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Biomedical subjects

F Rapp

Publications and source records attributed to F Rapp.

At least 73 records · Page 4Linked to original sources
PubMed1982

High efficiency latency and activation of herpes simplex virus in human cells.

Herpes simplex virus (HSV) exists in humans in a latent form that can be activated. To characterize the molecular basis of the cell-virus interactions and to analyze the state of the latent HSV genome, an in vitro model system was established. In this system a large fraction of the latently infected cells contain an HSV genome that can be activated. Cell survival was reduced minimally after repression of high multiplicity HSV type 1 (HSV-1) infection of human fibroblast cells with (E)-5-(2-bromovinyl)-2'-deoxyuridine in combination with human leukocyte interferon (IFN-alpha). A minimum of 1 to 3 percent of the surviving cells contained an HSV genome that could be activated either by human cytomegalovirus superinfection or reduction in incubation temperature.

Bromodeoxyuridine↗
PubMed1982

Activation of herpes simplex virus (HSV) type 1 genome by temperature-sensitive mutants of HSV type 2.

Previous studies have demonstrated that herpes simplex viruses (HSV) type 1 (HSV-1) and type 2 (HSV-2) can be maintained in a repressed form in human embryo lung cells. Reducing the incubation temperature or superinfecting with a heterologous herpesvirus, human cytomegalovirus (HCMV), results in activation of virus replication. We now report that superinfection with a partially homologous herpesvirus, HSV-2, also resulted in activation of HSV-1. To minimize excessive synthesis of infectious HSV-2 while allowing virus gene expression, repressed HSV-l-infected cultures were superinfected with HSV-2 temperature-sensitive (ts) mutants (tsF3, tsB5, or tsH9). The predominant virus replicated after HSV-2 ts mutant superinfection at a nonpermissive temperature was identified as activated parental-like HSV-1 by (i) plaquing efficiency at permissive (34 degrees) and nonpermissive (40.5 degrees) temperatures, (ii) sensitivity to inhibition by the HSV-l-specific antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine, and (iii) restriction endonuclease cleavage analysis. In addition, the fact that superinfection with HSV-2 tsB5 or tsH9, which are unable to synthesize virus DNA and express only early virus genes at nonpermissive temperature, resulted in synthesis of virus demonstrated that HSV-2 DNA synthesis is not required for activation. This system has provided the basis for further studies concerning the regulation of HSV gene expression in human cells.

Bromodeoxyuridine↗
PubMed1982

Restriction of virus-specific protein synthesis in a persistent paramyxovirus infection.

Synthesis of virus-specific proteins in a persistent, nonproductive paramyxovirus infection derived from the peripheral blood leukocytes of a patient with subacute sclerosing panencephalitis (SSPE) was investigated. The persistently infected cells expressed cytoplasmic virus-specific antigens and generated paramyxovirus nucleocapsids throughout long-term passage. When analyzed by specific immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, most of the virus structural proteins were synthesized in acutely infected cells, but only three of the proteins could be readily detected in persistently infected cells. The two structural proteins whose synthesis was most clearly restricted had molecular weights of 69,000 and 41,000 daltons and represented the putative HN and M virus proteins. The similarities between the restriction of virus protein synthesis in this system and that reported previously for other persistent paramyxovirus infections derived from SSPE suggest that a common mechanism may be involved in the maintenance of such infections.

Amnion↗
PubMed1982

Characterization of the Hallé SSPE measles virus isolate.

The Hallé subacute sclerosing panencephalitis (SSPE) measles virus isolate and its plaque-purified progeny were investigated to determine whether any unusual properties could be associated with its ability to cause persistent infection. Three types of plaque-purified progeny were isolated. One population appeared to be similar in biological and biochemical properties to laboratory-adapted measles virus and had the ability to induce syncytia (syn+). A second population (syn-) plaqued more efficiently at 39 degrees C than at 33 degrees C, did not cause normal cell fusion at either temperature, and produced particles that interfered with the replication of other measles virus isolates in vivo and in vitro. This syn- virus was further plaque-purified to eliminate the interfering particles, producing the syn- P2 virus. This virus also plaqued more efficiently at 39 degrees C than at 33 degrees C, but caused cell fusion only at 39 degrees C. Both syn- viruses and the parental virus were significantly less virulent in vivo than the syn+ virus and caused a more prolonged infection. Biochemical analysis showed that the syn- P2 population produced particles that banded at two different densities in potassium tartrate gradients; both densities were less than those of the standard laboratory measles virus and the syn+ virus. Although the syn- P2 virus did not cause cell fusion at 33 degrees C, [35S]methionine labelling demonstrated that the haemolysin/cell fusion protein was present in syn- P2 virions. The production of interfering particles, the inability to cause cell fusion at 33 degrees C, and the cold-sensitive nature of the syn- population appear to play a role in the ability of the Hallé SSPE virus to establish persistent infection.

