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Defective HIV-1 Proviruses Can Be Transcribed Upon Activation 1 Ho , Ya-Chi 1 Pollack , Ross 2 Yong , Patrick Robert F. 1,3 Siliciano 1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 2Yale University, New Haven, Connecticut; 3Howard Hughes Medical Institute, Baltimore, Maryland Email: [email protected] #392 Day 5 (21) (23) (13) +CTL Day 0 Day 5 78 (10) (19) (12) +CTL 80 (21) (23) (13) Mutated gag DNA 0 1 2 3 4 1000 100 10 1 5 100000000 2 3 4 1000000 100000 10000 1000 100 10 1 82 4 1000 100 10 1 0 5 85 1 2 3 4 gag RNA/gag DNA 100 10 1 3 4 Duration after activation (days) 3 4 100 10 1 0.1 p = 0.048 78 79 80 81 82 100 84 10 85 Median 1 0 1 2 3 4 p = 0.03 p = 0.004 2 3 4 78 81 100 82 10 84 85 1 Duration after activation (days) Hypermutated AAAAAAA Large deletion T cell activation AAAAAAA AAAAAAA AAAAAAA Median Day 1 2 3 4 5 CTL responses AAAAAAA AAAAAAA AAAAAAA HIV-1 DNA Trizol simultaneous DNA/RNA extraction DNA HIV-1 full-length sequencing qPCR (gag, RNaseP) gag targeted deep sequencing RNA DNase treatment, cDNA synthesis qPCR (gag) gag targeted deep sequencing HIV-1 may not be “transcriptionally silent” in resting memory CD4+ T cells. Low levels of HIV-1 RNA may be transcribed from both intact and defective HIV-1 proviruses in resting CD4+ T cells. HIV-1 DNA quantity per million cells stays unchanged: cells proliferate upon activation, but there is no significant preferential proliferation of HIV-1 containing cells. A small number of induced proviruses produce the majority of RNA, but defective proviruses also make a minority of RNA 85 Median Median 60% 40% 20% NON-mutated gag DNA No CTL 200% 78 80 82 100% 80% 60% 40% 20% 85 No CTL NON-mutated gag RNA 100% 78 79 80% 80 81 60% 82 40% 84 85 20% Median No CTL +CTL NON-Mutated gag RNA/gag DNA Mutated gag RNA/gag DNA DNA 100% 100% 100% 80% 60% 40% 20% 80% 60% 40% 20% +CTL 78 80% 79 80 60% 81 82 40% 84 20% 85 Median 0% 0% No CTL +CTL p = 0.025 p = 0.025 120% +CTL 0% No CTL +CTL Median 0% 0% 0% 84 50% Mutated gag RNA 120% 81 100% +CTL 140% 79 150% No CTL +CTL No CTL +CTL Duration after activation (days) Day 5 Day 3 80% 0% Induced Day 0 84 p = 0.03 80 1 85 0% Total gag RNA/gag DNA Figure 3. Changes of HIV-1 DNA and RNA quantities upon CD3/CD28 costimulation. The quantity of cells was calculated by RNaseP copy numbers. p value was calculated by two-tailed Wilcoxon rank sum test. HIV-1 RNA 100% No CTL 79 0 84 100% +CTL 120% 5 1000 5 82 200% Total gag RNA 1000 81 82 300% 0% No CTL 0.1 1 50% 5 10000 10000 1000 0 5 2 100000 0.01 2 100% NON-mutated gag RNA/gag DNA 10000 1000 1 1000000 5 p = 0.01 p = 0.02 p = 0.003 100000 150% Median 0 Mutated gag RNA/gag DNA p = 0.025 1 84 100 10000000 10000 p = 0.035 p = 0.004 0 81 1000 200% NON-mutated gag RNA 100000 3 79 80 5 gag RNA/million cells 10000000 gag RNA/gag DNA 1 p = 0.02 1000000 2 78 Mutated gag RNA Total gag RNA 1 100000 10000 81 Mutated gag DNA Total gag DNA 10 0 80 gag DNA/million cells fold change 1 0.1 Activate resting CD4+ T cells using antiCD3/CD28 costimulation under enfuvirtide 79 80 gag RNA/million cells fold change 10 10000 Remove CTLs by positive selection 78 79 gag RNA/gag DNA fold change 100 Isolate CTLs by negative selection CD4 activation for 3 days 78 Figure 2. Proportion of HIV-1 DNA and RNA containing inactivating G-to-A mutations upon anti-CD3/CD28 stimulation (day 0, 1, 3, 5) and CTL co-cultulre. gag DNA/million cells fold change 1000 CD4-CTL coculture for 2 days Isolate resting CD4+ T cells 0% gag RNA/million cells fold change 10000 10000 Median 20% NON-mutated gag DNA gag DNA/million cells gag DNA/million cells 100000 85 40% gag RNA/gag DNA fold change Total gag DNA 84 60% (12) Figure 1. Composition (A) and quantity (B) changes of clonal full-length HIV-1 proviruses upon anti-CD3/CD28 costimulation (day 0 and day 5) and CTL coculture in two patients (patient 78 and 80). Number in brackets: numbers of clones analyzed. Total gag RNA/gag DNA CTL activation for 6 days Day 0 82 RNA Patient ID 0 PBMC from HIV-1 infected individuals under suppressive antiretroviral therapy +CTL 80 (10) (19) Methods Activate