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.

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.

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.

ABSTRACT 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.

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.

ABSTRACT 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.

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.

A conformational conversion of the normal, protease-sensitive prion protein (PrP-sen or PrPC) to a protease-resistant form (PrP-res or PrPSc) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs), Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34–94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.

Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.

ABSTRACT It has been proposed that changes in cell surface concentrations of coreceptors may control infections by human immunodeficiency virus type 1 (HIV-1), but the mechanisms of coreceptor function and the concentration dependencies of their activities are unknown. To study these issues and to generate stable clones of adherent cells able to efficiently titer diverse isolates of HIV-1, we generated two panels of HeLa-CD4/CCR5 cells in which individual clones express either large or small quantities of CD4 and distinct amounts of CCR5. The panels were made by transducing parental HeLa-CD4 cells with the retroviral vector SFF-CCR5. Derivative clones expressed a wide range of CCR5 quantities which were between 7.0 × 10 2 and 1.3 × 10 5 molecules/cell as measured by binding antibodies specific for CCR5 and the chemokine [ 125 I]MIP1β. CCR5 was mobile in the membranes, as indicated by antibody-induced patching. In cells with a large amount of CD4, an unexpectedly low trace of CCR5 (between 7 × 10 2 and 2.0 × 10 3 molecules/cell) was sufficient for maximal susceptibility to all tested HIV-1, including primary patient macrophagetropic and T-cell-tropic isolates. Indeed, the titers as indicated by immunoperoxidase staining of infected foci were as high as the tissue culture infectious doses measured in human peripheral blood mononuclear cells. In contrast, cells with a small amount of CD4 required a much larger quantity of CCR5 for maximal infection by macrophagetropic HIV-1 (ca. 1.0 × 10 4 to 2.0 × 10 4 molecules/cell). Cells that expressed low and high amounts of CD4 were infected with equal efficiencies when CCR5 concentrations were above threshold levels for maximal infection. Our results suggest that the concentrations of CD4 and CCR5 required for efficient infections by macrophagetropic HIV-1 are interdependent and that the requirements for each are increased when the other component is present in a limiting amount. We conclude that CD4 and CCR5 directly or indirectly interact in a concentration-dependent manner within a pathway that is essential for infection by macrophagetropic HIV-1. In addition, our results suggest that multivalent virus-receptor bonds and diffusion in the membrane contribute to HIV-1 infections.

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution ofT-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4+ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4+ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4+ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4+ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4+ T cells.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of the brain results in viral replication primarily in macrophages and microglia. Despite frequent detection of viral genome and proteins in the brains of AIDS patients with and without HIV dementia, only 20% of AIDS patients become demented. To investigate the role of viral envelope gene variation in the occurrence of dementia, we examined regions of variability in the viral envelope gene isolated from brains of AIDS patients. Brain-derived HIV-1 V1-V2 envelope sequences from seven demented and six nondemented AIDS patients displayed significant sequence differences between clinical groups, and by phylogenetic analysis, sequences from the demented group showed clustering. Infectious recombinant viruses containing brain-derived V3 sequences from both clinical groups were macrophagetropic, and viruses containing brain-derived V1, V2, and V3 sequences from both clinical groups spread efficiently in macrophages. In an indirect in vitro neurotoxicity assay using supernatant fluid from HIV-1-infected macrophages, recombinant viruses from demented patients induced greater neuronal death than viruses from nondemented patients. Thus, the HIV-1 envelope diversity observed in these patient groups appeared to influence the release of neurotoxic molecules from macrophages and might account in part for the variability in occurrence of dementia in AIDS patients.

The tempo and intensity of retroviral neuropathogenesis are dependent on the capacity of the virus to invade the central nervous system. For murine leukemia viruses, an important determinant of neuroinvasiveness is the virus-encoded protein glycosylated Gag, the function of which in the virus life cycle is not known. While this protein is dispensable for virus replication, mutations which prevent its expression slow the spread of virus in vivo and restrict virus dissemination to the brain. To further explore the function of this protein, we compared two viruses, CasFrKP (KP) and CasFrKP41 (KP41), which differ dramatically in neurovirulence. KP expresses high early viremia titers, is neuroinvasive, and induces clinical neurologic disease in 100% of neonatally inoculated mice, with an incubation period of 18 to 23 days. In contrast, KP41 expresses early viremia titers 100- fold lower than those of KP, exhibits attenuated neuroinvasiveness, and induces clinical neurologic disease infrequently, with a relatively long incubation period. The genomes of these two viruses differ by only 10 nucleotides, resulting in differences at five residues, all located within the N-terminal cytoplasmic tail of glycosylated Gag. In this study, using KP as the parental virus, we systematically mutated each of the five amino acid residues to those of KP41 and found that substitution mutation of two membrane-proximal residues, E53 and L56, to K and P, respectively produced the greatest effect on early viremia kinetics and neurovirulence. These mutations disrupted the KP sequence E53FLL56, the leucine dipeptide of which suggests the possibility that it may represent a sorting signal for glycosylated Gag. Supporting this idea was the finding that alteration of this sequence motif increased the level of cell surface expression of the protein, which suggests that analysis of the intracellular trafficking of glycosylated Gag may provide further clues to its function.

