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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 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).
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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).

Mentions: HIV-1 capsid (CA) mutant N74D cannot recruit CPSF6 and is insensitive to depletion of HIV-1 cofactors Nup358 and TNPO3 suggesting it may utilize alternate cofactors for nuclear entry1-3. Furthermore, unlike wild type (WT) HIV-1, HIV-1 N74D cannot replicate in monocyte-derived macrophages (MDM) (Fig. 1a, Extended Data Fig. 2)2,4. Remarkably, an inability to replicate was accompanied by a burst of IFN-β detected 2-5 days after low multiplicity infection (Fig. 1b, Extended Data Fig. 2). The antiviral activity of IFN-β (Extended Data Fig. 3a)5 was revealed by rescuing HIV-1 N74D, but not WT replication with antibody to the IFNα/β receptor α chain (IFNAR2) (Fig. 1c,d, Extended Data Fig. 3b). Co-infection of MDM with WT and HIV-1 N74D led to suppression of WT replication (Fig. 1e), which was also rescued by IFNAR2 antibody (Extended Data Fig. 3c). This demonstrated that sensitivity to IFN-mediated restriction was not limited to the mutant virus.


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 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928559&req=5

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).
Mentions: HIV-1 capsid (CA) mutant N74D cannot recruit CPSF6 and is insensitive to depletion of HIV-1 cofactors Nup358 and TNPO3 suggesting it may utilize alternate cofactors for nuclear entry1-3. Furthermore, unlike wild type (WT) HIV-1, HIV-1 N74D cannot replicate in monocyte-derived macrophages (MDM) (Fig. 1a, Extended Data Fig. 2)2,4. Remarkably, an inability to replicate was accompanied by a burst of IFN-β detected 2-5 days after low multiplicity infection (Fig. 1b, Extended Data Fig. 2). The antiviral activity of IFN-β (Extended Data Fig. 3a)5 was revealed by rescuing HIV-1 N74D, but not WT replication with antibody to the IFNα/β receptor α chain (IFNAR2) (Fig. 1c,d, Extended Data Fig. 3b). Co-infection of MDM with WT and HIV-1 N74D led to suppression of WT replication (Fig. 1e), which was also rescued by IFNAR2 antibody (Extended Data Fig. 3c). This demonstrated that sensitivity to IFN-mediated restriction was not limited to the mutant virus.

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