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A3G interaction with HIV-1 RT in virions Suspensions of HIV-1 virions with packaged A3G_GFP, GFP_Vpr, GFP_CYPA or A3G_GFP and A3G_mCherry were immobilized on coverslips, fixed and stained with Cy3 labeled anti-RT or anti-CA Fab fragments. a) and b) Representative images show clusters of HIV-1 virions immobilized on fibronectin streaks with green fluorescence (left panel), red fluorescence (Cy3 or mCherry as indicated, right panel) and GFP lifetime as pseudo-colored images according to the indicated scale (as in Figure 3). White scale bars represent 10 μm. a) A3G_GFP demonstrates normal lifetime when packaged into HIV-1 virions. b) FRET is detected for the positive control of A3G_GFP and A3G_mCherry (upper left panel) and between A3G_GFP and Cy3 stained RT (lower right panel), but not between Vpr and RT, CYPA and RT, or A3G and CA (upper right panels). The absence of a signal for red fluorescence with HIV-1ΔRT virions confirmed the specificity of the anti-RT Fab fragments (lower left panel). c) Quantification of FRET efficiencies for n=5 areas. Individual measurements with their mean and standard deviation are shown.
Source publication
Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host-cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular antiviral effectors, called restriction or resistance factors. The host antivi...
Contexts in source publication
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... to Web version on PubMed Central for supplementary material. from one of six independent virus preparations. 'Low' or 'High' A3G refers to producer cell transfection ratios of 1:10 or 1:4, respectively (A3G expression plasmid to NL4.3/ΔVif). e) Single-cycle virion infectivity measured by β-galactosidase activity in challenged TZM-bl reporter cells. f) Quantitative PCR measuring cDNA abundance in CEM-SS cells at 4 h post- infection. For e) and f) the individual data points with their mean and standard deviation of eight independent infections from six virus preparations are shown. *** indicates p-value of <0.0001 in an unpaired, two-tailed t-test with Welch's correction performed in GraphPad Prism ® . g) Numbers of unique sequencing reads ending at each nt of the HIV-1 NL4.3 (-)sss cDNA were divided by the total read number (Supplementary Figure 4b) within each sample to show the relative abundance of cDNAs for each length between nt positions 23 and 182. Shown in dashed red lines are the percentages of reads carrying C to T/U mutations at that position (scale on the right y-axis). See Method section for analysis details. One representative experiment out of three independent repeats is shown. c) The abundance of (-)sss containing cDNA in CEM-SS cells at 4 h post-infection was measured by quantitative PCR. For b) and c) each viral preparation was used to infect TZM-bl or CEM-SS target cells with or without hUGI, black dots and grey squares respectively. The individual data points with their mean and standard deviation for three independent viral preparations and infections are shown. d) Sequencing reads from a MiSeq™ library run were analyzed and presented as in Figure 1g. The labeling to the right indicates whether the HEK293T producer cells (Prod) and/or the CEM-SS target (Target) cells expressed hUGI. No A3G indicates the absence of A3G in producer cells and high A3G refers to relative A3G content in the producer cells. Sequencing data are derived from one representative experiment out of two independent repeats. Co-immunoprecipiation analysis of A3G_HA binding to FLAG tagged HIV-1 RT. Transfected HEK293T cell lysates were subjected to anti-FLAG immunoprecipiation, recovered proteins were detected with anti-HA (for A3G), anti-RT or anti-FLAG antibodies. CD8_FLAG served as an irrelevant protein control. One representative experiment of three repeats is shown. *HC: immunoglobulin heavy chain b) RNase resistance of the A3G-RT complex. Shown are anti-FLAG immunoprecipitations after the bead bound proteins had been subjected to RNase A or RNase Mix treatment, at the indicated concentrations, followed by washing and immunoblotting. One representative experiment of three repeats is shown. Samples without RT_FLAG carry CD8_FLAG as an irrelevant tagged protein control. c) Surface plasmon resonance