HIV infection of its main target cells, macrophages and CD4+
T cells, does not induce cell-autonomous IFNs35
. We showed here that the host cytoplasmic exonuclease TREX1 helps HIV evade innate immunity by digesting reverse transcripts that are not imported into the nucleus and would otherwise induce IFNs. When TREX1
is inhibited by RNAi, HIV infection of primary cells triggers Type I IFN expression and secretion. The HIV-stimulated IFN response in cells deficient in TREX1, like the response to endogenous retroelement DNA and transfected DNA7
, is IRF3-dependent. IFN induction can be blocked by expressing enzymatically active TREX1 or by simultaneously suppressing IRF3 expression to interrupt IFN signaling. HIV-stimulated innate immune signaling also requires the adaptor protein STING and the protein kinase TBK1. Based on siRNA experiments, none of the known DNA sensors is involved. Therefore our working model of the innate immune pathway activated by cytosolic HIV DNA starts with an unknown sensor (that may preferentially recognize ssDNA) that signals through STING, TBK1 and IRF3 to activate IFN expression.
Type I IFNs inhibit HIV replication at multiple steps in the early phase of its life cycle and thereby suppress viral spreading. Failure to induce antiviral IFNs in infected T cells and macrophages may promote transmission by allowing the virus to spread from the initial nidus of infection to neighboring cells within genital tissue. However, testing TREX1's importance in transmission will require efficient methods to inhibit TREX1 expression in vivo in the immune cells that HIV infects. Such methods are not available, but are being developed. We used an MOI of 1 to achieve a reasonable frequency of infected cells. Since HIV DNA cytoplasmic accumulation and IFN triggering depend upon the MOI, the physiological relevance of our results to HIV transmission hinge upon what viral concentrations are achieved in vivo, which is unknown. Viral concentrations may reach high MOIs locally during the replicative burst that occurs within the genital tract during transmission, when a strong intrinsic antiviral immune response might prevent dissemination36
. Other settings of high viral concentration might be activated lymph nodes or gut-associated lymphoid tissues.
We did not examine whether TREX1 affects IFN production by pDCs, the major source of Type I IFNs during HIV infection. HIV replication is inefficient in DCs. IFN stimulation in pDCs appears to be triggered mostly by endocytosed virus, whose gRNA is recognized by TLR7 in endosomes37
. Productive HIV infection of macrophages and T cells, however, involves viral membrane fusion with the cell membrane and direct uncoating of the viral capsid into the cytosol, bypassing the endosomal compartment and TLR signaling. Nonetheless, it will be important to determine whether TREX1 modulates IFN signaling in pDC.
HIV-stimulated IFN activation in Trex1−/−
cells is eliminated by treating infected cells with an RT inhibitor, but not an IN inhibitor, suggesting that HIV DNA, but not gRNA, is the nucleic acid that triggers innate immunity. ssDNA is the nucleic acid most sensitive to TREX1 activity and is therefore likely its main substrate. IFN-β mRNA is normally detected 6-8 h after transfecting ISD or infection with a DNA virus6,21,38
. After HIV infection, IFN mRNA is not detected until 12 hpi; the lag in IFN-β expression is likely due to time needed to complete reverse transcription. The rapid decline in IFN-β mRNA expression after reaching its peak value suggests that a cell autonomous secondary mechanism tempers the innate immune response that, if unchecked, could be harmful to the host. Some HIV DNA accumulated in the cytoplasm of HIV-infected cells, even when TREX1 is normally expressed, but did not activate IFN expression. A cytoplasmic DNA threshold, which might vary in different cell types, may need to be exceeded to trigger innate immunity.
HMGB proteins have been proposed as innate immune sentinel proteins that facilitate nucleic acid recognition by cytosolic RNA and DNA sensors20
. Here HMGB2
siRNA experiments showed an opposite effect; HMGB2 helped suppress IFN induction by HIV in human cells. Although Hmgb2
RNAi in Trex1−/−
MEFs did not increase cytosolic HIV DNA, HMGB2
RNAi in human 293T cells, also treated with TREX1
siRNA, enhanced cytosolic HIV DNA and IFN-β and IFN-β induction. Therefore HMGB proteins may play a more complex role in innate immunity than originally suggested. In their role as foreign nucleic acids sentinels, they may facilitate the recognition of nucleic acids both by sensors that trigger innate immune responses as well as by proteins, such as TREX1, that inhibit IFN induction. Therefore the net effect of reduced HMGB proteins could be either to inhibit IFN induction (as in20
) or to enhance it, as shown here. We also found that HMGB2 can also act downstream of nucleic acid sensing at the IFN-β promoter to suppress IFN-β transcription, adding another layer of complexity. This transcriptional effect extends to non-HIV innate immune stimuli (poly(dA:dT) and MAVS over-expression). In the published study20
, poly(dA:dT)-stimulated IFN-β expression in Hmgb2−/−
MEFs was reduced compared to WT MEFs, whereas we found the opposite effect with Hmgb2
RNAi. The apparent discrepancy between the previous results and ours could be due to a difference in the consequences of complete or partial Hmgb2
elimination, especially if HMGB2 operates at multiple steps in IFN induction.
