HIV-1 evades innate immune recognition through specific cofactor recruitment

Abstract

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

Figure 1. HIV-1 CPSF6 binding mutant CA N74D is restricted in MDM due to induction of Type-I IFN

(a) Replication of WT HIV-1 or CA mutant N74D in MDM. (b) IFN-β levels in supernatants from (a). (c-d) Replication of HIV-1 CA N74D or WT HIV-1 with IFNAR2 or control antibody (cAb). (e) Replication of WT or WT plus CA N74D. Mean data and regression lines for biological replicates are shown in c-e. P values (2-way ANOVA) are given for (c-d) IFNAR2 blockade and (e) co-infection with CA mutant N74D. (f) Infection of MDM by HIV-1 measured at 48h (g) GAPDH normalized IP10 RNA levels expressed as fold change over untreated cells after infection with WT or HIV-1 mutants (Mean of 3 technical replicates ±SEM, f-g).

Figure 2. HIV-1 elicits a Type-1 IFN response that restricts replication in CPSF6 depleted MDM

(a) Protocol schema. (b) CPSF6/actin detected at time of infection (c). HIV-1 replication in MDM expressing shRNA targeting CPSF6 or control shRNA (d) IFN-β levels in supernatants from (c). (e-f) Infection of CPSF6 depleted or MDM expressing control shRNA with IFNAR2 or control antibody (cAb). P values (2-way ANOVA) are given for the effect of (e) CPSF6 depletion or (f) control shRNA on biological replicates. (g) IFN-β produced from shRNA expressing MDM or IFN-β treated MDM. (h) Infection of MDM by HIV-1 measured at 48h on CPSF6 depleted or control shRNA expressing MDM (Mean of 3 technical replicates ±SEM).

Figure 3. HIV-1 CypA binding mutant CA P90A is restricted in MDM due to induction of Type-I IFN

(a) Replication of WT HIV-1 or CA mutant P90A in MDM. (b) IFN-β levels in supernatants from (a). (c) Replication of HIV-1 CA P90A with IFNAR2 or control antibody (cAb). (d) as in Figure 1d. (e) Replication of WT or WT plus CA P90A. Mean data and regression lines are shown for biological replicates in c-e. P values (2-way ANOVA) are given for (c-d) IFNAR2 blockade and (e) co-infection with CA mutant P90A. (f) Infection of MDM by HIV-1 measured at 48h (g) GAPDH normalized IP10 RNA levels expressed as fold change over untreated cells after infection with WT or HIV-1 mutants (Mean of 3 technical replicates ±SEM, f-g).

Figure 4. NFκB/IRF3 are activated by mutant HIV-1 and SmBz-CsA treatment causes WT HIV-1 to trigger innate responses

(a, b) Mean (±SEM) nuclear:cytoplasmic ratios for NFκB or IRF3 in infected MDM (p<0.05, 2-way ANOVA). (c) Infection at 48h ±IKK inhibitor. (d, e) SmBz-CsA (green) complexed with CypA (gray), Cs (yellow) and calcineurin (orange/blue). (f) Replication of HIV-1 in MDM ±SmBz-CsA (g) IFN-β levels from (f). (h) MDM infected with WT HIV-1 plus SmBz-CsA and IFNAR2 antibody or cAb (mean data and regression lines). P value (2-way ANOVA) is given for IFNAR2 blockade of biological replicates. (i) Infection of MDM by WT HIV-1 ±SmBz-CsA at 48h (Mean of 3 technical replicates ±SEM).