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STING is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I Interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER–Golgi intermediate compartment. Here we found that deficiency in the Ca 2+ sensor STIM1 caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient suffering from combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and co-expression of full-length or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1–STING circuit that maintains the resting state of the STING pathway.

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

IntroductionChemokine receptor CCR5 is an attractive therapeutic target for inhibiting HIV-1, as it serves as a HIV-1 coreceptor and is essential for CCR5 tropic HIV-1 infection. [1][2][3][4] Blocking CCR5 expression should prevent HIV-1 infection at the initial stage of the viral life cycle. Individuals with a ⌬32/⌬32 homozygous mutation in the CCR5 gene do not express CCR5, are highly protected from HIV-1, and are apparently normal. [5][6][7] Recently, an HIV ϩ acute myelogenous leukemia patient was treated for leukemia and HIV infection by bone marrow transplantation using donated CCR5 ⌬32/⌬32 marrow. After the transplantation, nearly 100% of the patient's blood cells were replaced with donor cells. HIV DNA and RNA were undetectable at 20 months, even after the discontinuation of highly active antiretroviral therapy. 8 This evidence supports that long-term and stable reduction of CCR5 is a promising strategy for treating HIV-infected patients. The major limitation of this strategy is the difficulty of identifying human leukocyte antigen-matched CCR5 ⌬32/⌬32 homozygous donors as the mutation exists in approximately 1% of white populations and is rare in other ethnic populations. 9 Small interfering RNAs (siRNAs) induce sequence-specific degradation of mRNAs by RNA interference. 10 Many forms of siRNA have been used to inhibit HIV coreceptors and HIV-1 gene expression in in vitro and in vivo experimental settings. [11][12][13][14][15][16][17][18] To stably inhibit HIV replication, we and others developed lentiviral vectors that are capable of stably delivering short hairpin RNA (shRNA) in mammalian cells. [19][20][21][22][23][24][25] We demonstrated that expression of CCR5-specific shRNA in human primary T lymphocytes results in efficient CCR5-knockdown and protection of cells from HIV-1 infection in vitro. 22 However, we and others recognized that a high level of sustained shRNA expression may be toxic to cells because of competition with endogenous micro-RNA biogenesis, induction of interferon responses, and/or off-targeting effects. 23,[26][27][28][29][30][31][32][33] To stably reduce CCR5 expression without cytotoxicity, we identified a highly efficient shRNA (shRNA 1005) directed to human CCR5 mRNA using the enzymatic production of RNAi libraries (EPRIL) screening technique. 21,34 We expressed shRNA 1005 using the transcriptionally weak H1 promoter to stably reduce CCR5 expression without inducing cytotoxicity in human primary peripheral blood lymphocytes in vitro. 21,34 To test stable CCR5 reduction in vivo, we used a nonhuman primate hematopoietic stem cell transplantation model in which we were able to demonstrate stable reduction of CCR5 expression in peripheral blood lymphocytes in shRNA-transduced CD34 ϩ cell-transplanted rhesus macaques. 21 Because of a single nucleotide mismatch in the shRNA 1005 target sequence between human and rhesus macaque CCR5 mRNA, we mutated the human CCR5 shRNA 1005 so that it would be 100% homologous to the corresponding rhesus macaque CCR5 mRNA target sequence. This rhes...

SUMMARY In mice, clonal tracking of hematopoietic stem cells has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3–10 years) and likely represent self-renewing hematopoietic stem cells (HSCs), while the remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid or both lineages. Over time, the 4–10% of clones with robust dual lineage contribution predominated in repopulation capacity. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically-relevant model over a long time frame, and provides a substantial system-level dataset that is a reference point for future work.

The vast majority of new HIV infections result from relatively inefficient transmission1,2 of the virus across mucosal surfaces during sexual intercourse3. A consequence of this inefficiency is that small numbers of transmitted founder viruses initiate most heterosexual infections4. This natural bottleneck to transmission has stimulated efforts to develop interventions aimed at blocking this step of the infection process5. Despite the promise of this strategy, clinical trials of pre-exposure prophylaxis have had limited degrees of success in humans, due in part to lack of adherence to the recommended pre-exposure treatment regimens6,7. In contrast, a number of existing vaccines elicit systemic immunity that protects against mucosal infections, such as the vaccines for influenza8 and HPV9. We recently demonstrated the ability of vectored immunoprophylaxis (VIP) to prevent intravenous transmission of HIV using broadly neutralizing antibodies10. Here we demonstrate that VIP is capable of protecting humanized mice from intravenous as well as vaginal challenge with diverse viral strains, despite repeated exposures. Moreover, animals receiving VIP that expresses a modified VRC07 antibody were completely resistant to repetitive intravaginal challenge by a heterosexually transmitted founder HIV strain11, suggesting that VIP may be effective in preventing vaginal transmission of HIV between humans.

RNAi is a powerful method for suppressing gene expression that has tremendous potential for therapeutic applications. However, because endogenous RNAi plays a role in normal cellular functions, delivery and expression of siRNAs must be balanced with safety. Here we report successful stable expression in primates of siRNAs directed to chemokine (c-c motif) receptor 5 (CCR5) introduced through CD34؉ hematopoietic stem/progenitor cell transplant. After hematopoietic reconstitution, to date 14 months after transplant, we observe stably marked lymphocytes expressing siRNAs and consistent down-regulation of chemokine (c-c motif) receptor 5 expression. The marked cells are less susceptible to simian immunodeficiency virus infection ex vivo. These studies provide a successful demonstration that siRNAs can be used together with hematopoietic stem cell transplant to stably modulate gene expression in primates and potentially treat blood diseases such as HIV-1.short-hairpin RNA ͉ siRNA ͉ rhesus macaque ͉ gene therapy s iRNAs recognize cognate mRNAs and induce sequence specific RNA degradation through a highly conserved cellular mechanism (1). Because siRNAs have the potential for therapeutic application, a number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian cells in tissue culture and in animal model systems (2-7). The results of these studies indicate that expression of siRNAs can potentially be used to effectively down-regulate gene expression in vivo for therapeutic purposes; however, it is important to control for the negative effects of expressing siRNAs in mammalian cells.