HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production - PubMed (original) (raw)

. 2011 Jul 14;118(2):298-308.

doi: 10.1182/blood-2010-07-297721. Epub 2011 Mar 16.

Joey Lai, Stuart Turville, Shamith Samarajiwa, Lachlan Gray, Valerie Marsden, Sarah K Mercier, Kate Jones, Najla Nasr, Arjun Rustagi, Helen Cumming, Heather Donaghy, Johnson Mak, Michael Gale Jr, Melissa Churchill, Paul Hertzog, Anthony L Cunningham

Affiliations

HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production

Andrew N Harman et al. Blood. 2011.

Erratum in

Abstract

Many viruses have developed mechanisms to evade the IFN response. Here, HIV-1 was shown to induce a distinct subset of IFN-stimulated genes (ISGs) in monocyte-derived dendritic cells (DCs), without detectable type I or II IFN. These ISGs all contained an IFN regulatory factor 1 (IRF-1) binding site in their promoters, and their expression was shown to be driven by IRF-1, indicating this subset was induced directly by viral infection by IRF-1. IRF-1 and -7 protein expression was enriched in HIV p24 antigen-positive DCs. A HIV deletion mutant with the IRF-1 binding site deleted from the long terminal repeat showed reduced growth kinetics. Early and persistent induction of IRF-1 was coupled with sequential transient up-regulation of its 2 inhibitors, IRF-8, followed by IRF-2, suggesting a mechanism for IFN inhibition. HIV-1 mutants with Vpr deleted induced IFN, showing that Vpr is inhibitory. However, HIV IFN inhibition was mediated by failure of IRF-3 activation rather than by its degradation, as in T cells. In contrast, herpes simplex virus type 2 markedly induced IFNβ and a broader range of ISGs to higher levels, supporting the hypothesis that HIV-1 specifically manipulates the induction of IFN and ISGs to enhance its noncytopathic replication in DCs.

PubMed Disclaimer

Figures

Figure 1

Figure 1

HIV-1 and HSV-2 induction of IFNβ in MDDCs. Day 6 MDDCs were exposed to purified HIV-1BaL or HSV-2186 at MOI of 3 for 3-96 hours. (A) IFNβ mRNA expression was determined by qPCR at 3, 6, 12, and 24 hours after infection. (B) The level of IFNβ secreted into the supernatant fluid was determined by ELISA at 6, 12, 24, and 48 hours after infection. The mean data from 3 experiments are shown with standard error bars.

Figure 2

Figure 2

IRF protein expression after exposure to HIV. Day 6 MDDCs were treated with HIV-1BaL (MOI, 3) or mock-treated for 6-120 hours. (A) IRF-1 intracellular expression levels were determined by flow cytometry; the isotype control is shown as filled curve, mock-treated cells are shown with a solid line and HIV-1–treated cells are shown with a broken line. (B) The percentage of HIV-1–infected cells was determined by flow cytometry with the use of a PE-conjugated p24 antibody and (C) peak IRF-1 expression was determined in p24− cells and p24+ cells separately (means of 5 experiments shown with standard error bars). (D) IRF-1 expression was also determined by Western blot. (E) Panels A to C were repeated for IRF2, IRF-7, and IRF-8. Representative data are shown from 1 of 3 experiments.

Figure 3

Figure 3

In silico promoter analysis of identified up-regulated IFN-associated genes in MDDCs. RefSeq IDs and promoters were extracted with the use of the UCSC genome browser (

www.genome.ucsc.edu

) and sequences 3000 bp 5′ upstream from the transcription start site, plus the 5′ untranslated region were extracted. Transfac database was then used to obtain vertebrate TFBS matrices, and the Toucan2 tool MotifScanner was used to detect potential TFBSs in the sets of selected sequences. The prior (stringency level) was set to a value of 0.05. A small box indicates the location of identified potential IRF-1 binding sites.

Figure 4

Figure 4

IRF-1 ChIP assay. Day 6 MDDCs were treated with HIV-1BaL or IFNγ for 48 hours. A ChIP assay was then performed with an IRF-1 antibody, and qPCR primers were directed toward IRF-2 or IFIT5 promoter sequences to determine IRF-1 binding. (Lane 1) HIV-1–treated MDDCs no IRF-1 antibody, (lane 2) HIV-1–treated MDDCs plus IRF-1 antibody, (lane 3) IFNγ-treated MDDCs no IRF-1 antibody, (lane 4) IFNγ-treated MDDCs plus IRF-1 antibody, (lane 5) cell input DNA, (lane 6) PCR negative no DNA control, and (lane 7) ChIP reagents only negative control.

Figure 5

Figure 5

Deletion of the IRF-1/7 binding site (IRF-1 bs) from the HIV-1 LTR results in reduced infectivity. Day 2 MDDCs were treated with VSVG-pseudotyped HIV-1BaL-IRF-1 bs or HIV-1BaLwt at an MOI of 1 for 6-134 hours either alone in combination at various ratios. (A) The percentage of HIV-1–infected cells was determined by flow cytometry between 48 and 134 hours after infection. The mean data from 3 independent experiments are shown with standard error bars. There was a statistically significant difference between the rates of increase of the percentage of infected cells over time for HIV-1BaLwt versus HIV-1BaL-IRF-1 bs (P = .021 linear mixed-effects model). The mean difference in these infection rates was 0.028% per hour. (B) The greater the ratio of wild-type compared with mutant plasmid transfected, the greater the proportion of cells infected at 48 hours after infection. The mean data from 3 independent experiments are shown with standard error bars.

Figure 6

Figure 6

IFN induction in MDDCs by HIV viruses with Vpr but not Vif deleted from their genome. (A) Day 2 MDDCs were treated with VSVG-pseudotyped HIV-1NLAD8ΔVpr, HIV-1NLAD8ΔVif, HIV-1NLAD8ΔVpr,Vif, or HIV-1NLAD8 at an MOI of 1 for 48 and 96 hours. (A) IFNβ mRNA expression was determined by qPCR. The mean data from 3 independent donors are shown with standard error bars. (B-D) Day 6 MDDCs were treated with HIV-1BaL or mock-treated for 24 and 48 hours. (B) IRF-3 intracellular expression levels were determined by flow cytometry; the isotype control is shown as filled curve, mock treated cells are shown with a solid line, and HIV-1–treated cells are shown with a broken line. (C) The percentage of HIV-1–infected cells was determined by flow cytometry with the use of a PE-conjugated p24 antibody, and IRF-3 expression was determined in p24− cells and p24+ cells separately (mean of 5 experiments shown with standard error bars). (D-E) IRF-3 expression in response to HIV-1BaL was also determined by Western blot in both MDDCs (D) and SupT1 cells (E). (F) IRF3 cellular localization was determined by confocal microscopy in mock- and HIV-1–treated cells compared with Sendai virus–treated TZM-bl cells.

References

    1. Hu J, Gardner MB, Miller CJ. Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J Virol. 2000;74(13):6087–6095. -PMC -PubMed
    1. Sewell AK, Price DA. Dendritic cells and transmission of HIV-1. Trends Immunol. 2001;22(4):173–175. -PubMed
    1. Knight SC. Dendritic cells and HIV infection: immunity with viral transmission versus compromised cellular immunity? Immunobiology. 2001;204(5):614–621. -PubMed
    1. Turville SG, Cameron PU, Arthos J. Bitter-sweet symphony: defining the role of dendritic cell gp120 receptors in HIV infection. J Clin Virol. 2001;22(3):229–239. -PubMed
    1. Harman AN, Wilkinson J, Bye CR, et al. HIV induces maturation of monocyte-derived dendritic cells and Langerhans cells. J Immunol. 2006;177(10):7103–7113. -PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources