In vitro replication system mediated by NIb of Potato virus Y

doi:10.21203/rs.3.rs-3126691/v1

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In vitro replication system mediated by NIb of Potato virus Y

https://doi.org/10.21203/rs.3.rs-3126691/v1

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Potato virus Y (PVY) is a serious global virus. At present, PVY are mainly focused on strain analysis and protein interaction with host, but independent studies on PVY replication in vitro are still weak, the main problem is that there is no independent in vitro replication system for PVY. This study was focused on the creation of in vitro replication system mediated by NIb of PVY. The optimal expression concentration of MBP-NIb is 0.2 mM by the IPTG gradient experiment. Based on the solubility analysis, it is suggested that 37℃ is feasible and the soluble ratio of MBP-NIb is about 10%, which is enough for following affinity chromatography against MBP-tag. The purified MBP-NIb can specifically recognize the 3' terminal region of PVY plus or minus strand and perform in vitro replication. This is the first time that an in vitro replication system for PVY has been established. This in vitro replication system will facilitate the study of mechanism on PVY replication.

Potato virus Y

In vitro replication

NIb

RNA-dependent RNA polymerase

In vivo replication of RNA viruses is accomplished by multi-subunit replicase complexes, which consist of viral and host proteins (1–3). The core catalytic subunit of the replicase complexes is a RNA-dependent RNA polymerase (RdRp) encoded by RNA viruses, which specifically locate to and contact with the 3'-end of the viral template facilitating the replication of complementary strand (4, 5). For identifying the detailed mechanism how RdRp perform the replication of RNA viruses, in vitro replication systems specific to given RNA viruses have been set up using purified RdRp from viral-infected cells or heterologous expression systems (6–11).

The complete life cycle of plant RNA virus in host requires different processes, such as virus particle decapitation, virus protein translation, virus genome replication, virus particle assembly and so on. For a particular population of viruses in the host, these processes are almost simultaneous and can not be strictly distinguished. Therefore, a specific process of RNA virus needs to be studied qualitatively or quantitatively, it is necessary to establish an independent in vitro research system, such as in vitro replication, in vitro translation, in vitro packaging and so on. In vitro replication systems have been established for several plant RNA viruses (such as TVMV, TCV, CMV, TBSV, TBTV and so on.) (6, 9, 12), Combining mutation analysis and RNA structure analysis in vitro, we can locate the core RNA regulatory elements (such as TLS at the 3' end of CMV, PR at the 3' end of TCV) and the core amino acids in RdRp (13, 14). At the heart of establishing an in vitro replication system against plant RNA viruses is the acquisition of an active virus-encoded RdRp, a central component of the replicase complex required for plant RNA viruses to replicate their genomes in plants (1, 9). Active RdRps are generally obtained by 2 methods: heterologous expression systems such as protein purification after prokaryotic expression (6, 9, 15), and direct extraction from virus-infected plant cells or tissues (16, 17) .

Potato virus Y (PVY), a member of the genus Potyvirus in the family Potyviridae, is 3' polyadenylated and contains a 5' genome-linked protein VPg, which can infect plants comprising 495 species in 72 genera of 31 families (18), is the most prevalent virus infecting potato and tobacco. It can be transmitted mechanically, by aphids in a non-persistent manner, and by seed potato (19, 20). The genome of PVY is a positive-sense single-stranded RNA of 9700 nucleotides and contains a open reading frames (ORFs) (21). The ORF encodes a large polyprotein that can be cleaved by three self-encoded proteinases into 10 mature proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP. The additional P3N-PIPO protein may be produced by a transcriptional frameshift strategy (22). NIb protein has the characteristic of RdRp and may be involved in the pathogenicity of PVY (4). In addition, the pathogenicity of RNA viruses is the combined consequence of viral genome replication, viral protein translation, cell-to-cell and long distance movement and even viral particle packaging. In order to identify whether NIb of PVY is responsible for the replication of PVY genome and how perform the replication, an in vitro replication system need to be built up.

In this study, NIb fused with MBP-tag was expressed from prokaryotic system and purified through affinity chromatography against MBP-tag. The purified MBP-NIb specifically recognized the 3' region of PVY plus or minus strand and catalyzed in vitro synthesis of the complementary strand.

2.1 Construction of prokaryotic expression vectors containing PVY NIb and VPg

The primers PVY-NIb-BamH1-F (Supplement Table 1) and PVY-NIb-Sal1-R (Supplement Table 1) were designed based on PVY-GZ isolate (GenBank. MN381731) to amplify the NIb coding sequences of PVY GZ isolate.

