Limits...
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.

Show MeSH

Related in: MedlinePlus

A3G and A3F concentrations are similarly distributed between cells and VLPs by RNA. (A)293T cells were co-transfected at a 1:5 ratio with expression vectors for T7-tagged A3G or A3F, and Wt Gag. Cell lysates (CL) were kept for analysis. Supernatant from cells transfected with an empty plasmid (negative control) and VLPs were harvested and isolated by ultracentrifugation through a continuous sucrose gradient (20–60%). Fractions were harvested and p24Gag-containing fractions were identified by immunoblot. T7-tagged A3G was purified and quantified as specified in the material and methods. Cell lysates, fractions containing VLPs (or the corresponding fraction from the negative control) and purified A3G were analysed by immunoblot. Gag and HSP90 were visualised using anti-p24Gag and anti-HSP90 antibodies, respectively. A3G and A3F were visualised using an anti-T7 antibody. APOBEC3 proteins were quantified by densiometry. This figure shows a representative example. (B) Standard curve of purified T7-A3G visualised and quantified by densiometry of the immunoblot of Fig. 7A. (C) RNA was extracted from cell lysates (CL) and VLP samples and quantified. Total A3G/F protein in CL and VLPs was quantified using a standard curve. The RNA in the negative control was below the detection threshold of the method. (D) The total amount of A3G or A3F was divided by the total concentration of extracted RNA. The graph shows the average of 4 independent experiments. Error bars indicate standard deviation. There was no statistically significant difference between the ratios in lysates and VLPs (p>0.5).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4295846&req=5

ppat.1004609.g007: A3G and A3F concentrations are similarly distributed between cells and VLPs by RNA. (A)293T cells were co-transfected at a 1:5 ratio with expression vectors for T7-tagged A3G or A3F, and Wt Gag. Cell lysates (CL) were kept for analysis. Supernatant from cells transfected with an empty plasmid (negative control) and VLPs were harvested and isolated by ultracentrifugation through a continuous sucrose gradient (20–60%). Fractions were harvested and p24Gag-containing fractions were identified by immunoblot. T7-tagged A3G was purified and quantified as specified in the material and methods. Cell lysates, fractions containing VLPs (or the corresponding fraction from the negative control) and purified A3G were analysed by immunoblot. Gag and HSP90 were visualised using anti-p24Gag and anti-HSP90 antibodies, respectively. A3G and A3F were visualised using an anti-T7 antibody. APOBEC3 proteins were quantified by densiometry. This figure shows a representative example. (B) Standard curve of purified T7-A3G visualised and quantified by densiometry of the immunoblot of Fig. 7A. (C) RNA was extracted from cell lysates (CL) and VLP samples and quantified. Total A3G/F protein in CL and VLPs was quantified using a standard curve. The RNA in the negative control was below the detection threshold of the method. (D) The total amount of A3G or A3F was divided by the total concentration of extracted RNA. The graph shows the average of 4 independent experiments. Error bars indicate standard deviation. There was no statistically significant difference between the ratios in lysates and VLPs (p>0.5).

Mentions: Fig. 6 shows that A3G and A3F can be incorporated into VLPs when diverse RBDs are fused to Gag. This is consistent with our iCLIP data, demonstrating that A3G and A3F are able to bind to multiple diverse RNAs. One obvious question that is raised by these observations is whether A3G/F are preferentially encapsidated by assembling HIV-1 VLPs, or whether they are inevitably packaged as a natural consequence of their promiscuous RNA binding capabilities. To address this directly, we carefully quantified the ratios of A3G and A3F to RNA in the lysates of virus producing cells and the matching particle preparations (Fig. 7). Accordingly, 293T cells were transiently transfected with vectors expressing T7-tagged versions of A3G or A3F, as well as the wild type Gag expression vector. Cells were also transfected with an irrelevant plasmid to serve as a negative control. VLPs and cell lysates were collected at 48 h post transfection. VLPs were isolated through a continuous sucrose gradient and the fractions containing VLPs were identified by immunoblot (S6 Fig.). Protein quantities were then determined against a standard curve of recombinant T7-A3G (Fig. 7A and 7B), and RNA in cell lysates and VLPs were extracted and quantified by Qubit (Fig. 7C). Culture supernatant from cells transfected with an irrelevant plasmid was used to assess background, and RNA was not detected in these samples (threshold of detection, 20 pg/ml). Interestingly, the calculated ratios of A3G/F to RNA were similar in cells and in virus particles (Fig. 7D, mean of 4 independent experiments). In other words, there is no evident enrichment of A3G or A3F in virions relative to virus-producing cells. Taking all our findings together leads us to conclude that the packaging of A3G and A3F into HIV-1 particles is driven by RNA binding, and that multiple/diverse RNAs can fulfil this role provided they are themselves packaged.