Animals↗
PubMed1982

Protease inhibitors suppress fibrinolytic activity of herpesvirus-transformed cells.

Previous studies in this laboratory have quantified the fibrinolytic activity of herpesvirus-transformed cell lines and implicated the proteolytic capacity of cloned cell lines in the formation of primary and metastatic tumours. Because of the involvement of proteases in tumourigenesis in this system, we examined the effect of various protease inhibitors (alpha-1-antitrypsin, leupeptin and alpha-2-macroglobulin), as well as hamster serum containing acute phase proteins generated in response to physiological trauma, on the fibrinolytic capacity of a herpes simplex virus type 2-transformed hamster cell line. The effects of the various inhibitors on cell growth, fibrinolysis in vitro and tumourigenesis were examined. Leupeptin, alpha-1-antitrypsin, alpha-2-macroglobulin and hamster serum containing acute phase proteins were capable of inhibiting fibrinolysis in vitro, were not toxic to the cells and their action was reversible, while rejection of a mixture of protease inhibitor and transformed cells resulted in delayed tumour development.

Animals↗
PubMed1982

Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA.

RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.

Animals↗
PubMed1981

Repression and activation of the genome of herpes simplex viruses in human cells.

We have described previously a cell culture system in which the herpes simplex virus (HSV) type 2 (HSV-2) genome is maintained in a repressed form after treatment of infected cells with 1-beta-D-arabinofuranosylcytosine and increase of incubation temperature from 37 degrees C to 39.5 degrees C. Infectious HSV-2 production was activated by altering incubation temperature or by superinfecting with human cytomegalovirus. We now report the establishment of an analogous system utilizing HSV type 1 (HSV-1). Human embryo lung cells were infected with HSV-1 and treated with 1-beta-D-arabinofuranosylcytosine (25 micrograms/ml) for 7 days to minimize both synthesis of virus DNA and infectious virus while allowing expression of early virus genes. HSV-1 was maintained in an undetectable form for at least 72 days when the incubation temperature was raised from 37 degrees C to 40.5 degrees C after removal of the inhibitor. HSV-1 gene expression was then predictably turned on by superinfection with human cytomegalovirus or by reducing the incubation temperature. Virus replicated after activation was compared with the respective parental virus with regard to inhibition by the HSV-1-specific antiviral (E)-5-(2-bromovinyl)-2'-deoxyuridine and EcoRI, HindIII, and Xba I restriction endonuclease cleavage patterns. The results show activation of HSV gene expression in human cells by a human cytomegalovirus early gene function(s), followed by synthesis of parental-like HSV.

Animals↗
PubMed1981

Rescue of a cytopathic paramyxovirus from peripheral blood leukocytes in subacute sclerosing panencephalitis.

An infectious, cytopathic paramyxovirus was rescued from the peripheral blood leukocytes of a patient with subacute sclerosing panencephalitis (SSPE) by cocultivation procedures. Cells infected with the virus (1) demonstrated polykaryocytosis, (2) contained cytoplasmic inclusion bodies of paramyxoviral nucleocapsids, (3) hemadsorbed monkey erythrocytes, and (4) reacted with fluorescein-conjugated antisera to measles virus. On the basis of these observations, the virus was initially believed to be measles-like virus similar to those that have been rescued from other patients with SSPE. However, subsequent immunologic and molecular studies showed that the virus was distinct from measles virus and was more closely related to simian virus 5. These findings suggest that paramyxoviruses other than measles virus can be nonproductively carried by patients with SSPE and should not be designated as "measles like" or "measles related" solely on the basis of their biologic characteristics.