The 100 µL PCR reaction systems consist of 10 µL 10× PCR buffer (Mg^2་ plus), 2 µL dNTP (each 2.5 mmol/L), 2 µL PVY-NIb-BamH1-F and 2 µL PVY-NIb-Sal1-R, 1 µL pPVY-GZ (Dong, 2004), 1 µL Taq DNA polymerase (5U/µL). Then ddH₂O was added to make a final reaction volume of 100 µL. The PCR reaction process was pre-denaturation at 94 ℃ for 5 min, followed by 30 cycles reaction including denaturation at 94 ℃ for 40 s, annealing at 55 ℃ for 40 s, and extension at 72 ℃ for 2 min, and a final extension at 72 ℃ for 10 min.

The PCR products were recovered by DNA recovery kit, digested with BamH I and Sal I, followed by ligation with the same enzymes digested pMAL-C2X (NEB) under the action of T4 DNA ligase (Takara) at 16 ℃for at least 8 hrs. The ligation products were transformed into E. coli DH5α. The positive clones of pMAL-PVY-NIb were identified by colony PCR, restriction enzyme digestion and final DNA sequencing.

Prokaryotic expression vectors containing PVY VPg was constructed as pMAL-PVY-NIb. Detailed information of primers is listed in Supplement Table 1.

2.2 Prokaryotic expression of PVY NIb and VPg

The prokaryotic expression plasmids containing PVY NIb and VPg were respectively transformed into competent cells of E. coli Rosetta and plated with LB plates containing 100 mg / mL ampicillin. The Rossetta strain containing the prokaryotic expression plasmids were identified by colony PCR and then incubated overnight with 200 rpm shaking at 37 ℃. The overnight culture was added into 30 mL of fresh LB broth at a ratio of 1: 100 and incubated at 37 ℃ until OD₆₀₀ to 0.4–0.6. The cultures were placed on ice for 10 min and then distributed to finger tubes at 3 mL/ tube. IPTG was added to make a final concentration of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM and 2.0 mM, respectively. All tube were then incubated at 37 ℃ with 200 rpm for 4 h. 1mL culture from different tubes were collected at 13000 rpm for 30 s. 100 µL ddH₂O was added to suspend the supernatant through vortexing, and then an equal volume of 2×SDS protein loading buffer was added. Boiling treated protein samples were separated using 12.5% SDS-PAGE gel, followed by staining with coomassie blue and decolorization with acetic acid to detect the expression pattern under the induction of different concentration of IPTG.

2.3 Solubility Analysis of Prokaryotic Expression Proteins

According to the above steps of prokaryotic expression, 100 mL culture was incubated at 37 ℃ with the induction of optimal IPTG. Then 100 mL cultures were concentrated and resuspended using 10 mL of protein buffer (20 mM Tris-Cl(pH 8.0), 25 mM NaCl, 1 mM EDTA(pH 8.0), 10 mM β-mercaptoethanol, 10% glycerol). The iced protein samples were then treated using ultrasonic processing at the following condition: 400 W, 100 times with working 4s and interval 4 s. The ultrasonic treated samples were separated into supernatant and sediment by centrifugation with 13000 rpm for 15 min at 4℃. 100 µL of supernatant was mixed with 100 µL of 2×SDS protein loading buffer. Sediment was suspended using 200 µL of 1×SDS protein loading buffer. Supernatant and sediment samples were detected using SDS-PAGE electrophoresis.

2.4 Purification of MBP-Fused Protein by Affinity Column Chromatography

Protein fused with MBP was purified though amylose resin by affinity column chromatography. For ensuring the acceptable solubility of MBP-NIb, 500 mL culture was incubated at 37℃ with the induction of 0.2 mM IPTG. All culture was centrifuged and treated by ultrasonic processing as shown in 2.3. With a 0.45 µm water-based membrane filter, the supernatant was filtered into a 50 ml microcentrifuge tube, which was placed on ice waiting for affinity column chromatography.

Affinity column chromatography was performed at 4 ℃. 2 mL amylose resin (NEB E8021S) was added into the column with the package buffer running out. Then at least 50 mL protein buffer was used to balance the column. Protein samples was added into the balanced column and went through the column through gravity action. After loading of all proteins, at least 50 mL protein buffer was used to wash the column. Finally, 10 mL elution buffer (20 mM Tris-Cl(pH 8.0), 25 mM NaCl, 1 mM EDTA(pH 8.0), 10 mM β-mercaptoethanol, 10% glycerol, 10mM maltose)was used to elute the MBP-fused protein. All eluted products were collected using numbered tubes followed by SDS-PAGE electrophoresis to detect the quantity and quality of the MBP-NIb.

