N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro.

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.

Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein.

A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrP(C)) to a protease-resistant form (PrP-res or PrP(Sc)) 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.

Species-independent inhibition of abnormal prion protein (PrP) formation by a peptide containing a conserved PrP sequence.

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.

Neuronal death induced by brain-derived human immunodeficiency virus type 1 envelope genes differs between demented and nondemented AIDS patients.

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 neuroinvasiveness of a murine retrovirus is influenced by a dileucine-containing sequence in the cytoplasmic tail of glycosylated Gag.

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.

Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1.

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 x 10(2) and 1.3 x 10(5) molecules/cell as measured by binding antibodies specific for CCR5 and the chemokine [125I]MIP1beta. 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 x 10(2) and 2.0 x 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 x 10(4) to 2.0 x 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.

The cell tropism of human immunodeficiency virus type 1 determines the kinetics of plasma viremia in SCID mice reconstituted with human peripheral blood leukocytes.

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.

Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop.

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.

p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents.

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.

Mapping of independent V3 envelope determinants of human immunodeficiency virus type 1 macrophage tropism and syncytium formation in lymphocytes.

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.

In vitro effects of anti-HIV immunotoxins directed against multiple epitopes on HIV type 1 envelope glycoprotein 160.

We have used a panel of anti-gp160 MAbs to construct anti-HIV immunotoxins by coupling antibodies to ricin A chain (RAC). The ability of the immunotoxins to kill HIV-1-infected cells and halt the spread of infection was tested in tissue culture on persistently and acutely infected cell lines and primary lymphocyte cultures stimulated with phytohemagglutinin (PHA blasts). Laboratory strains and clinical isolates of HIV both were tested. The constitution and antigen-binding capacity of the immunotoxins were confirmed by ELISA and indirect immunofluorescence. Immunotoxins that bind epitopes exposed on the cell surface effectively killed persistently infected cells, although killing was not directly proportional to binding of immunotoxin to cell. The activity of anti-gp41, but not anti-gp120, immunotoxins was markedly enhanced in the presence of soluble CD4 or peptides corresponding to the CDR3 region of CD4. CD4-mediated enhancement of anti-gp41 immunotoxin activity was observed for laboratory strains neutralized by sCD4 and for clinical isolates that were resistant to neutralization by sCD4. Immunotoxin action was potentiated by brefeldin A, bafilomycin A1, cortisone, and an amphipathic fusion peptide, but not by cytochalasin D, nocodazol, monodansyl cadaverine, or trans-retinoic acid. Anti-HIV immunotoxins are useful tool with which to study the functional expression of gp120/gp41 antigens on the surface of HIV-infected cells, as well as potential AIDS therapeutics. Because these studies relate to the accessibility of viral antigens to antibody-mediated attack, these studies also have relevance for vaccine development.

Sequences regulating tropism of human immunodeficiency virus type 1 for brain capillary endothelial cells map to a unique region on the viral genome.

Two infectious molecular clones of human immunodeficiency virus type 1, NL4-3 and JR-CSF, differ in their abilities to productively infect human brain capillary endothelial (HBCE) cells. The phenotypes of recombinants between these two molecular strains were examined to identify viral sequences responsible for the difference in HBCE cell tropism between the two parental strains. Our results indicate that HBCE cell tropism maps to a region that encompasses the C1 region of env and includes overlapping reading frames for the accessory genes vpr, vpu, tat, and rev. This region was unique for HBCE cell tropism and did not cosegregate with either macrophage or T-cell line tropism. However, several recombinant clones displayed dual tropism for both HBCE cells and macrophages. These endothelial cell- and macrophage-tropic strains may have a unique pathogenic advantage by entering the brain via HBCE cells and subsequently infecting microglial cells with high efficiency, leading to the induction of human immunodeficiency virus dementia.

Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy.

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.

Human immunodeficiency virus envelope V1 and V2 regions influence replication efficiency in macrophages by affecting virus spread.

