P26-17. Increased binding to ALIX but no effect on viral growth for HIV-1 subtype C gag with PYKE insertion

Robert van Domselaar [1,2], Shambhu G. Aralaguppe [2], Ujjwal Neogi [2]
Affiliates: [1] Unit of Infectious Diseases and Dermatology, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden. [2] Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

Background
HIV-1 is a major health burden with over 36 million infected people and 1 million deaths worldwide in 2015. Recently, we identified a PYKE tetrapeptide insertion in the HIV-1 type C gag-P6 domain within a subgroup of infected patients. This domain is associated with ALIX-binding, a functional property lost in HIV-1 type C. Host cell protein ALIX is known to facilitate retroviral budding. In this study, we examined whether the PYKE insertion in the gag-P6 domain restored ALIX-binding for HIV-1 type C and whether it affected virion release.

Methods
We used in silico analysis to predict gag:ALIX interactions. Cellular localization of gag and ALIX was assessed by immunofluorescence. To assess the gag:ALIX interaction in vitro, GFP-tagged ALIX was co-expressed with one of the codon-optimized gag variants (gag_wt, gag_PYKE, gag_PYQE) in cells. Lysates were subjected to GFP-pull down and immunoblotting. Finally, viral growth kinetic assays were performed to determine the fitness of viruses with or without the PYKE insertion.

Results
In silico docking analysis showed that gag_PYKE could interact with ALIX using its positively charged amino acids and revealed a potential ubiquitination site at K485. Immunofluorescence showed predominant membrane localization for all gag variants and cytoplasmic localization for ALIX. Co-immunoprecipitation analysis showed increased binding to ALIX for gag_PYKE compared to gagwt. Abolishing the ubiquitination site by K485Q mutation (gag_PYQE) did not reverse the binding capacity compared to gag_PYKE. Using HIV-1 type C viruses expressing various patient-derived gag proteins showed no significant difference in viral fitness between viruses expressing gagPYKE compared to gagwt.

Conclusion
Although the PYKE insertion in the gag-domain of HIV-1 type C sequence did result in increased interaction of gag with ALIX, there was no clear significant difference in viral fitness between HIV-1 type C viruses with or without the PYKE insertion. Since PYKE insertion has also been associated with drug failure in HIV-1 patients, it will be clinically important to also evaluate drug responses against HIV-1 type C viruses with or without PYKE insertion.