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• W2053445583 abstract "•Human dendritic cells sense the cDNA of HIV-2 before integration•The HIV-1 capsid allows the virus to escape sensing of its cDNA by dendritic cells•Dendritic cells sense capsid-mutated HIV-1 without replication and stimulate T cells•The DNA sensor cGAS is essential in human dendritic cells for sensing HIV-1 and HIV-2 HIV-2 is less pathogenic for humans than HIV-1 and might provide partial cross-protection from HIV-1-induced pathology. Although both viruses replicate in the T cells of infected patients, only HIV-2 replicates efficiently in dendritic cells (DCs) and activates innate immune pathways. How HIV is sensed in DC is unknown. Capsid-mutated HIV-2 revealed that sensing by the host requires viral cDNA synthesis, but not nuclear entry or genome integration. The HIV-1 capsid prevented viral cDNA sensing up to integration, allowing the virus to escape innate recognition. In contrast, DCs sensed capsid-mutated HIV-1 and enhanced stimulation of T cells in the absence of productive infection. Finally, we found that DC sensing of HIV-1 and HIV-2 required the DNA sensor cGAS. Thus, the HIV capsid is a determinant of innate sensing of the viral cDNA by cGAS in dendritic cells. This pathway might potentially be harnessed to develop effective vaccines against HIV-1. HIV-2 is less pathogenic for humans than HIV-1 and might provide partial cross-protection from HIV-1-induced pathology. Although both viruses replicate in the T cells of infected patients, only HIV-2 replicates efficiently in dendritic cells (DCs) and activates innate immune pathways. How HIV is sensed in DC is unknown. Capsid-mutated HIV-2 revealed that sensing by the host requires viral cDNA synthesis, but not nuclear entry or genome integration. The HIV-1 capsid prevented viral cDNA sensing up to integration, allowing the virus to escape innate recognition. In contrast, DCs sensed capsid-mutated HIV-1 and enhanced stimulation of T cells in the absence of productive infection. Finally, we found that DC sensing of HIV-1 and HIV-2 required the DNA sensor cGAS. Thus, the HIV capsid is a determinant of innate sensing of the viral cDNA by cGAS in dendritic cells. This pathway might potentially be harnessed to develop effective vaccines against HIV-1. The human immune system mounts an immune response against HIV-1, but this response is not protective in most individuals. In contrast, the related lentivirus HIV-2, which is less pathogenic than HIV-1, has been proposed to be controlled by the immune system (Rowland-Jones and Whittle, 2007Rowland-Jones S.L. Whittle H.C. Out of Africa: what can we learn from HIV-2 about protective immunity to HIV-1?.Nat. Immunol. 2007; 8: 329-331Crossref PubMed Scopus (81) Google Scholar). Remarkably, coinfection with HIV-1 and HIV-2 results in a better outcome than HIV-1 infection alone, including delayed progression to AIDS, suggesting that the immune response against HIV-2 induces cross-reactive protection against HIV-1 pathology (Esbjörnsson et al., 2012Esbjörnsson J. Månsson F. Kvist A. Isberg P.E. Nowroozalizadeh S. Biague A.J. da Silva Z.J. Jansson M. Fenyö E.M. Norrgren H. Medstrand P. Inhibition of HIV-1 disease progression by contemporaneous HIV-2 infection.N. Engl. J. Med. 2012; 367: 224-232Crossref PubMed Scopus (67) Google Scholar). A key challenge is thus to understand how the immune system differs in its ability to induce immune responses against HIV-1 and HIV-2. Dendritic cells (DCs) play a major role in the induction of immune responses. Paralleling the pathophysiological differences between the two viruses, DCs differ in their ability to induce an innate immune response against HIV-1 and HIV-2 (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar, Manel and Littman, 2011Manel N. Littman D.R. Hiding in plain sight: how HIV evades innate immune responses.Cell. 2011; 147: 