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PubMed1981

Physical mapping of the genomes of lytic and temperate forms of phage theta.

Restriction maps of genomes of the lytic form and diverse temperate mutants of phage theta of Bacillus licheniformis were constructed. Most temperate mutants produced fragmentation patterns identical to that of the parent lytic form, theta c: in other mutants the only detectable change in the map was the deletion of a Bg/II restriction endonuclease site at 46.5% genome length. In the genomes of two other temperate mutants, theta 1 and theta 2, the central part of the genome was replaced by a piece of DNA of equal length, but with a different distribution of restriction sites; the maps of the two mutants failed to reveal any similarity in the location of restriction sites in the inserted DNA. It seems that any alteration comprising the locus around the coordinate 46.5% of the theta c genome, brings about a transition from the lytic to temperate phenotype, indicating the position of a regulatory gene responsible for positive control of phage replication.

Bacillus↗
PubMed1990

Interleukin 7 enhances cytolytic T lymphocyte generation and induces lymphokine-activated killer cells from human peripheral blood.

The effects of purified recombinant interleukin 7 (IL-7) on the generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) and on the induction of lymphokine-activated killer (LAK) cells in autologous cultures of human peripheral blood mononuclear cells were investigated. IL-7 was found to induce the generation of both CTL and LAK cells in bulk cultures. The appearance of peak CTL activity in MLC established with exogenous IL-7 was delayed in comparison with replicate cultures containing exogenous IL-2, but both cytokines stimulated quantitatively similar levels of antigen-specific lytic activity. An IL-2-neutralizing antiserum inhibited substantially, but not completely, the effect of IL-7 on CTL generation, implying the existence of both an indirect component of IL-7 activity via IL-2 utilization, as well as an IL-2-independent component. Cell surface phenotypic analysis of IL-2- or IL-7-generated CTL effector cells revealed that CD8+ cells were responsible for the vast majority of lytic activity. Limiting dilution analysis (LDA) revealed that essentially identical frequencies of CTL precursors (CTL-P) were capable of clonal expansion and/or differentiation in the presence of exogenous IL-2, IL-4, or IL-7, supporting the concept that all three of these cytokines are capable of exerting a major influence on T cell growth and differentiation. Approximately half of the CTL-P that responded in IL-7-supplemented LDA cultures did so in an IL-2-independent manner. IL-7 stimulated the development of LAK cells in autologous bulk cultures, but only weakly in comparison with IL-2. In contrast to its effects on CTL generation, the induction of LAK cells by IL-7 was virtually independent of IL-2. LAK cells induced by IL-7, like those induced by IL-2, were phenotypically heterogeneous and included CD8+, CD56+, and gamma/delta+ cells. Limiting dilution analysis indicated that IL-2 stimulated fivefold more LAK-P than IL-7 and 220-fold more than IL-4. Collectively, these data suggest that IL-7 has potent regulatory effects on human cytolytic cell populations and, either alone or in combination with other cytokines, could be important for the in vitro expansion of cells for adoptive immunotherapy.

Antigens, CD↗
PubMed2004

Dual infections of the central nervous system with Epstein-Barr virus.

We describe clinical and laboratory characteristics of 16 patients with central nervous system (CNS) infection caused by Epstein-Barr virus (EBV) and another pathogen. Seven of 10 immunocompromised patients had coinfection with viruses (3 with cytomegalovirus, 2 with JC virus, and 2 with varicella zoster virus) and 3 with nonviral pathogens (2 with pneumococcus and 1 with Cryptococcus species). Three of 6 immunocompetent patients had coinfections with viruses (1 each with herpes simplex virus, varicella zoster virus, and West Nile virus), and 3 had coinfections with nonviral pathogens (2 with Ehrlichia chaffeensis and 1 with Mycoplasma pneumoniae). The EBV load was similar in immunocompromised and immunocompetent patients and in patients with viral and nonviral coinfections. EBV lytic-cycle mRNA was detected in the cerebrospinal fluid of 5 of 6 tested samples, indicating EBV replication in the CNS during coinfection.

Adult↗
PubMed1996

Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy.