analysis of purified A3G and p51 on a Biacore T-200 instrument. Association and dissociation curves of p51_FLAG to immobilized A3G_6xHis at the indicated concentrations are shown. The sensorgram indicates specific binding between the two components, and the responses gave good fits to a single interaction binding model with a K d of ~1.6 μM. d)-f) Measurements of FRET efficiency using FLIM in HeLa cells expressing GFP and mCherry fusion proteins. Representative images with GFP fluorescence from multiphoton laser scanning microscopy (left panel) and corresponding wide field CCD camera images of mCherry fluorescence (right panels (e only)) are shown. The center panels represent pseudo-colored images of GFP lifetime (τ) (blue/green, normal/ longer GFP lifetime; yellow/red, shorter GFP lifetime indicating FRET). d)Control images demonstrating normal GFP lifetime in the absence of mCherry acceptor. White scale bars represent 10 μm. e) Co-expression of indicated GFP and mCherry fusion proteins and the fluorescence lifetime according to the scale in d) indicating the presence or absence of FRET. f) Dot plot of FRET efficiencies with their mean and standard deviation from n=7 cells each. expression plasmid to proviral plasmid during virus production. b) A3G-L35A, but not A3G-R24A, displays diminished HIV-1 inhibitory activity. A3G packaging was quantified by immunoblot density measurements and the different wild type A3G packaging levels were plotted over measured infectivity. The extent of infection inhibition exerted by the wild type protein at the empirically determined level of packaged mutant protein was then extrapolated (see Supplementary Fig 10). Inhibition levels, in % relative to the no A3G control, of wild type A3G (triangles) and L35A or R24A (circles) in eight (L35A) or seven (R24A) independent experiments are shown. A paired, two tailed student t test was performed in GraphPad Prism ® and * indicates p<0.05 (p=0.0223), ns: not significant c) As in b), but with (-)sss cDNA abundance measured by qPCR in cells 4 h post-infection for virions carrying wild type or mutant A3G. ** indicates p<0.005 (p=0.0028). d) Relative infectivity of n=5 independent virus preparations carrying the indicated wild type or mutant A3G at equal, 'high' levels as shown in the representative immunoblot in Supplementary Fig 10g. A paired, two tailed student t test was performed in GraphPad Prism ® . * indicates p<0.05 (p=0.0397 for A3G wt -L35A; p=0.0297 for C288S -C288S/L35A and p=0.0137 for L35A -C288S/L35A). e) Sequencing reads from a MiSeq™ library run were analyzed and presented as in Fig 1g. Labels to the right indicate the presence or absence of A3G proteins in ...
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... DNA extracts were then used to prepare libraries for sequencing ( Figure 1c). We first analyzed cDNA 3'-termini: unique reads for each (-)sss cDNA 3'-end were counted and divided by total read number, which ranged between 26500 (high A3G) and 81000 (no A3G) ( Supplementary Figure 4b), to obtain relative distributions of cDNA lengths. The top panel in Figure 1g depicts the profile for HIV-1ΔVif without A3G, with the most abundant species being the 180 nt (-)sss cDNA (>17% in this experiment). The rest of the profile was notably flat and evenly distributed, with some uplift in the abundance of shorter cDNAs. This pattern differs substantially from what is seen in reconstituted primer extension assays where DNA synthesis pauses at specific sites (Supplementary Figure ...
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... presence of A3G resulted in the appearance of five prominent peaks, each representing a specific 3'-terminus (Figure 1g, middle and bottom panels). Importantly, these sites do not match A3G-induced pause sites seen in reconstituted reactions containing purified A3G (Supplementary Figure 3a, lanes 6 to 9)22. Instead, these sites lie one nt 5' to cytidine-to- uridine mutations identified in longer DNA reads (dashed red lines), and occurring at consensus A3G editing sites5,16,38,39. These peaks also featured in first strand transfer sequences, but were absent when catalytically inactive A3G mutant proteins were tested ( Supplementary Figures 4a and 5)28. These observations suggested that A3G editing hotspots serve as cDNA cleavage ...