271-274Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). DCs do not normally get activated and efficiently infected by HIV-1 (Granelli-Piperno et al., 2004Granelli-Piperno A. Golebiowska A. Trumpfheller C. Siegal F.P. Steinman R.M. HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation.Proc. Natl. Acad. Sci. USA. 2004; 101: 7669-7674Crossref PubMed Scopus (224) Google Scholar, Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar), whereas DCs are naturally infected and activated by HIV-2 (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar). The absence of DC activation by HIV-1 is in part a consequence of the poor ability to infect these cells (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar), which is imposed by the restriction factor SAMHD1 (Hrecka et al., 2011Hrecka K. Hao C. Gierszewska M. Swanson S.K. Kesik-Brodacka M. Srivastava S. Florens L. Washburn M.P. Skowronski J. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.Nature. 2011; 474: 658-661Crossref PubMed Scopus (913) Google Scholar, Laguette et al., 2011Laguette N. Sobhian B. Casartelli N. Ringeard M. Chable-Bessia C. Ségéral E. Yatim A. Emiliani S. Schwartz O. Benkirane M. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.Nature. 2011; 474: 654-657Crossref PubMed Scopus (1123) Google Scholar), an Aicardi-Goutières Syndrome (AGS) susceptibility gene (Rice et al., 2009Rice G.I. Bond J. Asipu A. Brunette R.L. Manfield I.W. Carr I.M. Fuller J.C. Jackson R.M. Lamb T. Briggs T.A. et al.Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as regulator of the innate immune response.Nat. Genet. 2009; 41: 829-832Crossref PubMed Scopus (534) Google Scholar). In contrast, HIV-2 encodes the small accessory protein Vpx, which overcomes the SAMHD1 restriction in DCs. The Vpx protein is incorporated into the viral particles and released into the DCs after viral entry where it induces degradation of SAMHD1. When HIV-1 is complemented with the Vpx protein, DCs become readily infected (Goujon et al., 2006Goujon C. Jarrosson-Wuillème L. Bernaud J. Rigal D. Darlix J.L. Cimarelli A. With a little help from a friend: increasing HIV transduction of monocyte-derived dendritic cells with virion-like particles of SIV(MAC).Gene Ther. 2006; 13: 991-994Crossref PubMed Scopus (127) Google Scholar). Infected DCs in turn sense the virus and induce a concomitant innate immune response (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar). In this setting, sensing of HIV-1 requires genome integration and expression of the viral capsid (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar, Sunseri et al., 2011Sunseri N. O’Brien M. Bhardwaj N. Landau N.R. Human immunodeficiency virus type 1 modified to package Simian immunodeficiency virus Vpx efficiently infects macrophages and dendritic cells.J. Virol. 2011; 85: 6263-6274Crossref PubMed Scopus (91) Google Scholar) and requires interaction between the newly synthesized viral capsid and the cellular prolyl-isomerase Cyclophilin A (CypA) (Luban et al., 1993Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B.Cell. 1993; 73: 1067-1078Abstract Full Text PDF PubMed Scopus (703) Google Scholar, Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar). However, whether capsid or another viral component is the actual pathogen-associated molecular pattern (PAMP) in DCs is not known. Intriguingly, DCs do not sense the reverse-transcribed HIV-1 cDNA or the incoming capsid before viral integration occurs, even in the presence of Vpx. Whether DCs are competent for sensing HIV-1 before integration occurs or whether they lack the sensing machinery remains to be determined. Here, we focused on HIV-2 sensing by DCs to identify the mechanism of HIV-1 and HIV-2 sensing and the DC sensor. We set out to examine the role of the HIV-2 capsid in innate sensing by perturbing its affinity for CypA. We identify a striking capsid-mutated HIV-2 that activates DCs while