TRIM5α and TRIM22 are differentially regulated according to HIV-1 infection phase and compartment

Abstract

The antiviral role of TRIM E3 ligases in vivo is not fully understood. To test the hypothesis that TRIM5α and TRIM22 have differential transcriptional regulation and distinct anti-HIV roles according to infection phase and compartment, we measured TRIM5α, TRIM22, and type I interferon (IFN-I)-inducible myxovirus resistance protein A (MxA) levels in peripheral blood mononuclear cells (PBMCs) during primary and chronic HIV-1 infection, with chronic infection samples being matched PBMCs and central nervous system (CNS)-derived cells. Associations with biomarkers of disease progression were explored. The impact of IFN-I, select proinflammatory cytokines, and HIV on TRIM E3 ligase-specific expression was investigated. PBMCs from individuals with primary and chronic HIV-1 infection had significantly higher levels of MxA and TRIM22 than did PBMCs from HIV-1-negative individuals (P < 0.05 for all comparisons). PBMCs from chronic infection had lower levels of TRIM5α than did PBMCs from primary infection or HIV-1-uninfected PBMCs (P = 0.0001 for both). In matched CNS-derived samples and PBMCs, higher levels of MxA (P = 0.001) and TRIM5α (P = 0.0001) in the CNS were noted. There was a negative correlation between TRIM22 levels in PBMCs and plasma viral load (r = -0.40; P = 0.04). In vitro, IFN-I and, rarely, proinflammatory cytokines induced TRIM5α and TRIM22 in a cell type-dependent manner, and the knockdown of either protein in CD4(+) lymphocytes resulted in increased HIV-1 infection. These data suggest that there are infection-phase-specific and anatomically compartmentalized differences in TRIM5α and TRIM22 regulation involving primarily IFN-I and specific cell types and indicate subtle differences in the antiviral roles and transcriptional regulation of TRIM E3 ligases in vivo.

Importance: Type I interferon-inducible TRIM E3 ligases are a family of intracellular proteins with potent antiviral activities mediated through diverse mechanisms. However, little is known about the contribution of these proteins to antiviral immunity in vivo and how their expression is regulated. We show here that TRIM5α and TRIM22, two prominent members of the family, have different expression patterns in vivo and that the expression pattern depends on HIV-1 infection status and phase. Furthermore, expression differs in peripheral blood versus central nervous system anatomical sites of infection. Only TRIM22 expression correlated negatively with HIV-1 viral load, but gene silencing of both proteins enhances HIV-1 infection of target cells. We report subtle differences in TRIM5α and TRIM22 gene induction by IFN-I and proinflammatory cytokines in CD4(+) lymphocytes, monocytes, and neuronal cells. This study enhances our understanding of antiviral immunity by intrinsic antiviral factors and how their expression is determined.

FIG 1

Expression of antiviral factors according to HIV-1 infection status and compartment. (A to C) Expression of the IFN-I-responsive gene (MxA) (A), TRIM5α (B), and TRIM22 (C) in PBMCs from HIV-1-uninfected versus -infected subjects. (D to F) Expression of MxA (D), TRIM5α (E), and TRIM22 (F) in PBMCs versus CSF-derived cells from chronically infected patients. The samples from participants with primary infection were all collected within 12 months of infection. One time point, closest to 12 months postinfection, was used per participant (n = 28). For HIV-1-negative participants, only those who remained HIV-1 negative during the entire follow-up period (2 years) were included in this analysis (n = 19). The chronically infected group consisted of participants presenting with chronic meningitis (n = 26). Matched CSF samples and PBMCs from the same patients were analyzed (n = 26). Data are depicted as normalized ratios of MxA, TRIM5α, or TRIM22 to GAPDH. Median expression levels between HIV-negative and HIV-positive samples were compared. (G to I) Correlations (Pearson) between MxA and TRIM5α (G), TRIM22 and MxA (H), and TRIM22 and TRIM5α (I) in both CSF samples and PBMCs from patients with chronic HIV-1 infection.