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... abundance of cDNA along the (-)sss sequence in the main figures was calculated by dividing the number of total reads for each nt position by the number of total reads up to nt 182 (for total reads see Supplementary Fig 4b). The sole exception is Supplementary Figure 4a, which shows profiles beyond first strand transfer, where the read number was divided by the total read count in the entire sample. All figures displaying cDNA profiles (Fig 1g, 2d, 6e, and Supplementary Fig 2a, 4a, 5 and 7) show the relative abundance of HIV-1 cDNA molecules for each length between nt positions 23 and 182 of the HIV-1 NL4.3 (-)sss product (in blue histogram bars, scale on the left y-axis). All positions with cytosine bases in the HIV-1 NL4.3 (-)sss sequence were analyzed for the presence of cytosine versus thymine/uracil bases as described above; shown in dashed red lines is the percentage of reads, which carried C to T/U mutations at the indicated position (scale on the right y-axis). Labels to the right of the graphs describe the virions used for ...
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... abundance of cDNA along the (-)sss sequence in the main figures was calculated by dividing the number of total reads for each nt position by the number of total reads up to nt 182 (for total reads see Supplementary Fig 4b). The sole exception is Supplementary Figure 4a, which shows profiles beyond first strand transfer, where the read number was divided by the total read count in the entire sample. All figures displaying cDNA profiles (Fig 1g, 2d, 6e, and Supplementary Fig 2a, 4a, 5 and 7) show the relative abundance of HIV-1 cDNA molecules for each length between nt positions 23 and 182 of the HIV-1 NL4.3 (-)sss product (in blue histogram bars, scale on the left y-axis). All positions with cytosine bases in the HIV-1 NL4.3 (-)sss sequence were analyzed for the presence of cytosine versus thymine/uracil bases as described above; shown in dashed red lines is the percentage of reads, which carried C to T/U mutations at the indicated position (scale on the right y-axis). Labels to the right of the graphs describe the virions used for ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. Rather than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse ...
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... DNA extracts were then used to prepare libraries for sequencing ( Figure 1c). We first analyzed cDNA 3'-termini: unique reads for each (-)sss cDNA 3'-end were counted and divided by total read number, which ranged between 26500 (high A3G) and 81000 (no A3G) ( Supplementary Figure 4b), to obtain relative distributions of cDNA lengths. The top panel in Figure 1g depicts the profile for HIV-1ΔVif without A3G, with the most abundant species being the 180 nt (-)sss cDNA (>17% in this experiment). ...
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... these sites lie one nt 5' to cytidine-to- uridine mutations identified in longer DNA reads (dashed red lines), and occurring at consensus A3G editing sites5,16,38,39. These peaks also featured in first strand transfer sequences, but were absent when catalytically inactive A3G mutant proteins were tested ( Supplementary Figures 4a and 5)28. These observations suggested that A3G editing hotspots serve as cDNA cleavage sites. ...
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... also adapted our FRET-FLIM system for cell-free, bulk HIV-1 particles (Figure 4). A3G_GFP was packaged into virions by co-expression with HIV-1ΔVif, and these were purified and immobilized on coverslips, before immuno-staining and FRET analysis. ...
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... than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). ...
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... than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). ...
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... than using protein fusions that could interfere with viral assembly or particle, RT was detected using a Cy3-labelled anti-RT Fab fragment. A3G_GFP exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). ...
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... exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse transcription. ...
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... exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse transcription. ...
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... exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse transcription. ...
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... exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse transcription. ...
