HIV virions and HIV replication are unaffected by granulysin (2000)

Granulysin is a small peptide commonly found in the granules of CD8 T cells and natural killer (NK) cells [1,2] A role for granulysin has been implicated in the elimination of certain intracellular microorganisms by CD8 T cells [2] It is a member of the saposin-like protein family of lipid-binding proteins [1,3] Saposins interact with lipid membranes and activate lipid-degrading enzymes such as glucosylceramidase and sphingomyelinases, which can lead to apoptosis by initiating the caspase cascade [4–6] Recombinant granulysin shows a dose-dependent growth inhibition of several bacteria including Mycobacterium tuberculosis, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus[7] In addition, it can kill the fungi and parasite, Crypotococcus neoformans, Candida albicans, and Leishmania major. To date, however, nothing is known about the antiviral properties of granulysin. HIV is an enveloped virus that, like all viruses, replicates intracellularly. It is susceptible to elimination by cytotoxic T lymphocyte (CTL) killing of infected cells [8] However, little is known about whether the HIV-infected cells release infectious virions upon lysis by CTL. An agent that could cause the direct inactivation of the HIV virions would further control the spread of infection. Because granulysin is released by CD8 T cells upon antigen recognition, and has the above-mentioned antimicrobial properties, particularly its lipid membrane disrupting nature, we examined the potential antiviral activity of recombinant granulysin against HIV. To assess the possibility of a direct antiviral effect by granulysin on HIV particles, recombinant granulysin [9] was added to fluids containing 600 and 3000 TCID50 of HIV-1SF2[10] diluted in serum-free AIM-V medium (Gibco–BRL, Gaithersburg, MD, USA), to yield 5 mM of granulysin. The mixtures were incubated for 2–3 h at 37°C before being used to inoculate phytohemagluttinin-P (3 μg/ml) (Sigma Chemicals, St Louis, MO, USA)-stimulated CD4 cells from an HIV-seronegative control subject. After 1 h, the cells were washed and 3 × 105 infected cells were cultured in a 48-well culture plate (in triplicate) in complete medium, which consisted of RPMI 1640 medium containing 10% heat-inactivated (56°C for 30 min) fetal calf serum, 1% antibiotics (100 U/ml penicillin; 100 μg/ml streptomycin) 2 mM glutamine, and 100 U/ml recombinant IL-2 (Glaxo Wellcome, Research Triangle Park, NC). The cultures were passed and monitored for reverse transcriptase (RT) activity [11] every 3 days. Treatment of HIV virions with recombinant granulysin did not appreciably alter their infectivity, as indicated by similar peak levels of virus replication (Table 1) and replication kinetics (data not shown), relative to the control untreated virion cultures.Table 1: The effect of granulysin on HIV particle infectivitya. We next evaluated whether culturing acutely infected CD4 T cells in the continued presence of granulysin would affect HIV replication. Phytohemagglutinin-P-stimulated CD4 T cells were inoculated with approximately 3000 TCID50 of HIV-1SF2. After incubation for 1 h, the cells were washed and plated (in triplicate) in 96-well culture plates (105 cells/well) in complete medium. The infected CD4 cells were cultured alone or in the presence of various concentrations of granulysin (diluted in RPMI 1640 medium). Granulysin was added at the initiation of culture and replenished every 2 days thereafter upon cell passage. The amount of HIV replication in the cultures was determined every 2 days by measuring RT activity in culture fluid samples. In four separate experiments, granulysin showed no substantial effect on the levels of HIV production relative to the untreated control, and in general, no appreciable effect on cellular viability. In the different experiments, the peak levels of HIV replication reached between 4 × 105 and 2.5 × 106 cpm/0.1 ml of culture fluid by day 9. The amount of RT activity in the cultures treated with a series of granulysin concentrations did not differ from the untreated cultures by more than 20%. Continuous exposure of acutely infected CD4 T cells to granulysin thus did not affect their viability or reduce their ability to replicate HIV. The results of this study indicate that granulysin does not substantially affect HIV virion infectivity (Table 1), or ongoing virus replication in acutely infected CD4 T cells. The concentrations of granulysin examined in these experiments were sufficient to induce the various antimicrobial activities reported for this peptide [1,2] Although the external cell wall or membrane of various Gram-negative and Gram-positive bacteria, fungi, and the parasite Leishmania may be sensitive to disruption by granulysin, the envelope of HIV thus appears to be unaffected. The lack of any effect of granulysin on HIV replication in acutely infected cells indicates that granulysin does not prevent spread/reinfection in the cultures. However, because granulysin appears to require the presence of a molecule like perforin to enter the cells [9] these experiments did not address the possible intracellular activity of granulysin – an issue that may not be relevant because HIV does not acquire its envelope until it buds through the outer cell membrane. Therefore, this antimicrobial granule protein probably does not play a direct role in the control of HIV infection. Carl E. Mackewicz Susan Ridha Jay A. Levy Acknowledgement The authors would like to thank William Ernst and Robert Modlin for providing the granulysin.