HIV & AIDS Infectious Diseases Research Essay

HIV & AIDS Infectious Diseases Research Essay HIV & AIDS Infectious Diseases Research Essay ORDER NOW FOR CUSTOMIZED AND ORIGINAL NURSING PAPERS Unformatted Attachment Preview ARTICLE https://doi.org/10.1038/s41467-020-17753-w OPEN Clearance of HIV infection by selective elimination of host cells capable of producing HIV 1234567890():,; Min Li1, Wei Liu1, Tonya Bauch1, Edward A. Graviss Min Chen 5 & Jin Wang 1,6 ? 2, Roberto C. Arduino3, Jason T. Kimata 4, The RNA genome of the human immunode?ciency virus (HIV) is reverse-transcribed into DNA and integrated into the host genome, resulting in latent infections that are dif?cult to clear. Here we show an approach to eradicate HIV infections by selective elimination of host cells harboring replication-competent HIV (SECH), which includes viral reactivation, induction of cell death, inhibition of autophagy and the blocking of new infections. Viral reactivation triggers cell death speci?cally in HIV-1-infected T cells, which is promoted by agents that induce apoptosis and inhibit autophagy. SECH treatments can clear HIV-1 in >50% mice reconstituted with a human immune system, as demonstrated by the lack of viral rebound after withdrawal of treatments, and by adoptive transfer of treated lymphocytes into uninfected humanized mice. Moreover, SECH clears HIV-1 in blood samples from HIV-1-infected patients. Our results suggest a strategy to eradicate HIV infections by selectively eliminating host cells capable of producing HIV. 1 Immunobiology and Transplant Science Center, Houston Methodist Research Institute, Houston, TX 77030, USA. 2 Department of Pathology and Genomic Medicine, Houston Methodist Research Institute, Houston, TX 77030, USA. 3 Division of Infectious Diseases, Department of Internal Medicine, McGovern Medical School at The University of Texas Health Science Center, Houston, TX 77030, USA. HIV & AIDS Infectious Diseases Research Essay. 4 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA. 5 Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA. 6 Department of Surgery, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA. ?email: [email protected] NATURE COMMUNICATIONS | (2020)11:4051 | https://doi.org/10.1038/s41467-020-17753-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17753-w T he acquired immunode?ciency syndrome (AIDS) is caused by HIV, which infects and depletes CD4+ T cells in the patients1. The RNA genome of HIV type 1 (HIV-1) is reverse-transcribed into DNA and integrated into the host genome, resulting in persistent infections that are dif?cult to eradicate2. Combination antiretroviral therapy (cART) targeting different stages of the HIV-1 replication cycle can effectively inhibit viral replication and prevent the onset of AIDS3. The seeding of refractory latent viral reservoir takes place rapidly in human HIV-1 patients and in Simian immunode?ciency virus (SIV)-infected rhesus monkeys4,5. A stable HIV-1 proviral reservoir persists during cART6–8. Continuous cART is necessary to prevent new virus production from the HIV-1 reservoir9. Interestingly, two leukemia patients infected with HIV-1 have been reported to be cured of the virus through regimens of total body irradiation or chemotherapy plus antibody-mediated depletion of lymphocytes, followed by transplantation of hematopoietic stem cells from homozygous CCR5?32 donors10,11. However, a cure strategy for HIV-1 infections that is practical for people living with HIV in the general population remains to be developed. HIV-1 has evolved mechanisms to evade immune recognition. Inducing the expression of HIV-1 with latency reversal agents (LRAs) to display viral antigens may trigger immune responses against latently infected cells12. HIV & AIDS Infectious Diseases Research Essay. However, LRAs alone have not been shown to reduce or clear HIV infections13, suggesting the requirement for additional approaches. Using a long-acting