Active cell death in hormone-dependent cells was studied using cultured human mammary carcinoma cells (MCF-7) treated with the anti-estrogens (AEs) tamoxifen (TAM), 4-hydroxy-tamoxifen (OH-TAM) or ICI 164 384 (10(-8)-10(-5) M) as a model. The following results were obtained. (i) In untreated MCF-7 cells a wave of replication occurred in the first 5 days of culture. All three AEs caused a dose-dependent inhibition of cell replication. (ii) TAM and OH-TAM at 10(-5) M, but not ICI 164 384, caused lytic cell death (necrosis) within 24 h, which was not inhibited by estradiol (10(-9)-10(-6)M). (iii) Lower concentrations of TAM or OH-TAM (up to 10(-6) M) or ICI 164 384 induced a more gradual appearance of cell death beginning at day 3. This type of cell death was inhibited by estradiol (10(-9) M), indicating its active nature. (iv) Nuclei showed two distinct patterns of alteration: (a) apoptosis-like condensation and fragmentation of chromatin to crescent masses abutting the nuclear envelope; (b) condensation of the chromatin to a single, pyknotic mass in the center of the nucleus, detached from the nuclear envelope. Quantitative histological evaluation revealed the predominance of pyknosis. (v) Biochemical DNA analysis revealed that only a relatively small amount of the total DNA was finally degraded into low molecular weight fragments (20 kb and less). (vi) Active cell death, with both apoptotic and pyknotic nuclear morphology, was associated with extensive formation of autophagic vacuoles (AV).3-Methyladenine, a known inhibitor of AV formation, partially prevented cell death as detected by nuclear changes. (vii) ICI 164 384 was about 10 times more effective than TAM or OH-TAM at inhibiting DNA synthesis, but had equal potency in inducing active cell death. It is concluded that AEs have anti-proliferative and anti-survival effects on MCF-7 human mammary cancer cells in culture. These two effects are under separate control because they differ by kinetics, dose dependence and sensitivity to the various AEs. Active cell death in MCF-7 cells seems to be initiated by autophagy, in contrast to concepts of apoptosis, and thus corresponds to autophagic/ lysosomal or type II death as previously defined. This may be important because of biochemical and molecular differences between these various subtypes of active cell death.

Autophagy↗
PubMed2005

Vaccinia virus and oncolytic virotherapy of cancer.

Vaccinia viruses possess many of the key attributes necessary for an ideal viral backbone for use in oncolytic virotherapy. These include a short lifecycle, with rapid cell-to-cell spread. strong lytic ability, a large cloning capacity and well-defined molecular biology. In addition, although capable of replicating in human cells, they are not considered a natural health problem and are especially well characterized. having been delivered to millions of individuals during the campaign to eradicate smallpox. A variety of tumor-targeting mutations have been described in several different vaccinia strains and the expression of a variety of different transgenes has been studied. Early clinical results using either vaccine strains or genetically modified vaccinia strains have demonstrated antitumor effects. Future prospects for the development of these viruses will be discussed.

Antineoplastic Agents↗
PubMed2000

Mengovirus and encephalomyocarditis virus poly(C) tract lengths can affect virus growth in murine cell culture.

Many virulent aphthoviruses and cardioviruses have long homopolymeric poly(C) tracts in the 5' untranslated regions of their RNA genomes. A panel of genetically engineered mengo-type cardioviruses has been described which contain a variety of different poly(C) tract lengths. Studies of these viruses have shown the poly(C) tract to be dispensable for growth in HeLa cells, although the relative murine virulence of the viruses correlates directly and positively with tract length. Compared with wild-type mengovirus strain M, mutants with shortened poly(C) tracts grow poorly in mice and protectively immunize rather than kill recipient animals. In the present study, several murine cell populations were tested to determine whether, unlike HeLa cells, they allowed a differential amplification of viruses with long or short poly(C) tracts. Replication and cytopathic studies with four hematopoietically derived cell lines (CH2B, RAW 264.7, A20.J, and P815) and two murine fibroblast cell lines [L929 and L(Y)] demonstrated that several of these cell types indeed allowed differential virus replication as a function of viral poly(C) tract length. Among the most discerning of these cells, RAW 264.7 macrophages supported vigorous lytic growth of a long-tract virus, vMwt (C(44)UC(10)), but supported only substantially diminished and virtually nonlytic growth of vMC(24) (C(13)UC(10)) and vMC(0) short-tract viruses. The viral growth differences evident in all cell lines were apparent early and continuously during every cycle of virus amplification. The data suggest that poly(C) tract-dependent attenuation of mengovirus may be due in part to a viral replication defect manifest in similar hematopoietic-type cells shortly after murine infection. The characterized cultures should provide excellent tools for molecular study of poly(C) tract-mediated virulence.

Animals↗
PubMed1988

Activation of Epstein-Barr virus in latently infected cell lines.

Several methods that can switch between latency and replication of Epstein-Barr virus (EBV) have been developed. These methods made it possible to identify and characterize the majority of virus proteins associated with the virus replication and to develop new assays for diagnosis. A possible activator protein encoded from the BamHI Z fragment of EBV genome, which can disrupt latency in EBV-genome positive cell lines, has also been identified. The developments in activation of the virus lytic cycle will be reviewed in this paper.

Cell Line↗
PubMed1979

Role of virus variants and cells in maintenance of persistent infection by measles virus.

Hamster embryo fibroblasts persistently infected with a derivative of the Schwarz vaccine strain of measles virus spontaneously released virus particles with an average buoyant density considerably lower than that of the parental virus. The released virus contained all of the measles virus structural proteins and interfered with replication of standard virus. All of the virus structural proteins were associated with a membrane-free cytoplasmic extract from the persistently infected cells. Membrane-free cytoplasmic extracts prepared from Vero cells lytically infected with Schwarz strain measles contained little or no virus envelope structural protein. Maintenance of persistent infection may involve both the presence of virus variants and a defect in the ability of the infected cell to replicate the virus efficiently.

Animals↗
PubMed1993

Replicative viral DNA in Epstein-Barr virus associated Burkitt's lymphoma biopsies.

Epstein-Barr virus (EBV) is linked to a spectrum of human diseases including epithelial and lymphoid malignancies in which it exists predominantly in a latent state. EBV is capable of establishing replicative infection at oropharyngeal and genital sites. Replicative EBV infection also occurs in oral hairy leukoplakia, in EBV associated lymphoproliferative disorders, and to a minor degree in nasopharyngeal carcinomas. Recent evidence also suggests that EBV replication, also, may be associated with AIDS related lymphomas and Hodgkin's disease. However it is widely believed that virus in circulating B-lymphocytes and in B-cell malignancies is stringently latent. We now show that by Southern blot analysis we can detect replicative forms of virion DNA in 14.5% (8 of 55) of EBV-positive Burkitt's lymphoma biopsies. This may be the explanation for the elevation of the titres of lytic cycle EBV antigens that is associated with presentation and relapse of EBV associated Burkitt's lymphoma.

Biopsy↗
PubMed1999

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1.

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVDeltapac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVDeltapac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVDeltapac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVDeltapac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVDeltapac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD(50). Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Animals↗
PubMedDate not supplied

Conditionally replicating adenovirus-mediated gene therapy in bladder cancer: an orthotopic in vivo model.

INTRODUCTION: The effects of a conditionally replicating adenovirus on various bladder cancer lines were explored, a truncated bone sialoprotein (BSP) promoter controlling the E1a/b lytic-regulating sequence was used, since BSP protein is found in many osteotropic neoplasms, including bladder cancer. METHODS: Reverse transcriptase polymerase chain reaction analysis was used to determine expression patterns of BSP and Coxsackie adenovirus receptor, a receptor known to interact with adenovirus, on multiple lines of bladder cancer (253J, 253J B-V, RT4, transitional cell carcinoma, T24, UMUC3, and WH). Ad-BSP-E1a was tested in vitro for lytic activity on 4 of these cell lines. The 253J B-V cell line was used and inoculated into female nude mice either subcutaneously in the flank or orthotopically into the bladder, and treated with control or Ad-BSP-E1a virus. RESULTS: BSP is expressed in RT4, transitional cell carcinoma, and WH. Meanwhile, Coxsackie adenovirus receptor was expressed in all lines except T24. Ad-BSP-E1a had the most impact on 253J and 253J B-V cells; cell density declined significantly when compared to phosphate-buffered saline and Ad-BSP-TK "dummy" virus-treatment groups. The 253J B-V tumors treated with Ad-BSP-E1a revealed a decreased percent change of size in the subcutaneous model when compared to controls at week 3. The orthotopic murine model showed decreased end tumor mass in the Ad-BSP-E1a treated group over controls. Histologic examination of in vivo tumors showed evidence of fibrosis and apoptosis in the Ad-BSP-E1a treated groups using hematoxylin-eosin, trichrome, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining. Control groups only had viable tumor in in vivo models. CONCLUSION: Adenovirus therapy of orthotopic murine bladder tumors is feasible. Ad-BSP-E1a is effective in treating very aggressive yet sensitive bladder tumor cells. Further study of this conditionally replicating adenovirus treatment (Ad-BSP-E1a) with chemotherapeutic combination is warranted, and future translation of such combination therapy into human beings is a possibility.

