Effects of naturally-arising HIV Nef mutations on cytotoxic T lymphocyte recognition and Nef's functionality in primary macrophages

Although HIV can infect several cellular subsets, such as CD4⁺ T lymphocytes and macrophages, it remains unclear whether an HIV infection in macrophages supports cytotoxic T lymphocyte (CTL) escape. Here, we tested two naturally-arising mutations located in the well-conserved polyproline region of Nef for their effects on CTL recognition, Nef's functionality, and viral replication capacity in macrophages. These mutations were selected because they are known to cause CTL escape in the context of T lymphocytes. Monocyte-derived macrophages (MDMs) infected with the wild-type virus, but not with variant viruses, were efficiently killed by CTL clones targeting Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). The CTL-escape mutation, Arg⁷⁵Thr, or Arg⁷⁵Thr/Tyr⁸⁵Phe double mutation, reduced the HLA class I down-regulation activity and, interestingly, increased the susceptibility of virus-infected MDMs to recognition by CTLs targeting a different epitope. The same mutations reduced the CCR5, but not CD4, down-regulation activity. Moreover, the Nef variants were impaired for Hck activation and enhancement of viral replication in MDMs. These results suggest that HIV-infected MDMs are killed by CTLs targeting Nef epitopes, contributing to selection and adaptation of CTL-escape viral variants.