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Consequences of UDG inhibition on A3G antiviral phenotype and cDNA profiles a) Immunoblot analysis of HIV-1 virion lysates showing increasing amounts of packaged A3G_HA at constant CA levels for virions produced in the presence or absence of a codon optimized (humanized) uracil-DNA glycosylase inhibitor (hUGI). 'Low' or 'High' A3G refers to a producer cell transfection ratios of 1:10 or 1:1, respectively (A3G expression plasmid to NL4.3/ΔVif). One of three independent sets of virus preparations used for b) and c) is shown. b) Virion infectivity was evaluated by challenging TZM-bl cells and measurement of β-galactosidase activity. c) The abundance of (-)sss containing cDNA in
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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...
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... surprisingly, experimental system dependent differences extend to the effects of A3G on reverse transcription. Studies with reconstituted assays had indicated A3G-induced pausing of RT at specific sites22 (Supplementary Figure 3a), which helped formulate the "roadblock" model. Critically, our cell-based DNA sequencing approach provided no evidence for localized A3G-induced RT pausing, with the prominent A3G-dependent cDNA peaks being caused by UBER activity (Figure 2). These findings evoke a sequence-and site- independent mechanism for the suppression of RT by A3G, and imply: first, that there are differences in RT's behavior in the context of HIV-1 infection; and, second, that the A3G- induced pausing that is seen in reconstituted reactions reflects an assay-specific ...
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... of HIV-1 cDNA can also arise from the misincorporation of dUTP in place of dTTP during reverse transcription62, especially in non-dividing cells such as macrophages that have high dUTP:dTTP ratios63,64. UNG2-mediated recognition of the resulting uracilated viral cDNA suppresses infection, with restriction being most evident at the level of DNA integration65-68. Interestingly, and in contrast to observations made with A3G- induced uracilation (Figure 2, Supplementary Figure 7)25-27, UGI-mediated suppression of UNG2 in the face of misincorporation-driven uracilation provokes viral rescue65,66. The basis for this dichotomy remains to be determined, but could relate to variations in the extent of uracilation, or cell type dependent differences in the efficiency of UBER-mediated cDNA recognition or the fate/processing of uracilated ...
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... ligation-The barcoded adaptor carries a 5' phosphate group (PHO) and a 3' three carbon chain (C3) spacer (SpC3) on to the 3' hydroxyl group (5'-PHO- tgaagagcctagtcgctgttcannnnnnctgcccatagagagatcggaagagcacacgtct-SpC3-3') (Integrated DNA Technologies or MWG Eurofins) and was self annealed in T4 DNA ligase buffer by heating to 92°C followed by slow cooling to 16°C (2% slope on Eppendorf PCR machine). Ligation reactions were set up in 60 μl total volume, with 6 μl T4 DNA ligase buffer, 24 μl 50% PEG-8000 (Sigma), 6 μl 5 M betaine (Sigma), 4 μl pre-annealed adaptor (400 pmol total), 1.2 μl T4 DNA ligase (NEB, 2,000,000 units/ml) and 18.8 μl DNA sample. Reactions were incubated at 16°C overnight. As controls, instead of DNA samples, HTP control oligos (listed in Supplemental Figure 2) at 100 pmol/μl were mixed at equimolar ratios and then diluted 1 in 62,500 before being ligated as ...
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... test whether UDGs, specifically UNG240,41, mediate the processing of uridine- containing cDNA, leading to endonucleolytic cleavage, we utilized the bacteriophage uracil- DNA glycosylase inhibitor (UGI)42. CEM-SS target cells and HEK293T producer cells stably expressing codon optimized UGI (hUGI)25 were generated, the latter being required because UNG2 is packaged into virions43,44. Enzymatic analyses of cell lysates confirmed efficient UDG suppression (Supplementary Figure 6). HIV-1ΔVif was produced in control or hUGI expressing HEK293T cells in the absence or presence of A3G (Figure 2a). In bulk measurements of virus infectivity and cDNA abundance (Figures 2b and 2c), no differences were attributable to hUGI, irrespective of expression in producer, target or both cultures, reconfirming that UNG2 inhibition does not impact A3G anti-viral function25-27. Sequencing analysis revealed that UNG2 inhibition in target cells mitigated A3G's induction of foreshortened cDNAs, with the overall profiles of 3'-termini resembling those seen without A3G (Figure 2d; and Supplementary Figure 7). We therefore conclude: first, that target cell UNG2 can detect A3G-edited cDNAs leading to uracil removal and subsequent cleavage ( Figure 1a, pathway 2); second, that UBER- mediated cDNA fragmentation does not lead to their complete degradation, implying that a deamination-independent mechanism likely underlies the inhibition of cDNA accumulation by A3G; and, third, that A3G does not induce site-specific RT pausing, thus arguing against the aforementioned "roadblock" mechanism for RT ...
