Functional characterization of the in vitro folded human y(1) receptor in lipid environment.

We describe the recombinant production of the human Y(1) receptor from inclusion bodies of E. coli cultures. The in vitro refolding was carried out in the presence of lipids from bovine brain extracts. Y(1) receptors in brain lipids compete for cellular receptors in competitive binding experiments.

Activation of microglia cells is dispensable for the induction of rat retroviral spongiform encephalopathy.

In the course of retroviral CNS infections, microglia activation has been observed frequently, and it has been hypothesized that activated microglia produce and secrete neurotoxic products like proinflammatory cytokines, by this promoting brain damage. We challenged this hypothesis in a rat model for neurodegeneration. In a kinetic study, we found that microglia cells of rats neonatally inoculated with neurovirulent murine leukemia virus (MuLV) NT40 became infected in vivo to maximal levels within 9-13 days postinoculation (d.p.i.). Beginning from 13 d.p.i., degenerative alterations, i.e., vacuolization of neurons and neuropil were found in cerebellar and other brain-stem nuclei. Elevated numbers of activated microglia cells--as revealed by immunohistochemical staining with monoclonal antibody ED1--were first detected at 19 d.p.i. and were always locally associated with degenerated areas but not with nonaltered, yet infected, brain regions. Both neuropathological changes and activated microglia cells increased in intensity and numbers, respectively, with ongoing infection but did not spread to other than initially affected brain regions. By ribonuclease protection assays, we were unable to detect differences in the expression levels of tumor-necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in microglia cells nor in total brains from infected versus uninfected rats. Our results suggest that the activation of microglia in the course of MuLV neurodegeneration is rather a reaction to, and not the cause of, neuronal damage. Furthermore, overt expression of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 within the CNS is not required for the induction of retroviral associated neurodegeneration in rats.

Abundant defective viral particles budding from microglia in the course of retroviral spongiform encephalopathy.

A pathogenetic hallmark of retroviral neurodegeneration is the affinity of neurovirulent retroviruses for microglia cells, while degenerating neurons are excluded from retroviral infections. Microglia isolated ex vivo from rats peripherally infected with a neurovirulent retrovirus released abundant mature type C virions; however, infectivity associated with microglia was very low. In microglia, viral transcription was unaffected but envelope proteins were insufficiently cleaved into mature viral proteins and were not detected on the microglia cell surface. These microglia-specific defects in envelope protein translocation and processing not only may have prevented formation of infectious virus particles but also may have caused further cellular defects in microglia with the consequence of indirect neuronal damage. It is conceivable that similar events play a role in neuro-AIDS.

Suppression of rat bone marrow cells by Friend murine leukemia virus envelope proteins.

In a retroviral rat model, we have investigated the nontransforming effects of murine leukemia virus FB29 on the bone marrow. Upon intraperitoneal inoculation with murine leukemia virus FB29 of either neonatal or adult rats, bone marrow cells became massively infected within the first 12 days postinoculation. In neonatally inoculated rats, a persistent productive bone marrow infection was established, whereas in rats inoculated as adults, no infected bone marrow cells could be detected beyond 12 days postinoculation. Retroviral infection was most likely cleared by an antiviral immune response (Hein et al., 1995, Virology 211, 408-417). Exposure to virus irreversibly decreased numbers of bone marrow cells staining with monoclonal antibody OX7 by 10-30%. Reduction of OX7+ bone marrow cells by 20% was also observed in vitro, after bone marrow cells from uninfected adult rats had been co-incubated with virus. FB29-envelope proteins were sufficient alone to reduce numbers of OX7+ bone marrow cells, both in vivo and in vitro. According to results on incorporation of propidium iodide, decreased numbers of OX7+ cells were due to cell death. By flow cytometric analyses OX7+ bone marrow cells as well as monocytes/macrophages were identified to be major target cells for infection with FB29 within the bone marrow. Thus, the mechanism(s) responsible for death of OX7+ bone marrow cells might be due to direct toxicity of viral envelope proteins and/or to interactions of viral envelope proteins with cells of the monocytic lineage.