Background
HIV-1 replication is associated with the generation of various nucleic acid products that may act as ligands for cytosolic innate immune sensors including RIG-like receptors (RLRs). However, to induce an effective innate immune response, RLRs need to be primed by actin disturbances, sensed by R12C (Acharya et al., Cell 2022). While mounting evidence suggests that HIV-1 is sensed by RLRs, whether HIV-1 is capable of antagonizing this sensing mechanism is currently unclear.
Methods
Using wild-type and mutant clade B and C HIV-1 constructs, we quantified the impact of Nef-dependent actin remodeling on viral replication and the induction of antiviral and pro-inflammatory responses in macrophages, dendritic cells and CD4+T cells. The impact of immune sensing on viral replication was further examined by blocking IFN signaling. To define the key sensors and underlying mechanisms, we knocked-out MAVS in human primary macrophages (MDMs) and treated cells with HIV-1 inhibitors.
Results
We found that a single R191A or F191A mutation in the C-terminus of Nef, which specifically prevents Pak2-mediated phosphorylation of the actin modulator cofilin, doubled the level of MX1, CXCL10 and CCL5 induction in primary MDMs and dendritic cells (DCs) upon HIV-1 infection. Strikingly, infection of DCs with this mutant is also associated with an up to 10-fold increase in production of pro-inflammatory cytokines such as IP-10, IL-6 and IL-8. Using HIV inhibitors, we show that this response is dependent on integration. Furthermore, CRISPR-Cas9 KO of MAVS in primary human macrophages abrogated innate immune activation. We also support a role for the sensor MDA-5 as it is activated and ISGylated upon infection with the mutant virus. Finally, we show that Nef prevents recruitment of R12C to RLRs and thus maintains both MDA-5 and RIG-I in a phosphorylated, inactive state, similar to that of uninfected cells.
Conclusions
Our results present HIV-1 Nef as the first viral counter-mechanism of actin disturbances critical for effective immune sensing.