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... test whether UDGs, specifically UNG240,41, mediate the processing of uridine- containing cDNA, leading to endonucleolytic cleavage, we utilized the bacteriophage uracil- DNA glycosylase inhibitor (UGI)42. CEM-SS target cells and HEK293T producer cells stably expressing codon optimized UGI (hUGI)25 were generated, the latter being required because UNG2 is packaged into virions43,44. Enzymatic analyses of cell lysates confirmed efficient UDG suppression (Supplementary Figure 6). HIV-1ΔVif was produced in control or hUGI expressing HEK293T cells in the absence or presence of A3G (Figure 2a). In bulk measurements of virus infectivity and cDNA abundance (Figures 2b and 2c), no differences were attributable to hUGI, irrespective of expression in producer, target or both cultures, reconfirming that UNG2 inhibition does not impact A3G anti-viral function25-27. Sequencing analysis revealed that UNG2 inhibition in target cells mitigated A3G's induction of foreshortened cDNAs, with the overall profiles of 3'-termini resembling those seen without A3G (Figure 2d; and Supplementary Figure 7). We therefore conclude: first, that target cell UNG2 can detect A3G-edited cDNAs leading to uracil removal and subsequent cleavage ( Figure 1a, pathway 2); second, that UBER- mediated cDNA fragmentation does not lead to their complete degradation, implying that a deamination-independent mechanism likely underlies the inhibition of cDNA accumulation by A3G; and, third, that A3G does not induce site-specific RT pausing, thus arguing against the aforementioned "roadblock" mechanism for RT ...
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... test whether UDGs, specifically UNG240,41, mediate the processing of uridine- containing cDNA, leading to endonucleolytic cleavage, we utilized the bacteriophage uracil- DNA glycosylase inhibitor (UGI)42. CEM-SS target cells and HEK293T producer cells stably expressing codon optimized UGI (hUGI)25 were generated, the latter being required because UNG2 is packaged into virions43,44. Enzymatic analyses of cell lysates confirmed efficient UDG suppression (Supplementary Figure 6). HIV-1ΔVif was produced in control or hUGI expressing HEK293T cells in the absence or presence of A3G (Figure 2a). In bulk measurements of virus infectivity and cDNA abundance (Figures 2b and 2c), no differences were attributable to hUGI, irrespective of expression in producer, target or both cultures, reconfirming that UNG2 inhibition does not impact A3G anti-viral function25-27. Sequencing analysis revealed that UNG2 inhibition in target cells mitigated A3G's induction of foreshortened cDNAs, with the overall profiles of 3'-termini resembling those seen without A3G (Figure 2d; and Supplementary Figure 7). We therefore conclude: first, that target cell UNG2 can detect A3G-edited cDNAs leading to uracil removal and subsequent cleavage ( Figure 1a, pathway 2); second, that UBER- mediated cDNA fragmentation does not lead to their complete degradation, implying that a deamination-independent mechanism likely underlies the inhibition of cDNA accumulation by A3G; and, third, that A3G does not induce site-specific RT pausing, thus arguing against the aforementioned "roadblock" mechanism for RT ...