Adenoviridae↗
PubMed2003

Age-related increase in resistance to acute Trypanosoma cruzi infection in rats is associated with an appropriate antibody response.

Inoculation at weaning with Trypanosoma cruzi in inbred "l" rats resulted in a self-resolving acute infection characterized by marked parasitaemias, whereas challenge to adult rats revealed a mild disease with extremely low parasitaemias. To explore the mechanisms underlying such age-associated differences in disease outcome, we analysed the in vitro replication of T. cruzi, nitric oxide and tumour necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages (PMs), the serum concentrations of the specific immunoglobulins (Igs) IgM and IgG, antibodies exhibiting lytic activity against bloodstream forms of T. cruzi and circulating levels of nitrate, TNF-alpha and interferon-gamma (IFN-gamma). Macrophages from young rats were as effective as their adult counterparts for restraining intracellular parasite replication. When stimulated with IFN-gamma, culture supernatants from young PMs contained higher amounts of nitrite and TNF-alpha. Serum samples from 4 and 7 days post infection revealed easily detectable amounts of nitrate, with values being further augmented by day 7 post infection and significantly higher in the young group. TNF-alpha levels were only detected in the young group by day 7 post infection. Both groups had increased amounts of IFN-gamma in their sera, although in adult rats, this trend was followed by a significant drop at day 7, with young rats showing values still higher by the same time point evaluation. In contrast, young rats presented significantly lower levels of IgM and IgG antibodies during the first week of infection. Increased resistance in adult rats seems to be the result of a more appropriate antibody production.

Age Factors↗
PubMed2005

Oncolytic virotherapy: approaches to tumor targeting and enhancing antitumor effects.

The application of replicating viruses for the treatment of cancers represents a novel therapy that is distinct from traditional treatment modalities. It is apparent that the genetic changes that a virus produces within an infected cell in order to create an environment conducive to viral replication are often similar to the processes involved in cellular transformation. These include uncontrolled cellular proliferation, prevention of apoptosis, and resistance to host organism immune effector mechanisms. Deletions of viral genes involved in these processes have been exploited to produce viral mutants whose replication is selective for transformed cells. The use of tissue-specific transcriptional response or RNA stability elements to control the expression of critical viral genes has also resulted in targeted viruses. Work also is being undertaken to restrict or alter the tropism of viruses by altering their ability to infect certain cell types. Finally, the addition of exogenous genes can be used to increase the virus's lytic potential and/or bystander killing; to further induce the host's immune response against cancer cells; and/or to permit the controlled downregulation of viral replication if necessary. The combination of different tumor-targeting mutations in parallel with the expression of foreign genes has resulted in the evolution of second- and third-generation viruses that continue to become further distinct from their native parental strains. The movement of these viruses into the clinic has begun to demonstrate the potential of this approach in the treatment of cancers.

Apoptosis↗
PubMed2006

The BZLF1 homolog of an Epstein-Barr-related gamma-herpesvirus is a frequent target of the CTL response in persistently infected rhesus macaques.

Although CD8(+) T lymphocytes targeting lytic infection proteins dominate the immune response to acute and persistent EBV infection, their role in immune control of EBV replication is not known. Rhesus lymphocryptovirus (rhLCV) is a gamma-herpesvirus closely related to EBV, which establishes persistent infection in rhesus macaques. In this study, we investigated cellular immune responses to the rhLCV BZLF1 (rhBZLF1) homolog in a cohort of rhLCV-seropositive rhesus macaques. rhBZLF1-specific IFN-gamma ELISPOT responses ranging between 56 and 3070 spot-forming cells/10(6) PBMC were detected in 36 of 57 (63%) rhesus macaques and were largely mediated by CD8(+) T lymphocytes. The prevalence and magnitude of ELISPOT responses were greater in adult (5-15 years of age) rather than juvenile macaques (<5 years of age), suggesting that rhBZLF1-specific CTL increase over time following early primary infection. A highly immunogenic region in the carboxyl terminus of the rhBZLF1 protein containing overlapping CTL epitopes restricted by Mamu-A*01 and other as yet unidentified MHC class I alleles was identified. The presence of a robust CD8(+) T lymphocyte response targeting this lytic infection protein in both rhesus macaques and humans suggests that these CTL may be important for immune control of EBV-related gamma-herpesvirus infection. These data underscore the utility of the rhLCV-macaque model for studies of EBV pathogenesis.