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... exhibits a normal GFP lifetime in the absence of a fluorescent acceptor (Figure 4a) and a positive control of co- packaged A3G_GFP and A3G_mCherry demonstrates the suitability of this assay for detecting protein-protein interactions within virions (Figure 4b, left panel, top row; and Figure 4c). A substantial increase in FRET efficiency was evident when RT was detected with labeled Fab (Figure 4b, right panel, bottom row; and Figure 4c), which was not seen with other packaged proteins as acceptors (Vpr_GFP or cyclophilin A_GFP), the antibody- mediated detection of Capsid protein (p24 Gag ) (Figure 4b, top right panels; and Figure 4c), or for virions engineered to lack RT (Figure 4b, left panel, bottom row). These data demonstrate that A3G and RT interact in viral particles, and support the view that A3G is positioned at the site of reverse transcription. ...
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... abundance of cDNA along the (-)sss sequence in the main figures was calculated by dividing the number of total reads for each nt position by the number of total reads up to nt 182 (for total reads see Supplementary Fig 4b). The sole exception is Supplementary Figure 4a, which shows profiles beyond first strand transfer, where the read number was divided by the total read count in the entire sample. ...
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... abundance of cDNA along the (-)sss sequence in the main figures was calculated by dividing the number of total reads for each nt position by the number of total reads up to nt 182 (for total reads see Supplementary Fig 4b). The sole exception is Supplementary Figure 4a, which shows profiles beyond first strand transfer, where the read number was divided by the total read count in the entire sample. All figures displaying cDNA profiles (Fig 1g, 2d, 6e, and Supplementary Fig 2a, 4a, 5 and 7) show the relative abundance of HIV-1 cDNA molecules for each length between nt positions 23 and 182 of the HIV-1 NL4.3 (-)sss product (in blue histogram bars, scale on the left y-axis). ...
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... indicates p-value of <0.0001 in an unpaired, two-tailed t-test with Welch's correction performed in GraphPad Prism ® . g) Numbers of unique sequencing reads ending at each nt of the HIV-1 NL4.3 (-)sss cDNA were divided by the total read number (Supplementary Figure 4b) within each sample to show the relative abundance of cDNAs for each length between nt positions 23 and 182. Shown in dashed red lines are the percentages of reads carrying C to T/U mutations at that position (scale on the right y-axis). ...
Citations
... Among them, A3D, A3F, A3G and A3H are particularly effective as retrovirus restriction factors. They restrict viral replication by introducing cytosine-to-uracil hypermutations in viral complementary DNA, resulting in aberrant viral intermediates and impaired reverse transcription, a process reliant on their deaminase activity (83). ...
... Previous studies found that A3G prefers recognizing ssDNA and RNA with stem-loop structures. It restricts HIV infection primarily by directly binding to viral RNA or reverse transcriptase, thereby interfering with HIV-1 DNA synthesis in a manner distinct from its deaminase activity (83). Additionally, A3G inhibits HIV-1 integration by deaminating the 3' LTR, which increases integration site diversity (86). ...
Viral infectious diseases, caused by numerous viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), enterovirus (EV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), and human papillomavirus (HPV), pose a continuous threat to global health. As obligate parasites, viruses rely on host cells to replicate, and host cells have developed numerous defense mechanisms to counteract viral infection. Host restriction factors (HRFs) are critical components of the early antiviral response. These cellular proteins inhibit viral replication and spread by impeding essential steps in the viral life cycle, such as viral entry, genome transcription and replication, protein translation, viral particle assembly, and release. This review summarizes the current understanding of how host restriction factors inhibit viral replication, with a primary focus on their diverse antiviral mechanisms against a range of viruses, including SARS-CoV-2, influenza A virus, enteroviruses, human immunodeficiency virus, hepatitis B virus, and human papillomavirus. In addition, we highlight the crucial role of these factors in shaping the host-virus interactions and discuss their potential as targets for antiviral drug development.
... restriction factors against retroelements, retroviruses, and DNA viruses (2)(3)(4). APOBEC3 enzymes deaminate cytosine to uracil in single-stranded DNA replication intermedi ates, inducing mutations in or degradation of DNA, as well as inhibiting retroelement and retroviral reverse transcriptase through a deamination-independent mechanism (2)(3)(4)(5)(6). ...
