Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (21): 4440-4448.doi: 10.3864/j.issn.0578-1752.2020.21.012

• PLANT PROTECTION • Previous Articles     Next Articles

An in vitro Cap-Snatching System of Rice Stripe Tenuivirus Based on Crude Virion Preparations

LIN WenZhong(),WU Ran,JIN Jing,QIU Ping,ZHANG Jie,WU ZuJian(),DU ZhenGuo()   

  1. Plant Virus Research Institute, Fujian Agriculture and Forestry University/State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fuzhou 350002
  • Received:2020-06-15 Accepted:2020-07-13 Online:2020-11-01 Published:2020-11-11
  • Contact: ZuJian WU,ZhenGuo DU E-mail:linwenzhong1991@qq.com;wuzujian@126.com;duzhenguo1228@163.com

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Abstract:

【Background】Viruses of the order Bunyavirales and the family Orthomyxoviridae cleave host cellular mRNAs 10-20 nucleotides downstream of the cap structure and use the 5′ terminal cleavage product as a primer to initiate the transcription of their template RNAs. This process is called cap-snatching, the result of which is that all viral mRNAs contain a host-derived capped-RNA leader (CRL). Rice stripe tenuivirus (RSV) is a plant-infecting bunyavirus for which the mechanism of cap-snatching remains poorly characterized.【Objective】The objective of this study is to establish a convenient in vitro system to dissect the cap-snatching mechanisms of RSV, this study is designed to test whether crude preparations of RSV snatch CRLs from exogenously supplied mRNAs.【Method】Crude preparations of RSV were obtained by PEG precipitation combined with differential centrifugation. The protein components of the preparations were analyzed by SDS-PAGE and mass spectrometry. After adding rabbit reticulocyte lysate (RCL) rich in globin mRNAs, the preparations were used to establish an in vitro cap-snatching reaction mix. Total RNA was extracted from the reaction mix at the end of the reaction. RSV mRNAs with CRLs obtained from globin-α mRNAs were detected using nested RT-PCR. The products of the nested RT-PCR were cloned, and the sequences were analyzed.【Result】Two mL of crude RSV preparations was obtained from 100 g of virus-infected rice. The preparations contain chloroplast proteins including ribulose-1,5-bisphosphate carboxylase/oxygenase in addition to RSV virions. A 20 μL reaction system containing 2 μL RSV crude preparations, 8 μL RCL, 4 mmol·L-1 MgCl2, 2 mmol·L-1 of each NTP and 0.8 U·μL-1 RNase inhibitor was prepared and incubated at 30℃ for 1.5 h. Clearly discernable bands with expected sizes were obtained in the nested RT-PCR, indicating that RSV crude preparations can cleave globin-α mRNAs and use their CRLs. After adding the cap analogue m7G (5′) ppp (5′) G, the brightness of the bands was decreased and the extent of the decrease correlated with the concentration of the cap analogue, indicating that cap binding is essential for the cleavage. PCR bands with expected sizes were recovered and sequenced after cloning. Sequence analysis revealed a scenario reminiscent of that seen in vivo: RSV cleaves globin-α mRNAs after A or C to obtain CRLs capable of base-pairing with its template RNAs. In initiating transcription with these CRLs, RSV uses the prime-and-realign mechanism frequently and has a greater tendency to use this mechanism in synthesizing nucleoprotein gene (NP) than in synthesizing non-capsid protein gene (NCP) mRNAs.【Conclusion】The crude preparations of RSV can snatch CRLs from exogenously added mRNAs and use the CRLs to prime transcription in a way just like it does in vivo. Therefore, RSV crude preparations can be used to dissect the cap-snatching or the transcription of RSV.

Key words: rice stripe tenuivirus (RSV), cap-snatching, in vitro transcription

Table 1

The reagents and kits used in this study"

试剂名称
Reagents and kits		 来源
Manufacture		 用途
Usage
RCL（未经核酸酶处理
Not treated with nuclease）		 Promega 体外“抓帽”体系
In vitro cap-snatching
RNase inhibitor Promega
NTP (100 mmol·L-1 each) TaKaRa
m7G (5′) ppp (5′) G NEB
Prestained Protein Marker GenStar SDS-PAGE
RNA purification Kit (DP412) TIANGEN RSV mRNA的检测与克隆
Detection and cloning of RSV mRNAs
1st Strand cDNA Synthesis Kit Vazyme Biotech
2×ExTaq Mix TaKaRa
HiPure Gel Pure DNA Mini Kit Magen
pMD19-T TaKaRa

Fig. 1

Schematic diagram showing the nested RT-PCR used to detect RSV NP (transcribed from vRNA3) and NCP (transcribed from vcRNA4) mRNAs with capped-RNA leaders obtained from globin-α mRNA"

Fig. 2

SDS-PAGE detection of the RSV crude preparations The 4 protein bands subjected to mass spectrometry"

Fig. 3

The cap-snatching of crude RSV preparations from exogenously supplied mRNAs M：DNA marker。A：Untreated or heat inactivated RSV crude preparations were used in the in vitro cap-snatching assay. Nested RT-PCR was used to detect RSV NCP (lanes 1 and 3) or NP (lanes 2 and 4) mRNAs with capped-RNA leaders derived from globin-α. RT-PCR was used to detect a NP-corresponding region on RSV vRNA3 (lanes 5 and 7) or a NCP-corresponding region on RSV vcRNA4 (lanes 6 and 8);B：The cap analog m7G (5′) ppp (5′) G was added at a final concentration of 0.5, 1 or 1.5 mmol·L-1. Nested RT-PCR was used to detect RSV NCP with capped-RNA leaders derived from globin-α (lanes 1 to 4) and RT-PCR was used to detect a NCP-corresponding region on RSV vcRNA4"

Table 2

RSV NP mRNAs transcribed using capped-RNA leaders snatched from globin-α mRNA as primers"

行数
Row		 珠蛋白-α mRNA序列Globin-α mRNA sequence
m7G-ACACUUCUGGUCCAGUCCGACUGAG-		 克隆数
Clone number		 引发与重配
Priming and realignment
1 m7G-ACACUUCUGGUC12------------ACACAAAGUC- 1 +
2 m7G-ACACUUCUGGUCCAGUCC18------ACACAAAGUC- 7 +
3 m7G-ACACUUCUGGUC12-----AC-----ACACAAAGUC- 1 +
4 m7G-ACACUUCUGGUC12----ACAC----ACACAAAGUC- 1 +
5 m7G-ACACUUCUGGUC12-ACACACACAC-ACACAAAGUC- 3 +
6 m7G-ACACUUCUGGUCCA14----------CACAAAGUC- 5 -
7 m7G-ACACUUCUGGUCCA14------------CAAAGUC- 1 -

Table 3

RSV NCP mRNAs transcribed using capped-RNA leaders snatched from globin-α mRNA as primers"

行数
Row		 珠蛋白-α mRNA序列Globin-α mRNA sequence
m7G-ACACUUCUGGUCCAGUCCGACUGAG-		 克隆数
Clone number		 引发与重配
Priming and realignment
1 m7G-ACACUUCUGGUC12------------ACAAAGUC- 12 -
2 m7G-ACACUUCUGGUC12----------ACACAAAGUC- 3 +
3 m7G-ACACUUCUGGUC12--AC------ACACAAAGUC- 3 +
4 m7G-ACACUUCUGGUCCA14-----------CAAAGUC- 7 -
5 m7G-ACACUUCUGGUCCA14---------CACAAAGUC- 1 +
6 m7G-ACACUUCUGGUCCA14-C------ACACAAAGUC- 2 +

Fig. 4

Possible mechanisms by which RSV produces its own mRNAs using capped-RNA leaders snatched from globin-α mRNA A：Capped-RNA leaders base pair with the G2 of RSV template RNA;B：Capped-RNA leaders base pair with the U1 of RSV template RNA;C：Capped-RNA leaders base pair with the G2U3 of RSV template RNA"

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