HIV-1 rapidly induces the specific degradation of IRF3 in infected cells during acute infection of T cells and myeloid cells in vitro
and within mucosal T cells ex vivo
). Our studies (here and in the companion paper
]) reveal a novel role for Vpu in the control of innate antiviral immunity in HIV-1-infected cells. Our genetic and biochemical evidence suggests that Vpu is both necessary and sufficient for IRF3 degradation by HIV, in a lysosomal process similar to that previously described for tetherin/BST2 and CD4 downmodulation (12
). Here we addressed the functional outcome of infection of HIV-1 target cells in the absence of this potent Vpu-directed antagonism of the innate immune signaling response. We found a robust and potent induction of IRF3-dependent innate signaling and downstream production of ISGs when target cells were infected with Vpu-deficient viruses compared to wt Vpu-producing HIV-1 strains. This innate immune activation is retained in a number of different HIV-1 target cells, including primary macrophages, and in ex vivo
mucosal T cell cultures (). Our direct comparison of congenic Vpu-positive and -deficient HIV-1 strains uncovered a 5- to 10-fold increase in ISG induction by infection with the Vpu-deficient strain (), showing that HIV-1 can truly be recognized by PRRs to induce innate immune responses from host cells.
We also show that IRF3 phosphorylation and nuclear accumulation occur in host cells in response to infection with Vpu-deficient HIV-1 strains. IRF3 activation is downstream of pathogen sensing from a number of PRRs, including the RLR family, several TLRs, as well as several less well characterized responses to non-self-products, including cytoplasmic DNA (50
). Interestingly, we found that IRF3 activation and subsequent production of IRF3-dependent ISGs were unaffected by shRNA ablation of IPS-1 or MyD88, the essential adaptor molecules required for RLR and many TLR signaling responses, respectively. This suggests that the upstream recognition event of HIV-1 infection in these target cells is not reliant on RIG-I, MDA5, TLR7, or TLR9, all of which have been implicated in recognition of HIV-1 in different cellular contexts (2
). Thus, innate immune signaling within T cells or macrophages appears to be programmed differentially from that in plasmacytoid dendritic cells, which specifically utilize TLRs for HIV-1 recognition in an IRF3-independent program (2
). Our working model of IRF3 regulation by HIV-1 suggests that Vpu antagonizes IRF3 later in the viral life cycle, perhaps as replication intermediates serving as PAMPs accumulate and trigger signaling. We note that recent reports provide evidence of innate immune activation in other contexts that is consistent with this idea (11
). We do observe production of Vpu at times concurrent with this innate immune signaling that occurs in specific cell types; it is possible, however, that these processes reflect an additional early event of virus recognition not directly tied to the Vpu targeting of IRF3 that is still uncovered by the absence of Vpu. This process of PAMP triggering of innate immune signaling would be in contrast to host cell sensing of viral capsids, where incoming virions are sensed in an AP-1- and NF-κB-dependent mechanism (41
IRF3 activation is deleterious to HIV-1 infection and stimulates a robust and widely antiviral state within infected cells (9
). By interacting with IRF3 and targeting it to the lysosome, Vpu facilitates IRF3 proteolysis and prevents the expression of genes involved in innate immune defenses against HIV-1, including type I IFN, ISGs, and direct IRF3 target genes that mediate antiviral actions. Among the genes responsive to IRF3 are known HIV-1 restriction factors, including APOBEC3G, tetherin, ISG15, and others. Indeed, we show that HIV-1 suppression of IRF3 serves to enhance cell permissiveness for infection by relieving innate immune restriction of virus replication and cell spread during the critical stage of acute infection. It is important to note that our study has separated the Vpu-dependent IRF3 phenotypes from the known role of Vpu in antagonizing tetherin. Whenever possible, we utilized in our experiments cell types that are known to be deficient in tetherin expression (i.e., 293T cells), and when utilizing cells know to express tetherin (i.e., THP-1 cells), we have focused on evaluating only the early effects of Vpu deficiency and innate immune signaling responses dependent on IRF3 in order to avoid the confounding influences of tetherin-dependent late-egress phenotypes on HIV-1 production. For this reason, we are not able to determine the effects of IRF3 activation on HIV-1 spread in the current study. We find a global induction of ISGs by Vpu-deficient HIV-1 strains but find no regulation of NF-κB target genes, again underscoring the specific IRF3 activation in the absence of Vpu antagonism.
Our study is the first to reveal additional pathogen sensing of HIV-1 independent of RLR and MyD88 signaling, which drives IRF3-responsive gene expression within acutely infected immune cells from mucosal tissue (). This natural response does not require overcoming additional HIV-1 infection blockades, as has been reported for the cryptic sensing of HIV-1 in dendritic cells (32
), but is instead blocked downstream by antagonism of IRF3 signaling. IRF3 signaling is essential to promote cell expression of proinflammatory and immunomodulatory cytokines and chemokines from the site of infection that are required for effective adaptive immune responses (30
), and therefore, IRF3 regulation by HIV-1 would contribute to early immune dysfunction in HIV-1-infected patients. This early permissive environment may aid in seeding the initial infection, but in the absence of Vpu control, innate immune signaling may provide the necessary signals for effective control of the acute infection. Further characterization and study of innate immune responses against Vpu-deficient viruses may provide a platform for understanding the innate signatures necessary for HIV-1 suppression and enhancement of vaccine and adjuvant design.