Several APOBEC3 enzymes restrict HIV-1 by deaminating cytosine to form uracil in single-stranded proviral (−)DNA. However, HIV-1 Vif counteracts their activity by inducing their proteasomal degradation. This counteraction by Vif is incomplete, as evidenced by footprints of APOBEC3-mediated mutations within integrated proviral genomes of people living with HIV-1. The relative contributions of multiple APOBEC3s in HIV-1 restriction are not fully understood. Here, we investigated the activity of co-expressed APOBEC3F and APOBEC3G against HIV-1 Subtype B and Subtype C transmitted/founder viruses. We determined that APOBEC3F interacts with APOBEC3G through its N-terminal domain. We provide evidence that this results in protection of APOBEC3F from Vif-mediated degradation because the APOBEC3F N-terminal domain contains residues required for recognition by Vif. We also found that HIV-1 Subtype C Vifs, but not Subtype B Vifs, were less active against APOBEC3G when APOBEC3F and APOBEC3G were co-expressed. Consequently, when APOBEC3F and APOBEC3G were expressed together in a single cycle of HIV-1 replication, only HIV-1 Subtype C viruses showed a decrease in relative infectivity compared to when APOBEC3G was expressed alone. Inspection of Vif amino acid sequences revealed that differences in amino acids adjacent to conserved sequences influenced the Vif-mediated APOBEC3 degradation ability. Altogether, the data provide a possible mechanism for how combined expression of APOBEC3F and APOBEC3G could contribute to mutagenesis of HIV-1 proviral genomes despite the presence of Vif and provide evidence for variability in the Vif-mediated APOBEC3 degradation ability of transmitted/founder viruses.
IMPORTANCE
APOBEC3 enzymes suppress HIV-1 infection by inducing cytosine deamination in proviral DNA but are hindered by HIV-1 Vif, which leads to APOBEC3 proteasomal degradation. Moving away from traditional studies that used lab-adapted HIV-1 Subtype B viruses and singular APOBEC3 enzymes, we examined how transmitted/founder isolates of HIV-1 replicated in the presence of APOBEC3F and APOBEC3G. We determined that APOBEC3F interacts with APOBEC3G through its N-terminal domain and that APOBEC3F, like APOBEC3G, has Vif-mediated degradation determinants in the N-terminal domain. This enabled APOBEC3F to be partially resistant to Vif-mediated degradation. We also demonstrated that Subtype C is more susceptible than Subtype B HIV-1 to combined APOBEC3F/APOBEC3G restriction and identified Vif variations influencing APOBEC3 degradation ability. Importantly, Vif amino acid variation outside of previously identified conserved regions mediated APOBEC3 degradation and HIV-1 Vif subtype-specific differences. Altogether, we identified factors that affect susceptibility to APOBEC3F/APOBEC3G restriction.
... Some evidence indicates that A3 proteins neutralization by Vif is not always absolute [82][83][84]. In some cases, Vif loses its ability to effectively counteract A3 proteins, leading to an increase in G-to-A mutations in the viral genome [23]. ...
Despite its effectiveness in controlling plasma viremia, antiretroviral therapy (ART) cannot target proviral DNA, which remains an obstacle to HIV-1 eradication. When treatment is interrupted, the reservoirs can act as a source of viral rebound, highlighting the value of proviral DNA as an additional source of information on an individual’s overall resistance burden. In cases where the viral load is too low for successful HIV-1 RNA genotyping, HIV-1 DNA can help identify resistance mutations in treated individuals. The absence of treatment history, the need to adjust ART despite undetectable viremia, or the presence of LLV further support the use of genotypic resistance tests (GRTs) on HIV-1 DNA. Conventionally, GRTs have been achieved through Sanger sequencing, but the advances in NGS are leading to an increase in its use, allowing the detection of minority variants present in less than 20% of the viral population. The clinical significance of these mutations remains under debate, with interpretations varying based on context. Additionally, proviral DNA is subject to APOBEC3-induced hypermutation, which can lead to defective, nonviable viral genomes, a factor that must be considered when performing GRTs on HIV-1 DNA.
