Naturally arising HIV-1 Nef variants conferring escape from cytotoxic T lymphocytes influence viral entry co-receptor expression and susceptibility to superinfection

HIV-1 Nef is a key factor for pathogenesis and is known to down-regulate functionally important molecules, including viral entry co-receptor CCR5 and CXCR4, from the surface of HIV-infected cells. Some of these Nef activities are mediated by the well-conserved proline-rich region of Nef, and this region is highly targeted by cytotoxic T lymphocytes (CTLs). In the present study, we asked whether Nef variants selected under CTL-mediated selective pressure in vivo may constrain these important Nef activities. The analysis of autologous nef sequences isolated from a cohort of total 235 subjects in Japan revealed that the subjects showing amino acid variations, such as Arg75Thr and Tyr85Phe, located within the proline-rich region were significantly over-represented by those having HLA-B∗3501. CTL assays corroborated that these mutations conferred escape from HLA-B∗3501-restricted CTLs. The Arg75Thr variant Nef selectively impaired CCR5, but not CXCR4, down-regulation activity from the cell surface; whereas the Tyr85Phe variant Nef affected neither CCR5 nor CXCR4 down-regulation activity. Moreover, the cells expressing the Arg75Thr variant Nef significantly impaired protection from superinfection by CCR5-tropic, but not CXCR4-tropic, viruses. These results highlighted the importance of certain Nef-specific CTLs in modulation of viral co-receptor down-regulation activity and protection from HIV-1 superinfection, providing us with additional insight into vaccine design.

Research highlights
► A population level analysis reveals that HIV-specific CTL responses select CTL-escape-conferring variants even in a well-conserved functional region of HIV-1 Nef.
► Certain CTL-escape variants in Nef selectively impaired down-regulation activity of viral entry co-receptor CCR5 on the cell surface.
► The modulation of CCR5 down-regulation activity by the CTL-escape Nef variants causes the impairment of protection from HIV superinfection.
► Considering that the superinfection protection is an important capacity of viruses for efficient replication and persistence, the present study provides us with additional insight into vaccine design against HIV-1.