(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



LGP2 directly interacts with flavivirus NS5 RNA-dependent RNA polymerase and downregulates its pre-elongation activities [1]

['Zhongyuan Tan', 'The Joint Laboratory For Translational Precision Medicine', 'A. Guangzhou Women', 'Children S Medical Center', 'Guangzhou Medical University', 'Guangzhou', 'Guangdong', 'China', 'B. Wuhan Institute Of Virology', 'Chinese Academy Of Sciences']

Date: 2023-10

LGP2 is a RIG-I-like receptor (RLR) known to bind and recognize the intermediate double-stranded RNA (dsRNA) during virus infection and to induce type-I interferon (IFN)-related antiviral innate immune responses. Here, we find that LGP2 inhibits Zika virus (ZIKV) and tick-borne encephalitis virus (TBEV) replication independent of IFN induction. Co-immunoprecipitation (Co-IP) and confocal immunofluorescence data suggest that LGP2 likely colocalizes with the replication complex (RC) of ZIKV by interacting with viral RNA-dependent RNA polymerase (RdRP) NS5. We further verify that the regulatory domain (RD) of LGP2 directly interacts with RdRP of NS5 by biolayer interferometry assay. Data from in vitro RdRP assays indicate that LGP2 may inhibit polymerase activities of NS5 at pre-elongation but not elongation stages, while an RNA-binding-defective LGP2 mutant can still inhibit RdRP activities and virus replication. Taken together, our work suggests that LGP2 can inhibit flavivirus replication through direct interaction with NS5 protein and downregulates its polymerase pre-elongation activities, demonstrating a distinct role of LGP2 beyond its function in innate immune responses.

RNA-dependent RNA polymerases (RdRPs) are central components of RNA virus genome replication machinery. Host factors can regulate RNA virus genome replication through direct interactions with RdRPs, typically playing auxiliary roles. LGP2 is a host protein known to play critical roles in innate immune responses and has not been documented in participation of RNA virus genome replication. In this work, we reveal that LGP2 down-regulates flavivirus genome replication through direct interaction with viral RdRP and its RNA substrate, demonstrating a unique mechanism of RdRP regulation by a host factor.

Funding: This work was supported by the National Key Research and Development Program (2018YFA0507200 to P.G. and Z.Z.), China Postdoctoral Science Foundation (2020M672579 to Z.T.), National Natural Science Foundation of China (32000136 to J.W.; 32070185 to P.G.), Youth Innovation Promotion Association Program of Chinese Academy of Sciences (2022341 to J.W.), Creative Research Group Program of Natural Science Foundation of Hubei Province, China (2022CFA021 to P.G. and J.W.), and Key Biosafety Science and Technology Program of Hubei Jiangxia Laboratory (JXBS001 to P.G.). The funders had no role in study design, data collection, and interpretation, or submitting the work for publication.

Zika virus (ZIKV)/dengue virus (DENV) and tick-borne encephalitis virus (TBEV) are representatives of mosquito- and tick-borne flaviviruses, respectively [ 31 , 32 ]. Both ZIKV and TBEV can infect central nervous system (CNS) and replicate in multiple types of neural cells and abrogate neurogenesis, with ZIKV causing microcephaly in newborns and Guillain-Barre syndrome [ 33 ] and TBEV causing encephalitis [ 34 ]. DENV infection can lead to fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) [ 35 ]. Using ZIKV, DENV, and TBEV systems in this study, we find that LGP2 colocalizes with viral RC and directly interacts with the key replication protein NS5, and inhibits its polymerase function. This function is distinct from previously documented RLR-related functions in innate immune response, suggesting a unique route of antiviral regulation of LGP2 by directly targeting viral genome replication machinery.

