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Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.

Apolonia L, Schulz R, Curk T, Rocha P, Swanson CM, Schaller T, Ule J, Malim MH - PLoS Pathog. (2015)

Bottom Line: Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging.Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation.We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom.

ABSTRACT
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

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HIV-1 particles lacking genomic RNA package A3G and A3F. (A)293T cells stably expressing HA-tagged A3G or A3F were co-transfected with the packaging plasmid p8.91, and either with a vector coding for a package-competent lentiviral RNA (lt vector) or a mock plasmid. Vectors were harvested 48 h later and concentrated through a sucrose cushion. Gag proteins and A3G or A3F were visualised by immunoblot using anti-p24Gag and anti-HA antibodies. A representative immunoblot of 3 independent replicates is shown. (B) VLPs were produced by expression of the Gag protein of interest in 293T cells expressing HA-tagged A3G or A3F. Particles were concentrated and proteins detected as in panel A. Protein bands were quantified by densiometry using the Li-cor Odyssey infrared imaging and quantification software. Values obtained for A3G/A3F were divided by their respective p24Gag values. A representative immunoblot is displayed, together with a graph showing the average and standard deviation obtained for at least 3 experiments. (C) RNAs were harvested from VLPs, and genomic RNA was quantified by qPCR. The value obtained for Wt Gag-Pol VLPs was set to 1 and the other values compared to that. Values and error bars represent the average of 3 experiments and the standard deviation, respectively.
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ppat.1004609.g002: HIV-1 particles lacking genomic RNA package A3G and A3F. (A)293T cells stably expressing HA-tagged A3G or A3F were co-transfected with the packaging plasmid p8.91, and either with a vector coding for a package-competent lentiviral RNA (lt vector) or a mock plasmid. Vectors were harvested 48 h later and concentrated through a sucrose cushion. Gag proteins and A3G or A3F were visualised by immunoblot using anti-p24Gag and anti-HA antibodies. A representative immunoblot of 3 independent replicates is shown. (B) VLPs were produced by expression of the Gag protein of interest in 293T cells expressing HA-tagged A3G or A3F. Particles were concentrated and proteins detected as in panel A. Protein bands were quantified by densiometry using the Li-cor Odyssey infrared imaging and quantification software. Values obtained for A3G/A3F were divided by their respective p24Gag values. A representative immunoblot is displayed, together with a graph showing the average and standard deviation obtained for at least 3 experiments. (C) RNAs were harvested from VLPs, and genomic RNA was quantified by qPCR. The value obtained for Wt Gag-Pol VLPs was set to 1 and the other values compared to that. Values and error bars represent the average of 3 experiments and the standard deviation, respectively.

Mentions: One obvious RNA species that could potentially mediate the packaging of A3G into particles is the viral gRNA. Indeed, at least one previous report has considered this RNA to be essential for A3G packaging [34]. Since A3G and A3F are clearly able to bind to this RNA in infected cells, we performed packaging assays to test this hypothesis. First, we used lentiviral vectors with or without gRNA (Fig. 2A). Succinctly, 293T cells stably expressing HA-tagged A3G or A3F were co-transfected with the HIV-1-based packaging plasmid (p8.91) [43], the VSV glycoprotein envelope expression vector, and either the pHR’SIN-cPPT-SEW lentiviral vector plasmid (denoted lt vector) [44] that expresses gRNA with an intact packaging signal (Ψ) or a mock plasmid. Immunoblot analysis of particles harvested 48 h post transfection and isolated through a sucrose cushion shows that A3G and A3F were packaged into both viral vectors with almost identical efficiency, indicating that viral gRNA is not required for effective A3G or A3F packaging.


Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.

Apolonia L, Schulz R, Curk T, Rocha P, Swanson CM, Schaller T, Ule J, Malim MH - PLoS Pathog. (2015)

HIV-1 particles lacking genomic RNA package A3G and A3F. (A)293T cells stably expressing HA-tagged A3G or A3F were co-transfected with the packaging plasmid p8.91, and either with a vector coding for a package-competent lentiviral RNA (lt vector) or a mock plasmid. Vectors were harvested 48 h later and concentrated through a sucrose cushion. Gag proteins and A3G or A3F were visualised by immunoblot using anti-p24Gag and anti-HA antibodies. A representative immunoblot of 3 independent replicates is shown. (B) VLPs were produced by expression of the Gag protein of interest in 293T cells expressing HA-tagged A3G or A3F. Particles were concentrated and proteins detected as in panel A. Protein bands were quantified by densiometry using the Li-cor Odyssey infrared imaging and quantification software. Values obtained for A3G/A3F were divided by their respective p24Gag values. A representative immunoblot is displayed, together with a graph showing the average and standard deviation obtained for at least 3 experiments. (C) RNAs were harvested from VLPs, and genomic RNA was quantified by qPCR. The value obtained for Wt Gag-Pol VLPs was set to 1 and the other values compared to that. Values and error bars represent the average of 3 experiments and the standard deviation, respectively.
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ppat.1004609.g002: HIV-1 particles lacking genomic RNA package A3G and A3F. (A)293T cells stably expressing HA-tagged A3G or A3F were co-transfected with the packaging plasmid p8.91, and either with a vector coding for a package-competent lentiviral RNA (lt vector) or a mock plasmid. Vectors were harvested 48 h later and concentrated through a sucrose cushion. Gag proteins and A3G or A3F were visualised by immunoblot using anti-p24Gag and anti-HA antibodies. A representative immunoblot of 3 independent replicates is shown. (B) VLPs were produced by expression of the Gag protein of interest in 293T cells expressing HA-tagged A3G or A3F. Particles were concentrated and proteins detected as in panel A. Protein bands were quantified by densiometry using the Li-cor Odyssey infrared imaging and quantification software. Values obtained for A3G/A3F were divided by their respective p24Gag values. A representative immunoblot is displayed, together with a graph showing the average and standard deviation obtained for at least 3 experiments. (C) RNAs were harvested from VLPs, and genomic RNA was quantified by qPCR. The value obtained for Wt Gag-Pol VLPs was set to 1 and the other values compared to that. Values and error bars represent the average of 3 experiments and the standard deviation, respectively.
Mentions: One obvious RNA species that could potentially mediate the packaging of A3G into particles is the viral gRNA. Indeed, at least one previous report has considered this RNA to be essential for A3G packaging [34]. Since A3G and A3F are clearly able to bind to this RNA in infected cells, we performed packaging assays to test this hypothesis. First, we used lentiviral vectors with or without gRNA (Fig. 2A). Succinctly, 293T cells stably expressing HA-tagged A3G or A3F were co-transfected with the HIV-1-based packaging plasmid (p8.91) [43], the VSV glycoprotein envelope expression vector, and either the pHR’SIN-cPPT-SEW lentiviral vector plasmid (denoted lt vector) [44] that expresses gRNA with an intact packaging signal (Ψ) or a mock plasmid. Immunoblot analysis of particles harvested 48 h post transfection and isolated through a sucrose cushion shows that A3G and A3F were packaged into both viral vectors with almost identical efficiency, indicating that viral gRNA is not required for effective A3G or A3F packaging.

Bottom Line: Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging.Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation.We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom.

ABSTRACT
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

Show MeSH
Related in: MedlinePlus