Maria Pia Grossi

Suppression of BK virus replication and cytopathic effect by inhibitors of prokaryotic DNA gyrase

Nalidixic acid and oxolinic acid, two antibacterial agents known to inhibit bacterial DNA gyrase, are shown to suppress the replication, as well as the cytopathic effect, of BK virus in Vero cell cultures. The inhibition of virus replication was detectable at day 4 post infection in cultures which had been continuously exposed to drugs at concentrations as low as 0.02 to 0.04 mM of nalidixic acid and 0.2 mM of oxolinic acid. These active concentrations are inferior to plasma levels attained in the course of clinical use of the drugs for antibacterial chemotherapy. Also, under these circumstances, no cytotoxicity occurred. The inhibition of development of cytopathology and of virus-induced cell death was demonstrable in cultures treated for 12 days with the drugs. Under these circumstances of prolonged action, oxolinic acid proved to be slightly cytotoxic in that virus inhibitory doses reduced the viability of normal cells. No alterations in the topological conformation of the viral genome or accumulation of end products of viral DNA replication were detected. However, accumulation of viral DNA form I at 48 h post infection suggests that the drugs act through a mechanism involving DNA topoisomerase.

Transformation of human embryonic fibroblasts by BK virus, BK virus DNA and a subgenomic BK virus DNA fragment.

Human embryonic fibroblasts (HEF) have been transformed by BK virus (BKV) DNA and by u.v.-inactivated or live BKV alone or in association with methyl-cholanthrene (MTC). The transformed cells produced BKV large T and small t antigens as well as the cellular 53 kdal protein, detected by immunofluorescence and immunoprecipitation. After an initial phase of lysis and virus shedding, virus or its coat protein antigen could not be detected in transformed cells. All human transformed cell lines could be superinfected by BKV or BKV DNA, but their susceptibility to superinfection was 20- to 500-fold lower than normal HEF. BKV could be rescued by fusion of transformed cells with normal HEF or Vero cells and by transfection of normal HEF with total DNA and DNA extracted from the Hirt supernatant of transformed cells. Blot hybridization analysis of DNA from transformed cells showed a considerable amount of free BKV DNA in monomeric and polymeric forms. Integrated BKV DNA was absent in most cell lines but present in only small amounts in BKV-transformed cells treated with MTC. Analysis of free BKV DNA with various restriction endonucleases and by blot hybridization showed that monomeric forms were complete BKV genomes, whereas polymers contained both complete and defective or rearranged BKV DNA. Transformation of HEF was also obtained with a 3.7 kilobase (kb) fragment of the BKV genome, produced by sequential digestion of BKV with the restriction endonucleases HhaI and EcoRI. This fragment extends clockwise on the virus genome from 0 to 72.2 map units and contains the entire early region. Blot hybridization analysis of cells transformed by the HhaI/EcoRI 3.7 kb fragment showed two separate integrations of BKV sequences without free virus DNA.

Stable Transformation of Mouse, Rabbit and Monkey Cells and Abortive Transformation of Human Cells by BK Virus, a Human Papovavirus

Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.

Red blood cells as delivery system for recombinant HSV-1 glycoprotein B: immunogenicity and protection in mice

The immunotherapeutic potential of autologous red blood cells (RBC) coupled to the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) was examined with a mouse model of HSV-1 infection. C57BL/6 mice were immunized intraperitoneally with gB1s (0.05 microgram per dose) linked to RBC, or mixed with Freunds complete or bound to AlPO4 adjuvants (0.5 microgram per dose). Mice immunized with RBC coupled gB1s were protected against lethal and latent HSV-1 infection, and developed an anti-HSV antibody response, as measured by ELISA and HSV-1 neutralization assays, similar or higher than that elicited by the same antigen in Freunds complete adjuvant, which suggested that autologous RBC coupled to gB1s may provide an effective and safe method of immunization against HSV infection.

Inhibition of HIV-1 replication by a Tat transdominant negative mutant in human peripheral blood lymphocytes from healthy donors and HIV-1-infected patients

It was previously shown that a tat mutant (tat22) where cysteine 22 is substituted by glycine behaves as a transdominant negative mutant in Jurkat T cells lytically or latently infected by HIV-1. In this study we demonstrate that tat22 controls HIV-1 replication in primary cells. This effect was observed both after in vitro infection of peripheral blood mononuclear cells (PBMCs) from normal donors and after reactivation of the latent infection in PBMCs from seropositive patients. The antiviral effect of tat22 was limited to conditions of low virus production. The use of tat22 may be promising for a gene therapy approach to AIDS during the asymptomatic phase of the disease allowing control of virus replication in infected cells and inhibition of virus spread to uninfected cells.

Studies on the effect of the combined expression of anti-tat and anti-rev genes on HIV-1 replication

A series of retroviral vectors with potential anti-tat and anti-rev activity was developed. Vectors containing a tat transdominant negative mutant (tat22/37) and an RRE decoy in different positions, directed by the same promoter or by different promoters, were generated. Retroviral vectors containing tat22/37 and the RevM10 transdominant negative mutant were also constructed. Jurkat cells were transduced with the recombinant retroviruses to produce monoclonal and polyclonal cultures. In these cell lines the recombinant proviruses were correctly integrated and expression of the inserted genes was detected by Northern blot or RT-PCR analysis. However, infection of these cell lines with HIV-1 showed that none of these recombinant constructs inhibited virus replication at a high multiplicity of infection (MOI). At a low MOI, two cell clones containing tat22/37 and the RRE decoy in 3′ position showed a long lasting protection against virus replication, in comparison to control cultures expressing tat22/37 or RRE alone. Combination of tat and rev mutants was ineffective in inhibiting HIV-1 replication at both low and high MOIs. At a low MOI, HIV-1 replication was efficiently blocked in two cell clones expressing the RevM10 mutant alone. These results show a synergic effect of anti-tat and anti-rev molecules when the RRE sequence is cloned 3′ to tat22/37, suggesting the possibility of using this vector design to control HIV-1 replication.

New BK virus episomal vector for complementary DNA expression in human cells

SummaryThe properties of pRP-c, a new vector for complementary DNA (cDNA) expression, are described. The vector contains the early region and replication origin of BK virus (BKV), a human papovavirus. Due to the presence of these BKV sequences, pRP-c replicates in human cells allowing amplification of inserted cDNAs. The promoter, intron and polyadenylation region for cDNA expression are separated by unique restriction sites and can therefore be individually excised and substituted with different transcription signals. Coding sequences of the bacterial genes for chloramphenicol-acetyl transferase (CAT) or neomycin phosphotransferase (neo) were inserted into the cDNA cloning site of pRP-c and expressed in human cells in transient assays or stable clones. In both cases expression of the inserted sequences was significantly more efficient than by using the integration vectors pSV2CAT and pSV2neo, demonstrating the advantages of episomal expression vectors in human cells. Possible uses of pRP-c to express viral and cellular cDNAs in human cells are discussed.

Oncogenity of BK virus for immunosuppressed hamsters

SummaryTumors were induced by BK virus (BKV) inoculated intravenously in 3-week-old Syrian golden hamsters immunosuppressed with anti-lymphocyte serum or methylprednisolone acetate alone or in association with γ-radiation (60Co). The induced neoplasms were ependymoma, carcinoma of pancreatic islets, lymphoma, osteosarcoma, undifferentiated sarcoma, kidney and renal pelvis carcinoma, pheochromocytoma and hemangiosarcoma. High levels of insulin and glucagon and altered concentrations of glucose were detected in blood of animals with tumors of pancreatic islets. No antibodies to BKV tumor antigen (T Ag) and low levels of hemagglutination-inhibiting antibodies to BKV viral coat protein Ag were detected in hamster sera. BKV T Ag was found in tumors by complement fixation. Blot hybridization analysis of tumor DNA showed the presence of both free and integrated BKV genomes in tumor cells. BKV DNA inoculated intravenously and subcutaneously in immunosuppressed or immunocompetent hamsters was not oncogenic, whereas it was weakly oncogenic when inoculated intracerebrally.

Lack of Association between BK Virus and Ependymomas, Malignant Tumors of Pancreatic Islets, Osteosarcomas and Other Human Tumors

BK virus (BKV) DNA sequences were not detected in 142 human tumors analyzed by DNA-DNA reassociation kinetics and blot hybridization. The investigation was focused mainly on those rare types of human tumors (ependymomas, choroid plexus papillomas, tumors of pancreatic islets and osteosarcomas) that are induced with highest frequency by BKV in experimental animals. In addition, other tumors of the urinary apparatus and of the central nervous system were analyzed. BKV tumor (T) antigen was not detected in neoplastic tissues, and BKV T antibodies were not found in sera and cerebrospinal fluids from patients with neoplasms. Sequences homologous to BKV DNA were found in normal tissue from a kidney carrying a carcinoma. The neoplastic tissue from the same organ, however, had no sequences homologous to BKV DNA. Such DNA does not belong to BKV but probably to another papovavirus related to BKV.

The arrangement of integrated viral DNA is different in BK virus-transformed mouse and hamster cells

Abstract Secondary mouse and hamster cells were transformed with BK virus (BKV) or with purified BKV DNA. Restriction analysis of viral sequences present in transformed cell lines yielded complex patterns, indicating the presence in most lines of more than one viral intergration/cell and the existence of a large number of available integration sites. In spite of this variability, however, the arrangement of integrated viral sequences had specific features in the two types of transformed cells. Tandem insertions of full-size BKV genomes, which were a constant feature of transformed mouse lines, were not found in hamster lines. These appeared to contain no complete, intact BKV genomes, but only genomes interrupted by the insertion event or tandem integrations of incomplete viral sequences. Traces of free, full-size viral DNA were observed in all transformed mouse lines, but not in hamster lines. One transformed hamster line, after 84 passages in culture, showed the presence of tandemly integrated, full-size BKV DNA, which was not found in cells at 14 passages after the appearance of the transformed phenotype. This was the only BKV transformed line from which virus could be rescued by fusion with permissive cells. These results suggest that BKV DNA integration occurs differently in hamster (nonpermissive) and mouse (semipermissive) cells, and support the idea that excision of integrated viral DNA occurs usually via homologous recombination.