J. Peries

Study of human foamy virus proviral integration in chronically infected murine cells

This report describes integration sites of human foamy virus (HFV) in chronically infected BALB/c murine cells that we isolated by inverse PCR and characterized. We show that integration of HFV proviral genome mainly occurs in highly repetitive and/or transcriptionally active regions and leads to the formation of a 4-bp cellular direct repeat sequence at each provirus extremity. As non-random deletions were previously described in the HFV be/1 transactivator gene as well as in the long terminal repeats (LTRs), these regions were verified in integrated HFV. The analysis reveals that, in the studied chronic state, the defective interfering virus (delta HFV) is the main integrated proviral form and is always associated with a small LTR. Our results show that HFV can use a classic retroviral integration process to enter the host cell genome and stress the importance of delta HFV and the short LTRs in the establishment of the chronic state of infection.

Transient immunosuppressive effect induced in rabbits and mice by the human spumaretrovirus prototype HFV (human foamy virus)

Spumaviruses (foamy viruses) constitute one of the three retroviral genera isolated from man. Although spumaviruses have not been clearly linked to a given pathology in humans and other infected species, it is well established that they lead in vivo to chronic infections without detectable viral expression. We thought it of interest to investigate certain aspects of the pathology induced in laboratory animals by human foamy virus (HFV). In this work, we demonstrate that HFV infection of rabbits and mice gives rise to a transient immunosuppressive effect, as evaluated in vitro by lymphocyte transformation tests. This phenomenon occurs shortly after viral inoculation, at around 4-5 days, and regresses within thirty days.

In vitro studies on interferoninducing capacity and sensitivity to IFN of human foamy virus

We demonstrate in this article that human foamy virus (HFV) fails to induce interferon (IFN) production in two different human tissue culture cell lines: U373-MG and AV3. We also show the effect of human alpha-, beta- and gamma IFN on the multiplication cycle of HFV. Treatment of cells with 100 IU/ml of any IFN led to strong inhibition of an HFV-induced cytopathic effect. This effect was associated with a significant diminution of reverse transcriptase activity in supernatant fluids of IFN-treated infected cultures, and a substantial decrease in viral particle production, as detected by electron microscopy. All these effects were accompanied by strong inhibition of both viral proteins and RNA synthesis, as well as almost total disappearance of free and integrated proviral DNA. In light of our data, human IFN action on HFV seems to be mediated by a mechanism which differs from that observed in the case of other retroviruses (type C and D for instance); however, it evokes that described for HIV.

Effects of human recombinant alpha and gamma and of highly purified natural beta interferons on simian Spumavirinae prototype (simian foamy virus 1) multiplication in human cells

The present study demonstrates the inhibitory effect of human recombinant interferons (r-Hu-IFN) alpha and gamma, and that of highly purified natural human interferon beta on the replication of simian foamy virus type 1 (SFV1) in human AV3-cell cultures. All IFN led to strong inhibition of the SFV1 cytopathic effect. Electron microscopy showed a 70 to 95% decrease in viral particles. Significant inhibition of virus-associated reverse transcriptase activity was found in supernatant fluids of infected IFN-treated cultures. Metabolic labelling of the virus confirmed the inhibition of extracellular release of SFV1. PAGE analysis of immunoprecipitates indicated a reduction in viral-specific protein bands. Altogether, these results indicate that the mechanism of inhibition of Spumavirinae infection by interferon differs from that described for the other Retroviridae, and particularly for types B, C and D viruses. Our data is of therapeutic interest since Spumavirinae have been linked to pathological processes such as de Quervain thyroiditis.

Studies on in vitro interferon induction capacity and interferon sensitivity of Simian foamy viruses

SummaryWe demonstrate that Simian Foamy viruses (SFV) types 1, 2, 4 and 10 do not induce Interferon (IFN) production in mouse and primate (simian and human) cell lines, but that their cytopathogenic effect is blocked by this viral inhibitor. The mechanisms of action of IFN seems to be different from that of other Retroviridae. No trapping of virions appears in treated cells examined by ectron microscopy. Moreover, neither precursor nor mature virus particles were observed in infected cultures submitted to IFN treatment.

