Novel Mechansims for P^k (Gb₃) Inhibition of HIV Infection.

Many blood group antigens belong to a class of lipid-sugar conjugate molecules known as glycosphingolipids (GSLs), including ABH, Lewis and P blood group systems. GSLs are implicated in HIV-host cell fusion, and several bind to HIV-gp120 in vitro. In addition, inhibiting glycolipid biosynthesis has been shown to prevent HIV membrane fusion. The P^k blood group antigen, also known as globotriaosylceramide (Gb₃), has been shown to augment HIV fusion in in vitro models; however its exact role in HIV infection is not known. We have previously developed a soluble analogue of P^k, a highly effective gp120 ligand, which inhibits HIV-1 and HIV-2 membrane fusion. We hypothesize that P^k influences susceptibility to HIV infection, inhibiting at the level of membrane fusion and entry. We have presently investigated the effects of soluble and expressed P^k on HIV-1 infection in vitro. Soluble P^k effects were considered by pre-treating monocyte (M)-tropic HIV-1_Ba-L or T-cell (T)-tropic HIV-1_IIIB with soluble P^k prior to infection of PHA or PHA/IL2 activated peripheral blood-derived mononuclear cells (PBMCs) or Jurkat T-cells. Productive M-tropic and/or T-tropic HIV-1 infection of PBMCs and Jurkat cells was inhibited by more than 90%. Inhibition of membrane fusion and entry was visualised by transmission electron microscopy. Soluble P^k had minimal effects on cell viability or HIV receptor (CD4) and co-receptor (CCR5 or CXCR4) expression as measured by flow cytometry. Exogenously introduced P^k in Jurkat T-cells was accomplished by liposome fusion, and susceptibility to T-tropic HIV-1_IIIB infection was determined. P^k transfer in Jurkat T-cells also reduced productive HIV-1 infection by 50%, without affecting cell viability or HIV receptor and co-receptor expression. Effects of differential P^k expression on HIV susceptibility was investigated using PBMCs with P^k accumulation, from patients with Fabry disease and P₁^k blood group individuals, or which lack P^k, from p blood group individuals. PBMCs representative of these two groups were infected with M-tropic HIV-1_Ba-L or T-tropic HIV-1_IIIB. PBMCs with P^k accumulation from patients with Fabry disease were resistant to productive M-tropic, but not T-tropic HIV-1 infection. These PBMCs had significantly decreased expression levels of the M-tropic HIV-1 co-receptor CCR5. P^k accumulation in P₁^k blood group PBMCs shows resistance to T-tropic and M-tropic HIV-1 infection, unrelated to levels of chemokine receptors. PBMCs lacking P^k from p blood group individuals were hypersensitive to M-tropic HIV-1 infection, while T-tropic HIV-1 infection levels were variable. Interestingly, p blood group PBMCs showed significantly higher levels of the CCR5 co-receptor. Our findings suggest P^k is inhibitory to HIV infection, which may translate into natural resistance in individuals that express high levels of P^k. This may be linked to interactions with CCR5, through receptor trafficking to the cell surface, or associations within lipid rafts. Furthermore, soluble P^k or potential mechanisms to increase cellular P^k expression, provide novel strategies for prevention of HIV infection.