178-M. Low Quantity of SU on HIV Is Due to Low Incorporation of Envelope Glycoproteins and not to Shedding

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Session 38 Poster Session
Viral and Cellular Proteins on the Virion Surface
Session Time: 4:30-6:30 pm
Room 4E-F

178-M.

Low Quantity of SU on HIV Is Due to Low Incorporation of Envelope Glycoproteins and not to Shedding
E. Chertova*¹, J. Bess¹, B. Crise¹, R. Sowder II¹, T. Schaden¹, J. Hilburn¹, J. Hoxie², L. Henderson¹, and L. O. Arthur¹
¹SAIC, Frederick, MD and ²Univ. of Pennsylvania, Philadelphia

Background: Our program has focused on the design and synthesis of whole inactivated virion particle vaccines, specifically, using methods to irreversibly inactivate live virus while retaining native and functional envelope (Env) proteins. To study the contribution of SU density to the efficacy of the vaccine we focus on viral incorporation and retention of Env proteins.
Methods: To determine the amounts of Gag and Env glycoproteins retained on purified virus and to calculate the ratio of Gag /Env proteins, we have employed microscale HPLC methods. Eluted proteins were analyzed by SDS-PAGE, immunoblot analysis, mass spectrometry, and protein sequence analysis. Physical parameters that contribute to the average surface density of Env complexes on purified virus have been explored. These include temperature and freeze-thaw cycles, and effect of sCD4 on SU retention.
Results: All HIV-1 and SIV isolates examined, except one SIV clone, had surface envelope SU glycoprotein to transmembrane ™ glycoprotein molar ratios of 1:1. This ratio suggests that SU does not generally shed from the virus once TM is inserted into the membrane of the virus. All HIV-1 and most SIV isolates examined had Gag:Env ratios of approximately 60:1, corresponding to only 7-14 SU containing envelope trimers per virion, assuming that 1200-2500 Gag molecules per virion. In contrast, SIV purified from the biological clone E11S contained levels of SU at least 10-fold greater than SU from HIV-1 isolates. The quantity of SU bound to the virus and the SU-to-TM ratios were not significantly changed during multiple freeze-thaw cycles and purification through sucrose gradients. Exposure of HIV and SIV to temperatures of 55°C or greater for 1 hour resulted in loss of most of the SU from the virus but retention of TM. Incubation of purified virus with soluble CD4 at 37°C resulted in no appreciable loss of SU from SIV or HIV.
Conclusions: These studies suggest that the SU-TM interactions are highly stable and incorporation of TM into the viral membrane may be the primary factor determining the quantity of SU associated with SIV and HIV-1.