The chemokine receptor CCR5 acts as an essential cofactor for cell entry by macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains, whereas CXCR4 acts as an essential cofactor for T-cell-line-adapted strains. We demonstrated that the specific amino acids in the V3 loop of the HIV-1 envelope protein that determine cellular tropism also regulate chemokine coreceptor preference for cell entry by the virus. Further, a strong correlation was found between HIV-1 strains classified as syncytium inducing in standard assays and those using CXCR4 as a coreceptor. These data support the hypothesis that progressive adaptation to additional coreceptors is a key molecular basis for HIV-1 phenotypic evolution in vivo.

Antigen capture enzyme-linked immunosorbent assay (ELISA) for quantitation of the p24 gag protein of human immunodeficiency virus type-1 (HIV-1) is currently the most common method used to demonstrate virus replication both in vivo and in vitro. The present paper describes an ELISA employing readily available inexpensive reagents and gives detailed suggestions for optimizing the variables for specific purposes. The assay is as sensitive as commercial kits (25 pg/ml) and has a linear dose response over a wide range of p24 concentrations (25-1000 pg/ml). For these reasons, as well as its low cost, this assay has proven useful in a variety of research applications. This assay also has been found to be effective in detecting the gag protein of human immunodeficiency virus type-2 and simian immunodeficiency virus.

Multidrug-resistant human immunodeficiency virus type 1 (HIV-1) strains with reverse transcriptase (RT) mutations at codons A62-->V, V75-->I, F77-->L, F116-->Y, and Q151-->M have been reported in patients receiving combination therapy with zidovudine (AZT) and didanosine (ddI). Infectious clones with each mutation alone, all five mutations together, and various combinations of mutations were created by site-directed mutagenesis. Mutation Q151-->M conferred partial resistance to AZT, ddI, zalcitibine, and stavudine, whereas a combination of four mutations conferred increased resistance to AZT, ddI, zalcitibine, and stavudine. The positions of residues 75, 77, and 151 in the three-dimensional crystal structure of HIV-1 RT suggest that these residues may affect the ability of the enzyme to discriminate between deoxynucleoside triphosphates and nucleoside analog RT inhibitors. Replication experiments showed that clones with mutation F77-->L but without V75-->I (HIV-1(77), HIV-1(77,151), and HIV-1(77,116,151) had attenuated growth compared with that of the original HIV-1NL4-3 strain and strains containing mutations at both positions 75 and 77 (HIV-1(75,77,151) and HIV-1(75,77,116,15)). Sequence analysis of viral RNA and proviral DNA from several patients indicated that RT mutations developed in a sequential and cumulative pattern over the course of a 2- to 4-year observation period. The present results suggest that drug resistance and viral replicative capacity both may play a role in selection of HIV-1 RT mutations.

The V3 region of the human immunodeficiency virus type 1 (HIV-1) envelope protein is known to have a major influence on macrophage tropism as well as the ability to cause syncytium formation or fusion in CD4-positive lymphocyte cultures. Using infectious molecular HIV-1 clones, a series of mutant clones was created which allowed detailed mapping of V3 amino acid positions involved in these properties. In these experiments the non-syncytium-inducing phenotype in T cells did not always correlate with macrophage tropism. Macrophage tropism appeared to depend on the presence of certain combinations of amino acids at five specific positions within and just outside of the V3 loop itself, whereas syncytium formation in lymphocytes was influenced by substitution of particular residues at two to four positions within V3. In most cases, different V3 amino acid positions were found to independently influence macrophage tropism and syncytium formation in T cells and position 13 was the only V3 location which appeared to simultaneously influence both macrophage tropism and syncytium formation in lymphocytes.