Many of our experiments used genetically deficient mouse cells to demonstrate that Trex1 helps HIV evade innate immune detection and define the HIV DNA-stimulated IFN signaling pathway. Knockout mouse cells are powerful tools for HIV research8,39,40
. Once the block in entry in mouse cells is overcome by VSV-G-pseudotyping, most early steps of HIV replication, including reverse transcription, integration and LTR-driven transcription, are similar in human and mouse cells. Furthermore, human TREX1 is 73.3% identical to its mouse homolog in amino acid sequence, and is 71.4%/100%/86.7% identical in its three exonuclease motifs15
. Human and mouse TREX1 have the same enzymatic activity and can substitute for each other15
. Therefore mouse cells are well suited for studying TREX1 function in HIV replication. Nonetheless, human immune cells susceptible to HIV can differ from MEF in their ability to activate innate immune pathways. For example, IFN-β was induced by HIV in human immune cells, but not MEFs. The differential role of HMGB2 in HIV DNA accumulation in human macrophages and MEFs may be another case in point. We validated all key findings in primary human cells, including HIV DNA accumulation, IFN induction and inhibition of HIV replication when TREX1
was inhibited by RNAi, and efficient rescue by co-suppressing IRF3
Our data shed light on the fate of non-productive or non-integrated HIV DNA in the cell. At an MOI of 1, HIV infection produces many reverse transcripts (although only one per incoming gRNA), but very few copies manage to integrate into the host chromosome. The remaining HIV DNA is cleared by TREX1, since cytosolic HIV DNA builds up when TREX1 function is deficient or inhibited and can be removed by expression of enzymatically active TREX1. As a consequence, WT TREX1 fully rescues the HIV infection block in Trex1−/− MEFs and the D18N mutant fails to rescue. Other host nucleases might also help digest cytosolic HIV DNA. It is unclear why the excess HIV DNA that accumulates in Trex1−/− cells does not lead to more chromosomal integration. Sequencing these excess HIV DNAs may reveal whether they are capable of integration and what prevents them from integrating. These excess HIV DNAs may be mostly non-productive RT products.
TREX1 promotes HIV replication in two ways – it inhibits autointegration and suppresses the IFN response. Several models might explain the dual function of TREX1 on HIV DNA. One possibility is that TREX1 might sort productive vs non-productive HIV RT products. HIV RT is error-prone and often produces incomplete products. TREX1 recognizes ssDNA or dsDNA with single strand overhangs – the kind of DNA in failed RT products. TREX1 might bind to HIV DNA nonspecifically in the cytosol, but as an exonuclease can only efficiently digest HIV DNA that contains broken ends or single strand overhangs. HIV integrase binds to the ends of reverse transcripts that are capable of chromosomal integration and might protect them from TREX1 digestion. Incomplete RT products, however, would not bind integrase and therefore would be susceptible to TREX1 degradation. Autointegration requires the full-length RT product and active DNA ends bound by integrase, which catalyzes autointegration. Although TREX1 likely binds to full-length integration-competent products, as well as to transcripts that are not competent for integration, its exonuclease activity might be inhibited in the full-length transcript by lack of some DNA feature that facilitates digestion (such as shielding by integrase). Another possibility is that TREX1 is inhibited by components of the SET complex that also bind to the HIV PIC2
. Another DNase in the SET complex, NM23-H1, is inhibited by SET protein and is only activated when Granzyme A cleaves SET41
. TREX1 is an abundant protein that is not exclusively in the SET complex. One could also imagine that two subpopulations of TREX1 are involved in different actions: the SET complex-associated TREX1 inhibits autointegration; while TREX1 outside the SET complex is enzymatically active and removes excess HIV DNA. This model would also explain why siRNAs against most other SET complex genes do not induce IFN, but do protect against autointegration. Further studies are needed to test these ideas.
TREX1 mutations that interfere with TREX1 enzymatic function or localization are associated with SLE and other autoimmune/inflammatory diseases3-5
. Patients with SLE are underrepresented in HIV-infected populations42
. It would be worth evaluating whether TREX1 polymorphisms or autoimmune disease are associated with reduced HIV transmission or a more benign disease course. The innate immune pathway uncovered in this study will improve understanding of how HIV intersects with innate immunity and may also shed light on autoimmune and inflammatory syndromes linked to TREX1