APOBEC3 family members and their associated roles in exogenous viruses and endogenous retroelements restriction

Data are compiled from [27, 77, 87, 90, 126-140].<p><b>Copyright information:</b></p><p>Taken from "Uracil within DNA: an actor of antiviral immunity"</p><p>http://www.retrovirology.com/content/5/1/45</p><p>Retrovirology 2008;5():45-45.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2427051.</p><p></p

Biosynthesis pathways of ribonucleotides and deoxyribonucleotides in mammalian cells and the possible consequence of the misincorporation and repair of uracil residues in DNA

synthesis of AMP, CMP, GMP and UMP ribonucleotides allows the formation of dATP, dCTP, dGTP, dTTP and dUTP deoxyribonucleotides, which can be readily incorporated in DNA by cellular DNA polymerases. Note that dTTP derives from dUTP hydrolysis. Abbreviations: A, adenine; C, cytosine; G, guanine; T, thymine; U, uracil; MP, monophosphate; DP, diphosphate; TP, triphosphate; rNDP, ribonucleotide diphosphate; NMPK, nucleotide monophosphate kinase; NDPK, nucleotide diphosphate kinase.<p><b>Copyright information:</b></p><p>Taken from "Uracil within DNA: an actor of antiviral immunity"</p><p>http://www.retrovirology.com/content/5/1/45</p><p>Retrovirology 2008;5():45-45.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2427051.</p><p></p

Characterization of the recovered viruses.

<p>Replication of recovered viruses was assessed using a combination of several criteria: (i) the presence or absence of cytopathic effect (CPE) is highlighted in green or red, respectively, (ii) the amount of viral RNAs in cell supernatant at the second passage, assessed using a real-time RT-PCR assay is reported as mean Log₁₀ copies/mL ±SD (iii) the infectious titer of each of the rescued virus at the second passage is expressed as a mean log₁₀ TCID₅₀/mL ±SD.</p

Specificity for ATP as the initiating nucleotide.

<p>(<b>A</b>) Specific pppAG dinucleotide formation by NS5Pol_DV in the presence of Mg²⁺ on DV₁₀3′- templates (ACUAACAA-CU) with varying last nucleotides: lane 1 -CU, lanes 2 and 3 -CC, lane 4 -CA and lane 5 -CG) in presence of Mg²⁺. Corresponding initiating NTPs and GTP were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 500 nM NS5Pol_DV, 10 µM template, 500 µM of initiating NTPs, and 100 µM GTP (containing αGTP). For the reaction on the -CC template, 300 µM (lane 2) and 600 µM GTP (lane 3) was used. Reactions were started by the addition of MgCl₂ and incubated for 2 h. Samples were analyzed using PAGE and autoradiography. (<b>B</b>) pppAG dinucleotide formation by NS5Pol_DV in the presence of Mn²⁺. Reaction mixtures contained 2 mM MnCl₂, 500 nM NS5Pol_DV, 500 µM GTP, and 100 µM ATP (containing αATP) and either no template (lane 1), 1 µM DV₁₀3′+ (lane 2), or 1 µM DV₁₀3′- (lane 3). Reactions were started by the addition of MnCl₂ and incubated for 2 h. The identity of product bands is given on the right. (<b>C</b>) Specific non-templated pppAG dinucleotide formation and non-specific NG dinucleotide formation on DV₁₀3′- template variants (see under <b>A</b>) in the presence of Mn²⁺. Reaction mixtures contained 2 mM MnCl₂, 500 nM NS5Pol_DV, 1 µM template, 500 µM of NTPs, which were not labeled, and 100 µM GTP (containing either αGTP or γGTP as outlined below the gel) and either no template or DV₁₀3′- variants (given below the gel). Reactions were started by the addition MnCl₂ and samples were taken at given time points. The identity of product bands is given on the right side of the reaction kinetics.</p

Role of NS5Pol_DV residues His798 and Trp795 as initiation platform.

