1. Volume 21, Issue 4, Pages 494-506.e4 (April 2017)
Defective HIV-1 Proviruses Are Expressed and Can Be Recognized by Cytotoxic T Lymphocytes, which Shape the Proviral Landscape 
Ross A. Pollack, R. Brad Jones, Mihaela Pertea, Katherine M. Bruner, Alyssa R. Martin, Allison S. Thomas, Adam A. Capoferri, Subul A. Beg, Szu-Han Huang, Sara Karandish, Haiping Hao, Eitan Halper-Stromberg, Patrick C. Yong, Colin Kovacs, Erika Benko, Robert F. Siliciano, Ya-Chi Ho 
Cell Host & Microbe 
Volume 21, Issue 4, Pages e4 (April 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 21, 494-506. e4DOI: (10. 1016/j. chom. 2017
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3. Figure 1 The Landscape of HIV-1 Proviruses May Be Dynamic
Correlation between the duration from diagnosis to HIV-1 proviral landscape analysis (enrollment) and the proportion of different subsets of HIV-1 proviruses containing (A) intact genome, (B) Ψ/MSD deletions/mutations, (C) hypermutations, and (D) large internal deletions from ART-treated individuals. R and p values are calculated by Pearson correlation. See also Figure S1.
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4. Figure 2
HIV-1 Proviruses Can Bypass MSD Defects by Activating Novel SDSs and Splicing into Canonical SASs In Vitro and Ex Vivo
(A) Spliced HIV-1 RNA transcripts from reconstructed patient-derived proviruses. Numbers on the right indicate the number of isolates from cloning of PCR products.
(B–F) Sequences of canonical (Ocwieja et al., 2012; Schwartz et al., 1990a), known alternative (Ocwieja et al., 2012), and novel splice sites from proviruses with ψ deletion (45E6, B; and 39G2, C), MSD mutation (42B6, D), hypermutation causing MSD mutation (31G4, E), and 5’ genome deletion causing MSD deletion (E44E11, F).
(G) Spliced HIV-1 RNA from patient resting CD4+ T cells upon ex vivo activation.
(H) Sequences of novel splice sites in participant 85. Purple box, short sequence repeats likely related to the 245 bp deletion encompassing MSD. See also Tables S1–S5; Figures S2–S6.
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5. Figure 3 HIV-1hypermut Can Be Transcribed Ex Vivo
Transcription of HIV-1hypermut in vivo from rCD4s and upon ex vivo anti-CD3/CD28 activation. Resting CD4+ T cells from ART-suppressed individuals were treated with or without anti-CD3/CD28 activation for 3 days in the presence of enfuvirtide. Levels of nonhypermutated and hypermutated HIV-1 gag RNA were measured as the frequency of total HIV-1 gag (by qRT-PCR) times the proportion of hypermutated HIV-1 gag RNA (as measured by targeted gag deep sequencing).
(A) Location of hypermutation hotspots (TGG) which will cause lethal missense start codon mutation or lethal nonsense mutations.
(B and C) Proportion of hypermutated HIV-1 RNA in resting (B) and activated (C) CD4+ T cells from ART-treated aviremic HIV-1-infected individuals. Green, nonhypermutated HIV-1 RNA. Red, hypermutated HIV-1 RNA containing lethal mutations.
(D and E) Frequency of non-hypermutated (D) and hypermutated (E) HIV-1 RNA in resting and activated CD4+ T cells. Data represent mean ± SEM.
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6. Figure 4
Cells Containing HIV-1def Can Be Recognized by HIV-1-Specific CTLs
Flow cytometry showing percent degranulated CTLs (CD8+CD107a+) from two Gag-specific clones (B27-Gag-IK9, A; and B07-Gag-HA9, B), two Nef-specific clones (Cw08-Nef-AL9, C; and B62-Nef-RA9, D), and a CMV-specific clone (E) upon coculture with autologous CD4+ T cells transfected with HIV-1def plasmids. NL4-3 has an anchor residue mutation (K162Q) at the IK9 epitope making it not susceptible to CTL recognition (A). Each target was tested in independent quadruplets shown in mean ± SEM. p values were calculated by one-way ANOVA with Dunnett’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < See also Figure S7.
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7. Figure 5
Cells Containing HIV-1def Compete with Cells Containing Intact Proviruses for CTL Recognition
(A) Negative control using CD4+ T cells transfected with the vector plasmid.
(B) Positive control using CD4+ T cells transfected with the vector plasmid and loaded with cognate peptide.
(C–E) A cold-target inhibition assay showing HIV-1-specific CTL recognition of cells containing defective HIV-1 proviruses containing ψ deletion (45E6, C), hypermutation (31G4, D), and nonsense mutation (19B3, E). Six paired replicates per condition were shown. p values were calculated by paired Student’s t test. See also Figure S7.
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8. Figure 6
Cells Containing HIV-1hypermut Can Be Recognized by CD8+ T Cells Ex Vivo
Resting CD4+ T cells were activated with anti-CD3/CD28 in the presence of enfuvirtide and cocultured with autologous CD8+ T cells prestimulated with a Gag peptide mixture, a CMV peptide mixture, or a HLA-A2-restricted SL9 peptide. Targeted gag RNA deep sequencing and qRT-PCR were used to measure the proportion and the frequency of HIV-1 RNA containing lethal hypermutations.
(A–D) Proportion (A–C) and frequency (D) of HIV-1 RNA containing lethal hypermutations from CD4+ T cells upon coculture with autologous (A) CMV-, (B) Gag-, and (C) SL9-prestimulated CD8+ T cells.
(E–G) Proportion of HIV-1 RNA with or without lethal hypermutations 5′ to wild-type or escaped CTL epitopes upon coculture with autologous (E) CMV-, (F) Gag-, and (G) SL9-prestimulated CD8+ T cells. Data represent mean ± SEM. ∗p value < 0.05 by two-tailed Student’s t test.
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9. Figure 7
Proposed Model of the Scope of Potential Targets of HIV-1-Specific CTLs Compared with the Size of the Latent Reservoir
The provirus population includes intact (yellow and pink) and defective (blue) proviruses as described (Ho et al., 2013). Following reversal of latency by endogenous stimuli or latency-reversing agents, cells with certain types of HIV-1def may express HIV-1 RNA and protein and may become targets for HIV-1-specific CTLs.
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