Adolescent↗
PubMed1981

Latency in vitro using irradiated herpes simplex virus.

Human embryonic fibroblasts infected with u.v.-irradiated herpes simplex virus type 2 (HSV-2, strain 186) and maintained at 40.5 degrees C did not yield detectable virus. Virus synthesis was induced by temperature shift-down to 36.5 degrees C. The induced virus grew very poorly and was inactivated very rapidly at 40.5 degrees C. Non-irradiated virus failed to establish latency at 40.5 degrees C in infected cells. Enhanced reactivation of HSV-2 was observed when latently infected cultures were superinfected with human cytomegalovirus (HCMV) or irradiated with a small dose of u.v. light at the time of temperature shift-down. HCMV did not enhance synthesis of HSV-2 during a normal growth cycle but did enhance synthesis of u.v.-irradiated HSV-2. These observations suggest that in this in vitro latency system, some HSV genomes damaged by u.v. irradiation were maintained in a non-replicating state without being destroyed or significantly repaired.

Cell Line↗
PubMed1981

Involvement of an early human cytomegalovirus function in reactivation of quiescent herpes simplex virus type 2.

We have previously described an in vitro system in which the function lacking for herpes simplex virus type 2 (HSV-2) replication can be induced by human cytomegalovirus (HCMV). The mechanism of this reactivation of quiescent HSV-2 by HCMV has been further defined. The HCMV function(s) responsible for HSV-2 stimulation was examined temporally, and the fraction of cells in quiescent cultures producing HSV-2 after superinfection was determined. Using independent biological, genetic and molecular techniques we have made the following observations. (i) As early as 12 h after HCMV superinfection, HSV-2 RNA was expressed in latently infected cells. (ii) At 24 h after HCMV superinfection, a time when newly synthesized HCMV was not yet apparent, infectious HSV-2 was produced by reactivated cultures. (iii) Four HCMV temperature-sensitive mutants, which are DNA-negative at nonpermissive temperature and represent four different complementation groups, induced reactivation of HSV-2 at 39.5 degrees C. (iv) Early after HCMV superinfection, 1.6% of quiescent cells could be induced to transcribe HSV-2 information. (v) Early after HCMV superinfection, 0.3% of cells in the quiescent cultures could be induced to yield infectious HSV-2. The finding that a significant interaction can occur between HCMV and quiescent HSV-2 in an in vitro model is noteworthy in light of the knowledge that both of these herpesviruses often reside simultaneously in the human host.

Cell Line↗
PubMed1981

Enhanced capacity of DNA repair in human cytomegalovirus-infected cells.

Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants.

Animals↗
PubMed1981

Tumorigenicity of herpesvirus-transformed cells correlates with production of plasminogen activator.

Our studies first demonstrated that established hamster cell lines transformed in vitro by herpesviruses activate plasminogen more effectively than normal hamster fibroblasts. This ability is probably due to increased levels of the enzyme plasminogen activator (PA). In the studies described here, the 333-8-9 cell line, originally transformed by herpes simplex virus type 2 strain 333, was used to derive subclonal lines that maintained stable PA phenotypes over the course of long in vitro passage. We were interested in correlating tumor formation by the subclones with their fibrinolytic capacity. Cells were, therefore, single-cell subcloned twice, and resulting cultures were tested for ability to activate plasminogen in vitro. PA activity was then quantitated by [125I]fibrin lysis assay, and high- and low-activity subclones were isolated; these retained high- or low-activity phenotypes. Syngeneic newborn hamsters were inoculated with these subclones and observed for the appearance of palpable tumors. A strong correlation between enzyme activity and tumor formation was observed in four separate trials; animals receiving high-PA subclones developed tumors more rapidly than those inoculated with the parental cell line. Tumors were also excised from test animals, and the cell lines established from the tumors were tested in vitro at different passages for their ability to activate plasminogen. These tumor cells were then reinoculated into syngeneic animals to confirm the tumorigenicity of cell lines with high fibrinolytic activity. In these experiments, the positive correlation between PA production and tumorigenicity was confirmed.

Animals↗