The V3 hypervariable region of the HIV-1 envelope protein is a major determinant of viral tropism for macrophages. However, the replication of macrophage-tropic HIV-1 strains varies considerably in macrophages, and this variability has been linked to the V1 and V2 envelope regions. In the present study, recombinant HIV clones were generated by inserting V1 and V2 sequences from the Ba-L HIV isolate, which has a high macrophage replication level, into the genomic background of a macrophage-tropic clone with a low macrophage replication level. Infection of macrophages with varying multiplicities of infection and direct detection of the number of infected macrophages per culture showed that the Ba-L V1 and V2 envelope sequences enhanced the ability of virus to spread in the cultures. In contrast, macrophage-tropic clones with low replication efficiency infected macrophages initially but showed no evidence of spread to additional cells during the culture period. This effect on virus spread appeared to be macrophage-specific as it was not observed in cultures of T lymphocytes. Comparison of recombinant clones containing V1, V2, and V3 envelope sequences from high-efficiency Ba-L and JR-FL strains indicated that markedly different V1 and V2 sequences could impart the same rapidly spreading phenotype in macrophages.

A 60-kDa prion protein (PrP) with properties of both the normal and scrapie-associated forms of PrP.

Scrapie is a transmissible spongiform encephalopathy of sheep and other mammals in which disease appears to be caused by the accumulation of an abnormal form of a host protein, prion protein (PrP), in the brain and other tissues. The process by which the normal protease-sensitive form of PrP is converted into the abnormal protease-resistant form is unknown. Several hypotheses predict that oligomeric forms of either the normal or abnormal PrP may act as intermediates in the conversion process. We have now identified a 60-kDa PrP derived from hamster PrP expressed in murine neuroblastoma cells. Peptide mapping studies provided evidence that the 60-kDa PrP was composed solely of PrP and, based on its molecular mass, appeared to be a PrP dimer. The 60-kDa PrP was not dissociated under several harsh denaturing conditions, which indicated that it was covalently linked. It was similar to the disease-associated form of PrP in that it formed large aggregates. However, it resembled the normal form of PrP in that it was sensitive to proteinase K and had a short metabolic half-life. The 60-kDa PrP, therefore, had characteristics of both the normal and disease-associated forms of PrP. Formation and aggregation of the 60-kDa hamster PrP occurs in uninfected mouse neuroblastoma cells, which suggests that hamster PrP has a predisposition to aggregate even in the absence of scrapie infectivity. Similar 60-kDa PrP bands were identified in scrapie-infected hamster brain but not in uninfected brain. Therefore, a 60-kDa molecule might participate in the scrapie-associated conversion of protease-sensitive PrP to protease-resistant PrP.

Contrasting effects from a single major histocompatibility complex class II molecule (H-2E) in recovery from Friend virus leukemia.

Resistance to erythroleukemia induced by infection with the Friend virus complex (FV) has been mapped to several genes residing both within and outside the murine major histocompatibility complex (MHC). MHC genes located in the A, D, and Qa/Tla regions of the murine H-2 complex have been shown to affect disease resistance through their capacity to regulate various aspects of the host immune response to viral antigens. This study establishes H-2E as the fourth MHC locus controlling immunological resistance to FV. Our investigation into the role of H-2E molecules revealed two distinct and opposite effects on recovery from Friend disease. H-2b/b mice normally lack a functional E gene product and are resistant to high doses of FV. The expression of H-2E molecules in H-2 recombinant or transgenic mice of this genotype resulted in a significant decrease in spontaneous recovery from FV-induced leukemia. In contrast, H-2E expression also appeared to influence recovery from Friend disease in a positive manner, since blocking these molecules with anti-E antibodies in vivo significantly decreased recovery from Friend disease. The data indicate that the positive effects of H-2E molecules derive from their function as restriction elements for helper T-cell recognition of the viral envelope glycoprotein, and we postulate that the negative effects are due to H-2E-dependent deletion in the T-cell repertoire during development.

Differences in CD4 dependence for infectivity of laboratory-adapted and primary patient isolates of human immunodeficiency virus type 1.

