Analysis of two monoclonal antibodies reactive with envelope proteins of murine retroviruses: one pan specific antibody and one specific for Moloney leukemia virus.

Many monoclonal antibodies (MAbs) reactive with various proteins of murine leukemia viruses (MuLVs) have been developed. In this report two additional MAbs with differing and unusual specificities are described. MAb 573 is reactive with the envelope protein of all MuLVs tested including viruses in the ecotropic, xenotropic, polytropic and amphotropic classes. Notably, MAb 573 is one of only two reported MAbs that react with the envelope protein of amphotropic MuLVs. This MAb appears to recognize a conformational epitope within the envelope protein, as it reacts strongly with live virus and live infected cells, but does not react with formalin-fixed or alcohol-fixed infected cells or denatured viral envelope protein in immunoblots. In contrast, Mab 538 reacts only with an epitope unique to the envelope protein of the Moloney (Mo-) strain of MuLV, a prototypic ecotropic MuLV that is the basis for many retroviral tools used in molecular biology. MAb 538 can react with live cells and viruses, or detergent denatured or fixed envelope protein. The derivation of these antibodies as well as their characterization with regard to their isotype, range of reactivity with different MuLVs and utility in different immunological procedures are described in this study.

Neurovirulence of polytropic murine retrovirus is influenced by two separate regions on opposite sides of the envelope protein receptor binding domain.

Changes in the envelope proteins of retroviruses can alter the ability of these viruses to infect the central nervous system (CNS) and induce neurological disease. In the present study, nine envelope residues were found to influence neurovirulence of the Friend murine polytropic retrovirus Fr98. When projected on a three-dimensional model, these residues were clustered in two spatially separated groups, one in variable region B of the receptor binding site and the other on the opposite side of the envelope. Further studies indicated a role for these residues in virus replication in the CNS, although the residues did not affect viral entry.

Increased proinflammatory cytokine and chemokine responses and microglial infection following inoculation with neural stem cells infected with polytropic murine retroviruses.

Proinflammatory cytokines and chemokines are often detected in brain tissue of patients with neurological diseases such as multiple sclerosis (MS), HIV-associated dementia (HAD) and Alzheimer's disease (AD). We have utilized a mouse model of retrovirus-induced neurological disease to examine how these proinflammatory responses contribute to neuropathogenesis. In previous studies with this model, a correlation was found between neurovirulence and cytokine and chemokine expression. However, it was unclear whether the induction of these cytokines and chemokines was in response to specific virus envelope determinants or was regulated by the level of virus infection in the brain. In the current study, we demonstrated that multiple polytropic retroviruses induced cytokine and chemokine mRNA expression following increased virus levels in the brain. Increased virus levels of polytropic viruses also correlated with increased neuropathogenesis. In contrast, the ecotropic retrovirus, FB29, did not induce cytokine or chemokine mRNA expression or neurological disease, despite virus levels either similar to or higher than the polytropic retroviruses. As polytropic and ecotropic viruses utilize different receptors for entry, these receptors may play a critical role in the induction of these innate immune responses in the brain.

Role of low CD4 levels in the influence of human immunodeficiency virus type 1 envelope V1 and V2 regions on entry and spread in macrophages.

Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to infect macrophages. Previous experiments have mapped viral determinants of macrophage infectivity to the V3 hypervariable region of the HIV-1 envelope glycoprotein. In our earlier studies, V1 and V2 sequences of HIV-1 were also shown to alter the ability of virus to spread in macrophage cultures, whereas no effect was seen in lymphocyte cultures. In the present study, determinants that allowed certain HIV-1 clones to infect and spread in macrophages were primarily mapped to the V2 region and were found to act by influencing early events of viral infection. By an assay of viral entry into macrophages, it was shown that viruses with the V2 region from the Ba-L strain of HIV-1 had >10-fold-higher entry efficiency than viruses with the V2 region derived from the NL4-3 strain. V1 region differences between these groups caused a twofold difference in entry. The known low expression of CD4 on macrophages appeared to be important in this process. In entry assays conducted with HeLa cell lines expressing various levels of CD4 and CCR5, low levels of CD4 influenced the efficiency of entry and fusion which were dependent on viral V1 and V2 envelope sequences. In contrast, no effect of V1 or V2 was seen in HeLa cells expressing high levels of CD4. Thus, the limited expression of CD4 on macrophages or other cell types could serve as a selective factor for V1 and V2 envelope sequences, and this selection could in turn influence many aspects of AIDS pathogenesis in vivo.

