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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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A3G and A3F are incorporated in VLPs when Gag is fused to different RNA-binding domains.Gag ΔNC was fused to RNA-binding domains (RBD) of hnRNP C1, hnRNP K, SRSF2 and Staufen-1. These constructs were co-transfected into 293T cells with vectors expressing either Wt Gag or Gag ΔNC at a ratio of 5:1 and also with HA-tagged A3G or A3F. VLPs were harvested and concentrated, and proteins were visualised by immunoblot. A representative blot of 3 independent experiments is shown.
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ppat.1004609.g006: A3G and A3F are incorporated in VLPs when Gag is fused to different RNA-binding domains.Gag ΔNC was fused to RNA-binding domains (RBD) of hnRNP C1, hnRNP K, SRSF2 and Staufen-1. These constructs were co-transfected into 293T cells with vectors expressing either Wt Gag or Gag ΔNC at a ratio of 5:1 and also with HA-tagged A3G or A3F. VLPs were harvested and concentrated, and proteins were visualised by immunoblot. A representative blot of 3 independent experiments is shown.

Mentions: These expression constructs were co-transfected with A3G/F into 293T cells together with vectors for wild type Gag or Gag ΔNC, and VLPs analysed by immunoblot (Fig. 6). Remarkably, all four RBDs readily rescued packaging of A3G and A3F in mixed virions with Gag ΔNC with (generally) similar efficiency as the wild type Gag or NC itself when reconnected to Gag ΔNC (compare lanes 6, 8, 10 and 12 with 1, 2 and 4; and 18, 20, 22 and 24 with 13, 14 and 16). Given that A3G and A3F exhibit very broad RNA binding characteristics (Fig. 1), we conclude that a multitude of such RNA substrates, if packaged, can serve to draw A3G/F into VLPs.


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)

A3G and A3F are incorporated in VLPs when Gag is fused to different RNA-binding domains.Gag ΔNC was fused to RNA-binding domains (RBD) of hnRNP C1, hnRNP K, SRSF2 and Staufen-1. These constructs were co-transfected into 293T cells with vectors expressing either Wt Gag or Gag ΔNC at a ratio of 5:1 and also with HA-tagged A3G or A3F. VLPs were harvested and concentrated, and proteins were visualised by immunoblot. A representative blot of 3 independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295846&req=5

ppat.1004609.g006: A3G and A3F are incorporated in VLPs when Gag is fused to different RNA-binding domains.Gag ΔNC was fused to RNA-binding domains (RBD) of hnRNP C1, hnRNP K, SRSF2 and Staufen-1. These constructs were co-transfected into 293T cells with vectors expressing either Wt Gag or Gag ΔNC at a ratio of 5:1 and also with HA-tagged A3G or A3F. VLPs were harvested and concentrated, and proteins were visualised by immunoblot. A representative blot of 3 independent experiments is shown.
Mentions: These expression constructs were co-transfected with A3G/F into 293T cells together with vectors for wild type Gag or Gag ΔNC, and VLPs analysed by immunoblot (Fig. 6). Remarkably, all four RBDs readily rescued packaging of A3G and A3F in mixed virions with Gag ΔNC with (generally) similar efficiency as the wild type Gag or NC itself when reconnected to Gag ΔNC (compare lanes 6, 8, 10 and 12 with 1, 2 and 4; and 18, 20, 22 and 24 with 13, 14 and 16). Given that A3G and A3F exhibit very broad RNA binding characteristics (Fig. 1), we conclude that a multitude of such RNA substrates, if packaged, can serve to draw A3G/F into VLPs.

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