antiviral therapy and adenovirus-mediated delivery of CRISPRCas9 to excise integrated HIV-1, it was shown that HIV-1 was cleared from two human cell-implanted mice14. It has been shown that only a small portion (<3%) of integrated HIV-1 is capable of producing the infectious virions, whereas most integrated proviruses are defective and pose no risks in producing the virus15. Thus, the majority of the infected cells, which are nonproductively infected, do not need to be cleared to achieve a cure for HIV-1. So far, the task of ?nding the needle in a haystack to sort out and destroy the HIV-1 reservoir capable of producing the virus has been challenging. Killing cells harboring intact HIV-1 proviruses would be ideal for clearing the HIV-1 reservoir. HIV-1 can trigger different cell death pathways in T cells16–23. Interestingly, productive HIV-1 infection induces caspase-dependent apoptosis in host cells, whereas abortive HIV-1 infection leads to pyroptosis24. We and others have found that cell types important for immunological memory, including memory B cells, and CD4+, and CD8+ memory T cells, depend on autophagy for their long-term survival25–28. Because CD4+ memory T cells are the major reservoir for latent HIV-1, we hypothesize that targeting autophagy would facilitate the elimination of latent HIV-1 infection. By a selective elimination of host cells harboring replication-competent HIV (SECH) approach with the combination of latency reversal, inhibition of autophagy and induction of apoptosis, we show that it is feasible to clear host cells harboring replication-competent HIV-1 in humanized mice in vivo, as well as in blood samples from HIV-1-infected patients in vitro. Results Inhibition of autophagy promotes host cell apoptosis. As autophagy is important for the protection of memory T cells, we tested whether inhibition of autophagy can help to reduce the HIV-1 reservoir in these cells. We used CD3+CD4+CD45RO +CCR7+ central memory T cells (CMT; Supplementary Fig. 1a) for HIV-1 infection and culture in the presence of CCL19 to establish HIV latent infection29. Four days after infection with CXCR4-tropic HIV-1 NL4-330 at 0.1 multiplicity of infection 2 (MOI), CMT showed no detectable expression of HIV-1 p24 protein (Fig. 1a, Supplementary Fig. 1b). Stimulation with phytohemagglutinin (PHA) led to latency reversal of HIV-1 as shown by the induction of p24 (Fig. 1a, Supplementary Fig. 1b). HIV & AIDS Infectious Diseases Research Essay. Latency reversal was also observed by the expression of HIV-1 mRNA after stimulation with PHA or ingenol-3,20-dibenzoate (IDB), a non-tumorigenic protein kinase c-? activator31 (Supplementary Fig. 1c). Interestingly, inhibition of autophagy in T cells by silencing the expression of an essential autophagy gene, Atg732, reduced the number of HIV-1 p24+ cells after PHA stimulation (Fig. 1a). Consistently, SAR405, an autophagy inhibitor that prevents autophagy initiation by suppressing VPS3433, decreased the number of HIV-1 p24-producing T cells after latency reversal (Fig. 1b, Supplementary Fig. 2a). Chloroquine (CQ), another autophagy inhibitor that blocks the progression of autophagolysomes34, also reduced the number of HIV-1 p24-producing T cells after latency reversal (Fig. 1b). We also established HIV latency in CMT infected with CCR5-utilizing HIV-1 AD835 at 1 MOI, and induced latency reversal with IDB (Supplementary Fig. 1c). Induction of HIV-1 p24 expression by IDB-induced latency reversal was also inhibited by SAR405 (Supplementary Fig. 2b). These data suggest that inhibition of autophagy can reduce the numbers of cells capable of producing HIV-1. Inhibition of autophagy may reduce HIV-1 p24+ cells after latency reversal by affecting HIV-1 reverse transcription, integration of viral DNA into the host genome, viral reactivation or host cell survival. To distinguish between these possibilities, we measured early and late products of reverse transcription by R/U5 and