<p>(<b>A</b>) Activity of wt NS5Pol_DV and its mutants W795A and H798A was determined on a specific minigenomic template. Reaction mixtures in the presence of [³H]-UTP were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a>. Initial velocities in cpm/min determined using filter-binding assays and liquid scintillation counting are compared in the presence of Mg²⁺ (left panel) and Mn²⁺ (right panel). (<b>B</b>) Reaction kinetics of wt NS5Pol_DV and its mutants W795A and H798A on the minigenomic template were analyzed on an agarose-formaldehyde gel. Reaction mixtures in the presence of [α-³²P]-UTP and Mg²⁺ ions were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a>. Product bands are labeled on the right sight of the gel. (<b>C</b>) <i>De novo</i> initiation of wt NS5Pol_DV, deletion mutant TGGK and mutants W795A and H798A was followed using either DV₁₀3′- (1 µM in the presence of Mn²⁺ and 10 µM in the presence of Mg²⁺), in the absence of a template or 1 µM DV₁₀3′+ (from top to bottom as indicated). Reaction mixtures contained 2 mM MnCl₂ or 5 mM MgCl₂ as indicated, 500 nM enzyme, 500 µM of ATP, and 100 µM GTP (containing αGTP). Reactions were started by addition of catalytic ions and samples were taken at given time points. Identities of labeled product bands are given on the left side of the gels. pppGA internal <i>de novo</i> initiation product on DV₁₀3′+ is labeled by an asterisk.</p

<i>De novo</i> initiation by NS5Pol_DV on oligonucleotides corresponding to the 3′-ends of dengue virus genome and antigenome.

<p>(<b>A</b>) <i>De novo</i> initiation in the presence of Mg²⁺ ions. Reaction mixtures were prepared as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 10 µM template, 500 nM NS5Pol_DV and the given concentrations of NTPs. Radiolabeled GTP (αGTP) was used. Reactions were started by the addition of MgCl₂ and reactions incubated for indicated time periods. Samples were analyzed by PAGE and autoradiography. Markers in lanes 1 and 2 are labeled DV₁₀3′+ and DV₁₀3′-, respectively. DV₁₀3′+ (lanes 3 to 10) and DV₁₀3′- (lanes 11 to 18) were used as templates. Nucleotide sequences are given above the panel. Identities of labeled product bands are given on the right and left side of the gel. Full-length products are labeled by an asterisk. (<b>B</b>) <i>De novo</i> initiation in the presence of Mn²⁺ ions. Reaction mixtures were prepared as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 2 mM MnCl₂, 1 µM template, 500 nM NS5Pol_DV and the given concentrations of NTPs. Radiolabeled ATP (γATP) was used. Reactions were started by the addition of MnCl₂, incubated for given time periods, and analyzed by PAGE and autoradiography. Markers in lanes 1 to 4 include an oligoG-ladder (lanes 1 and 2), labeled DV₁₀3′+ (lane 3) and DV₁₀3′- (lane 4). DV₁₀3′+ (lanes 5 to 12) and DV₁₀3′- (lanes 13 to 20) were used as templates. Identities of labeled product bands are given on the right and left side of the reaction lanes. Full-length products are labeled by an asterisk.</p

Role of the predicted priming loop T794-A799 in correct <i>de novo</i> initiation.