... In the course of viral infections, ADARs can modulate cellular responses by acting directly through hypermutation of viral RNA or indirectly by editing host transcripts [5, 8,9]. While APOBECs utilize C-to-U hypermutation or non-enzymatic pathways that interfere with reverse transcription to target viral genomes, typically DNA intermediates [10][11][12][13]. Since the translational machinery recognizes inosine (I) as guanosine (G) and uracil (U) as thymine (T), RNA editing in the coding regions of the genome may result in amino acid substitutions that alter the function of the protein [2]. ...
RNA editing is increasingly recognized as a post-transcriptional modification that directly affects viral infection by regulating RNA stability and recoding proteins. the duck hepatitis A virus genotype 3 (DHAV-3) infection is seriously detrimental to the Asian duck industry. However, the landscape and roles of RNA editing in the susceptibility and resistance of Pekin ducks to DHAV-3 remain unclear. Here, we profiled dynamic RNA editing events in liver tissue and investigated their potential functions during DHAV-3 infection in Pekin ducks. We identified 11,067 informative RNA editing sites in liver tissue from DHAV-3-susceptible and -resistant ducklings at three time points during virus infection. Differential RNA editing sites (DRESs) between S and R ducks were dynamically changed during infection, which were enriched in genes associated with vesicle-mediated transport and immune-related pathways. Moreover, we predicted and experimentally verified that RNA editing events in 3′-UTR could result in loss or gain of miRNA–mRNA interactions, thereby changing the expression of target genes. We also found a few DRESs in coding sequences (CDSs) that altered the amino acid sequences of several proteins that were vital for viral infection. Taken together, these data suggest that dynamic RNA editing has significant potential to tune physiological processes in response to virus infection in Pekin ducks, thus contributing to host differential susceptibility to DHAV-3.
... First, the binding of A3F and A3G proteins to HIV-1 genomic RNA blocks the elongation of reverse transcription directly [14,[49][50][51]55]. Second, a direct interaction between the A3G protein and HIV-1 Reverse transcriptase causes the disruption of cDNA synthesis [20,60]. Third, an interesting notion is that the A3B protein promotes stress granule formation through a protein kinase R signaling pathway that mediates translational shutdown in cells infected with diverse RNA viruses, such as Sendai virus, Polio virus, and Sindbis virus [61]. ...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations since its emergence. Analyses of the SARS-CoV-2 genomes from infected patients exhibit a bias toward C-to-U mutations, which are suggested to be caused by the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) cytosine deaminase proteins. However, the role of A3 enzymes in SARS-CoV-2 replication remains unclear. To address this question, we investigated the effect of A3 family proteins on SARS-CoV-2 replication in the myeloid leukemia cell line THP-1 lacking A3A to A3G genes. The Wuhan, BA.1, and BA.5 variants had comparable viral replication in parent and A3A-to-A3G-null THP-1 cells stably expressing angiotensin-converting enzyme 2 (ACE2) protein. On the other hand, the replication and infectivity of these variants were abolished in A3A-to-A3G-null THP-1-ACE2 cells in a series of passage experiments over 20 days. In contrast to previous reports, we observed no evidence of A3-induced SARS-CoV-2 mutagenesis in the passage experiments. Furthermore, our analysis of a large number of publicly available SARS-CoV-2 genomes did not reveal conclusive evidence for A3-induced mutagenesis. Our studies suggest that A3 family proteins can positively contribute to SARS-CoV-2 replication; however, this effect is deaminase-independent.
... Notably, human immunodeficiency virus type 1 (HIV-1) is the best characterized substrate for A3 family proteins. In primary CD4 + T cells, at least four A3 enzymes (A3D, A3F, A3G, and only stable A3H) restrict HIV-1 replication by deaminating viral cDNA intermediates and physically blocking reverse transcription [13][14][15][16][17][18][19][20][21][22]. A3 enzymes recognize specific dinucleotide motifs for deamination, such as 5′-CC for A3G or 5′-TC for other A3 enzymes at target cytosine bases, which appear as 5′-AG or 5′-AA mutations in the genomic strand [15,17,23,24]. ...