CTLs using group M consensus Gag peptide mixture in PBMC for 6 days Day 5 gag RNA/gag DNA Conclusions. Hypermutated HIV-1 proviruses can be transcribed both in resting CD4 T cells and upon T cell activation, which may cause an overestimation of latency reversal of intact proviruses using cell-associated RNA measurements. We propose that CTLs eliminate only a small number of cells which produce significant amount of HIV-1 RNA, presumably from cells containing intact LTR. The RNA level decreases after CTL co-culture but the DNA level remains unchanged because the fraction of cells eliminated may be too small compared with the large quantities of integrated HIV-1 proviruses. Day 0 gag RNA/million cells + # of clones gag DNA/million cells Results. The effect of T cell activation on defective HIV-1 proviruses. We found that around 20% of the HIV-1 DNA and around 10% HIV-1 RNA contain inactivating mutations in the gag region, from both resting and activated CD4+ T cells. Upon activation, the quantity of gag DNA per million cells remains unchanged, while the quantity of gag RNA per million cells increases significantly. The increase of HIV-1 RNA is most prominent when HIV-1 RNA quantity is normalized to HIV-1 DNA quantity (p <0.05). Using deep sequencing of the full-length gag genome to quantify the proportion of hypermutated defective sequences, we found that the quantity of hypermutated and nonhypermutated HIV-1 DNA remains unchanged. Both hypermutated and non-hypermutated HIV-1 RNA increases significantly (p <0.05). This indicates that defective HIV-1 can be transcribed, both at resting state and upon activation. The effect of CTLs on defective HIV-1 proviruses. Addition of CTLs does not decrease the quantity of hypermutated or non-hypermutated HIV-1 proviruses. However, both hypermutated and non-hypermunated HIV-1 RNA decreases significantly after CTL co-culture. The quantity of non-hypermutated HIV-1 RNA decreased (~1 log reduction) more than that of the hypermutated HIV-1 RNA (~0.5 log reduction). +CTL 78 gag RNA/million cells Methods. To understand how T cell activation affects the transcription of HIV-1 proviruses, resting CD4+ T cells from aviremic patients under suppressive antiretroviral therapy were activated with anti-CD3/CD28 costimulation under enfuvirtide to prevent new rounds of in vitro infection. To examine whether cells containing intact or defective HIV-1 can be eliminated by CTLs, peripheral blood mononuclear cells (PBMC) containing autologous CTLs were stimulated with group M consensus Gag peptide mixture in the presence of interleukin-2. After 3 days of CD4+ T cell activation and 6 days of CTL activation, pre-stimulated autologous CTLs (magnetic purified) and activated CD4+ T cells were co-cultured at 1:1 ratio. CTLs were removed by magnetic bead depletion from the CTL-CD4 coculture before simultaneous DNA and RNA extraction by Trizol reagent. Cell-associated RNA and proviral DNA from cells which are activated for 0, 1, 3, and 5 days and cocultured with CTLs for 2 days was subjected to quantitative PCR and deep-sequencing of the gag region. The full-length gag contains the start codon and the 9 TGG sequences encoding tryptophan residues. These sites are hotspots APOBEC-mediated G-to-A hypermutations. Any G-to-A mutation in these sites will lead to either a missense methionine-to-isoleucine change of the start codon or a nonsense mutation of the tryptophan residues into premature stop codons, and render the provirus defective. Day 5 81 Day 0 Day 1 Day 3 Day 5 +CTL Day 0 80 Day 0 Day 1 Day 3 Day 5 +CTL 1 79 Day 0 Day 1 Day 3 Day 5 +CTL 0% 78 80% gag DNA/million cells fold change 20% 10 0% gag RNA/million cells fold change Hypothesis. We hypothesize that some defective proviruses can be transcribed and translated upon T cell activation, but only a minority of them can be eliminated by CTLs. Understanding of the dynamics of defective HIV-1 transcription upon activation and elimination by CTLs can facilitate the correct measurement of the efficacy of latency reversing agents. Intact Point mutation Packaging signal deletion Hypermut Deletion gag