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... sequencing approach also allowed us to examine the effects of UBER on viral cDNA following exposure to A3G. Although, we reveal UNG2-mediated uridine excision from deaminated cDNA, our measurements show ensuing endonucleolytic cleavage is far from complete since substantial proportions of cytidine-to-uridine edited cDNAs remained intact. (Figures 1, 2 and 6). Appreciating this inefficiency helps to reconcile the apparent discrepancy between the predicted degradative fate of deaminated cDNAs ( Figure 1a, mechanism 2) and the lack of persuasive evidence for UDG involvement in A3G function. Nevertheless, despite the absence of an evident anti-viral effect in single round infections (Figure 2b), inefficient UBER recognition of edited cDNA could still play a role in the interplay between HIV-1 and infected cells, for instance through sensing of aberrant cDNA fragments as a pathogen-associated molecular pattern1,60. How HIV-1 may circumvent this (aside from Vif induced A3 destruction in virus producing cells) is a matter of conjecture, but we note that HIV-1 Vpr and its interacting CUL4 ubiquitin ligase induces UNG2 ...
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... main information extracted from each read were: first, the last nucleotide of the HIV-1 sequence adjacent to the fixed adaptor sequence, which was ligated to the viral cDNAs (see Figure 1c and Supplementary Figure 1b). This represents the open 3'-terminus of the viral cDNA at time of harvest and, second, the base variation of all bases, in particular C to T mutations. For this purpose FASTQ files were subjected to in house analysis. Adaptors were trimmed and sequences that were duplicated (including the barcode) were removed as PCR artifacts. The remaining sequences were aligned to the HIV-1 sequence using Bowtie (http:// bowtie-bio.sourceforge.net/index.shtml), allowing a maximum of 3 base mismatches, and the position of 3'-termini for each read was determined from the alignment position. Mutation rates from the template sequence for each base were also calculated. Where required, a linear length-dependent correction factor was calculated from synthesized oligos control library (see Supplementary Figure 2) and applied to the dataset to correct for differences in sequencing efficiency of longer ...
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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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... data supporting this study and custom software are available from the corresponding author upon reasonable request. There are no restrictions to data availability. Raw MiSeq® sequencing files analyzed in this study (presented in Fig 1g, 2d, 6e as well as Supplementary Fig 2,
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... analyses of cell lysates confirmed efficient UDG suppression (Supplementary Figure 6). HIV-1ΔVif was produced in control or hUGI expressing HEK293T cells in the absence or presence of A3G (Figure 2a). In bulk measurements of virus infectivity and cDNA abundance (Figures 2b and 2c), no differences were attributable to hUGI, irrespective of expression in producer, target or both cultures, reconfirming that UNG2 inhibition does not impact A3G anti-viral function25-27. ...
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... was produced in control or hUGI expressing HEK293T cells in the absence or presence of A3G (Figure 2a). In bulk measurements of virus infectivity and cDNA abundance (Figures 2b and 2c), no differences were attributable to hUGI, irrespective of expression in producer, target or both cultures, reconfirming that UNG2 inhibition does not impact A3G anti-viral function25-27. Sequencing analysis revealed that UNG2 inhibition in target cells mitigated A3G's induction of foreshortened cDNAs, with the overall profiles of 3'-termini resembling those seen without A3G (Figure 2d; and Supplementary Figure 7). ...
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... bulk measurements of virus infectivity and cDNA abundance (Figures 2b and 2c), no differences were attributable to hUGI, irrespective of expression in producer, target or both cultures, reconfirming that UNG2 inhibition does not impact A3G anti-viral function25-27. Sequencing analysis revealed that UNG2 inhibition in target cells mitigated A3G's induction of foreshortened cDNAs, with the overall profiles of 3'-termini resembling those seen without A3G (Figure 2d; and Supplementary Figure 7). We therefore conclude: first, that target cell UNG2 can detect A3G-edited cDNAs leading to uracil removal and subsequent cleavage ( Figure 1a, pathway 2); second, that UBER- mediated cDNA fragmentation does not lead to their complete degradation, implying that a deamination-independent mechanism likely underlies the inhibition of cDNA accumulation by A3G; and, third, that A3G does not induce site-specific RT pausing, thus arguing against the aforementioned "roadblock" mechanism for RT inhibition. ...