LTR-gag reverse transcription polymerase chain reaction (RT-PCR), respectively36. We observed that the production of early and late HIV-1 transcripts, which indicates the level of reverse transcription, was not affected by silencing of Atg7 or treatment with SAR405 (Fig. 1c, d). We also found that inhibition of autophagy did not affect HIV-1 integration into the host genome by Alu-gag PCR37 (Fig. 1e). Induction of HIV-1 mRNA expression from latently infected cells was also unaffected by inhibition of autophagy (Fig. 1f). Moreover, induction of HIV-1 mRNA in latently infected peripheral blood mononuclear cells (PBMCs) from ART-treated HIV-1 patients was not changed by SAR405 (Fig. 1g), indicating that autophagy is not required for the reactivation of latently infected HIV-1. Together, these data suggest that inhibition of autophagy does not have a direct effect on reverse transcription, integration, and reactivation of latent HIV-1. HIV & AIDS Infectious Diseases Research Essay. Interestingly, we observed that latency reversal with IDBinduced cell death in HIV-1-infected CMT, as shown by annexin V staining (Fig. 1h), and increased caspase-3 activities measured by cleavage of DEVD (Fig. 1i). Moreover, IDB-induced cell death in HIV-1-infected CMT was promoted by autophagy inhibitors SAR405 and CQ (Fig. 1h, i). These data suggest that inhibition of autophagy promotes host cell death during latency reversal. Speci?c killing of host cells by HIV-1 reactivation. We found that IDB-mediated latency reversal induced the activation of caspase-9, caspase-3, caspase-6, and caspase-7 in HIV-1-infected T cells, as shown by the appearance of active processed forms of these caspases (Fig. 2a). This is consistent with the possibility that latency reversal by IDB induces cell death in HIV-1-infected host cells (Fig. 1h, i). Treatment with IDB did not change the expression of antiapoptotic Bcl-2, but increased the expression of antiapoptotic Bcl-xL and Mcl-1 in CD4+ T cells with or without HIV-1 infections (Fig. 2a). The increase in Bcl-xL expression in HIV-1-infected cells was greater than in uninfected controls (Fig. 2a). This indicates that HIV replication may synergize with IDB in inducing antiapoptotic Bcl-xL, reminiscent of the roles for NATURE COMMUNICATIONS | (2020)11:4051 | https://doi.org/10.1038/s41467-020-17753-w | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17753-w f kD 100 75 50 37 10 5 Atg7 Control 0 HIV-1 mRNA 6 (copies/10 cells) 15 Atg7 Control % p24+ cells siRNA 20 Atg7 Actin 104 103 102 101 100 – SAR405 – – + + PHA siRNA 104 103 102 101 100 HIV-1 mRNA 6 (copies/10 cells) a Control Atg7 – – + + PHA 10 siRNA Control Atg7 30 20 10 0 3 12 24 48 6 CQ 20 10 0 3 No virus 10 101 100 + SAR405 – – + IDB + HIV-1 20 100 siRNA 15 10 0 24 48 Control Atg7 – SAR405 CQ 75 50 25 0 – + + No virus HIV-1 HIV-1 Control No virus HIV-1 IDB i 3 6 12 24 48 Hours LTR-gag – SAR405 75 50 25 0 3 24 48 Hours 100 75 50 25 0 + Alu-gag – SAR405 – + + HIV-1 % DEVD-FITC 100 ND siRNA Control Atg7 Relative value LTR-gag 400 300 200 100 10 5 0 Relative value d Relative expression 2 h Hours Hours – Alu-gag – SAR405 30 + e R/U5 40 0 ND R/U5 40 5 – Relative value Relative expression c Relative expression – 0 10 – SAR405 103 HIV-1 mRNA 6 (copies/10 cells) 20 g 15 % Annex V+ 30 SAR405 % p24+ cells b LC3 punctates/cell siRNA 100 75 – SAR405 CQ 50 25 0 No virus HIV-1 Control No virus HIV-1 IDB Fig. 1 Regulation of host cell survival, but not HIV-1 reverse transcription and integration into the host genome by inhibition of autophagy. a CMT transfected with Atg7 siRNA were infected with HIV-1 (NL4-3, 0.1 MOI) and cultured for 4 d to establish latency. After latency reversal by PHA, p24+ cells (n = 3 biologically independent samples) and Atg7 expression (representative of two biologically independent experiments) were determined. *p = 0.011. b CMT latently infected with HIV-1 were stimulated with IDB with SAR405 (2 ?M) or CQ (10 ?M). p24 staining and the number of LC3 punctate per cells were analyzed. p24+ cells (n = 3 biologically independent experiments), p = 0.010 (SAR405) and 0.0098 (CQ); LC3 punctate/cell (n = 30 cells from two biologically independent experiments), p = 0.0001 (no virus) and 0.0001 (HIV-1). c, d Atg7 siRNA-transfected CMT were infected with HIV-1. Alternatively, CMT infected with HIV-1 were cultured with SAR405. Cells at different time after infection were collected (n = 3 biologically independent samples) for RT-PCR for R/U5 c and LTR-gag d. e Alu-gag PCR for genomic DNA from CMT as in a, CMT treated with SAR405 as in b or uninfected CMT. Data were normalized against ?-globin. ND: not detectable. Data are presented as mean ± SD (n = 3 biologically independent samples). f, g CMT cultured as in a, b were reactivated with PHA for 24 h f (n = 3 biologically independent samples). PBMCs from ART-treated HIV-1-infected patients (n = 5 patients) were stimulated with IDB in the presence or absence of SAR405 for 24 h g. HIV-1 mRNA was determined by RT-PCR. Data are presented as mean ± SD. The dashed line indicates detection limit. h, i CMT latently infected with HIV-1 (NL4-3, 1 MOI) as in a were reactivated with 100 nM IDB for 24 h. Annexin V h or DEVD i staining were analyzed by ?ow cytometry (n = 3 biologically independent samples). SAR405 vs. control, p = 0.0012 h, 0.0024 i; CQ vs. control, p = 0.0164 h, 0.0088 i. Source data are provided as a Source Data ?le. viral components in the regulation of Bcl-xL38. Moreover, IDB also induced the expression of LC3 in T cell with or without HIV1 infections (Fig. 2a), indicating that IDB promotes autophagy in T cells. Although IDB can induce virus production in T cells harboring latent HIV-1 to trigger apoptosis, the upregulation of antiapoptotic molecules and autophagy by IDB would counteract apoptosis signaling. This may explain in part why the use of LRAs alone is not suf?cient to clear HIV-1-infected cells. Nevertheless, the induction of antiapoptotic molecules and autophagy by IDB may have the advantage by conferring resistance of uninfected T cells to the induction of cell death. Killing of host cells by targeting apoptosis and autophagy. Virus reactivation by IDB-induced cell death in HIV-1-infected T cells as shown by staining with Annexin V and cleavage of DEVD, whereas uninfected cells were relatively resistant (Fig. 2b, c). Inhibition of autophagy with SAR405 increased IDB-induced killing of T cells latently infected by HIV-1 (Fig. 2b, c). Because latency reversal by IDB also showed the unintended effect of increasing antiapoptotic molecules (Fig. 2a), targeting antiapoptotic molecules in addition to reactivating the virus is potentially advantageous in promoting the killing of HIV-infected cells. Indeed, an inhibitor of Bcl-2 and Bcl-xL, ABT-26339, increased IDB-mediated cell death in latently infected T cells, as shown by staining with Annexin V and DEVD-FITC (Fig. 2b, c). ABT-263 signi?cantly increased the loss of viable HIV-1 p24+ cells in IDB-stimulated HIV-1-infected cells (Fig. 2d). This suggests that counteracting antiapoptotic molecules and inhibiting autophagy can promote the killing of HIV-1-infected T cells induced by latency reversal. To eradicate HIV infections, all T cells that are capable of producing infectious HIV-1 need to be cleared. We found that combining SAR405 with ABT-263 and IDB further increased cell death in HIV-infected T cells (Fig. 2b, c). Moreover, over 95% of NATURE COMMUNICATIONS | (2020)11:4051 | https://doi.org/10.1038/s41467-020-17753-w | www.nature.com/naturecommunications 3 ARTICLE a NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17753-w – + – HIV-1 – + IDB kD 37 Caspase-9 Bcl-xL 50 Mcl-1 37 50 37 25 37 25 Caspase-3 25 Bcl-2 20 25 25 Cleaved Caspase-3 20 37 20 Bax 15 37 25 20 25 20 15 Caspase-6 25 20 15 Bak LC3-I LC3-II 10 37 25 20 15 Caspase-7 50 Actin d HIV-1 No virus 100 75 50 25 0 – – – + – – – + – + + – – – + + – + – – – + – – – + – + + – – – + + ABT-263 – SAR405 + CQ 100 75 50 25 0 – – – + – – – + – + + – – – + + ABT-263 – SAR405 + CQ % p24+ cells remaining 37 c % Total T cell death b HIV-1 – + IDB 20 Cleaved Caspase-9 25 + 25 37 39 – % killing of p24+ cells kD 50 – 100 75 50 25 0 – – – + – – – + – + + – – – + + ABT-263 – SAR405 + CQ % DEVD+ Annexin V+ cells e No virus 60 HIV-1 40 20 0 – – – + – – + + – + – + + + + IDB ABT-263 SAR405 Fig. 2 Induction of caspase activation and cell death in HIV-1-infected T cells. a CD4+ T cells from PBMCs with or without infection by HIV-1 (NL4-3, 1 MOI) were cultured for 4 days to establish latency, followed by stimulation with IDB for 24 h. Cell lysates were used for western blot (representative of two biologically independent experiments). Arrows indicate cleaved caspases. b CMT with or without infection by HIV-1 (NL4-3, 1 MOI) were cultured for 4 days to establish latency. The cells were stimulated with 0.1 ?M IDB. ABT-263 (0.2 ?M) and SAR405 (2 ?M) and chloroquine (CQ, 10 ?M) were added as indicated. The cells were cultured for 48 h, followed by incubation with DEVD-FITC, staining with APC-Annexin V, and intracellular staining with PE-antiHIV p24. c Total cell death for cells treated in b was calculated (n = 3 biologically independent samples). Data are presented as mean ± SD. p values for control vs. ?ve treatment groups in sequence: 0.0001, 0.0011, 0.0002, 0.0049, and 0.0001 (one-way ANOVA with unpaired two-tailed t test). d The combinations of ABT-263 and SAR405 or CQ in the killing of IDB-stimulated HIV-1-infected T cells in b was calculated. The remaining viable HIV-1 p24+ cells (negative for staining by APC-annexin V and DEVD-FITC) in b were also calculated. Data are presented as mean ± SD (n = 3 biologically independent samples). p values for control vs. four treatment groups in sequence (for killing of p24+ cells and for p24+ cells remaining): 0.0001, 0.0006, 0.0001, 0.0011, and 0.0001 (one-way ANOVA with unpaired two-tailed t test). e T cells latently infected with HIV-1 as in Fig. 1a were mixed with CellTrace violetlabeled uninfected T cells, and cultured with IDB, SAR405, and ABT-263 as in b, in the presence of 0.2 ?M BMS-626529 for 48 h. The cells were stained with DEVD-FITC and APC-Annexin V, followed by ?ow cytometry analysis. Data are presented as mean ± SD and are representative of ?ve independent experiments. p values for control vs. four treatment groups (HIV-1-infected samples) in sequence: 0.0059, 0.0005, 0.0004, and 0.0001 (one-way ANOVA with unpaired two-tailed t test). Source data are provided as a Source Data ?le. 4 NATURE COMMUNICATIONS | (2020)11:4051 | https://doi.org/10.1038/s41467-020-17753-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17753-w HIV-1-p24+ cells in HIV-1-infected T cells could be killed within 2 days in vitro (Fig. 2d), whereas uninfected T cells were relatively resistant (Fig. 2b, c). Use of CQ instead of SAR405 also achieved similar results (Fig. 2b–d). These results suggest that latency reversal in combination with inhibition of autophagy and induction of apoptosis could ef?ciently kill HIV-1-infected T cells. HIV-1 can establish latency in both resting and activated T cells40. In addition to memory T cells, certain levels of autophagy are present in T cells at all developmental stages41. We therefore examined whether inhibition of autophagy might affect the survival of HIV-1-infected CD4+ T cells with different differentiation and activation status in addition to CMT, including CD3+CD4+CD45RO+CCR7? effector memory T cells (EMT), CD3+CD4+CD45RO?CCR7+ naive T cells (Supplementary Fig. 1a), as well as CD4+ T cells activated with PHA and IL-2. We found that the combination of IDB-induced latency reversal with inhibition of autophagy and promotion of ap … Purchase answer to see full attachment Get a 10 % discount on an order above $ 100 Use the following coupon code : NURSING10

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