losing its ability to productively infect the cell. We find that DC activation by HIV-2 requires the viral cDNA and occurs in the cytosol, before nuclear entry and integration. We show that DCs can also sense the HIV-1 cDNA before integration when the analogous mutation is introduced in its capsid and Vpx is present. DCs response to capsid-mutated HIV-1 leads to activation of CD4+ and CD8+ T cells in the absence of productive infection, which is reminiscent of the adjuvant activity of live attenuated vaccines. Finally, we find that the cytosolic DNA sensor cGAS is essential in human DCs for innate sensing of HIV-1 and HIV-2. In HIV-1, the interaction of capsid with CypA is required for DC activation (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar). We reasoned that altering the capsid-CypA interaction might allow us to gain mechanistic insight into sensing by DCs; in particular, the natural sensing of HIV-2. We noticed that two residues in HIV-1 capsid (His87-Ala88) located in the CypA-binding loop (Yoo et al., 1997Yoo S. Myszka D.G. Yeh C. McMurray M. Hill C.P. Sundquist W.I. Molecular recognition in the HIV-1 capsid/cyclophilin A complex.J. Mol. Biol. 1997; 269: 780-795Crossref PubMed Scopus (225) Google Scholar), align to only one residue in HIV-2 capsid (Pro86) (Figure 1A), which binds less efficiently to CypA (Price et al., 2009Price A.J. Marzetta F. Lammers M. Ylinen L.M. Schaller T. Wilson S.J. Towers G.J. James L.C. Active site remodeling switches HIV specificity of antiretroviral TRIMCyp.Nat. Struct. Mol. Biol. 2009; 16: 1036-1042Crossref PubMed Scopus (75) Google Scholar). In HIV-1, His87-Ala88 position the Pro90, the substrate of the isomerizing activity, into the catalytic site of CypA. Strikingly, in the aligned capsid of HIV-2, the homologous Pro88 is not positioned correctly in the catalytic site of CypA (Figures 1C and 1D). This raised the possibility that introducing His-Ala in place of Pro86 in HIV-2 (Figure 1B) would position Pro88 in the catalytic site and increase the affinity of HIV-2 capsid for CypA (HIV-2 affinity-enhanced capsid, HIVac-2), potentially perturbing innate sensing by DCs. Expression of HIVac-2 was similar to its WT counterpart, and the mutation did not alter expression of CypA in virus-producing cells (Figure 1E). Examination of particles by transmission electron microscopy showed a typical lentiviral ultrastructure (data not shown). However, HIVac-2 particles encapsidated 28 times more CypA than HIV-2 WT and 3 times more than HIV-1 (Figure 1E; see also Figure S1A available online). We measured the binding of recombinant capsid proteins (N-terminal domains) to recombinant CypA (Figures S1B and S1C). With a coprecipitation assay, HIVac-2 capsid remained better associated to CypA compared to HIV-2 WT capsid. The association was inhibited by Cyclosporin A, an inhibitor of the capsid-CypA interaction that targets the active site of CypA. Next, microscale thermopheresis (MST) was used to estimate the dissociation constant between CypA and the capsids. The affinity of HIV-2 WT capsid to CypA was too low to be determined in this assay, whereas the affinity of HIVac-2 capsid to CypA was 2.6 μM (Figure S1D). Thus, HIVac-2 capsid bound CypA with a higher affinity than the HIV-2 WT capsid. We examined the ability of HIV-2 WT and HIVac-2 to infect and activate DCs. We used a single-round HIV-2 vector that expressed GFP in DCs after integration to measure infection. Because innate sensing of HIV-1 with Vpx was independent of the viral envelope used (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar), we used VSV-G-pseudotyped particles. As previously shown, HIV-2 WT was able to infect and activate DCs (Figure 2A) in a dose-dependent manner (Figure 2B; Figure S2A). Remarkably HIVac-2 infection was completely blocked in DCs (Figures 2A and 2B; Figure S2A). Unexpectedly, the block did not prevent DC sensing of HIVac-2, and HIVac-2 activated DCs more efficiently than HIV-2 WT (Figure 2B). Addition of virus-like particles (VLPs) from the macaque simian immunodeficiency virus (SIV), a close relative of HIV-2, which provides an increased amount of Vpx and boosts abrogation of SAMHD1 restriction (Hrecka et al., 2011Hrecka K. Hao C. Gierszewska M. Swanson S.K. Kesik-Brodacka M. Srivastava S. Florens L. Washburn M.P. Skowronski J. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.Nature. 2011; 474: 658-661Crossref PubMed Scopus (913) Google Scholar, Laguette et al., 2011Laguette N. Sobhian B. Casartelli N. Ringeard M. Chable-Bessia C. Ségéral E. Yatim A. Emiliani S. Schwartz O. Benkirane M. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.Nature. 2011; 474: 654-657Crossref PubMed Scopus (1123) Google Scholar), enhanced activation by HIVac-2, but infection was still blocked (Figure S2A). DC activation was characterized by production of type I interferon (IFN) (Figure 2C) and the type I IFN response gene IP-10 (CXCL10) (Figure 2D) by the DCs (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar). Neutralizing reagents against type I IFN effectively inhibited expression of IP-10, while CD86 expression was less dependent on type I IFN (Figure S2B). However, neutralizing type I IFN did not rescue the infection by HIVac-2, indicating an intrinsic effect of the mutation. Thus, mutations of the HIV-2 capsid reveal that DCs are competent for innate sensing in the absence of productive viral infection. We dissected the early steps of the viral cycle to identify the determinants of HIV-2 that are sensed in DCs (Figure S3A). To determine whether the viral genetic material was required for sensing, we generated HIV-2 and HIVac-2 VLPs in which abundance of the viral genomic RNA was reduced by combining two previously described deletions that disrupt the encapsidation signal (ΔΨ) (Griffin et al., 2001Griffin S.D. Allen J.F. Lever A.M. The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity.J. Virol. 2001; 75: 12058-12069Crossref PubMed Scopus (78) Google Scholar, Poeschla et al., 1998Poeschla E. Gilbert J. Li X. Huang S. Ho A. Wong-Staal F. Identification of a human immunodeficiency virus type 2 (HIV-2) encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors.J. Virol. 1998; 72: 6527-6536Crossref PubMed Google Scholar). The mutation did not prevent Gag maturation in the particles (Figure S3B) but reduced viral infectivity (Figure S3C). Despite their residual infectivity, HIV-2 and HIVac-2 VLPs with reduced RNA encapsidation were not efficient at activating DCs (Figure S3D). Thus, intact encapsidation of the genomic RNA in the particles is required for sensing. Next, to determine whether viral DNA generated after reverse transcription was required for sensing, we produced particles lacking Vpx that are unable to abrogate the SAMHD1 restriction (ΔVpx) and are compromised for reverse transcription. HIV-2 and HIVac-2 particles lacking Vpx were not infectious and were unable to activate DCs (Figure S3E). To confirm that a viral cDNA was required for sensing, we added the reverse transcriptase inhibitor AZT at the time of infection. AZT prevented infection and sensing by HIV-2 (Figures 3A and 3B ). Thus, synthesis of a viral cDNA is required for sensing. We next examined whether integration was required for sensing by using Raltegravir, an inhibitor of integrase. As expected, Raltegravir blocked infection of HIV-2 WT (Figure 3A). However, Raltegravir did not prevent DC activation by HIV-2 WT and HIVac-2 (Figures 3A and 3B). To confirm that sensing occurred in the absence of integration, we inactivated the integrase (IN) by introducing a D116A mutation in its catalytic site in HIV-2 and HIVac-2 (Figure S3A). D116A-mutated viral particles displayed a normal profile of mature capsid protein (Figure S3B). HIV-2 IN D116A and HIVac-2 IN D116A maintained the ability to activate