-responsive genes: BMLF1, BHLF1, BMRF1, BaRF1, and BLRF2. All CRBEs in the promoters of the five genes conform to the proposed recognition sequence GNCCN9GGNG, where N is any nucleotide and N9 represents a sequence of nine nucleotides. Nonetheless, CRBEs varied markedly in their abilities to bind Rta in electrophoretic mobility shift assays. Not all CRBEs bound or responded to Rta. Binding affinities of the CRBEs and the capacity to be activated by Rta in reporter assays were strongly correlated. The CRBEs from the BMLF1 and BHLF1 promoters conferred the greatest response. The response of the BMRF1, BaRF1, and BLRF2 CRBEs was less robust. By creation of chimeras, inversions, and point mutations, differences in binding affinities and transcriptional activation levels could be attributed to N9 sequence variation. The length of N9 was also critical for a maximal response. In Raji and BZLF1-knockout cells, the mRNAs of the five Rta-responsive lytic-cycle genes differed dramatically in kinetics of expression, abundance, and synergistic responses to ZEBRA and Rta. Affinities of Rta response elements for Rta are likely to play an important role in temporal regulation and the level of lytic-cycle EBV gene expression.

Binding Sites↗
PubMed1990

trans activation of the thymidylate synthase promoter of herpesvirus saimiri.

Herpesvirus saimiri has been shown to possess a thymidylate synthase (TS) gene that is unusual in its transcriptional regulation. Although TS is believed to be required for viral DNA synthesis, the TS-specific 2.5-kb mRNA was found most abundantly during the late phases of asynchronous virus replication in permissive cultures. To study the kinetics of gene activation, the TS promoter and regulatory sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. No CAT expression or transcripts were found after transfection of fusion genes into permissive owl monkey kidney (OMK) cells. However, the promoter was strongly activated when CAT plasmids were cotransfected with intact herpesvirus saimiri virion DNA or were transferred to OMK cells that were lytically infected with herpesvirus saimiri or a related herpesvirus, herpesvirus ateles. CAT was expressed at reduced levels in cultures when viral DNA replication was inhibited by phosphonoacetic acid; this indicates that the gene is activated during the delayed-early phase. However, the highest amounts of mRNA were present in the late period of replication. Deletion analyses localized essential response elements for trans activation in the promoter upstream region between nucleotides -311 and -56; they consisted of related tandem repeats and perfect palindromes. A sequence with two overlapping palindromes of 16 and 18 bp was found to be a major target for activation of the herpesvirus saimiri TS promoter. These palindromes did not have any significant homologies with known sequences of herpesviruses or cellular DNA; the 18-bp palindrome had, however, a certain structural similarity with a conserved sequence of the E2-responsive cis sequence that is required for transcription regulation of early papillomavirus genes.

Animals↗
PubMed2005

Topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm.

Infection of mammalian cells with several positive-strand RNA viruses induces double-membraned vesicles whose cytosolic surfaces serve as platforms for viral RNA replication. Our recent publication (Jackson et al. PLoS Biol 2005; 3:861-71) chronicled several similarities between poliovirus-induced membranes and autophagosomes, including induced co-localization of GFP-LC3 and LAMP1. Occasionally, the cytosolic lumen of these structures also contains viral particles; this likely results from wrapping of cytosol, which can contain high viral concentrations late in infection, by newly formed double membranes. Interestingly, RNAi treatment to reduce LC3 or Atg12p concentrations reduced yields of extracellular virus even more than intracellular virus. It is often assumed that exit of non-enveloped viruses such as poliovirus requires cell lysis. However, we hypothesize that autophagosome-like double-membranes, which can become single-membraned upon maturation, provide a long-sought mechanism for the observed non-lytic release of cytoplasmic viruses and possibly other cytoplasmic material resistant to the environment of maturing autophagosomes.

Animals↗
PubMed2000

Lytic infection by double-strand DNA viruses and cell cycle alterations.

Polyomaviruses, papillomaviruses, adenoviruses and herpesviruses are double-stranded DNA viruses that replicate in the nucleus of the cells they infect and have evolved various strategies to create a cellular environment that is optimally conducive to their replication. One of these strategies consists of activating cellular genes, mostly S-phase genes that are required for the replication of the viral genome. Concomitantly, they encode one or several proteins that negatively regulate the response of the cell to viral infection, notably cell cycle arrest and/or apoptosis. As a result, these viruses profoundly alter the biochemical pathways that normally control cellular growth, and may thus promote uncontrolled cell proliferation. This review describes some well-known mechanisms of cell cycle alteration induced by these viruses.

Adenoviridae Infections↗
PubMed2003

Interaction of EBV latent origin of replication with the nuclear matrix: identification of S/MAR sequences and protein components.

During latency, Epstein Barr virus (EBV) genome, as an episome, is attached to the nuclear matrix (NM) via the latent origin of replication ori P. Within this element, we have found that a region, 580 bp long, encompassing the replicator DS element, shows the strongest affinity for the NM. In addition, by cross-linking with cis-diamminedichloroplatinum, we have identified two NM proteins with an apparent molecular weight of 85 and 60 kDa that, with high affinity and specificity, bind ori P. These proteins are not induced by EBV infection, but their interaction with ori P is lost upon induction of EBV lytic cycle. These data strongly suggest that the binding of ori P to specific components of the NM is required for EBV latent replication.

Binding Sites↗