SAMHD1-independent inhibition of HIV-1 DNA accumulation by endogenous CCL2 neutralization: possible role of APOBEC3A

Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Furthermore, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. We found that CCL2 neutralization potently reduced viral DNA accumulation and the number of p24 Gag+ cells during the course of either a productive or a single cycle infection with HIV-1BaL or (VSV-G) HIV-1, respectively. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that SAMHD1 expression was not affected by CCL2 neutralization. Furthermore, CCL2 blocking reduced the percentage of p24 Gag+ cells both in the absence and in the presence of exogenous dNTPs, and restricted the transduction of macrophages with Vpx+ lentiviral particles, which determined a strong down-modulation of SAMHD1 expression. These results demonstrated that altered SAMHD1 expression or function cannot account for the CCL2 neutralization-mediated restriction of HIV-1 replication in macrophages. Conversely, a strong and selective induction of APOBEC3A transcript and protein expression was associated with the inhibition of HIV-1 replication mediated by CCL2 neutralization. Interestingly, the level of CCL2 blocking induced APOBEC3A expression was comparable to those of freshly isolated monocytes. Finally, induction of A3A was type I IFN independent, since neutralizing Abs to IFN-α or IFN-β did not abolish CCL2 blocking-mediated A3A expression. Overall, these data demonstrate that neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle likely involving the up-regulated expression of APOBEC3A. These results highlight a novel mechanism which may contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defenses against HIV-1 and protecting macrophages from infection.