DCs (Figures 3C and 3D). Thus, DCs sense HIV-2 before integration, and the viral cDNA is required. Sensing of HIV-2 might occur in the cytosol or in the nucleus. We measured the relative abundance of the “Late RT” HIV-2 cDNA product, the “2LTR circles” (a hallmark of nuclear entry of the viral DNA), and the “integrated” proviral DNA after infection of DCs (Figure S4A). HIV-2 WT generated all forms of DNA in an AZT-sensitive manner (Figure 4A). HIVac-2, which activates DCs in the absence of infection, produced normal amounts of Late RT viral cDNA, but not a substantial amount of 2LTR circles or integrated DNA (Figure 4A, red arrows). Treatment of HIV-2 WT with Raltegravir reduced the amount of integrated DNA and also increased the numbers of 2LTR circles, but this was not the case for HIVac-2. This indicates that sensing occurs before the formation of the 2LTR circles, thus presumably in the cytosol. Next, we determined whether nuclear entry was required for sensing. We mutated the central polypurine-tract (cPPT), which is required for nuclear import of the viral cDNA in dendritic cells (Rivière et al., 2010Rivière L. Darlix J.L. Cimarelli A. Analysis of the viral elements required in the nuclear import of HIV-1 DNA.J. Virol. 2010; 84: 729-739Crossref PubMed Scopus (53) Google Scholar, Zennou et al., 2000Zennou V. Petit C. Guetard D. Nerhbass U. Montagnier L. Charneau P. HIV-1 genome nuclear import is mediated by a central DNA flap.Cell. 2000; 101: 173-185Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar) (Figure S4A). The mutation did not modify the Gag maturation profile of viral particles, incorporation of CypA and Vpx, or efficiency of RT (Figures S4B and S4C). We confirmed that the cPPT mutation reduced infectivity of HIV-2 in nondividing DCs (Figure 4B) and the abundance of 2LTR circles (Figure S4C). Sensing of cPPT-mutated HIVac-2 and HIV-2 was maintained (Figure 4C). Taken together, these results show that sensing of viral cDNA occurs in the cytosol. Unlike HIV-2 or HIVac-2, DCs do not sense the HIV-1 cDNA before integration (Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar). We hypothesized that in HIV-1 with Vpx the wild-type (WT) capsid masked the viral DNA, preventing innate sensing up to integration. To examine whether the HIV-1 cDNA can stimulate an innate response, we mutated the CypA-binding loop of HIV-1 capsid to the corresponding sequence from HIVac-2 (Figure 5A). HIVac-1 particles incorporated more CypA (Figures S5A and S5B), and the HIVac-1 capsid had a higher affinity for CypA than WT HIV-1 (Figures S5C and S5D). Similar to HIVac-2, HIVac-1 retained the ability to activate DCs and induce IP-10 in the presence of Vpx and showed a reduced infectivity (Figures 5B–D). Blockade of residual HIVac-1 integration with Raltegravir did not inhibit innate activation and IP-10 induction (Figures 5B–5D). Genetic inactivation of integrase in HIVac-1 also maintained the ability to activate DCs and induce IP-10 (Figure S5E). Thus, the HIV-1 capsid prevents sensing of the viral cDNA, escaping innate sensing before integration, but capsid mutations lead to cDNA-dependent sensing in the cytosol similarly to HIV-2 and HIVac-2. The ability of HIVac to stimulate the innate immune response induced by HIV-2 in the absence of productive infection prompted us to test whether HIVac could activate T cells. First, to test whether DCs activated by HIVac could provide functional costimulatory signals to T cell activation, we examined the polyclonal proliferation of cocultured naive CD4+ T cells in the presence of suboptimal concentrations of anti-CD3 antibody (Figure S6A). DCs were exposed to HIVac-2 particles, leading to DC activation in the absence of infection (Figure 6A; Figure S6B). In the presence of a limiting dose of an anti-CD3 antibody, cocultured primary naive CD4+ T cells proliferated when DCs had been exposed to HIVac-2 (Figure 6A; Figure