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... with reconstituted assays had indicated A3G-induced pausing of RT at specific sites22 (Supplementary Figure 3a), which helped formulate the "roadblock" model. Critically, our cell-based DNA sequencing approach provided no evidence for localized A3G-induced RT pausing, with the prominent A3G-dependent cDNA peaks being caused by UBER activity (Figure 2). These findings evoke a sequence-and site- independent mechanism for the suppression of RT by A3G, and imply: first, that there are differences in RT's behavior in the context of HIV-1 infection; and, second, that the A3G- induced pausing that is seen in reconstituted reactions reflects an assay-specific epiphenomenon. ...
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... this inefficiency helps to reconcile the apparent discrepancy between the predicted degradative fate of deaminated cDNAs ( Figure 1a, mechanism 2) and the lack of persuasive evidence for UDG involvement in A3G function. Nevertheless, despite the absence of an evident anti-viral effect in single round infections (Figure 2b), inefficient UBER recognition of edited cDNA could still play a role in the interplay between HIV-1 and infected cells, for instance through sensing of aberrant cDNA fragments as a pathogen-associated molecular pattern1,60. How HIV-1 may circumvent this (aside from Vif induced A3 destruction in virus producing cells) is a matter of conjecture, but we note that HIV-1 Vpr and its interacting CUL4 ubiquitin ligase induces UNG2 degradation23,44,61. ...
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... recognition of the resulting uracilated viral cDNA suppresses infection, with restriction being most evident at the level of DNA integration65-68. Interestingly, and in contrast to observations made with A3G- induced uracilation (Figure 2, Supplementary Figure 7)25-27, UGI-mediated suppression of UNG2 in the face of misincorporation-driven uracilation provokes viral rescue65,66. The basis for this dichotomy remains to be determined, but could relate to variations in the extent of uracilation, or cell type dependent differences in the efficiency of UBER-mediated cDNA recognition or the fate/processing of uracilated cDNA. ...
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... were incubated at 16°C overnight. As controls, instead of DNA samples, HTP control oligos (listed in Supplemental Figure 2) at 100 pmol/μl were mixed at equimolar ratios and then diluted 1 in 62,500 before being ligated as above. ...
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... rates from the template sequence for each base were also calculated. Where required, a linear length-dependent correction factor was calculated from synthesized oligos control library (see Supplementary Figure 2) and applied to the dataset to correct for differences in sequencing efficiency of longer products. ...
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... 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). ...
Citations
... The roadblock model, a well-known deaminase-independent mechanism, involves A3G protein physically blocking viral reverse transcription and reducing the accumulation of reverse transcription products [34-37,39,40,66]. The direct interaction of A3G protein with HIV-1 reverse transcriptase also blocks reverse transcription [155,156]. Notably, the antiviral activity of A3F and A3H protein mainly arises in a deaminase-independent manner [120,[157][158][159]. Importantly, A3G and A3F proteins interfere with viral genome integration by disrupting the structural integrity of the HIV-1 preintegration complex to inhibit proviral DNA integration into the host genome and by directly interacting with HIV-1 integrase to inhibit provirus formation [160,161] or compromising viral integration efficiency by affecting the processing of long extremities for viral long terminal repeats (LTRs) [162]. Additional nonediting activities of A3 proteins include the A3F protein, and, to a lesser extent, the A3G protein, remaining associated with the viral preintegration complex as it traffics into the host nucleus [163], altering proviral DNA integration site selection to avoid gene coding sequences and/or favoring integration into short interspersed nuclear elements, oncogenes, or transcription-silencing non-B DNA [160], potentially promoting more latent HIV-1 expression profiles (Figure 1). ...
The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3/A3) family of cytosine deaminases serves as a key innate immune barrier against invading retroviruses and endogenous retroelements. The A3 family’s restriction activity against these parasites primarily arises from their ability to catalyze cytosine-to-uracil conversions, resulting in genome editing and the accumulation of lethal mutations in viral genomes. Additionally, non-editing mechanisms, including deaminase-independent pathways, such as blocking viral reverse transcription, have been proposed as antiviral strategies employed by A3 family proteins. Although viral factors can influence infection progression, the determinants that govern A3-mediated restriction are critical in shaping retroviral infection outcomes. This review examines the interactions between retroviruses, specifically human immunodeficiency virus type 1 and human T-cell leukemia virus type 1, and A3 proteins to better understand how editing and non-editing activities contribute to the trajectory of these retroviral infections.
... 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.