2.5 In vitro Transcription with T7 RNA Polymerase

With the plasmid as template, PCR was performed to amplify different PCR fragments corresponding 5' terminal regions, internal regions and 3' terminal regions of PVY-GZ plus or minus strand. Detailed information about primers and corresponding fragments were listed in Supplement Table 1.Purified PCR products were used to do in vitro transcription with T7 RNA polymerase (NEB). The 100 µL reaction consists of DNA template 2 µg, 100 mM DTT 10 µL, 20 mM ATP 2.5 µL, 20 mM GTP 2.5 µL, 20 mM CTP 2.5 µL, 20 mM UTP 2.5 µL, T7 RNA polymerase 5µL, 10X T7 buffer 10 µL and ddH₂O. In vitro transcription was performed at 37 ℃ for 2 hrs. The target RNAs were separated through 1.5% agarose gel electrophoresis and purified by glass wool.

2.6 In vitro replication reaction

The 50 µL reaction systems consist of 1 µg of RNA templates, 1 M Tris-Cl (pH 8.2) 5 µL, 1 M MgCl₂ 0.5 µL, 1 M DTT 0.5 µL, 1 M KCl 5 µL, 10 mg/mL yeast tRNA 1.75 µL, 20 mM ATP 2.5 µL, 20 mM GTP 2.5 µL, 20 mM CTP 2.5 µL, 1 mM UTP 0.5 µL, α-³²P-UTP 0.5 µL, 12.5 µg of tested protein and ddH₂O. In vitro replication reaction was performed at 20 ℃ for 1.5 h. After the reaction, 70 µL ddH₂O and 120 µL of phenol/chloroform were added followed by centrifugation at 13000 rpm. The supernatant was precipitated by adding 2.4 times volume of NH₄Ac / isopropanol (5M NH4Ac: isopropanol = 1: 5) and placed at -80 ℃ for at least 2 h. The precipitation was resuspended using 10 µL RNA loading buffer and separated in 5% PAGE gel containing 8M urea with 1500 mA for 1.5 h. Then, the gel was dried and exposed to a phosphorimager screen, followed by detection with the Typhoon FLA-7000(GE Healthcare).

3.1 Construction of prokaryotic expression vector pMAL-PVY-NIb

With the plasmid pPVY-GZ as template, 1.6 kb PCR products were amplified using primers PVY-NIb-BamH1-F and PVY-NIb-Sal1-R (Fig. 1A). The size of this PCR product is consistent with the length of the coding sequence of PVY NIb (1557 nt). Both the purified PCR fragments and pMAL-C2X were digested with BamH I and Sal I and ligated. After ligation, transformation and colony PCR, positive plasmid was digested to confirm the insertion of NIb coding sequences into pMAL-C2X. After digestion with BamH I and Sal I, plasmid produced 6.0 kb and 1.6 kb of fragments (Fig. 1B), which correspond with basic vector and NIb coding fragment, respectively. The positive plasmid pMAL-PVY-NIb was also confirmed by DNA sequencing.

3.2 Optimization of Prokaryotic expression of MBP-NIb fusion protein

Compared with the negative control, different concentration (0.2 to 2.0 mM) of IPTG can induce the expression of MBP-NIb fused protein with the molecular weight of about 100 kD (Fig. 2), which is consistent with the sum of the molecular weight of MBP ( 40 kD) and NIb (60 kD). Moreover, it is suggested that 0.2 mM IPTG is optimal to induce the expression of MBP-NIb at 37℃ (Fig. 2).

Solubility analysis was also performed to identify the 37℃of prokaryotic expression, which could ensure the acceptable solubility of MBP-NIb to meet the requirement of affinity chromatography. At 37℃, the ratio of soluble MBP-NIb is about 10% (Fig. 3), which could meet the requirement of affinity chromatography. In addition, it can as far as possible ensure the activity of purified proteins due to the avoidance of extra process of the denaturation.