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 concentrations are similarly distributed between cells and VLPs by RNA. (A)293T cells were co-transfected at a 1:5 ratio with expression vectors for T7-tagged A3G or A3F, and Wt Gag. Cell lysates (CL) were kept for analysis. Supernatant from cells transfected with an empty plasmid (negative control) and VLPs were harvested and isolated by ultracentrifugation through a continuous sucrose gradient (20–60%). Fractions were harvested and p24Gag-containing fractions were identified by immunoblot. T7-tagged A3G was purified and quantified as specified in the material and methods. Cell lysates, fractions containing VLPs (or the corresponding fraction from the negative control) and purified A3G were analysed by immunoblot. Gag and HSP90 were visualised using anti-p24Gag and anti-HSP90 antibodies, respectively. A3G and A3F were visualised using an anti-T7 antibody. APOBEC3 proteins were quantified by densiometry. This figure shows a representative example. (B) Standard curve of purified T7-A3G visualised and quantified by densiometry of the immunoblot of Fig. 7A. (C) RNA was extracted from cell lysates (CL) and VLP samples and quantified. Total A3G/F protein in CL and VLPs was quantified using a standard curve. The RNA in the negative control was below the detection threshold of the method. (D) The total amount of A3G or A3F was divided by the total concentration of extracted RNA. The graph shows the average of 4 independent experiments. Error bars indicate standard deviation. There was no statistically significant difference between the ratios in lysates and VLPs (p>0.5).
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004609.g007: A3G and A3F concentrations are similarly distributed between cells and VLPs by RNA. (A)293T cells were co-transfected at a 1:5 ratio with expression vectors for T7-tagged A3G or A3F, and Wt Gag. Cell lysates (CL) were kept for analysis. Supernatant from cells transfected with an empty plasmid (negative control) and VLPs were harvested and isolated by ultracentrifugation through a continuous sucrose gradient (20–60%). Fractions were harvested and p24Gag-containing fractions were identified by immunoblot. T7-tagged A3G was purified and quantified as specified in the material and methods. Cell lysates, fractions containing VLPs (or the corresponding fraction from the negative control) and purified A3G were analysed by immunoblot. Gag and HSP90 were visualised using anti-p24Gag and anti-HSP90 antibodies, respectively. A3G and A3F were visualised using an anti-T7 antibody. APOBEC3 proteins were quantified by densiometry. This figure shows a representative example. (B) Standard curve of purified T7-A3G visualised and quantified by densiometry of the immunoblot of Fig. 7A. (C) RNA was extracted from cell lysates (CL) and VLP samples and quantified. Total A3G/F protein in CL and VLPs was quantified using a standard curve. The RNA in the negative control was below the detection threshold of the method. (D) The total amount of A3G or A3F was divided by the total concentration of extracted RNA. The graph shows the average of 4 independent experiments. Error bars indicate standard deviation. There was no statistically significant difference between the ratios in lysates and VLPs (p>0.5).
Mentions: Fig. 6 shows that A3G and A3F can be incorporated into VLPs when diverse RBDs are fused to Gag. This is consistent with our iCLIP data, demonstrating that A3G and A3F are able to bind to multiple diverse RNAs. One obvious question that is raised by these observations is whether A3G/F are preferentially encapsidated by assembling HIV-1 VLPs, or whether they are inevitably packaged as a natural consequence of their promiscuous RNA binding capabilities. To address this directly, we carefully quantified the ratios of A3G and A3F to RNA in the lysates of virus producing cells and the matching particle preparations (Fig. 7). Accordingly, 293T cells were transiently transfected with vectors expressing T7-tagged versions of A3G or A3F, as well as the wild type Gag expression vector. Cells were also transfected with an irrelevant plasmid to serve as a negative control. VLPs and cell lysates were collected at 48 h post transfection. VLPs were isolated through a continuous sucrose gradient and the fractions containing VLPs were identified by immunoblot (S6 Fig.). Protein quantities were then determined against a standard curve of recombinant T7-A3G (Fig. 7A and 7B), and RNA in cell lysates and VLPs were extracted and quantified by Qubit (Fig. 7C). Culture supernatant from cells transfected with an irrelevant plasmid was used to assess background, and RNA was not detected in these samples (threshold of detection, 20 pg/ml). Interestingly, the calculated ratios of A3G/F to RNA were similar in cells and in virus particles (Fig. 7D, mean of 4 independent experiments). In other words, there is no evident enrichment of A3G or A3F in virions relative to virus-producing cells. Taking all our findings together leads us to conclude that the packaging of A3G and A3F into HIV-1 particles is driven by RNA binding, and that multiple/diverse RNAs can fulfil this role provided they are themselves packaged.

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