IFN-inducible IFITM proteins (IFITM1, 2, and 3) inhibit the replication of various viruses including HIV-1 through poorly understood mechanisms. Here, we further analyzed characteristics of these newly identified HIV-1 restriction factors. Firstly, in contrast to other anti-HIV-1 proteins, such as tetherin and APOBEC3G, IFITMs were resistant to a down-regulation of surface expression or degradation by HIV-1 proteins. Secondly, the enforced expression of IFITMs reduced the production of HIV-1 viruses from cells transfected with proviral plasmids containing whole viral sequences. Although their inhibitory activities were modest when compared to that of tetherin, IFITMs, but not tetherin, directly reduced the expression of HIV-1 proteins including Gag, Vif and Nef. Of importance, however, IFITMs had no inhibitory effect when these viral proteins were expressed by codon-optimized cDNAs that bypassed the viral-specific expression machinery. Indeed, our results supported the idea that IFITMs interfere with viral protein expression mediated by double-stranded viral RNAs, such as RRE and TAR. Finally, the S-palmitoylation of IFITMs, which is crucial for their anti-influenza virus activity, was not required for their anti-HIV-1 activity, indicating that IFITMs restrict these viruses at different steps. These characteristics lead to a better understanding of the mechanism by which IFITMs restrict HIV-1 and other viruses.

HIV-1 proteins, including Tat, gp120, and Nef, activate macrophages (MΦ), which is consistent with the fact that HIV-1 infection is characterized by sustained immune activation. Meanwhile, MΦ are functionally classified into two types: proinflammatory M1-MΦ and anti-inflammatory M2-MΦ. We show that HIV-1 proteins, particularly Nef, preferentially activate M2-MΦ. Extracellular Tat, gp120, and Nef activated MAPK and NF-κB pathways in human peripheral blood monocyte-derived MΦ. However, the activation was marked in M-CSF–derived M2-MΦ but not GM-CSF–derived M1-MΦ. Nef was the most potent activator, and its signaling activation was comparable to that by TNF-α. Indeed, Nef was internalized more rapidly by M2-MΦ than by M1-MΦ. The myristoylation and proline-rich motif of Nef were responsible for the observed signaling activation. Consistent with the activation of MAPK/NF-κB pathways, Nef stimulated the production of a number of proinflammatory cytokines/chemokines by M2-MΦ. However, Nef reduced the expression of CD163 and phagocytosis, the characteristic markers of M2-MΦ, indicating that Nef drives an M2-like to M1-like phenotypic shift. Because the differentiation of most tissue MΦ depends on M-CSF and its receptor, which is the essential axis for the anti-inflammatory M2-MΦ phenotype, the current study reveals an efficient mechanism by which HIV-1 proteins, such as Nef, induce the proinflammatory MΦ.