3.3 PVY NIb-mediated in vitro replication system

To identify the activity and specificity of purified MBP-NIb through affinity chromatography, in vitro replication reaction was performed using 6 different RNA fragments as template. For PVY plus strand, MBP-NIb can specifically recognized the 3' fragment, subsequently catalyzed the corresponding transcription of complementary strands, while MBP-NIb did not catalyze the in vitro replication of 5' fragment and internal fragment (Fig. 4). For PVY minus strand, MBP-NIb can specifically recognized the 3' fragment in the presence of Vpg, subsequently catalyzed the corresponding transcription of complementary strands, while MBP-NIb did not catalyze the in vitro replication of 5' fragment and internal fragment in the presence of Vpg, and MBP-NIb can't specifically recognized the 3' fragment in the absence of Vpg. In addition, MBP did not catalyze the in vitro replication of the 3' fragment of PVY plus or minus strand (Fig. 4). It is suggested that NIb of PVY specifically preformed in vitro replication of the 3' fragment of PVY.

The core component for setting up in vitro replication system is the replicase, which can be obtained from viral-infected cells or heterologous expression systems (6–9). Replicase purified from viral-infected cells possess the characteristic of posttranslational protein-modification such as phosphorylation, glycosylation and ubiquitination, while replicase proteins purified from heterologous expression systems such as E.coli easily possess high purity. In this study, we obtained the RdRp (NIb protein) from E.coli through prokaryotic expression. The whole process is easily handled and repeated, meanwhile the purified RdRp presented the catalytic activity and RNA specificity (Fig. 4).

PVY was first discovered in potato in 1931, and has been spreading worldwide (23). Much research has been done on the pathogenesis of PVY (24, 25), However, the detailed mechanism how PVY perform the genome replication is unclear. In this study, purified MBP-NIb presented higher specificity on 3' fragment than other fragments of PVY (Fig. 4), which is also shown in research of other viruses, But most of these studies focus on the transcription of sense to antisense strands, and there is relatively little research on the transcription of sense from antisense strands (10, 11, 26), In this study, we established for the first time an experimental system for the mutual transcription of PVY positive and negative sense strands in vitro (Fig. 4). For picornaviruses, VPg serves as a primer to synthesize the nascent viral RNA and exists at the 5′ end (27). In this study, for PVY minus strand, NIb can specifically recognized the 3' fragment in the presence of Vpg, subsequently catalyzed the corresponding transcription of complementary strands, while NIb can't specifically recognized the 3' fragment in the absence of Vpg. However, for PVY plus strand does not require the Vpg, which is different from animal RNA viruses, and may be caused by the difference between plant and animal replication systems.This also explains how Vpg is added to the 5' end of the plus strand. Using this in vitro replication system, further research will be performed on the core elements involved in replication of PVY.

PVY

Potato virus Y

TVMV

Tobacco vein mottling virus

TCV

Turnip mosaic virus

CMV

Cucumber mosaic virus

TBSV

Tomato bushy stunt virus

TBTV

Tobacco bushy top virus

TLS

tRNA-like Structure

proximal hairpin

MBP

maltose binding protein

IPTG

Isopropyl β-D-Thiogalactoside

Luria-Bertani

RdRp

RNA-dependent RNA polymerase

VPg

Viral Protein Genome—linked

UTR

untranslated region

E.coli

Escherichia coli.

Acknowledgments

We are grateful to Prof. Xiangdong Li and Prof. Xuefeng Yuan (Shandong Agricultural University) for providing the plasmid pPVY-GZ.

Funding

This work was funded by National Natural Science Foundation of China (32100132), the Shandong Province Natural Sciences Foundation of China (ZR2021QC008), and “Youth Innovation team program” in College of Shandong province of China (2022KJ119).

Author Contributions

JF contributed to prokaryotic expression and in vitro replication assay. ZL contributed to construction of prokaryotic expression vectors and data analysis. ML contributed to the data analysis and drafting the manuscript. GG contributed to the design of the study, data analysis and drafting the manuscript. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Guowei Geng.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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In vitro replication system mediated by NIb of Potato virus Y

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Construction of prokaryotic expression vectors containing PVY NIb and VPg

2.2 Prokaryotic expression of PVY NIb and VPg

2.3 Solubility Analysis of Prokaryotic Expression Proteins

2.4 Purification of MBP-Fused Protein by Affinity Column Chromatography

2.5 In vitro Transcription with T7 RNA Polymerase

2.6 In vitro replication reaction

3. Results

3.1 Construction of prokaryotic expression vector pMAL-PVY-NIb

3.2 Optimization of Prokaryotic expression of MBP-NIb fusion protein

3.3 PVY NIb-mediated in vitro replication system

4. Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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