Purification and characterization of simian foamy virus type I structural core polypeptides

SummaryThe present study concerns the purification and partial characterization of simian foamy virus type 1 (SFV 1) structural core polypeptides. The obtention of SFV 1 cores separated from envelope components after viral disrupture was verified by electron microscopy (EM), density gradient, and polyacrylamide gel electrophoresis (PAGE). Multistep purification by column chromatography, verified by PAGE, enabled us to separate the structural core polypeptides from the 80,000 molecular weight reverse transcriptase. Two species of structural core polypeptides were identified with apparent molecular weights of 51 and 15 kd. By affinity chromatography on a double-stranded DNA-cellulose column, the main internal protein, p51, was shown to be composed of a major 30 kd protein and a minor 19 kd polypeptide, which binds to double stranded DNA. The p15 internal protein was shown to have a ribonucleotide binding nature.

In vitro induction of early mouse embryo intracisternal particles (ε particles) in cultured cell lines

Summary— The experimental induction of ε particles, retrovirus‐like structures corresponding to the small IA particles of the mouse, was studied by electron microscopy in rodent‐cultured cell lines. Among the chemicals tested, only IdUr was shown to be an effective inducer, but not cycloheximide, puromycine, deoxy‐fluorouracile or 5‐azacytine. However, only two mouse‐derived cell lines: KiBALB and FG 10, among 27 cell lines of mouse, rat and mink origins tested, expressed ε particles upon IdUr treatment. Epsilon particles thus respond to chemical inducers very differently in comparison with large IAP. Moreover, the addition of interferon previously shown to attenuate IAP production, had no effect on that of ε particles.

Biological and molecular studies on Syrian hamster intracisternal R-type particles

SummaryRetrovirus-like intracisternal R-type particles (IRP) are structures present in Syrian hamster (Mesocricetus auratus) cells cultured in vitro where they appear either spontaneously or under chemical induction conditions. We have tested several chemical inducers and ten different cell lines, looking for the best IRP induction conditions. BHK21 cl. 13 showed the highest inducibility one day after a 24 h treatment with 1 µg/ml of 5-aza-2′-deoxycytidine. Using detergent treatments and sucrose gradients, we obtained semi-purified IRP cores. A 7.2 kb RNA associated with the core fraction was revealed by hybridization with total Syrian hamster genomic DNA, but not with Syrian hamster intracisternal A particle (IAP) specific probes. This suggests that the IRP genes are distinct from IAP ones.

Cell transfection with DNA from simian foamy virus type 1 producing cultures generates equivalent infectious virions

Summary The transfection of canine and murine cells with DNA from simian foamy virus type 1 cell cultures led to the appearance of a cytopathic effect (CPE) similar to that induced by the original SFV1. High reverse transcriptase activity was present in supernatants of transfected cultures. Inoculation of these supernatants into permissive cells induced a syncytial CPE and high RNA-dependent DNA polymerase activity. These two effects were specifically inhibited by immune serum against SFV1. By electron microscopy, inoculated cultures showed the presence of virions homologous to those of SFV1.

In vitro growth properties and PCR ras gene analysis of a murine embryonal carcinoma cell line harboring an amplified C-Ki-ras gene

Ras proto‐oncogenes are thought to be involved in both proliferation and malignant processes. We examined some growth properties of a murine embryonal carcinoma cell line (ECC), PCC4, that have been shown previously to be amplified for the c‐KI‐ras gene. Our results show that doubling time and plating efficiency are not significantly affected by such amplification. To examine the possible link between malignant behavior and c‐Ki‐ras alteration, we investigated activating mutations in this PCC4 cell line as well as in other ECC. Analysis of the in vitro amplified Ki‐ras gene by PCR technology has not revealed point mutations in any of the ECC examined.