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma differentiation antigen 5 (MDA5), and laboratory genetics and physiology 2 (LGP2), are cytoplasmic viral RNA sensors that recognize and bind viral dsRNA to trigger antiviral innate immune responses [ 18 ]. They all comprise of a DExD/H helicase domain and a C-terminal domain (CTD) that is also known as regulatory domain (RD) [ 19 ]. The helicase module has ATPase activity and the RD domain plays key roles in RNA recognition and binding, as supported by studies characterizing purified RLRs RD domains in vitro [ 20 ], and LGP2 binds to dsRNA or single-stranded RNA (ssRNA) with higher affinity than either RIG-I or MDA5 [ 21 , 22 ]. However, lacking the N-terminal two caspase activation and recruitment domains (CARDs) compared with RIG-I and MDA5, LGP2 only functions as a regulator of RIG-I and MDA5, not directly triggering RLR signaling pathway by activating the downstream adaptor protein mitochondrial antiviral signaling (MAVS) [ 19 ]. Previous studies suggest that LGP2 negatively or positively regulates RIG-I and MDA5 in response to infection of different viruses [ 23 , 24 ]. For example, LGP2 inhibits IFN-stimulated regulatory element- and NF-ƙB-dependent pathways induced by Sendai virus (SEV) and Newcastle disease virus (NDV) infection [ 25 ]. Hepatitis C virus (HCV) infection promotes the interaction of LGP2 with MDA5 and facilitates MDA5 recognition of HCV RNA that induced IFN signaling [ 26 ]. The transgenic mouse overexpressing LGP2 decreases inflammatory mediators and leukocyte infiltration into the bronchoalveolar airspace during influenza A virus (IAV) infection [ 27 ]. The production of IFNβ significantly decreases in response to various RNA viruses, such as encephalomyocarditis virus (EMCV), mengovirus, Japanese encephalitis virus (JEV), SEV, and vesicular stomatitis virus (VSV), in an LGP2-knockout cell line [ 28 ]. Therefore, LGP2 plays an important role in RLRs signaling in response to various viral infections. Except for acting as a regulator of RIG-I and MDA5, LGP2 is also involved in other processes by interacting with key proteins of virus or host. For example, LGP2 binds to the dsRNA binding sites of TAR-RNA binding protein (TRBP), resulting in inhibition of pre-miRNA binding and recruitment by TRBP [ 29 ]. The leader protease (Lpro) of foot-and-mouth disease virus (FMDV) interacts with LGP2 and targets LGP2 for cleavage at the an RGRAR sequence [ 30 ].

It has been documented that some host proteins involved in the viral RC may provide auxiliary functions related to efficiency and specificity for flavivirus replication. For example, reticulon 3.1 (RTN 3.1A) [ 7 ], fatty acid synthase (FASN) [ 8 ], heat shock protein 40 (HSP40) chaperon protein DNAJC14 [ 9 ], vimentin [ 10 ], transmembrane protein 41B (TMEM41B) [ 11 ], and atlastins (ATLs) [ 12 ] are recruited to RC through interactions with viral proteins. These host proteins mainly facilitate RC membrane shaping and offer a scaffold for RC central components NS5 and NS3. However, a few host proteins also directly interact with NS5. Tripartite motif (TRIM) protein TRIM79α [ 13 ], signal transducer and activator of transcription 2 (STAT2) [ 14 ], Golgi brefeldin A resistance factor (GBF1) [ 15 ], cyclophilin A (CyPA) [ 16 ], and protein kinase G (PKG) [ 17 ] interact with flavivirus NS5 and regulate viral replication. However, host proteins that are directly involved in RC and downregulate viral genome replication have not been well documented in flaviviruses or even in all positive-strand RNA viruses.

The Flavivirus genus of Flaviviridae family includes a large number of arthropod-borne human pathogens, posing a serious threat to human health [ 1 ]. The flavivirus genome is a positive-sense RNA that encodes a single polyprotein precursor. The polyprotein is processed into three structural proteins and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by viral and host proteases [ 2 ]. Flavivirus genome replication takes place in an endoplasmic reticulum (ER) membrane-associated viral replication complex (RC) including viral non-structural proteins, viral RNA, and host proteins [ 3 ]. NS5 and NS3 are central components of RC on a scaffold created by the other five transmembrane proteins, and they are together responsible for all enzymatic activities required to amplify the viral RNA [ 4 ]. In the first step of genome replication, the positive-sense genomic RNA serves as the template, and the negative-sense RNA is synthesized by NS5 RNA-dependent RNA polymerase (RdRP) module, forming a double-stranded RNA (dsRNA) replication intermediate. The negative-sense RNA then serves as the template for NS5 to synthesize adequate positive-sense RNA, which is 5′-capped and methylated with the cooperation of NS5 methyltransferase (MTase) and NS3 NTPase/triphosphatase modules [ 5 ]. NS3 also possesses RNA helicase and protease functions, playing key roles in resolving RNA tertiary structures during RdRP synthesis and viral polyprotein processing [ 6 ]. Therefore, NS5 and NS3 play critical roles in flavivirus replication and their dysfunction may strongly impact the virus life cycle.

Results

LGP2 colocalizes with the replication complex of ZIKV Bir A is a 35-kD DNA-binding biotin protein ligase in Escherichia coli (E. coli) that regulates the biotinylation and acts as a transcriptional repressor for biotin biosynthetic operon. Bir A mutant (R118G, Bir A*) can result in promiscuous protein biotinylation because its free bioAMP readily reacts with primary amines [42]. In order to identify LGP2 interacting viral proteins, we constructed the transfer plasmid of fusion protein LGP2-BirA*, and packaged lentiviruses in 293T cell line by using a third-generation lentivirus packaging system. The stably LGP2-BirA*-overexpressing cell line was established using the lentiviruses to infect CCF-STTG1 cells. Then, we infected the stably overexpressing cell line with ZIKV and cultured it in 1640 culture medium with 50 μM biotin. Cell lysates were incubated with streptavidin-conjugated magnetic beads, immunoprecipitated by the biotinylated protein, and detected by Western blot using horseradish peroxidase (HRP)-streptavidin or ZIKV NS5/NS3 antibodies. Western blot data indicate that ZIKV NS5 and NS3 were biotinylated by LGP2-BirA* and immunoprecipitated by magnetic beads. These results demonstrate that LGP2 is spatially adjacent to ZIKV NS5 and NS3 (Fig 3A). Our data therefore suggest that LGP2 may be an important host protein regulating ZIKV RC. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. LGP2 colocalizes with the RC of ZIKV. (A) The affinity capture of biotinylated viral proteins. LGP2-BirA*-overexpressing CCF-STTG1 cells were infected by ZIKV (MOI = 0.1) and incubated in complete media supplemented with 1 μg/mL puromycin and 50 μM biotin for 48 h. The supernatants of cell lysates were used for Co-IP and Western blot analysis. Three independent experiments were performed and images from one experiment were shown. (B) A competitive Co-IP assay of LGP2 to NS5 and NS3. 2 μg LGP2 and (0, 0.5, 1 μg) ZIKV NS3, or 2 μg ZIKV NS3 and (0, 0.5, 1 μg) LGP2-overexpressing plasmids were transfected into 293T cells with 2 μg ZIKV NS5-overexpressing plasmids. The supernatants of cell lysates were used to perform Co-IP analysis, and immunoprecipitates were analyzed by Western blot. Three independent experiments were performed and images from one experiment were shown. (C) Cellular localizations of LGP2, ZIKV NS5, and viral dsRNA during ZIKV infection. HeLa cells were transfected with LGP2-GFP-overexpressing plasmids and infected by ZIKV (MOI = 5) after 16–20 h. At 24 hpi, the cells were fixed and analyzed by confocal immunofluorescence. Cellular nuclei (Blue), LGP2-GFP fusion protein (Green), ZIKV NS5 (Red) and viral dsRNA (Purple) were stained and excited with 405/488/555/633 nm lasers, respectively. Image processing and colocalization analysis was performed by LAS X and Imaris. The representative four channels, merged, and colocalization images were shown, and the insets at the top-right corner of each images showed the magnified 2× view of the area marked and the scale bars showed at the bottom-right corner. (D) Pairwise MCCs of two-channel in (C) (ZIKV NS5+dsRNA, LGP2+dsRNA, LGP2+ZIKV NS5) were shown. (E) Pairwise MCCs of two-channel each other in (C) (LGP2+ZIKV NS5+dsRNA) were shown. M1 = ZIKV NS5+dsRNA/ZIKV NS5, M2 = ZIKV NS5+dsRNA/dsRNA, M3 = LGP2+dsRNA/LGP2, M4 = LGP2+dsRNA/dsRNA, M5 = LGP2+ZIKV NS5/ LGP2, M6 = LGP2+ZIKV NS5/ ZIKV NS5. At least 10 cells were randomly selected for analysis. https://doi.org/10.1371/journal.ppat.1011620.g003 Flavivirus NS3-NS5 interactions have been documented in several studies [43,44]. In order to understand the relationship between NS3, NS5, and LGP2, we performed a competitive Co-IP assay. The results indicate that the change of ZIKV NS3 amount does not much affect the Co-IP of LGP2 and ZIKV NS5, but change of LGP2 amount has an apparent effect on the Co-IP of ZIKV NS3 and NS5 (Fig 3B). These data suggest that LGP2 can impair NS3 binding to NS5, but not vice versa. Confocal immunofluorescence microscopy was further used to analyze the cellular localization of LGP2, NS5, and dsRNA. HeLa cells were transfected with LGP2-GFP-overexpressing plasmids and infected with ZIKV, and then the cellular localization of LGP2, NS5, and dsRNA was examined. LGP2-GFP and dsRNA were localized exclusively in the cytoplasm, whereas NS5 was localized both in the cytoplasm and nuclear regions. In the two-channel colocalization images, the colocalization of LGP2-dsRNA, ZIKV NS5-dsRNA and LGP2-ZIKV NS5 are visualized in the cytoplasm (Fig 3C) with Manders’ colocalization coefficients (MCCs) greater than 0.5 (Fig 3D). In the three-channel colocalization image, the colocalization of LGP2-dsRNA-ZIKV NS5 is also visualized in the cytoplasm around cell nucleus (Fig 3C) with MCCs of each two channels greater than 0.5 (Fig 3E). These data further indicate that LGP2 colocalizes with ZIKV NS5 and dsRNA that are both key components of the ZIKV RC. Altogether, these data demonstrate the colocalization of LGP2 and RC during ZIKV infection, and LGP2 could possibly interact with NS5.

LGP2 may interact with the RdRP module of NS5 To further dissect flavivirus RC regulation by LGP2, LGP2 was overexpressed in A549 cells, and then the cells were infected by ZIKV. An endogenous Co-IP assay showed that LGP2 could Co-IP with endogenous ZIKV NS5 (Fig 4A). Therefore, these results show that the interaction of LGP2 with ZIKV NS5 could occur in ZIKV infection. Meanwhile, a series of Co-IP assays were performed by co-transfection. These data indicate that LGP2 also co-immunoprecipitates with TBEV NS5 (Fig 4B). We next compared the Co-IP of LGP2 with NS5 RdRP or MTase modules, respectively. The results indicate that LGP2 only co-immunoprecipitates with the RdRP but not the MTase (Fig 4C). Since both LGP2 and NS5 can bind to RNA, the possible interaction between LGP2 and NS5 may be mediated by RNA bridging. We therefore compared the Co-IP results with and without the treatment of the cell lysates by a mixture of two single-stranded RNA ribonucleases RNase A and RNase I, and the results indicate that RNA degradation by this RNase mixture does not affect the Co-IP of LGP2 and NS5, but weakens the Co-IP of LGP2 and Dicer (Fig 4D) [45]. These Co-IP data together suggest that LGP2 is involved in viral RC and likely interact with NS5 RdRP module and viral RNA. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. LGP2 interacts with the RdRP module of NS5. (A) A Co-IP assay of LGP2 and ZIKV NS5 during viral infection. LGP2-overexpressing plasmid was transfected into A549 cells and infected with ZIKV (MOI = 0.1) after 16–20 h. At 48 hpi, the supernatants of cell lystates were used for Co-IP analysis. (B-C) A series of Co-IP assays of ZIKV NS5, TBEV NS5, MTase module, or RdRP module with LGP2 by plasmid co-transfection in 293T cells. After 24 hpi, the supernatant of the cell lysates was used for Co-IP analysis. (D) A Co-IP assay of LGP2 and ZIKV NS5 with treatment of an RNase A-RNase I mixture. Two plasmids co-transfected into 293T cells, and the supernatants of cell lysates were pre-treated by the RNase mixture prior to Co-IP analysis. All of the Co-IP samples were analyzed by using Western blot. A Co-IP assay of LGP2 and Dicer was performed as a positive control. Three independent experiments were performed and images from one experiment were shown. https://doi.org/10.1371/journal.ppat.1011620.g004

The regulatory domain of LGP2 interacts with NS5 To verify the critical NS5 binding site of LGP2, we performed a series of truncations at LGP2 C-terminus (537–678 residue) (Fig 5A), and used Co-IP and luciferase assay experiments to evaluate the impact of these truncations on NS5 interaction and virus replication. We found that the LGP2 1–646 mutant can still interact with NS5 but its ability to inhibit viral replication is weakened. LGP2 1–596 mutant can no longer interact with NS5 and its ability to inhibit viral replication is decreased if compared with LGP2 WT (Fig 5B and 5C). These results suggest that the primary interaction sites of LGP2 with NS5 is likely within the 596–678 region of RD and the interaction plays a key role in the inhibition of viral replication. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. LGP2 RD directly interacts with NS5. (A) Schematic diagram of LGP2 truncation mutants. (B) A Co-IP assay of LGP2 or its truncation mutants with ZIKV NS5. Two plasmids were co-transfected into 293T cells and the supernatants of cell lysates were used for Co-IP and Western blot analysis after 24 h. Three independent experiments were performed and images from one experiment were shown. (C) Relative luciferase activities of ZIKV replicon inhibited by LGP2 or its truncation mutants. 400 ng LGP2 WT- or its truncation mutant-overexpressing plasmids were co-transfected with 100 ng viral replicons in 293T cells. Luciferase activities were detected and relative luciferase activities of replicon transfected with vector were set to 100%. Data collected from three independent experiments were shown as Means ± SD (Student’s t-test; **: p<0.01, ***: p<0.001). The protein expressions of LGP2 were determined by Western blot. (D-G) Protein binding studies of biotinylated LGP2 RD with ZIKV/TBEV/DENV2 NS5, HSA (negative control), respectively. Biotinylated LGP2 RD protein was diluted (50 μg/mL) with assay buffer. ZIKV/TBEV/DENV2 NS5 and HSA proteins were diluted in a series of concentrations (20, 40, 80, 160 nM). Streptavidin-coated biosensors were used to detect the signal by ForteBio Octet RED system. Data were fitted using Prism. Three independent experiments were performed and results from one representative experiment were shown. https://doi.org/10.1371/journal.ppat.1011620.g005 To further verify the interaction of LGP2 with NS5, we purified the RD of LGP2 (LGP2 RD, residues 537–678) [21] as well as ZIKV/TBEV/dengue virus serotype 2 (DENV2) NS5 proteins, and used the biolayer interferometry to measure the equilibrium dissociation constant (K d ) of LGP2 RD and NS5. The association and dissociation processes between immobilized LGP2 RD and flowing ZIKV NS5 (Fig 5D), TBEV NS5 (Fig 5E), DENV2 NS5 (Fig 5F) or human serum albumin (HSA, negative control) (Fig 5G) at a series of concentrations were monitored in real time. ZIKV, TBEV, and DENV2 NS5 produced enough optical response to indicate the interaction with LGP2 RD, while HSA did not. The K d values were 88±11 nM, 1.2±0.8 μM, and 75±14 nM for RD-ZIKV NS5, RD-TBEV NS5, and RD-DENV2 NS5 interactions, respectively. These results again support direct interaction between LGP2 RD and flavivirus NS5.

LGP2 inhibits RdRP activity of NS5 To understand the impact of LGP2 RD on the polymerase function of NS5, we carried out an in vitro RdRP assay to dissect the underlying mechanisms following methods established in our DENV2 and TBEV NS5 studies, and we chose DENV2 instead of ZIKV because we have established RdRP assays in the former [46,47]. LGP2 RD was provided at different molar ratios to NS5 and a pre-incubation was performed to ensure its binding to NS5. Then, ATP and UTP were provided as the only NTP substrates for the production of 9-mer product (P9) directed by a T30/P2 RNA construct comprising a 30-mer template (T30) and a G-G dinucleotide primer with a 5′-monophosphate (P2) (Fig 6A). We performed the in vitro RdRP assay as shown in Fig 6B. The results indicate that the amount of P9 products synthesized by NS5 is decreased when LGP2 RD is added and is positively correlated with the amount of LGP2 RD added for both DENV2 (Fig 6C, compare lanes 7–8, 13–14 to 10–11, 16–17, respectively) and TBEV NS5 (Fig 6D, compare lanes 1, 3, 7–9, 13–15 to 4, 6, 10–12, 16–18, respectively). Intercellular adhesion molecule 1 (ICAM-1) acting as the negative control protein can’t inhibit P9 products synthesized by DENV2 NS5 (S5A Fig). These data suggest that LGP2 RD can inhibit polymerase activities of DENV2 and TBEV NS5. Since LGP2 is likely not very abundant in viral RC, the relatively high molar ratio (5:1) of LGP2 RD:NS5 required for observation of the inhibitory effect in DENV2 highlights the difference between the RdRP assay and in vivo situation, with respect to components involved in the RNA synthesis and solution conditions. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. LGP2 RD inhibits NS5 RdRP activities. (A) A diagram of T30/P2 RNA construct used in NS5 polymerase assay and the reaction scheme to synthesize a 9-mer product (P9). (B) The reaction flow chart of P2-to-P9 conversion. (C-D) Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the P2-to-P9 conversion by DENV2 NS5 (C) and TBEV NS5 (D) in the absence (Mock) or presence (LGP2 RD) of LGP2 RD. Top panels: Representative gel images. M: marker, a chemically synthesized 8-mer RNA (P8). This RNA migrates slower than the P9 product bearing a 5′-phosphate as documented in a previous study [46]. Bottom panels: The intensity values were acquired by ImageJ and the relative intensity of P9 at 10 min in the absence of LGP2 RD was set to 1.0. Mean intensities and standard deviations were derived from three independent experiments (Student’s t-test; ns: no significant difference, *: p<0.05, **: p<0.01). https://doi.org/10.1371/journal.ppat.1011620.g006

LGP2 inhibits flavivirus RdRP in pre-elongation stages To further dissect the mechanism of LGP2 RD inhibition of NS5 RdRP synthesis, we investigated LGP2 RD effect at polymerase pre-elongation and elongation stages in two different assays. In the DENV2 system, an elongation complex (EC) is formed upon the synthesis of the P9 product using the T30/P2 construct [46,48]. This P9-containing EC (EC9) is largely insoluble under low-salt condition (e.g., 20 mM NaCl) optimal for RdRP initiation, but becomes much more soluble under high-salt condition (e.g., 200 mM NaCl). Therefore, we removed ATP and UTP used in EC9 assembly by centrifugation and pellet wash. Then, EC9 in the pellet was resuspended in a high-salt buffer, and CTP was added to allow single-nucleotide addition to synthesizing a 10-mer product (P10) (S6A and S6B Fig). The P9-to-P10 conversion thus allows the probing of polymerase elongation activities. With LGP2 RD provided at a 5:1 molar ratio to NS5, we did not observe inhibitory effect of LGP2 RD in P10 production (S6C Fig). These data therefore suggest that LGP2 RD may not interfere with RdRP elongation process. We then carried out an assay to study whether LGP2 RD can impair pre-elongation activities of NS5 RdRP. Same as above, LGP2 RD at different molar ratio to NS5 was pre-incubated with NS5 before the only NTP substrate, ATP, was added. A 3-mer product (P3) was synthesized, as directed by the T30/P2 construct (Fig 7A). We performed the in vitro pre-elongation activity assay as shown in Fig 7B and observed that the accumulation of P3 products was reduced when LGP2 RD was added for both DENV2 (Fig 7C, compare lanes 1, 3, 7–9, 13–15 to 4, 6, 10–12, 16–18, respectively) and TBEV NS5 (Fig 7D, compare lanes 2–3, 7–8, 14–15 to 5–6, 10–11, 17–18 respectively). ICAM-1 acting as the negative control protein cannot inhibit P3 production by DENV2 NS5 (S5B Fig). Since P2-to-P3 conversion is a multiple turnover process, NS5-RNA rebinding could still occur even if a pre-incubation is carried out. Hence, LGP2 RD impacting on P2-to-P3 conversion cannot be attributed solely to its regulation on RdRP initiation or on NS5-RNA binding. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. LGP2 inhibits flavivirus RdRP in pre-elongation stages. (A) A diagram of construct T30/P2 used in the P2-to-P3 conversion assay and the reaction scheme. (B) The reaction flow chart of P2-to-P3 conversion. (C-D) Denaturing PAGE analysis of the P2-to-P3 conversion by DENV2 NS5 (C) and TBEV NS5 (D) polymerase assays in the absence (Mock) or presence (LGP2 RD) of LGP2 RD. Representative gel images and the band intensity quantitation were shown as in Fig 6. M: marker, a mixture of chemically synthesized 10-mer (5′-hydroxyl), 3-mer (5′-phosphate) and 2-mer (5′-phosphate) RNAs (P10, P3 and P2). The intensity values were acquired by ImageJ and the relative intensity of P3 at 10 min in the absence of LGP2 RD was set to 1.0. Mean intensities and standard deviations were derived from three independent experiments (Student’s t-test; ns: no significant difference, *: p<0.05, **: p<0.01). https://doi.org/10.1371/journal.ppat.1011620.g007

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011620

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.