... First, the binding of A3F and A3G proteins to HIV-1 genomic RNA blocks the elongation of reverse transcription directly [14,[49][50][51]55]. Second, a direct interaction between the A3G protein and HIV-1 Reverse transcriptase causes the disruption of cDNA synthesis [20,60]. Third, an interesting notion is that the A3B protein promotes stress granule formation through a protein kinase R signaling pathway that mediates translational shutdown in cells infected with diverse RNA viruses, such as Sendai virus, Polio virus, and Sindbis virus [61]. ...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations since its emergence. Analyses of the SARS-CoV-2 genomes from infected patients exhibit a bias toward C-to-U mutations, which are suggested to be caused by the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) cytosine deaminase proteins. However, the role of A3 enzymes in SARS-CoV-2 replication remains unclear. To address this question, we investigated the effect of A3 family proteins on SARS-CoV-2 replication in THP-1 cells lacking A3A to A3G genes. The Wuhan, BA.1, and BA.5 variants had comparable viral replication in parent and A3A-to-A3G-null THP-1-ACE2 cells. On the other hand, the replication and infectivity of these variants were abolished in A3A-to-A3G-null THP-1-ACE2 cells in a series of passage experiments over 20 days. In contrast to previous reports, we observed no evidence for A3-induced SARS-CoV-2 mutagenesis in the passage experiments. Furthermore, our analysis of a large number of publicly available SARS-CoV-2 genomes did not reveal conclusive evidence for A3-induced mutagenesis. Taken together, our studies suggest that A3 family proteins can positively contribute to SARS-CoV-2 replication, however this effect is deaminase-independent.
... The A3 proteins are cytidine deaminases and deamination of the minus-strand DNA by A3G/F/D/H during reverse transcription induces G-to-A substitutions in the viral genome that cause missense and stop-codon mutations 6,9-12 . A3s can also block viral replication by binding viral RNA or reverse transcriptase and inhibit integration by blocking 3′ processing of the viral DNA ends by integrase [13][14][15][16][17][18][19][20][21][22] . To overcome A3 restriction, HIV-1 Vif targets A3 proteins for polyubiquitination and proteasomal degradation 4,7,23-32 by interacting with cellular cofactor core-binding factor beta (CBFβ) 33,34 and recruiting the cellular cullin-RING ligase 5 (CRL5) E3 ubiquitin ligase complex composed of cullin 5 (Cul5), elongin B and elongin C complexes (EloB and EloC) and RING-box subunit 2 (RBX2) 24,27,31 . ...
... The A3 proteins are cytidine deaminases and deamination of the minus-strand DNA by A3G/F/D/H during reverse transcription induces G-to-A substitutions in the viral genome that cause missense and stop-codon mutations 6,[9][10][11][12] . A3s can also block viral replication by binding viral RNA or reverse transcriptase and inhibit integration by blocking 3′ processing of the viral DNA ends by integrase [13][14][15][16][17][18][19][20][21][22] . To overcome A3 restriction, HIV-1 Vif targets A3 proteins for polyubiquitination and proteasomal degradation 4,7,23-32 by interacting with cellular cofactor core-binding factor beta (CBFβ) 33,34 and recruiting the cellular cullin-RING ligase 5 (CRL5) E3 ubiquitin ligase complex composed of cullin 5 (Cul5), elongin B and elongin C complexes (EloB and EloC) and RING-box subunit 2 (RBX2) 24,27,31 . ...
