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HIV-1 evades innate immune recognition through specific cofactor recruitment.

Rasaiyaah J, Tan CP, Fletcher AJ, Price AJ, Blondeau C, Hilditch L, Jacques DA, Selwood DL, James LC, Noursadeghi M, Towers GJ - Nature (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University College London, Medical Research Council Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 90 Gower Street, London WC1E 6BT, UK.

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.

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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).
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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).

Mentions: Given that CA mutation N74D prevents recruitment of CPSF61,3 we hypothesized that CPSF6 depletion would induce WT HIV-1 to trigger IFN responses in MDM. In fact, CPSF6 depletion by shRNA expression in MDM (Fig. 2a,b, Extended Data Fig. 2) completely abrogated HIV-1 replication (Fig. 2c) due to a burst of IFN-β. MDM expressing a non-targeting shRNA did not produce IFN-β on HIV-1 infection (Fig. 2d). The restrictive role of IFN was confirmed by rescue of infectivity with IFNAR2 antibody (Fig. 2e). Neither the IFNAR2 nor isotype control antibody had any effect on HIV-1 replication in control shRNA expressing MDM (Fig. 2f). Importantly, shRNA expression itself did not induce IFN-β production (Fig. 2g). We conclude that the defect in WT HIV-1 replication after CPSF6 depletion in MDM was largely due to type-1 IFN production. In line with observations made with HIV-1 N74D, CPSF6 depletion also reduced single round infectivity in MDM by a few fold, 3.5-fold versus 5-fold (Fig. 2h).


HIV-1 evades innate immune recognition through specific cofactor recruitment.

Rasaiyaah J, Tan CP, Fletcher AJ, Price AJ, Blondeau C, Hilditch L, Jacques DA, Selwood DL, James LC, Noursadeghi M, Towers GJ - Nature (2013)

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).
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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).
Mentions: Given that CA mutation N74D prevents recruitment of CPSF61,3 we hypothesized that CPSF6 depletion would induce WT HIV-1 to trigger IFN responses in MDM. In fact, CPSF6 depletion by shRNA expression in MDM (Fig. 2a,b, Extended Data Fig. 2) completely abrogated HIV-1 replication (Fig. 2c) due to a burst of IFN-β. MDM expressing a non-targeting shRNA did not produce IFN-β on HIV-1 infection (Fig. 2d). The restrictive role of IFN was confirmed by rescue of infectivity with IFNAR2 antibody (Fig. 2e). Neither the IFNAR2 nor isotype control antibody had any effect on HIV-1 replication in control shRNA expressing MDM (Fig. 2f). Importantly, shRNA expression itself did not induce IFN-β production (Fig. 2g). We conclude that the defect in WT HIV-1 replication after CPSF6 depletion in MDM was largely due to type-1 IFN production. In line with observations made with HIV-1 N74D, CPSF6 depletion also reduced single round infectivity in MDM by a few fold, 3.5-fold versus 5-fold (Fig. 2h).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University College London, Medical Research Council Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 90 Gower Street, London WC1E 6BT, UK.

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.

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Related in: MedlinePlus