Background: Recent studies have demonstrated the significance of cell surface HSPGs in HIV-1 infection, as well as the cellular uptake of Tat, the transactivator protein of HIV-1. In this study we examined: The hypothesis that HIV-1 in the process of viral budding incorporates HSPG molecules; and the role of perlecan, the only HSPG expressed on the cell surface of the colon carcinoma cell line WiDr, in HIV-1 Tat uptake and internalization.   

Methods:  CEM cells (10⁶) were infected with 1 ng p24 antigen of the T-tropic HIV-1 strain NL4-3. Cells were then metabolically labeled with 40 mCi of Na₂³⁵SO₄ and transferred to ELISA plates, in RPMI-1640 medium supplemented with 10% FBS at 37°C. 7 days post-infection virus-containing supernatant was collected, and HIV-1 was purified by column chromatography using CL-2B Sepharose. Eluted fractions were ³⁵S scintillation counted, and analyzed for HIV-1 p24 antigen. ³⁵S-HSPG molecules incorporated into HIV-1 particles were further purified by DEAE chromatography and analyzed by SDS gel followed by autoradiography.  WiDr cells were incubated in serum-free medium with ¹²⁵I-Tat at 37°C for 5 and/or 21 hours. Tat cellular uptake and internalization was analyzed by quantitation of surface-bound (heparin-releasable), intracellular (heparin-resistant), and degraded ¹²⁵I-Tat.

Results:  Purified HIV-1 particles that were generated from ³⁵SO₄ -labeled CEM cells are highly sulfated, and further analysis by DEAE-chromatography and SDS-PAGE provides strong support that sulfation of HIV-1 is due to incorporation of ³⁵S-HSPG molecules, which may take place during viral budding.  WiDr cells are able to bind and internalize substantial amounts of extracellular ¹²⁵I-Tat, consistent with a role for the perlecan HSPG. A minority of the internalized ligand underwent degradation, while most of ¹²⁵I-Tat remained in the cells even after 21 hours.

Conclusions: The presence of ³⁵S-molecules on HIV-1 particles strongly suggests the incorporation of HSPGs into the envelope during viral budding. Further characterization of these molecules will provide valuable information and more complete understanding of viral pathogenicity.  In addition our results provide strong support for the role of perlecan, the only HSPG expressed on the cell surface of the colon carcinoma cell-line WiDr, in HIV-1 Tat uptake and internalization. Further experimentation is required to fully characterize the role of HSPG in extracellular HIV-1 Tat uptake.