HIV-1 Vif recruits host cullin-RING-E3 ubiquitin ligase and CBFβ to degrade the cellular APOBEC3 antiviral proteins through diverse interactions. Recent evidence has shown that Vif also degrades the regulatory subunits PPP2R5(A–E) of cellular protein phosphatase 2A to induce G2/M cell cycle arrest. As PPP2R5 proteins bear no functional or structural resemblance to A3s, it is unclear how Vif can recognize different sets of proteins. Here we report the cryogenic-electron microscopy structure of PPP2R5A in complex with HIV-1 Vif–CBFβ–elongin B–elongin C at 3.58 Å resolution. The structure shows PPP2R5A binds across the Vif molecule, with biochemical and cellular studies confirming a distinct Vif–PPP2R5A interface that partially overlaps with those for A3s. Vif also blocks a canonical PPP2R5A substrate-binding site, indicating that it suppresses the phosphatase activities through both degradation-dependent and degradation-independent mechanisms. Our work identifies critical Vif motifs regulating the recognition of diverse A3 and PPP2R5A substrates, whereby disruption of these host–virus protein interactions could serve as potential targets for HIV-1 therapeutics.
... Although the deamination of adenosine to inosine in tRNAs is a well-characterized deamination event, cytosine deamination has not been documented (93). Based on the ability of A3 enzymes to regulate HIV-1 reverse transcriptase by binding to the RNA template or the enzyme itself, the roles of A3G and A3H may be to regulate the activity of other enzymes by binding the tRNA (94,95). This may relate to an antiviral role if A3G and A3H can temporarily slow or shut down protein synthesis during a viral infection, which would facilitate immune clearance. ...
Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein–protein and protein–RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein–protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.
... To keep pace with the virus, some of the host proteins gain antiviral functions (reviewed in (36)). Most of these factors are frequently induced by interferon (IFN) signaling in response to viral infections (36,69). Several reports demonstrated that NUP98 is an interferon-inducible protein and is implicated as an antiviral factor for viruses including poliovirus, cardiovirus, and influenza virus (40, [70][71][72][73][74]. ...
Nucleoporins (NUPs) are cellular effectors of human immunodeficiency virus-1 (HIV-1) replication that support nucleocytoplasmic trafficking of viral components. However, these also non-canonically function as positive effectors, promoting proviral DNA integration into the host genome and viral gene transcription, or as negative effectors by associating with HIV-1 restriction factors, such as MX2, inhibiting the replication of HIV-1. Here, we investigated the regulatory role of NUP98 on HIV-1 as we observed a lowering of its endogenous levels upon HIV-1 infection in CD4⁺ T cells. Using complementary experiments in NUP98 overexpression and knockdown backgrounds, we deciphered that NUP98 negatively affected HIV-1 long terminal repeat (LTR) promoter activity and lowered released virus levels. The negative effect on promoter activity was independent of HIV-1 Tat, suggesting that NUP98 prevents the basal viral gene expression. ChIP-qPCR showed NUP98 to be associated with HIV-1 LTR, with the negative regulatory element (NRE) of HIV-1 LTR playing a dominant role in NUP98-mediated lowering of viral gene transcription. Truncated mutants of NUP98 showed that the attenuation of HIV-1 LTR-driven transcription is primarily contributed by its N-terminal region. Interestingly, the virus generated from the producer cells transiently expressing NUP98 showed lower infectivity, while the virus generated from NUP98 knockdown CD4⁺ T cells showed higher infectivity as assayed in TZM-bl cells, corroborating the anti-HIV-1 properties of NUP98. Collectively, we show a new non-canonical function of a nucleoporin adding to the list of moonlighting host factors regulating viral infections. Downregulation of NUP98 in a host cell upon HIV-1 infection supports the concept of evolutionary conflicts between viruses and host antiviral factors.
... Although deamination of adenosine to inosine in tRNAs is a well characterized deamination event, cytosine deamination has not been documented (93). Based on the ability of A3 enzymes to regulate HIV-1 reverse transcriptase by binding to the RNA template or the enzyme itself, the roles of A3G and A3H may be to regulate activity of other enzymes by binding the tRNA (94,95). This may relate to an antiviral role if A3G and A3H can temporarily slow or shut down protein synthesis during a viral infection, which would facilitate immune clearance. ...
Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence is not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and map a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology.