Exploring the Dynamics of Four RNA-Dependent RNA Polymerases by a Coarse-Grained Model

In this article, we present a hybrid ENM/MARTINI coarse-grained model and examine the impact of reduced chemical detail on both static and dynamic properties by comparing against explicit atomistic simulations. This methodology complements the advanced molecular characterization and dynamics of proteins for medical and bioengineering applications by developing a fundamental understanding of how the motion and molecular characteristics of proteins, viruses, their precursors, and their interactions with the environment govern their behavior in different populations. As an example, we explore the dynamics of RNA-dependent RNA polymerases (RdRPs) from the following viruses: poliovirus, Coxsackie virus B3, human rhinovirus 16, and foot-and-mouth-disease virus. The hybrid coarse-grained model allows the microsecond time scales of interest for biological functions to be explored. Additionally, the ENM/MARTINI model captures the main features obtained from atomistic MD simulations for each of the RdRPs studied herein, including the higher flexibility of the pinky finger and thumb regions, as well as collective motions that might contribute significantly to the conformational transition between the open and closed states

Kinetic Analysis of Human PrimPol DNA Polymerase Activity Reveals a Generally Error-Prone Enzyme Capable of Accurately Bypassing 7,8-Dihydro-8-oxo-2′-deoxyguanosine

Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg²⁺ as a cofactor, PrimPol binds primer-template DNA with low affinity <i>K</i>_d,DNA values (∼200–1200 nM). DNA binding is enhanced 34-fold by Mn²⁺ (<i>K</i>_d,DNA = 27 nM). The pol activity of PrimPol is increased 400–1000-fold by Mn²⁺ compared to Mg²⁺ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100–2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg²⁺ is the cofactor compared to Mn²⁺. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn²⁺) to 6-fold (Mg²⁺) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn²⁺, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription

Binding by the Hepatitis C Virus NS3 Helicase Partially Melts Duplex DNA

Binding of NS3 helicase to DNA was investigated by footprinting with KMnO₄, which reacts preferentially with thymidine residues in single-stranded DNA (ssDNA) compared to those in double-stranded DNA (dsDNA). A distinct pattern of reactivity was observed on ssDNA, which repeated every 8 nucleotides (nt) and is consistent with the known binding site size of NS3. Binding to a DNA substrate containing a partial duplex was also investigated. The DNA contained a 15 nt overhang made entirely of thymidine residues adjacent to a 22 bp duplex that contained thymidine at every other position. Surprisingly, the KMnO₄ reactivity pattern extended from the ssDNA into the dsDNA region of the substrate. Lengthening the partial duplex to 30 bp revealed a similar pattern extending from the ssDNA into the dsDNA, indicating that NS3 binds within the duplex region. Increasing the length of the ssDNA portion of the partial duplex by 4 nt resulted in a shift in the footprinting pattern for the ssDNA by 4 nt, which is consistent with binding to the 3′-end of the ssDNA. However, the footprinting pattern in the dsDNA region was shifted by only 1–2 bp, indicating that binding to the ssDNA–dsDNA region was preferred. Footprinting performed as a function of time indicated that NS3 binds to the ssDNA rapidly, followed by slower binding to the duplex. Hence, multiple molecules of NS3 can bind along a ssDNA–dsDNA partial duplex by interacting with the ssDNA as well as the duplex DNA

MALDI-TOF results of proteins associating with the EVA71 3D polymerase.

<p>MALDI-TOF results of proteins associating with the EVA71 3D polymerase.</p

Co-precipitation of UGGT1 and the EVA71 3D viral polymerase.

<p>(A) At 6 hours post-infection, lysates from EVA71-infected or mock-infected cells were immunoprecipitated with anti-3D monoclonal antibody, and the precipitates were separated using SDS-PAGE, after which silver staining was applied for visualization. The seven labeled bands were excised, digested with trypsin, and analyzed by MALDI-TOF MS. (B) EVA71-infected and mock-infected cells were harvested and subjected to co-IP assays with anti-3D antibody (lanes 3 and 4) or mouse IgG (lanes 5 and 6); or anti-UGGT1 antibody (lanes 7 and 8) or rabbit IgG (lanes 9 and 10). The precipitates were analyzed using Western blotting with anti-UGGT1, anti-3D, anti-VP2, and anti-actin antibodies. (C) Cells were harvested at 6 h post-transfection, and lysates were treated with RNase A prior to being used in co-IP assays with an anti-3D antibody. Actin served as a loading control. Degradation of RNA was confirmed by RNA gel analysis. The precipitates were analyzed using Western blotting with anti-UGGT1, anti-ILF3, and anti-actin antibodies. (D) Membrane protein fractions were purified from EVA71-infected and mock-infected cells, and immunoprecipitation results with anti-3D antibody were analyzed by Western blotting with anti-3D, anti-3A, anti-VP2, and anti-UGGT1 antibodies. Expression of UGGT1, 3D, 3CD, 3AB, and 3A in the input lysate are shown. (E) EVA71-infected and mock-infected cells were fixed and stained with anti-UGGT1 and anti-3D antibodies at 6 h post-infection. An anti-UGGT1 antibody was used in panels 1 and 5, which were examined using a FITC filter. An anti-3D antibody was used in panels 2 and 6, which were examined using a rhodamine filter. Panels 3 and 7 display Hoechst 33258 staining results, and were examined using a 4’,6-diamidino-2-phenylindole (DAPI) filter. Panels 4 and 8 display merged rhodamine, FITC, and DAPI images. (F) EVA71-infected or mock-infected cells were fixed and stained with antibodies against UGGT1 and double strand RNA. Results with the anti-double strand RNA antibody are shown in panels 1 and 5, which were examined using a rhodamine filter. Anti-UGGT1 antibody was used for panels 2 and 6, which were examined using an FITC filter. Panels 3 and 7 display Hoechst 33258 staining results, which were examined using a DAPI filter. Panels 4 and 8 display merged rhodamine, FITC, and DAPI images.</p

<i>In vivo</i> interaction between 3D polymerase and UGGT1.

<p>Uninfected RD cells were co-transfected with pFLAG-UGGT1 or pHA-3D DNA, and total cell lysates were harvested at 48 h after transfection for co-IP assays. (A) IP was performed with anti-HA, and the precipitates were analyzed by Western blotting (WB) with anti-FLAG. (B) IP was performed with anti-FLAG, and the precipitates were analyzed by WB with anti-HA. (C) Uninfected RD cells were transfected with vector or pHA-3D, and cell lysate proteins that were immunoprecipitated with anti-HA were subjected to WB with anti-UGGT1. The expression of FLAG-UGGT1 and HA-3D in the input lysates is indicated.</p

UGGT1 deploys from the ER lumen to the cytosol during EVA71 infection.

<p>(A) Mock-infected or EVA71-infected cells were fixed and stained with anti-3D, anti-UGGT1, and anti-CNX antibodies at 2, 4, and 6 h post-infection. Use of an anti-3D antibody is shown in panels 1, 6, 11, and 16, which were examined using a cy5 filter. Use of an anti-UGGT1 antibody is shown in panels 2, 7, 12, and 17, which were examined using an FITC filter. Use of an anti-CNX antibody is shown in panels 3, 8, 13, and 18, which were examined using a rhodamine filter. Panels 4, 9, 14, and 19 indicate Hoechst 33258 staining, and were examined with a DAPI filter. Panel 21 displays enlargement zone 1 from panel 5. Panel 22 displays enlargement zone 2 from panel 20. (B) Percentage of UGGT1 and CNX signal overlap as calculated with ImageJ JACoP plugins from images shown in (A). The data shown represent the average and standard deviation of ten randomly selected images. ***P < 0.001, as calculated by two-tailed unpaired Student’s t-test. (C) RD cells were infected with EVA71, harvested at 6 h post-infection, separated into cytosol and microsome fractions, and subjected to Western blot analysis using anti-UGGT1 antibody. The same blot was also probed with anti-calnexin, anti-3D, and anti-3A antibodies. The results are representative of at least three independent and reproducible experiments.</p

UGGT1 facilitates EVA71 replication and propagation.

<p>(A) Confocal microscopy results of UGGT1 and 3D expression in NC or UGGT1 siRNA-treated RD cells that were infected with EVA71 and subjected to immunostaining at 6 h post-infection. Panels 1, 5, 9, and 13 were stained with anti-UGGT1 and examined using a FITC filter; panels 2, 6, 10, and 14 were stained with anti-3D and examined with a rhodamine filter; and panels 3, 7, 11, and 15 were subjected to Hoechst 33258 staining and examined with a DAPI filter. (B) and (C) RD cells were transfected with NC or UGGT1 siRNA for 48 h, and then challenged with EVA71 at an MOI of 10 or 0.1. A plaque assay was performed to measure viral propagation rates at various timepoints post-infection. The left panels show the knockdown of uggt1 following siRNA treatment. (D) RD cells were transfected with 1, 2, or 4 μg of pFLAG-UGGT1 or pFLAG-vector for 48 h, and then challenged with EVA71 at an MOI of 10. A plaque assay was performed to measure viral yields at 6 h post-infection. (E) UGGT1 was overexpressed by respectively transfecting 1, 2, or 4 μg of plasmid pFLAG-UGGT1 to RD cells, and the panels show the corresponding increase in UGGT1 levels following overexpression, with actin serving as a loading control. ***P < 0.001, **P < 0.01, and *P < 0.05, as calculated by two-tailed unpaired Student’s t-test.</p

Viral yield, neurological symptoms, and lethality in UGGT1 heterozygous knockout mice infected with EVA71.

<p>(A) Phenotypes of UGGT1+/+ wild-type (WT) mice and UGGT1+/− heterozygous knockout mice. (B) UGGT1 expression levels in brain tissue homogenates of UGGT1+/+ WT mice and UGGT1+/− heterozygous knockout mice, as detected by western blotting. (C) and (D) 10-day-old WT or Uggt1 heterozygous knockout mice were injected with 10⁵ PFU/mouse of EVA71 strain MP4, and on Day 3 after infection, EVA71 virus was extracted from brain and muscle tissues and quantitated. (E) CNS-like hind limb paralysis and (F) Survival rates in 10-day-old WT and Uggt1 heterozygous knockout mice injected with 10⁵ PFU/mouse of the EVA71 MP4 strain were evaluated, and one-way ANOVA on ranks (Kruskal-Wallis H test) was used to determine statistical significance. The number (n) of mice in each group is shown.</p

UGGT1 enhances 3D viral polymerase levels in a viral protein 3A-associated membrane fraction.

<p>(A) RD cells were co-transfected with pFLAG-3A and pFLAG-3D; pFLAG-3AB and pFLAG-3D; or pFLAG-3D only, and harvested at 48 h post-transfection. Membrane fractions were isolated and subjected to Western blot analysis. (B) Cells were transfected with pFLAG-3A, pFLAG-3AB, and pFLAG-vector and harvested at 48 h post-transfection. Membrane fractions were separated and subjected to Western blot analysis. (C) Cells were transfected with NC or UGGT1 siRNA for 48 h, and then co-transfected with pFLAG-3A and pFLAG-3D, or pFLAG-3AB and pFLAG-3D. Cells were harvested at 48 h post-transfection, and the membrane fractions were extracted and subjected to Western blot analysis. Anti-UGGT1, anti-3D and anti-3A antibodies were used. The same blot was probed with anti-CNX and anti-HSP90 antibodies. (D) Cells were transfected with NC or UGGT1 siRNA for 48 h, and then co-transfected with pFLAG-3D. Cells were harvested at 48 h post-transfection, and the membrane fractions were extracted and subjected to Western blot analysis. Anti-3D, anti-UGGT1 and anti-CNX antibodies were used. Results are representative of at least three independent experiments.</p