S6B). This proliferation was blocked when DC activation was inhibited by AZT, and it was increased when sensing of the virus was enhanced by providing an additional dose of Vpx with VLPs. Thus, DC activation by HIVac leads to functional costimulation to CD4+ T cell activation. Next, we tested whether monocyte-derived DCs obtained from HIV-1-infected subjects were still competent for sensing HIVac. We superinfected the DCs with HIV-1 WT or HIVac-1 in the presence of Vpx and Raltegravir (Figure S6C). DCs were activated by HIVac-1, but not by the WT virus, and HIVac-1 sensing was AZT-sensitive (Figure S6D). Thus, DCs from HIV-1-infected subjects remain competent for innate activation by the virus. Because noninfectious HIV particles can provide antigen (Buseyne et al., 2001Buseyne F. Le Gall S. Boccaccio C. Abastado J.P. Lifson J.D. Arthur L.O. Rivière Y. Heard J.M. Schwartz O. MHC-I-restricted presentation of HIV-1 virion antigens without viral replication.Nat. Med. 2001; 7: 344-349Crossref PubMed Scopus (137) Google Scholar, Manel et al., 2010Manel N. Hogstad B. Wang Y. Levy D.E. Unutmaz D. Littman D.R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells.Nature. 2010; 467: 214-217Crossref PubMed Scopus (341) Google Scholar), we examined whether this would increase stimulation of autologous CD8+ T cells (Figure S6C). DCs exposed to HIVac-1 with Vpx and Raltegravir increased the proliferation of CD8+ T cells as compared to HIV-1 WT. (Figures 6B and 6C). This effect was blocked by AZT treatment in DCs, indicating that it required innate sensing. Thus, HIVac-1 can stimulate CD8+ T cells from HIV-1-infected subjects in the absence of integration. These results indicate that a cytosolic DNA sensor might be required for HIV sensing. A large number of candidate DNA sensors have been proposed in recent years in various cell types and models (Paludan and Bowie, 2013Paludan S.R. Bowie A.G. Immune sensing of DNA.Immunity. 2013; 38: 870-880Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). In the transformed human cell line THP-1, the sensors cGAS, DDX41, and IFI16 were found to be required for DNA sensing, leading to type I IFN production (Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339: 786-791Crossref PubMed Scopus (2508) Google Scholar, Unterholzner et al., 2010Unterholzner L. Keating S.E. Baran M. Horan K.A. Jensen S.B. Sharma S. Sirois C.M. Jin T. Latz E. Xiao T.S. et al.IFI16 is an innate immune sensor for intracellular DNA.Nat. Immunol. 2010; 11: 997-1004Crossref PubMed Scopus (1184) Google Scholar, Zhang et al., 2011Zhang Z. Yuan B. Bao M. Lu N. Kim T. Liu Y.J. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells.Nat. Immunol. 2011; 12: 959-965Crossref PubMed Scopus (640) Google Scholar). We screened whether these sensors could be implicated in HIV sensing by DCs derived from primary monocytes by using pooled shRNA. Reducing DDX41 or IFI16 expression did not inhibit CD86 induction by HIVac-2 (Figures S7A and S7B). In contrast, inhibition of cGAS expression suppressed CD86 induction by HIVac-2. We confirmed that the shRNA inhibited cGAS protein expression by transducing individual shRNA (data not shown) and selected two shRNA: shRNA #2, which modestly decreased cGAS expression, and shRNA #4, which efficiently decreased cGAS expression (Figure 7A). We stimulated shRNA-transduced DCs with HIV-1, HIV-2, HIVac-2, and the soluble RNA molecule poly(I:C) as control. cGAS targeting by RNA interference prevented induction of CD86 by HIV-1, HIV-2, and HIVac-2, but not sensing of poly(I:C) (Figure 7B). Titrated infections confirmed that cGAS is required for induction of CD86 by HIV-1, HIV-2, and HIVac-2 (Figure 7C). cGAS targeting by RNA interference also prevented IP-10 production after stimulation by HIV-2 and HIVac-2 (Figure S7C). Altogether, these data demonstrate that DC sensing of HIV-1 and HIV-2 is mediated by the DNA sensor cGAS (Figure S7D). We investigated how human DCs sense HIV-1 and HIV-2. The HIV-1 capsid and its interaction with Cyclophilin A were previously found to play an essential role in innate sensing of the virus in DCs with Vpx. However, the actual function of capsid in innate sensing, the genuine PAMP and the identity of its sensor were not known. By manipulating the interaction between the viral capsid and CypA through mutation (HIVac), we were able to dissociate innate sensing from productive infection of DCs. By using HIVac as an investigation tool, we report that DC sensing of HIV-2 requires reverse transcription of the viral cDNA, but not nuclear entry and integration. In contrast to HIV-2, we find that DCs cannot sense HIV-1 before integration because the viral capsid prevents cDNA sensing. Capsid-mutated HIV-1 revealed that DCs are nonetheless competent for HIV-1 sensing of the viral cDNA before integration. Such capsid-mutated particles elicit innate immunity in DCs and activate T cells in the absence of viral replication. Finally, we demonstrate that human DCs use the DNA sensor cGAS to sense the viral cDNA. Thus, our results establish that, in DCs, the cytosolic viral cDNA is an HIV PAMP and that the viral capsid dictates whether this cDNA will escape sensing or will be sensed through cGAS. Various candidate DNA sensors have been proposed in the recent years (Paludan and Bowie, 2013Paludan S.R. Bowie A.G. Immune sensing of DNA.Immunity. 2013; 38: 870-880Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar) that seem to operate differently between human and mouse, between cell types and between DNA stimuli (Abe et al., 2013Abe T. Harashima A. Xia T. Konno H. Konno K. Morales A. Ahn J. Gutman D. Barber G.N. STING recognition of cytoplasmic DNA instigates cellular defense.Mol. Cell. 2013; 50: 5-15Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, Brunette et al., 2012Brunette R.L. Young J.M. Whitley D.G. Brodsky I.E. Malik H.S. Stetson D.B. Extensive evolutionary and functional diversity among mammalian AIM2-like receptors.J. Exp. Med. 2012; 209: 1969-1983Crossref PubMed Scopus (155) Google Scholar, Cavlar et al., 2013Cavlar T. Deimling T. Ablasser A. Hopfner K.P. Hornung V. Species-specific detection of the antiviral small-molecule compound CMA by STING.EMBO J. 2013; 32: 1440-1450Crossref PubMed Scopus (131) Google Scholar, Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339: 786-791Crossref PubMed Scopus (2508) Google Scholar, Unterholzner et al., 2010Unterholzner L. Keating S.E. Baran M. Horan K.A. Jensen S.B. Sharma S. Sirois C.M. Jin T. Latz E. Xiao T.S. et al.IFI16 is an innate immune sensor for intracellular DNA.Nat. Immunol. 2010; 11: 997-1004Crossref PubMed Scopus (1184) Google Scholar, Zhang et al., 2011Zhang Z. Yuan B. Bao M. Lu N. Kim T. Liu Y.J. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells.Nat. Immunol. 2011; 12: 959-965Crossref PubMed Scopus (640) Google Scholar), and this complexity, which might reflect functional redundancy, has not been resolved. In human myeloid dendritic cells, the sensor(s) that are required for sensing cytosolic DNA are not yet known. One of the DNA sensors, cGAS, synthetizes cyclic GMP-AMP (cGAMP) upon DNA sensing. cGAMP was recently detected in DCs infected by HIV-1 in the presence of Vpx (Gao et al., 2013Gao D. Wu J. Wu Y.T. Du F. Aroh C. Yan N. Sun L. Chen Z.J. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.Science. 2013; 341: 903-906Crossref PubMed Scopus (690) Google Scholar), but whether cGAS or any other DNA sensor is actually required for innate sensing in DCs was not evaluated. By using RNA interference, we demonstrate that" @default.
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• W2053445583 title "The Capsids of HIV-1 and HIV-2 Determine Immune Detection of the Viral cDNA by the Innate Sensor cGAS in Dendritic Cells" @default.
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