FIG 2

TRIM5α and TRIM22 expression in different immune cells. (A) Representative flow cytometry plots showing the gating strategy employed to define different cell populations. (Top) Cells were first gated for singlets (forward scatter height [FSC-H] versus forward scatter area [FSC-A]), and CD4⁺ T cells were defined from the lymphocyte gate (side scatter area [SSC-A] versus FSC-A) as CD3⁺ CD4⁺ events. (Middle) Flow cytometry plots illustrate the gating for B cells as CD3⁻ CD19⁺ cells and monocytes as CD3⁻ CD19⁻ CD56⁻ CD14⁺ cells. (Bottom) NK cells were defined as CD3⁻ CD56⁺ CD16⁺ cells. (B) Data were compared between each subset of cells in the CSF and PBMCs only by using the paired t test. Magnetic cell sorting was employed to isolate CD4 cells and monocytes from 6 fresh HIV-1-negative PBMC samples. Natural killer cells were isolated by using the Easy Sep negative-selection human NK cell enrichment kit (Stemcell Technologies). (C and D) Different cell populations were analyzed for TRIM5α (C) and TRIM22 (D) mRNA expression. Data are depicted as normalized ratios of TRIM5α or TRIM22 to GAPDH.

FIG 3

Association of antiviral gene expression with markers of disease progression in primary and chronic HIV-1 infection. (A and B) Differences between samples from participants with primary infection closest to 12 months postinfection (n = 28) and samples from chronically infected patients (n = 28) were compared in terms of CD4 T cell counts and viral loads. (C) Viral loads in plasma and CSF samples from the chronically infected group were compared. (D to F) Pearson correlations were performed for MxA (D), TRIM5α (E), and TRIM22 (F) expression levels and viral loads in the CSF or peripheral blood compartments in the chronically HIV-1-infected group.

FIG 4

Regulation of TRIM5α and TRIM22 by IFN-α and select proinflammatory cytokines in immune cells. Shown is the effect of IFN-α stimulation and proinflammatory cytokine stimulation of CD4 cells and monocytes on antiviral gene expression. Cells from healthy donors were stimulated for 24 h. (A and B) Total cellular RNA from cells was then subjected to real-time RT-PCR to measure mRNA levels using primers specific for TRIM5α (A) and TRIM22 (B) and GAPDH. (C to E) Protein expression analyses of TRIM5α (C and D) and TRIM22 (C and E) are shown. Results shown in panels A, B, D, and E are the means of three independent experiments (with bars indicating ranges), each performed in duplicate.

FIG 5

siRNA-mediated silencing of TRIM5α or TRIM22 in CD4 cells. To determine the functional impact of TRIM5α and TRIM22 on HIV-1 replication in CD4 cells, gene knockdown experiments were performed by transducing the cells with siRNA against TRIM5α or TRIM22 or a nontargeting (scramble) control siRNA, with or without IFN-α stimulation for 24 h. (A to F) Knockdown of TRIM5α or TRIM22 by siRNA in the absence or presence of IFN-α was validated by mRNA RT-PCR (A and B) and Western blotting (C to F). Cells were then challenged with a VSV-G-pseudotyped HIV-1 laboratory strain (JRCSF) (600 ng of p24/ml) for 48 h. (G and H) Culture lysates were collected and assessed for HIV p24 antigen levels by an enzyme-linked immunosorbent assay. Data are depicted as fold changes, where the levels of p24 antigen in knockdown cells were divided by the levels in control cells. TRIM22.2 siRNA knockdown efficiency was inconsistent, and the data are thus excluded from the graphs.

FIG 6

siRNA-mediated silencing of TRIM5α or TRIM22 in neuroblasts. To determine the functional impacts of TRIM5α and TRIM22 on HIV-1 infection in neuroblasts, gene knockdown experiments were performed by transducing the cells with siRNA against TRIM5α or TRIM22 or a nontargeting (scrambled) control siRNA, with or without IFN-α stimulation for 24 h. (A to D) Knockdown of TRIM5α or TRIM22 by siRNA in the absence or presence of IFN-α was validated by mRNA RT-PCR (A and B) and Western blotting (C and D). Cells were then challenged with a VSV-G-pseudotyped HIV-1 laboratory strain (JRCSF) (600 ng of p24/ml) for 48 h. (E and F) Culture lysates were collected and assessed for HIV p24 antigen levels by an enzyme-linked immunosorbent assay. Data are depicted as fold changes, where levels of p24 antigen in knockdown cells were divided by levels in control cells.