CD4 is known to be an important receptor for human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes and macrophages. However, the limiting steps in CD4-dependent HIV-1 infections in vivo and in vitro are poorly understood. To address this issue, we produced a panel of HeLa-CD4 cell clones that express widely different amounts of CD4 and quantitatively analyzed their infection by laboratory-adapted and primary patient HIV-1 isolates. For all HIV-1 isolates, adsorption from the medium onto HeLa-CD4 cells was inefficient and appeared to be limiting for infection in the conditions of our assays. Adsorption of HIV-1 onto CD4-positive peripheral blood mononuclear cells was also inefficient. Moreover, there was a striking difference between laboratory-adapted and primary T-cell-tropic HIV-1 isolates in the infectivity titers detected on different HeLa-CD4 cells. Laboratory-adapted HIV-1 isolates infected all HeLa-CD4 cell clones with equal efficiencies regardless of the levels of CD4, whereas primary HIV-1 isolates infected these clones in direct proportion to cellular CD4 expression. Our interpretation is that for laboratory-adapted isolates, a barrier step that preceeds CD4 encounter was limiting and the subsequent CD4-dependent virus capture process was highly efficient, even at very low cell surface concentrations. In contrast, for primary HIV-1 isolates, the CD4-dependent steps appeared to be much less efficient. We conclude that primary isolates of HIV-1 infect inefficiently following contact with surfaces of CD4-positive cells, and we propose that this confers a selective disadvantage during passage in rapidly dividing leukemia cell lines. Conversely, in vivo selective pressure appears to favor HIV-1 strains that require large amounts of CD4 for infection.

Foreign PrP expression and scrapie infection in tissue culture cell lines.

PrP expression and scrapie infectivity was studied in a group of tissue culture cell lines from mice, hamsters, rats and humans. Cell surface PrP protein was detected in seven of ten cell lines from a variety of tissues and showed both cell surface and cytoplasmic perinuclear distributions. Mouse N2a neuroblastoma cells could be persistently infected with scrapie in vitro, but OBL-21 mouse neuroma cells were never successfully infected. Foreign PrP molecules including hamster PrP, mutant hamster PrP with leucine at amino acid 102, and mutant mouse PrP expressing a hamster-encoded PrP antigen epitope were expressed in mouse N2a cells. However, none of these cell lines could be successfully infected with the 263K hamster scrapie agent. Furthermore, expression of these foreign PrP molecules did not lead to spontaneous generation of protease-resistant PrP or a transmissible scrapie-like agent in vitro.

Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.

Previous experiments indicate that the V3 hypervariable region of the human immunodeficiency virus (HIV) envelope protein influences cell tropism of infection; however, so far no consistent V3 sequence can account for macrophage or T-cell tropism. In these experiments, we studied infectious recombinant HIV clones constructed by using V3 region sequences of HIV isolates from 16 patients to search for sequences associated with cell tropism. Remarkable homology was seen among V3 sequences from macrophage-tropic clones from different patients, and a consensus V3 region sequence for patient-derived macrophage-tropic viruses was identified. In contrast, V3 sequences of T-cell-tropic clones from different patients were highly heterogeneous, and the results suggested that sequence diversity leading to T-cell tropism might be generated independently in each patient. Site-specific mutations identified amino acids at several positions on each side of the GPGR motif at the tip of the V3 loop as important determinants of tropism for T cells and macrophages. However, a wide variety of mutant V3 sequences induced macrophage tropism, as detected in vitro. Therefore, the homogeneity of macrophage-tropic patient isolates appeared to be the result of selection based on a biological advantage in vivo.

Detailed mapping of the Rfv-1 gene that influences spontaneous recovery from Friend retrovirus-induced leukaemia.

Using H-2 recombinant and mutant mice, the Rfv-1 gene influencing spontaneous recovery from Friend retrovirus (FV)-induced leukaemia was mapped in the D locus. Two Db alleles were required for full recovery, and a single Dd transgene did not convey increased susceptibility to FV in the presence of homozygous Db/b genotype. The results suggest that an increase in the expression of Db may lead to more effective stimulation of FV-specific CTL.