Negative factor from SIV binds to the catalytic subunit of the V-ATPase tointernalize CD4 and to increase viral infectivity

The accessory protein negative factor (Nef) from human immunodeficiency vir us (HIV) and simian immunodeficiency virus (SIV) is required for optimal vi ral infectivity and the progression to acquired immunodeficiency syndrome ( AIDS). Nef interacts with the endocytic machinery, resulting in the down-re gulation of cluster of differentiation antigen 4 (CD4) and major histocompa tibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex th at is required for the internalization of proteins from the plasma membrane , could be demonstrated. In this study we investigated the relevance of dif ferent motifs in Nef from SIVmac239 for its internalization, CD4 down-regul ation, binding to components of the trafficking machinery, and viral infect ivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subseque nt studies on Nef mutant viruses revealed that the flexible loop is essenti al for optimal viral infectivity. Therefore, our data demonstrate how Nef c ontacts the endocytic machinery in the absence of its direct binding to AP- 2 and suggest an important role for subunit H of the V-ATPase in viral infe ctivity.