Amino Acid Sequence↗
PubMed2005

KSHV viral cyclin inactivates p27KIP1 through Ser10 and Thr187 phosphorylation in proliferating primary effusion lymphomas.

Kaposi sarcoma herpesvirus (KSHV) infection is consistently associated with primary effusion lymphomas (PELs) that are non-Hodgkin lymphomas of B-cell origin. All PEL cells are latently infected with KSHV and express latent viral proteins such as the viral cyclin (v-cyclin), which has previously been implicated in down-regulation of cell-cycle inhibitor p27(KIP1) levels via phosphorylation on Thr187. PEL cells retain high levels of p27(KIP1) but yet proliferate actively, which has left the biologic significance of this p27(KIP1) destabilization somewhat elusive. We have recently demonstrated that v-cyclin and p27(KIP1) stably associate in PEL cells. Here we demonstrate that v-cyclin together with its kinase partner CDK6 phosphorylates the associated p27(KIP1) in PEL cells, which represent a biologically relevant model system for KSHV pathobiology. During latent viral replication p27(KIP1) was phosphorylated by v-cyclin-CDK6 predominantly on Ser10, which enhances its cytoplasmic localization. Interestingly, upon reactivation of KSHV lytic cycle, v-cyclin-CDK6 phosphorylated p27(KIP1) on Thr187, which resulted in down-regulation of p27(KIP1) protein levels. These findings indicate that v-cyclin modulates the cell-cycle inhibitory function of p27(KIP1) by phosphorylation in PELs, and also suggest a novel role for v-cyclin in the lytic reactivation of KSHV.

Cell Proliferation↗
PubMed1987

The AT-rich sequence of the SV40 control region influences the binding of SV40 T antigen to binding sites II and III.

During lytic infection SV40 T antigen binds specifically to three different regions of the SV40 DNA to initiate DNA replication and to regulate early and late transcription. We constructed plasmids containing either 23-bp synthetic oligonucleotides representing site I or II or SV40 DNA fragments with combinations of binding sites II and III with or without SV40 specific flanking regions. These plasmids were used to determine which sequences are sufficient for specific binding to isolated regions II and III. Under identical conditions T antigen bound in a sensitive in vitro binding assay efficiently to site I but not to the corresponding oligonucleotide of site II. Binding to site II could only be observed in the presence of the adjacent 17-bp AT-rich region of the SV40 DNA. On account of the markedly low affinity for binding site II, T antigen concentrations were required which exceeded those necessary to achieve saturation of binding to site I. The very low affinity for isolated site III could be slightly raised by the same AT-rich region. An increased binding to site II at 37 degrees compared to 0 degree in the presence of this region points to an indirect influence on the DNA structure of the binding site.

Animals↗
PubMed2003

The persistence of bovine viral diarrhea virus.

Bovine viral diarrhoea virus (BVDV) has a unique capacity to cause persistent infections of foetuses exposed within the first 150 days of gestation. Preventing foetal BVDV infection will aid in improved control. This unique ability gives BVDV a selective advantage allowing continual mutation and antigenic variation within cattle populations. Therefore, BVDV has become widespread and causes economic losses due to respiratory, reproductive and enteric disease. Vaccination (modified-live or killed) can provide some protection from acute disease and the development of persistently infected foetuses. However, vaccination programmes alone cannot control or eliminate BVDV. In naturally exposed and vaccinated herds, BVDV infections are not self-limiting and may persistent over time. This underscores the ability of the BVDV genome to remain fluid and adapt under selective pressures. Factors influencing persistence of BVDV infections in cattle populations include: non-lytic infections; evasion of host immune responses; foetal infections; acute infections; management practices; contaminated biologics; secondary hosts; defective replicated intermediates; antigenic variation; and replication in privileged anatomical sites.

Animals↗
PubMed1998

Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein.

The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H. The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints. The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).

Amino Acid Sequence↗