Unintegrated HIV-1 DNA recruits cGAS via its histone-binding domain to escape innate immunity

Abstract

To ensure optimal replication and spread, viruses have evolved countermeasures to evade type 1 IFN-mediated antiviral activity. During the early viral replication cycle steps until uncoating, the HIV-1 core protects viral pathogen associated molecular patterns (viral RNA and reverse transcription products) from recognition by innate immune sensors, including cGAS. However, after capsid uncoating, unintegrated viral DNA (uvDNA) becomes accessible. Here, we show that HIV-1 uses chromatin-mediated cGAS inactivation as a mechanism to protect its uvDNA from innate immune activation.

Keywords: HIV; cGAS; chromatin; innate immunity.

Fig. 1.

POLE3 knockdown results in cGAS sensing of unintegrated HIV-1 DNA. (A) Experimental scheme: primary CD4⁺ T cells isolated from four healthy donors were activated with PHA/IL2, electroporated with the indicated siRNA, and infected with VSV-G- HIV-Luc IN^D116A in duplicate (Upper panel). Representative immunoblot of POLE3 and cGAS expression (Left panel). IFIT1 mRNA expression (normalized to GAPDH mRNA expression) was measured by qPCR at 24 hpi (Right panel). (B) Experiment performed as described in (A), except that NT and POLE3 siRNA-transfected CD4+ T cells were treated with DMSO or the cGAS inhibitor (G140) 3 h before infection with VSV-G- HIV-Luc IN^D116A. IFIT1 mRNA expression was quantified as described in A. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Bonferroni’s multiple comparisons test (ANOVA test).

Fig. 2.

HIV-1 DNA-mediated cGAS activation in POLE3 KO cells reduces viral replication. HIV-1 replication kinetics in POLE3 KO HeLa-P4 (white dots) and NT control (black dots) cells treated with the cGAS inhibitor G140 (squares and dashed lines) or DMSO (circles and continuous lines) were measured by quantifying p24 antigen in the culture supernatant every 3 d post infection. Each kinetic plot represents 2 independent experiments with duplicates. *P < 0.05, independent Student’s t test

Fig. 3.

uHIV-1 DNA tethers cGAS via chromatin binding. (A) Western blot analysis of HeLa KO cells stably transduced with Flag-HA EV or cGAS-FHA with the indicated antibodies. (B and C) ChIP analysis of cGAS binding to uHIV-1 DNA. HeLa-EV and HeLa-cGAS-FHA were infected with VSV-G- HIV-Luc IN^D116A with or without NVP treatment. Chromatin was prepared 24 hpi. ChIP was performed with an α-HA antibody. qPCR analysis was performed with the indicated primers. Data are presented relative to cGAS-FHA with HIV-specific primers: Nuc1 (B) or luc (C). Mean ± SD values of four independent experiments. (D) Western blot analysis of HeLa KO cells stably transduced with EV, cGAS-FHA, or the cGAS mutant (cGAS-FHA R236E R255E) using the indicated antibodies. (E and F) ChIP analysis of cGAS binding to histones H2A-H2B on uHIV-1 DNA. HeLa-EV, HeLa-cGAS-FHA, and HeLa-cGAS-FHA R236E R255E cells were infected with VSV-G- HIV-Luc IN^D116A. ChIP was performed as described in B and C. Data are presented relative to cGAS-FHA with HIV-specific primers: Nuc1 (E) or luc (F). Mean ± SD values of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; dependent Student’s t test.