Epigenetics, N-myrystoyltransferase-1 and casein kinase-2-alpha modulates the increased replication of HIV-1 CRF02_AG, compared to subtype-B viruses

Abstract

HIV subtypes distribution varies by geographic regions; this is likely associated with differences in viral fitness but the predictors and underlying mechanisms are unknown. Using in-vitro, in-vivo, and ex-vivo approaches, we found significantly higher transactivation and replication of HIV-1-CRF02_AG (prevalent throughout West-Central Africa), compared to subtype-B. While CRF02_AG-infected animals showed higher viremia, subtype-B-infected animals showed significantly more weight loss, lower CD4+ T-cells and lower CD4/CD8 ratios, suggesting that factors other than viremia contribute to immunosuppression and wasting syndrome in HIV/AIDS. Compared to HIV-1-subtype-B and its Tat proteins(Tat.B), HIV-1-CRF02_AG and Tat.AG significantly increased histone acetyl-transferase activity and promoter histones H3 and H4 acetylation. Silencing N-myrystoyltransferase(NMT)-1 and casein-kinase-(CK)-II-alpha prevented Tat.AG- and HIV-1-CRF02_AG-mediated viral transactivation and replication, but not Tat.B- or HIV-1-subtype-B-mediated effects. Tat.AG and HIV-1-CRF02_AG induced the expression of NMT-1 and CKII-alpha in human monocytes and macrophages, but Tat.B and HIV-1-subtype-B had no effect. These data demonstrate that NMT1, CKII-alpha, histone acetylation and histone acetyl-transferase modulate the increased replication of HIV-1-CRF02_AG. These novel findings demonstrate that HIV genotype influence viral replication and provide insights into the molecular mechanisms of differential HIV-1 replication. These studies underline the importance of considering the influence of viral genotypes in HIV/AIDS epidemiology, replication, and eradication strategies.

Figure 1

Increased replication of primary HIV-1 CRF02_AG isolates in human PBMC and MDM, compared to clade-B viruses. Reverse transcriptase activity in primary human PBMC (A–D) and MDM (E,F) infected with AG and clade-B viruses. RT activity assessed at day-12 (A,B) and day-15 (C,D) p.i. in PBMC, and from day-5 to day-18 p.i. in MDM (E,F). ^#P < 0.0001. Errors bars represent SEM.

Figure 2

Increased immune suppression in animals infected with HIV-1 clade-B compared to CRF02_AG. (A,B) Body weight of control (PBS), HIV-1 CRF02_AG (AG), and clade-B (B) infected animals before (Pre-E) and after (Post-E) engraftment, before and after HIV-1 infection. (C–H) levels of human (h)CD4+ (C,D) and hCD8+ (E,F) T-cells in the blood of control, AG- and clade-B-infected animals pre-infection and at week-1, -2, and -3 post-infection; and hCD4/hCD8 ratios (G,H). *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.0001. For all panels, errors bars represent SD.

Figure 3

Increased viremia in the blood and tissues of animals infected with HIV-1 CRF02_AG (AG) compared to clade-B-infected animals. (A,B) Plasma HIV-1 p24 levels at week-1, -2, and -3 p.i. (C–F) quantitative PCR showing copies number of HIV-1 LTR (C), pol (D), tat (E), and gag (F) in animal’s heart, kidney, liver, lungs, and spleen. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.0001. For all panels, errors bars represent SD.

Figure 4

Data normalized to tissues hCD4 levels confirmed increased viremia in tissues of animals infected with HIV-1 CRF02_AG (AG) compared to clade-B-infected animals. (A–D) quantitative PCR showing copies number of HIV-1 LTR (A), pol (B), tat (E), and gag (D) in animal’s heart, kidney, liver, lungs, and spleen. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.0001. For all panels, errors bars represent SD.

Figure 5

Tat.AG and HIV-1 CRF02_AG isolates induced higher LTR transactivation and increased HAT activity compared to Tat.B and HIV-1 clade-B. (A,B) Levels of CAT enzyme (A) and HAT activity (B) in U38 cells treated for 48 h with Tat.AG or Tat.B (10 to 1000 ng/ml). HI-Tat: heat-inactivated Tat proteins. Comparative p-values, or p-values compared to cells treated with HI-Tat proteins are shown. (C–F) HAT activity in primary human monocytes (C,D) and MDM (E,F) exposed to HIV-1 CRF02_AG or clade-B for 6 to 24 h. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.0001. (A–C,E) errors bars represent SD. (D,F) errors bars represent SEM.

Figure 6

Increased acetylation of histone H3 and H4 by Tat.AG compared to Tat.B. (A) Schematic structure of the integrated HIV-1 LTR promoter; the position of nucleosomes (Nu)-0, Nu-1, and the nucleosome-free regions (NFRs) are shown, as well as the position of transcriptional elements known to control HIV transactivation. The blue arrow indicates the transcription start site. Brown arrows indicate the positions of the forward (F’) and reverse (R’) primers used for PCR amplification. (B) Quantification of acetylated H3 and H4 by PCR of crosslinked DNA in U38 cells, following 48 h treatment with Tat.AG, Tat.B or HI-Tat (all at 100 ng/ml), and ChIP with anti-acetylated histone H3, anti-acetylated histone H4, or control IgG antibodies.

Figure 7

Silencing the NMT1 or CKIIα genes blocked Tat.AG but not Tat.B-induced HIV-1 transactivation in U38 (A–C) and TZM-bl (D–F) cells. (A,D) RT-PCR confirmation of NMT1, NMT2, CKIIα and CKIIβ genes silencing. (B,E) Western blot confirmation of NMT1, NMT2, CKIIα and CKIIβ genes silencing. Control: non-transduced cells. S: cells transduced with scrambled shRNA. (C,F) Silencing the NMT1 or CKIIα genes blocked Tat.AG-induced increase in CAT enzyme levels (C) and luciferase activity (F), but had no effect on Tat.B-induced viral transactivation. Silencing the NMT2 or CKIIβ genes had no effect on Tat.AG or Tat.B-induced viral transactivation. *P < 0.05, **P < 0.01, ^#P < 0.0001, compared to cells treated with similar concentrations of scrambled (S) shRNA and Tat proteins. Errors bars represent SD.

Figure 8

Silencing the NMT1 gene blocked the infection of human MDM by HIV-1 CRF02_AG isolates, but had no effect on clade-B HIV-1 infection. Silencing of NMT1, NMT2, CKIIα and CKIIβ genes in primary human MDM was confirmed by RT-PCR (A) and Western blot analyses (B). For panels A and B, controls are non-transduced MDM. “S” are MDM transduced with scrambled shRNA. Following NMT1 and NMT2 gene silencing, MDM were infected with CRF02_AG or clade-B HIV-1 isolates (3 different isolates for each subtype, each isolate tested in triplicate) and RT activity measured from day-5 to day-21 p.i. (C,D) RT activity in MDM with NMT1 gene silenced. (E,F) RT activity in MDM with NMT2 gene silenced. For panels C–F, controls are non-HIV infected (mock-infected) MDM, “S” are MDM transduced with scrambled shRNA; “sh” are MDM transduced with NMT1 or NMT2 shRNA. **P < 0.01; ^#P < 0.0001, compared to infected MDM treated with similar concentrations of scrambled shRNA (S).

Figure 9

Silencing the CKIIα gene blocked the infection of human MDM by HIV-1 CRF02_AG isolates, but had no effect on clade-B HIV-1 infection. (A–D) Following silencing of CKIIα and CKIIβ genes, MDM were infected with CRF02_AG or clade-B HIV-1 isolates (3 different isolates for each subtype, each isolate tested in triplicate) and RT activity measured from day-5 to day-21 p.i. (A,B) RT activity in MDM with CKIIα gene silenced. (C,D) RT activity in MDM with CKIIβ gene silenced. Controls are non-HIV infected (mock-infected) MDM, “S” are MDM transduced with scrambled shRNA; “sh” are MDM transduced with CKIIα or CKIIβ shRNA. *P < 0.05, ^#P < 0.0001, compared to infected MDM transduced with similar concentrations of scrambled shRNA (S).

Figure 10

Tat.AG and HIV-1 CRF02_AG, but not Tat.B or clade-B viruses, induced NMT1 expression in primary human monocytes and MDM. (A–D) Exposure of human monocytes (A,C) and MDM (B,D) to Tat.AG induced a dose-dependent (A,B) and time-dependent (C,D) increase in NMT1 expression and secretion. (A,B) cells exposed to Tat proteins for 24 h; (C,D) Tat proteins used at 100 ng/ml. (E–H) Exposure of human monocytes (E,F) and MDM (G,H) to HIV-1 CRF02_AG isolates induced a time-dependent increase in NMT1 expression and secretion. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.0001. P-values are in comparison to HI-Tat or Tat.B (A–D), or clade-B viruses (E–H).

Figure 11

Tat.AG and HIV-1 CRF02_AG, but not Tat.B or clade-B viruses, induced CKIIα expression in primary human monocytes and MDM. (A–D) Exposure of human monocytes (A,C) and MDM (B,D) to Tat.AG induced a dose-dependent (A,B) and time-dependent (C,D) increase in CKIIα expression. (A,B) cells exposed to Tat proteins for 24 h; (C,D) Tat proteins used at 100 ng/ml. (E–H) Exposure of human monocytes (E,F) and MDM (G,H) to HIV-1 CRF02_AG isolates induced a time-dependent increase in CKIIα expression. *P < 0.05, **P < 0.01, ^#P < 0.0001. P-values are in comparison to HI-Tat or Tat.B (A–D), or clade-B viruses (E–H).