Separate sequences in a murine retroviral envelope protein mediate neuropathogenesis by complementary mechanisms with differing requirements for tumor necrosis factor alpha.

The innate immune response, through the induction of proinflammatory cytokines and antiviral factors, plays an important role in protecting the host from pathogens. Several components of the innate response, including tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein 1, interferon-inducible protein 10, and RANTES, are upregulated in the brain following neurovirulent retrovirus infection in humans and in animal models. However, it remains unclear whether this immune response is protective, pathogenic, or both. In the present study, by using TNF-alpha(-/-) mice we analyzed the contribution of TNF-alpha to neurological disease induced by four neurovirulent murine retroviruses, with three of these viruses encoding portions of the same neurovirulent envelope protein. Surprisingly, only one retrovirus (EC) required TNF-alpha for disease induction, and this virus induced less TNF-alpha expression in the brain than did the other retroviruses. Analysis of glial fibrillary acidic protein and F4/80 in EC-infected TNF-alpha(-/-) mice showed normal activation of astrocytes but not of microglia. Thus, TNF-alpha-mediated microglial activation may be important in the pathogenic process initiated by EC infection. In contrast, TNF-alpha was not required for pathogenesis of the closely related BE virus and the BE virus induced disease in TNF-alpha(-/-) mice by a different mechanism that did not require microglial activation. These results provide new insights into the multifactorial mechanisms involved in retrovirus-induced neurodegeneration and may also have analogies to other types of neurodegeneration.

Human immunodeficiency virus type 1 envelope-mediated neuronal death: uncoupling of viral replication and neurotoxicity.

Although brain tissue from patients with human immunodeficiency virus (HIV) and/or AIDS is consistently infected by HIV type 1 (HIV-1), only 20 to 30% of patients exhibit clinical or neuropathological evidence of brain injury. Extensive HIV-1 sequence diversity is present in the brain, which may account in part for the variability in the occurrence of HIV-induced brain disease. Neurological injury caused by HIV-1 is mediated directly by neurotoxic viral proteins or indirectly through excess production of host molecules by infected or activated glial cells. To elucidate the relationship between HIV-1 infection and neuronal death, we examined the neurotoxic effects of supernatants from human 293T cells or macrophages expressing recombinant HIV-1 virions or gp120 proteins containing the V1V3 or C2V3 envelope region from non-clade B, brain-derived HIV-1 sequences. Neurotoxicity was measured separately as apoptosis or total neuronal death, with apoptosis representing 30 to 80% of the total neuron death observed, depending on the individual virus. In addition, neurotoxicity was dependent on expression of HIV-1 gp120 and could be blocked by anti-gp120 antibodies, as well as by antibodies to the human CCR5 and CXCR4 chemokine receptors. Despite extensive sequence diversity in the recombinant envelope region (V1V3 or C2V3), there was limited variation in the neurotoxicity induced by supernatants from transfected 293T cells. Conversely, supernatants from infected macrophages caused a broader range of neurotoxicity levels that depended on each virus and was independent of the replicative ability of the virus. These findings underscore the importance of HIV-1 envelope protein expression in neurotoxic pathways associated with HIV-induced brain disease and highlight the envelope as a target for neuroprotective therapeutic interventions.

Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrP(Sc)) into contiguous membranes.

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease-sensitive PrP isoform (PrP-sen) to the protease-resistant isoform (PrP-res). A cell-free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP-sen and brain microsomes from scrapie-infected mice as a source of PrP-res. Interestingly, DRM-associated PrP-sen was not converted to PrP-res until the PrP-sen was either released from DRMs by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). PEG-assisted conversion was optimal at pH 6--7, and acid pre-treating the DRMs was not sufficient to permit conversion without PI-PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP-res during TSE infection requires (i) removal of PrP-sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP-res into the raft domains of recipient cells.