<p>(<b>A</b>) 3D-structural model of NS5Pol_DV used in this study (DV serotype 2 strain New Guinea C) derived from the structure of serotype 3 NS5Pol_DV (PDB code 2J7W <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#ppat.1002912-Yap1" target="_blank">[16]</a>). NS5Pol_DV adopts the typical closed right-hand structure of RdRps containing the palm (light green), fingers (light blue) and thumb (red) subdomains. Between fingers and thumb subdomains the template tunnel runs down to the active site harbored mainly by the palm subdomain. The side chains of the three conserved catalytic residues D533 (motif A), D663 and D664 (motif C) in the active site are shown in sticks (C-atoms light green, O-atoms red). The priming loop emerges from the thumb subdomain and closes the dsRNA exit tunnel and the active site. The close-up shows aromatic residues W795 and H798 (in sticks) within the putative priming loop T794 to A799. In the mutant TGGK the priming loop was replaced by two glycines situated between T793 and K800 (in sticks). (<b>B</b>) Activity of wt NS5Pol_DV and its deletion mutant TGGK NS5Pol_DV was determined on a specific minigenomic template. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a>. Initial velocities in cpm/min determined by filter-binding assays in the presence of [³H]-UTP and liquid scintillation counting, are compared in the presence of Mg²⁺ (left panel) and Mn²⁺ (right panel). The center panel shows agarose-formaldehyde gel analysis of reaction kinetics in the presence of [α-³²P]-UTP and Mg²⁺ ions. Product bands are labeled on the right sight of the gel. (<b>C</b>) <i>De novo</i> initiation of wt NS5Pol_DV and its deletion mutant TGGK was followed in the presence of Mn²⁺ using either 1 µM DV₁₀3′-, in the absence of a template, or 1 µM DV₁₀3′+ (from left to right as indicated). Reaction mixtures also contained 2 mM MnCl₂, 500 nM enzyme, 500 µM of NTPs, which were not labeled, and 100 µM labeled NTP (containing αGTP or γATP as indicated). Reactions were started by addition of MnCl₂ and samples were taken at given time points. Identities of labeled product bands are given on the right and left side of the reaction kinetics. pppGA and pppGAA internal <i>de novo</i> initiation products on DV₁₀3′+ are labeled by an asterisk.</p

Dengue virus RdRp conserves the correct 5′- and 3′-ends of the genome.

<p>DV RdRp conducts strict ATP-specific <i>de novo</i> initiation in the absence of a template and in the presence of the correct template using the indicated catalytic ions Mn²⁺ or Mg²⁺. The pppAG primer is then elongated. When DV RdRp encounters templates with incorrect 3′-end nucleotides it refuses <i>de novo</i> initiation (when Mg²⁺ is present) or corrects the error by preferentially generating and elongating pppAG (using Mn²⁺ as catalytic ion). The structure of the DV2 RdRp domain is shown in the background.</p

pppAG-elongation on the correct antigenome 3′-end and on variants with an incorrect last nucleotide.

<p>pppAG-elongation by NS5Pol_DV on DV₁₀3′- templates (ACUAACAA-CN) varying the last nucleotide (correct -CU versus -CC, -CA and -CG). Control reactions were included without template. (<b>A</b>) pppAG elongation in the presence of Mn²⁺. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 2 mM MnCl₂, 500 nM NS5Pol_DV, and 1 µM template. Reactions were started by addition of MnCl₂ and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the left, the identity of product bands is given on the right. (<b>B</b>) pppAG-elongation in the presence of Mg²⁺. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 5 µM NS5Pol_DV, and 1 µM template. Reactions were started by addition of MgCl₂ and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the right; the identity of product bands is given on the left.</p

pppAG-formation on the correct DV₁₀3′- and on variant templates having an incorrect last nucleotide.

<p>Dinucleotide formation by NS5Pol_DV on DV₁₀3′- templates (ACUAACAA-CN) varying the last nucleotide (correct -CU versus -CC, -CA and -CG). Control reactions were included without template. (<b>A</b>) pppAG dinucleotide formation in the presence of Mn²⁺. Only ATP (500 µM) and GTP (100 µM containing αGTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 2 mM MnCl₂, 500 nM NS5Pol_DV, and 1 µM template. Reactions were started by the addition of MnCl₂ and samples were taken at given time points. Samples were analyzed by PAGE and autoradiography. pppGG is marked by an asterisk for clarity. (<b>B</b>) pppAG dinucleotide formation in the presence of Mg²⁺. Only ATP (500 µM) and GTP (100 µM containing αGTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 5 µM NS5Pol_DV, and 1 µM template. Reactions were started by addition of MgCl₂ and samples were taken at given time points. Samples were analyzed as under A. (<b>C</b>) pppNG dinucleotide formation in the presence of Mn²⁺. All NTPs were given as substrates at equal concentration (100 µM). Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 2 mM MnCl₂, 500 nM NS5Pol_DV, and 1 µM template. 1 µM overall RNA concentration was used when all templates were present. Radiolabelled αGTP was used. Reactions were started by addition of MnCl₂ and samples were taken at given time points and analyzed as in A.</p