Selection and characterization of aptamers targeting the Vif-CBFß-ELOB-ELOC-CUL5 complex.

The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFß-ELOB-ELOC-CUL5 (VßBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. While recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VßBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VßBCC complex. These aptamers not only bind to the VßBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VßBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VßBCC complex, offering potential avenues for therapeutic intervention.

Direct inhibition of human APOBEC3 deaminases by HIV-1 Vif independent of the proteolysis pathway.

HIV-1 Vif is known to counteract the antiviral activity of human apolipoprotein B mRNA-editing catalytic polypeptide-like (A3), a cytidine deaminase, in various ways. However, the precise mechanism behind this interaction has remained elusive. Within infected cells, Vif forms a complex called VßBCC, comprising CBFß and the components of E3 ubiquitin ligase, Elongin B, Elongin C, and Cullin5. Together with the ubiquitin-conjugating enzyme, VßBCC induces ubiquitination-mediated proteasomal degradation of A3. However, Vif exhibits additional counteractive effects. In this study, we elucidate that VßBCC inhibits deamination by A3G, A3F, and A3B independently of proteasomal degradation. Surprisingly, we discovered that this inhibition for A3G is directly attributed to the interaction between VßBCC and the C-terminal domain of A3G. Previously, it was believed that Vif did not interact with the C-terminal domain. Our findings suggest that inhibiting the interaction between VßBCC and the C-terminal domain, as well as the N-terminal domain known to be targeted for ubiquitination, of A3G may be needed to prevent counteraction by Vif.

NMR characterization of the structure of the intrinsically disordered region of human origin recognition complex subunit 1, hORC1, and of its interaction with G-quadruplex DNAs.

Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1413-511). Here, we investigate the structural characteristics of hORC1413-511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1413-511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1413-511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin.

An insight into the dependence of the deamination rate of human APOBEC3F on the length of single-stranded DNA, which is affected by the concentrations of APOBEC3F and single-stranded DNA.

BACKGROUND: APOBEC3F (A3F), a member of the human APOBEC3 (A3) family of cytidine deaminases, acts as an anti-HIV-1 factor by deaminating deoxycytidine in the complementary DNA of the viral genome. A full understanding of the deamination behavior of A3F awaits further investigation. METHODS: The real-time NMR method and uracil-DNA glycosylase assay were used to track the activities of the C-terminal domain (CTD) of A3F at different concentrations of A3F-CTD and ssDNA. The steady-state fluorescence anisotropy measurement was used to examine the binding between A3F-CTD and ssDNA with different lengths. The use of the A3F-CTD N214H mutant, having higher activity than the wild-type, facilitated the tracking of the reactions. RESULTS: A3F-CTD was found to efficiently deaminate the target deoxycytidine in long ssDNA in lower ssDNA concentration conditions ([A3F-CTD] ≫ [ssDNA]), while the target deoxycytidine in short ssDNA is deaminated efficiently in higher ssDNA concentration conditions ([A3F-CTD] ≪ [ssDNA]). This property is quite different from that of the previously studied A3 family member, A3B; the concentrations of the proteins and ssDNA had no effect. CONCLUSIONS: The concentrations of A3F-CTD and ssDNA substrates affect the ssDNA-length-dependence of deamination rate of the A3F-CTD. This unique property of A3F is rationally interpreted on the basis of its binding characteristics with ssDNA. GENERAL SIGNIFICANCE: The discovery of the unique property of A3F regarding the deamination rate deepens the understanding of its counteraction against HIV-1. Our strategy is applicable to investigate the other aspects of the A3 activities, such as those involved in the cancer development.

The C-terminal cytidine deaminase domain of APOBEC3G itself undergoes intersegmental transfer for a target search, as revealed by real-time NMR monitoring.

APOBEC3G (A3G), an anti-human immunodeficiency virus 1 factor, deaminates cytidines. We examined deamination of two cytidines located separately on substrate ssDNA by the C-terminal domain (CTD) of A3G using real-time NMR monitoring. The deamination preference between the two cytidines was lost when either the substrate or non-substrate ssDNA concentration increased. When the non-substrate ssDNA concentration increased, the deamination activity first increased, but then decreased. This indicates that even a single domain, A3G-CTD, undergoes intersegmental transfer for a target search.

Characterization of the Deamination Coupled with Sliding along DNA of Anti-HIV Factor APOBEC3G on the Basis of the pH-Dependence of Deamination Revealed by Real-Time NMR Monitoring.

Human APOBEC3G (A3G) is an antiviral factor that inactivates HIV. The C-terminal domain of A3G (A3G-CTD) deaminates cytosines into uracils within single-stranded DNA (ssDNA), which is reverse-transcribed from the viral RNA genome. The deaminase activity of A3G is highly sequence-specific; the third position (underlined) of a triplet cytosine (CCC) hotspot is converted into CCU. A3G deaminates a CCC that is located close to the 5' end of ssDNA more effectively than ones that are less close to the 5' end, so-called 3' → 5' polarity. We had developed an NMR method that can be used to analyze the deamination reaction in real-time. Using this method, we previously showed that 3' → 5' polarity can be explained rationally by A3G-CTD's nonspecific ssDNA-binding and sliding direction-dependent deamination activities. We then demonstrated that the phosphate backbone is important for A3G-CTD to slide on the ssDNA and to exert the 3' → 5' polarity, probably due to an electrostatic intermolecular interaction. In this study, we investigate the pH effects on the structure, deaminase activity, and 3' → 5' polarity of A3G-CTD. Firstly, A3G-CTD was shown to retain the native structure in the pH range of 4.0-10.5 by CD spectroscopy. Next, deamination assaying involving real-time NMR spectroscopy for 10-mer ssDNA containing a single CCC revealed that A3G-CTD's deaminase activity decreases as the pH increases in the range of pH 6.5-12.7. This is explained by destabilization of the complex between A3G-CTD and ssDNA due to the weakened electrostatic interaction with the increase in pH. Finally, deamination assaying for 38-mer ssDNA having two CCC hotspots connected by a long poly-adenine linker showed that A3G-CTD retains the same pH deaminase activity preference toward each CCC as that toward the CCC of the 10-mer DNA. Importantly, the 3' → 5' polarity turned out to increase as the pH decreases in the range of 6.5-8.0. This suggests that A3G-CTD tends to continue sliding without abortion at lower pH, while A3G-CTD tends to dissociate from ssDNA during sliding at higher pH due to the weakened electrostatic interaction.

Catalytic analysis of APOBEC3G involving real-time NMR spectroscopy reveals nucleic acid determinants for deamination.

APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3'→5' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5'-end is deaminated more effectively than one less close to the 5'-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2's activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3'→5' polarity. Examination of the effects of different salt concentrations on the 3'→5' polarity indicated that the higher the salt concentration, the less prominent the 3'→5' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.