RNA /gag DNA fold change 40% 100 20% Day 0 Day 1 Day 3 Day 5 +CTL 60% 40% Day 0 Day 1 Day 3 Day 5 +CTL 80% 60% Day 0 Day 1 Day 3 Day 5 +CTL 1000 100% DNA Day 0 Day 1 Day 3 Day 5 +CTL B 80% Day 0 Day 1 Day 3 Day 5 +CTL A HIV-1 DNA /million cells Background. Human immunodeficiency virus type-1 (HIV-1) persists in the latent reservoir, primarily resting memory CD4+ T cells, as the major barrier to cure. HIV-1 proviruses reside in the resting memory CD4+ T cells as three types: induced proviruses, intact noninduced proviruses and defective proviruses. The defective provirus is comprised of ~32% of APOBEC-mediated G-to-A hypermutations, ~46% of large internal deletions, ~6% of packaging signal deletions/mutations, and ~4% of inactivating point mutations. Since these defective proviruses lack epigenetic silencing and may have intact long terminal repeat (LTR) promoter function, it is possible that they can be transcribed and translated upon activation, such as by antigen stimulation, by homeostatic proliferation and by latency reversing agents used in the shock-and-kill strategy for HIV-1 eradication. Transcription of defective proviruses may complicate the correct estimation of the efficacy of latency reversal of inducible proviruses using cell-associated RNA measurements. Whether cells containing defective proviruses can be eliminated by cytotoxic T lymphocytes (CTLs) upon activation remains unknown. %gag containing inactivating G-to-A mutations Results Abstract CTLs may eliminate a small number of cells which produce the majority of HIV-1 RNA. Therefore, the total HIV-1 DNA quantity does not decrease, as the proportion is too small to make significant changes.. The decrease of hypermutated HIV-1 RNA suggests that cells containing defective proviruses may be recognized and eliminated by CTLs. Figure 5. Proposed dynamics of defective HIV-1 proviruses upon T cell activation and CTL elimination. Figure 4. Changes of HIV-1 DNA and RNA quantities upon CTL co-culture. The quantity of cells was calculated by RNaseP copy numbers. p value was calculated by two-tailed Wilcoxon rank sum test. Conclusions 1. The effect of T cell activation on hypermuted/non-hypermuted HIV-1: 1) HIV-1 DNA level (copies per million cells, as measured by gag DNA/RNaseP) remains unchanged in cells containing hypermuted and non-hypermuted proviruses. 2) HIV-1 RNA level (copies per million cells, as measured by gag RNA/RNaseP) increases in both hypermuted and non-hypermuted samples, indicating that even hypermutated samples can be transcribed upon T cell activation. This effect is most prominent when measured as gag RNA copies/gag DNA copies, which reflects HIV-1 RNA reactivation from comparable numbers of HIV-1 proviruses in each sample. 2. The effect of CTL on cells containing hypermutated/non-hypermutated HIV-1: 1) HIV-1 DNA level (copies per million cells) remains unchanged in cells containing hypermut and non-hypermut proviruses. 2) HIV-1 RNA level (copies per million cells) decreases in hypermutated gag RNA (~3-fold reduction) and non-hypermutated gag RNA (~10-fold reduction). 3. We propose that CTLs eliminate only a small number of cells which produce significant amount of HIV-1 RNA. The fraction of cells eliminated may be too small in proportion to all the integrated HIV-1 DNA. Therefore, RNA level decreases but DNA levels remain unchanged. It cannot be distinguished thus far whether it is the induced, intact noninduced, or defective proviruses with large internal deletions which produce the non-hypermutated RNA. Acknowledgements We thank all study participants. We thank Dr. Joel N. Blankson for his critical advice, Adam Longwich for study subject recruitment and coordination, and Dr. Haiping Hao for deep sequencing. This work was supported by the Martin Delaney CARE and DARE Collaboratories (NIH grants AI096113 and 1U19AI096109), by an ARCHE Collaborative Research Grant from the Foundation for AIDS Research (amFAR 108165-50-RGRL), by the Johns Hopkins Center for AIDS Research (P30AI094189), by NIH grant 43222, by the Howard Hughes Medical Institute and by the Bill and Melinda Gates Foundation.