量子点标记的流感病毒样颗粒用于病毒脱壳过程的研究

Chong Qin; Xiaowei Zhang; Zongqiang   Cui

doi:10.21769/BioProtoc.3366

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Peer-reviewed

Production of Quantum Dots-containing Influenza Virus Particles for Studying Viral Uncoating Processes

量子点标记的流感病毒样颗粒用于病毒脱壳过程的研究

CQ Chong Qin

XZ Xiaowei Zhang

Zongqiang Cui email

发布: 2019年09月20日第9卷第18期 DOI: 10.21769/BioProtoc.3366 浏览次数: 5800

评审: George William CarnellWelsch Charles JeremyAnonymous reviewer(s)

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Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

Feb 2019

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Abstract

The genome of influenza A virus (IAV) comprises eight pinlike genomic segments called vRNPs enclosed in viral capsid. During infection, uncoating is the key step for viral replication and represents an antiviral therapeutic target, but it is difficult to observe the transient and dynamic event in detail. Here, we report a protocol for production of quantum dots-containing influenza virus particles by encapsulating quantum dot-conjugated vRNPs during viral assembly. These labeled virions can be used for monitoring viral trafficking in real time and studying viral uncoating processes.

Keywords: Influenza A virus (甲型流感病毒)

vRNP (vRNP)

Quantum dots (量子点)

Viral assembly (病毒装配)

Uncoating (脱壳)

Background

Influenza A virus (IAV) is one of the most important human pathogens. Viral entry before virus-endosome fusion has been widely studied by various methods, including single-particle tracking. However, the viral uncoating process following endocytosis remains elusive. Robust live imaging strategies that offer high temporal and spatial resolution are essential to study this dynamic and transient event. However, the influenza RNA genome is intolerant to insertion of large genetic materials, and rescue of viruses using fluorescent protein fused to viral genomic core proteins have been limited (Lakdawala et al., 2014). This has delayed progress in the field of influenza-uncoating and vRNPs-dynamics live-imaging studies. Although purified vRNPs have been dye-labeled and microinjected into cells to track their intracellular transportation and nuclear import process in living cells in real time (Babcock et al., 2004), this method cannot capture the real infection process of incoming virions or determine vRNP behaviors during uncoating. Fluorescence in situ hybridization (FISH) and colocalization analyses with single-molecule resolution have also been used to study the behavior of vRNPs in infected cells (Chou et al., 2013), but the fixation procedures are not compatible with imaging vRNP dynamics in living cells. Semiconductor quantum dots (QDs) have unique optical properties, such as remarkable brightness and superior photostability, and are well-suited to single-particle tracking of viral infection. Single-particle tracking of virions with QDs-labeled genetic material provides an opportunity for tracking viral uncoating and post-uncoating genetic behaviors in real time. As a subunit of polymerase complex, PA protein participates in the composition of vRNP-genetic core of IAV. In PNAS, Qin et al. (2019) developed a nanotechnology that labels IAV viral ribonucleoprotein complexes (vRNPs) with QDs. Briefly, the biotin acceptor peptide (AP tag) was genetically fused to the PA protein to construct a recombinant PR8 strain. The rescued rPR8-PA-AP virus was then cultured in BirA-expressing MDCK cells in the presence of biotin. Streptavidin-coated QDs (SA-QDs) were introduced into these cells by lipofection to allow noncovalent binding of the QDs to biotinylated PA. QDs-containing influenza virus particles were thus generated. This approach will advance the mechanistic understanding of influenza virus uncoating using live fluorescence microscopy.

Materials and Reagents

Materials
1. 1.5 ml graduated microcentrifuge tubes (QSP, catalog number: 509-GRD-Q)
2. 2 ml graduated microcentrifuge tubes (QSP, catalog number: 508-GRD-Q)
3. SW32 Ultra-Clear Tubes (Beckman Coulter, catalog number: 344058)
4. SW41 Ultra-Clear Tubes (Beckman Coulter, catalog number: 344059)
5. 0.1-10 µl pipette tips (QSP, catalog number: T104RS-Q)
6. 10-200 µl pipette tips (QSP, catalog number: TTW110RS-Q)
7. 100-1250 µl pipette tips (QSP, catalog number: T112NXLRS-Q)
8. 12-well plates (Corning Costar, catalog number: 3513)
9. 6-well plates (Thermo Scientific, catalog number: 140675)
10. V-bottom 96-well plates (Solarbio, catalog number: YA0590)
11. Scalpel Blade (Hong Yue, catalog number: GC-HY00388)
  (link: https://www.casmart.com.cn/product-details/page/103/11044449)
12. 100 mm Nunc EasYDishes (Thermo Scientific, catalog number: 150464)
13. 75 cm² Nunc EasYFlasks (Thermo Scientific, catalog number: 156472)
14. 25 cm² Nunc EasYFlasks (Thermo Scientific, catalog number: 156340)
15. 0.45 μm filter (Millex-HV, catalog number: SLHU033RB)
16. Filter paper (Biorad, catalog number: 1703932)
17. 3 ml Pasteur pipettes (Nest, catalog number: 318314)
18. 1 ml syringes (Gemtier, catalog number: JT-001)
19. 10 ml syringes (Gemtier, catalog number: JT-010)
20. 15 ml centrifuge tubes (Nest, catalog number: 601052)
21. 50 ml centrifuge tubes (Nest, catalog number: 602052)
Cell lines
1. 293T cells (Boster, catalog number: CX0008; ATCC, catalog number: ACS-4500)
2. MDCK cells (Boster, catalog number: CX0206; ATCC, catalog number: CRL-2936)
Plasmids
Note: All plasmids are available from our lab. BirA is an enzyme that catalyzes ligation of Biotin and AP tag.
1. Eight plasmids (pHW2000) each containing individual cDNA segment of the influenza virus strain A/Puerto Rico/8/34 (H1N1) (Hoffmann et al., 2000)
2. pcDNA3.1(+)-BirA
Primers
Linearized Vector-F: GTGCTACTATTTGCTATCCATACTGTCC
Linearized Vector-R: ACATCTTCTCCAATCTCATCGAGCTCTATC
Insert-F1: GAAAATCGAATGGCACGAATAGTCTAGAAAACTGCTTCTTATCGTTCAGG
Insert-R1: TGGACAGTATGGATAGCAAATAGTAGCAC
Insert-F2: GATATCTTCGAAGCTCAGAAAATCGAATGGCACGAATAGTCT
Insert-R2: TGGACAGTATGGATAGCAAATAGTAGCAC
Insert-F3: GAGTGGTTCAGGAGGCGGTCTGAACGATATCTTCGAAGCTCAGAAAATCGAAT
Insert-R3: TGGACAGTATGGATAGCAAATAGTAGCAC
Insert-F4: GATAGAGCTCGATGAGATTGGAGAAGATGT
Insert-R4: GTTCAGACCGCCTCCTGAACCACTCAATGCATGTGTAAGGAAGGAGTTG
Insert-F5: GATAGAGCTCGATGAGATTGGAGAAGATGT
Insert-R5: TGGACAGTATGGATAGCAAATAGTAGCAC
PA-F: AGCGAAAGCAGGTACTGATCCAAAATG
PA-R: AGTAGAAACAAGGTACTTTTTTGGACAGTATG
Note: All primers were synthesized by Sangon Biotech.
Reagents
1. SPF embryonated chicken eggs (Merial-vital)
2. Competent cells DH5α (TIANGEN, catalog number: CB101-01)
  (Link: http://www.tiangen.com/?productShow/t1/6/id/160.html)
3. 70% ethanol (Amresco, catalog number: E505)
4. 2 kb Plus DNA ladder (TransGen, catalog number: BM121)
5. Ampicillin (MDBio, catalog number: 69-52-3)
6. SA-QD625 (Invitrogen, catalog number: A10196)
7. KOD FX DNA polymerase enzyme (TOYOBO, catalog number: KFX-101)
8. T4 DNA polymerase (NEB, catalog number: M0203S)
9. 50x TAE buffer (Sangon Biotech, catalog number: B548101-0500)
10. Agarose G-10 (BIOWESTE, catalog number: 111860)
11. Omega Cycle-Pure Kit (OMEGA, catalog number: D6492-01)
12. Omega Viral RNA Kit (OMEGA, catalog number: R6874-02)
13. Omega gel extraction kit (OMEGA, catalog number: D2500-02)
14. Omega Plasmid Mini kit (OMEGA, catalog number: D6943-02)
15. One Step SYBR PrimeScript PLUS RT-PCR Kit (TaKaRa, catalog number: RR096A)
16. DMEM (Gibco, catalog number: 11965118)
17. MEM (Genom, catalog number: GNM41500)
18. DMEM Powder (Gibco, catalog number: 12100061)
19. Low gelling temperature Agarose (Sigma-Aldrich, catalog number: A9045)
20. FBS (Gibco, catalog number: 1787602)
21. 7.5% BSA (Solarbio, catalog number: H1130)
22. TPCK-trypsin (Sigma-Aldrich, catalog number: T1426)
23. 1x PBS (Gibco, catalog number: 10010049)
24. Lipofectamine 2000 (Invitrogen, catalog number: 11668019)
25. Biotin (Sigma-Aldrich, catalog number: B4639)
26. Sucrose (HUSHI, catalog number: 10021418)
  (Link: https://www.reagent.com.cn/goodsDetail/94d364146ee0495d955089dbd33dade3)
27. NaCl (HUSHI, catalog number: 10019318)
  (Link: https://www.reagent.com.cn/goodsDetail/d8517dae2fd14870aeeaddbb8b0a13f4)
28. Tryptone (OXOID, catalog number: LP0042)
29. Yeast extract (OXOID, catalog number: LP0021)
30. LB medium with 100 μg/ml ampicillin (see Recipes)
31. LB agar plate with 100 μg/ml ampicillin (see Recipes)
32. Cell culture medium (see Recipes)
33. Infection medium (see Recipes)
34. 0.5% Chicken red blood cell (RBC) (see Recipes)
35. 2x plaque medium (see Recipes)
36. 1.6% agar (see Recipes)
37. Sucrose solution (see Recipes)

Equipment

Eppendorf Pipettes (0.1-10 µl, 10-200 µl, 100-1250 µl)
6 W UV transilluminator (UVP, model: UVLM-26)
Rotating incubator (FUMA, model: QYC-200)
Centrifuge (Eppendorf, model: 5810R)
Ultracentrifuge (Beckman, model: OPTIMA XE-100)
Water bath (YIHENG17, model: DK-8D)
Beckman SW32 rotor
Beckman SW41 rotor
PCR machine (GnenAmp, PCR System 9700)
100 kV Transmission electron microscope (Hitachi, model: H-7000 FA)

Procedure

English

中文翻译

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如何引用

Qin, C., Zhang, X. and Cui, Z. (2019). Production of Quantum Dots-containing Influenza Virus Particles for Studying Viral Uncoating Processes. Bio-protocol 9(18): e3366. DOI: 10.21769/BioProtoc.3366.