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Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System
宿主调控的乙型肝炎病毒衣壳在无哺乳动物细胞系统中的组装   

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Journal of Virology
Jun 2016

Abstract

The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions. We have recently developed a mammalian cell-free system based on the rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological concentrations and assembles into capsids under near-physiological conditions. This system has already revealed HBc assembly requirements that are not anticipated based on previous assembly systems. Furthermore, capsid assembly in this system is regulated by endogenous host factors that can be readily manipulated. Here we present a detailed protocol for this cell-free capsid assembly system, including an illustration on how to manipulate host factors that regulate assembly.

Keywords: Hepatitis B virus (乙型肝炎病毒), Cell-free capsid assembly system (无细胞衣壳组装系统), Rabbit reticulocyte lysate (兔网织红细胞裂解液), Phosphorylation (磷酸化), RNA binding (RNA结合)

Background

The hepatitis B virus (HBV) is an important global human pathogen that chronically infects hundreds of millions of people worldwide and represents a major cause of viral hepatitis, liver cirrhosis, and liver cancer (Seeger et al., 2013; Trepo et al., 2014). HBV replicates its genomic DNA, a relaxed circular, partially duplex DNA (RC DNA), via reverse transcription of an RNA intermediate, the so-called pregenomic RNA (pgRNA), within a nucleocapsid (NC) (Summers and Mason, 1982; Hu and Seeger, 2015; Hu, 2016), which packages a copy of pgRNA together with the virally encoded reverse transcriptase (RT) protein (Bartenschlager and Schaller, 1992; Hu and Lin, 2009). It is within NCs that the RT converts pgRNA into RC DNA.

The icosahedral HBV capsid shell consists of multiple copies of a single viral protein, the HBV core (capsid) protein (HBc). HBc is composed of an N-terminal domain (NTD, aa 1-140) and a C-terminal domain (CTD, 150-183 or 185 depending on strains), which are connected by a linker region (141-149). In heterologous overexpression systems including bacteria and insect cells and in vitro assembly reactions using high concentrations of purified HBc and/or salt, NTD alone, without CTD, is sufficient for capsid assembly; thus it is also called the assembly domain (Gallina et al., 1989; Birnbaum and Nassal, 1990; Lanford and Notvall, 1990; Wingfield et al., 1995). Though not required for capsid assembly in these systems, the highly basic and arginine-rich CTD shows non-specific nucleic acid binding activities (Hatton et al., 1992) and plays important roles in viral RNA packaging (Nassal, 1992), DNA synthesis (Nassal, 1992; Yu and Summers, 1994), and nuclear import of capsids (Liao and Ou, 1995; Liu et al., 2015), all of which is further regulated by the dynamic phosphorylation state of CTD controlled by host factors (Kann and Gerlich, 1994; Kann et al., 1999; Daub et al., 2002; Ludgate et al., 2012; Liu et al., 2015). Whether CTD, and its state of phosphorylation, play a role in capsid assembly under physiological conditions remained unclear.

To study HBV capsid assembly under more physiological conditions, we have recently developed a mammalian cell-free assembly system based on the commonly used mammalian cell extract, rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological (low) concentrations (25-50 nM, monomer) and assembles into capsids under near-physiological conditions (Ludgate et al., 2016). This system allowed us to reveal an unexpected role of CTD in capsid assembly, which is further subjected to regulation by the state of CTD phosphorylation as controlled by endogenous host factors (Ludgate et al., 2016). This protocol is adapted from Ludgate et al. (2016) and more detailed information on this cell-free capsid assembly system is included, and different treatments are applied to address the roles of viral and host factors, such as RNA-binding activities of CTD and host phosphatases, in HBV capsid assembly under near-physiological conditions. This protocol will facilitate detailed studies on capsid assembly and host regulation under physiological conditions and identification of novel antiviral agents targeting HBc.

Materials and Reagents

  1. Pipette tips (Denville Scientific, catalog numbers: P1096-FR , P1121 , P1122 , P1126 )
  2. 1.5 ml microcentrifuge tube (Denville Scientific, catalog number: C2170 )
  3. Gloves and lab coat (Denville Scientific, catalog number: G4162 ; Medline Industries, catalog number: 83044QHW )
  4. Proteinase K, RNA grade (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25530049 )
  5. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509-1KG )
  6. Phenol solution (Sigma-Aldrich, catalog number: P4557 )
  7. Chloroform (Fisher Scientific, catalog number: BP1145-1 )
  8. Sodium acetate (Sigma-Aldrich, catalog number: S2889-1KG )
  9. Ethyl alcohol (EtOH) (AmericanBio, catalog number: AB00515-00100 )
  10. UltraPureTM DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  11. Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15510027 )
  12. TNT® Coupled Rabbit Reticulocyte Lysate (RRL) (Promega, catalog number: L4610 )
  13. EasyTagTM L-[35S]-Methionine (PerkinElmer, catalog number: NEG709A001MC )
  14. RNasin® Plus Ribonuclease Inhibitor (Promega, catalog number: N2611 )
  15. RNaseZapTM RNase Decontamination Solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
  16. RNase A, DNase and protease-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0531 )
  17. NEBuffer 3 (New England Biolabs, catalog number: B7003S )
  18. Alkaline Phosphatase, Calf Intestinal (CIAP) (New England Biolabs, catalog number: M0290S )
  19. Tris Base (Fisher Scientific, catalog number: BP152-10 )
  20. Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: E5134 )
  21. Sodium fluoride (Sigma-Aldrich, catalog number: S7920 )
  22. Sodium pyrophosphate tetrabasic decahydrate (Sigma-Aldrich, catalog number: S6422 )
  23. β-Glycerophosphate (Sigma-Aldrich, catalog number: G6251 )
  24. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  25. cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 04693132001 )
  26. TE buffer (see Recipes)
  27. 10x phosphatase inhibitors (PPI) (see Recipes)
  28. 25x protease inhibitor (see Recipes)

Equipment

  1. Pipettes (Gilson, P1000, P200, P20, P2)
  2. Fume hood (e.g., Protector Xstream Laboratory Hood, Labconco)
  3. 30 °C/37 °C Oven (SciGene, Robbins Scientific, model: Model 400 )
  4. Microcentrifuge (Fisher Scientific, FisherbrandTM, model: accuSpinTM Micro 17 )
  5. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM1000 )

Procedure

  1. Make RNase-free plasmids for in vitro translation in rabbit reticulocyte lysate
    1. Treat plasmid DNA with Proteinase K and SDS to remove any contaminating RNases in plasmid DNA. As shown in the table below, incubate HBc-expressing plasmids with SDS (final concentration 0.6% w/v) and Proteinase K (final concentration 0.4 µg/µl) at 50 °C for 30 min.

      Notes:
      1. Always dilute 20% of SDS in the TE buffer first before adding Proteinase K.
      2. Any plasmid vector containing a bacteriophage promoter (T7 in the example below) that can be used for in vitro transcription using the cognate phage RNA polymerase can be used for coupled transcription and translation in this system. 
    2. Add an equal volume (200 µl) of phenol to the digestion reaction, mix well and spin at the maximal speed (17,000 x g) for 5 min at room temperature. Transfer the aqueous (top) layer to a new tube and repeat this step.
    3. Add an equal volume of chloroform and extract once as described in Step A2.
    4. Measure the aqueous solution and add an appropriate volume of 3 M of sodium acetate (final concentration at 0.3 M) and 100% of ethanol (final concentration at 70%), mix well and incubate at -20 °C for 30 min. Centrifuge at the maximal speed (17,000 x g) for 20 min at 4 °C and wash the pellet 3 times each with 1 ml of 70% ethanol, with spinning at the maximal speed for 10 min at 4 °C.
    5. Resuspend DNA with 30 µl of RNase-free water. Measure DNA concentration with a spectrophotometer (NanoDropTM 1000) and adjust the DNA concentration to 1 µg/µl and then verify DNA integrity by agarose gel electrophoresis.

  2. In vitro translation in rabbit reticulocyte lysate
    A TNT® Coupled Rabbit Reticulocyte Lysate (RRL) in vitro translation system (Promega) is used to express the HBc protein as recommended by the manufacturer.
    1. Preparation of the translation kit
      1. Put TNT® RNA Polymerase (T7) on ice.
      2. Quickly thaw the TNT® Rabbit Reticulocyte Lysate by hand-warming and immediately put on ice.
      3. Thaw the other components at RT (25 °C) and put on ice.
      Notes:
      1. The TNT® Reaction Buffer may contain a precipitate after thawing and sitting on ice. Redissolve the precipitate by vortexing at room temperature for 30 sec.
      2. Do not freeze-thaw the lysate more than twice.
      3. Any unused lysate should be refrozen in a dry ice/ethanol bath as soon as possible after thawing to minimize loss of translational activity.
    2. Assemble the reaction components in a 1.5 ml microcentrifuge tube following the table below.

      Note: Small-scale reactions may be performed by reducing all components proportionally.
    3. Incubate reaction at 30 °C for 90 min.
    4. Put the reactions on ice and proceed to the assembly steps or store the reactions at -80 °C for future use.
      Notes:
      1. Except for the actual translation incubation, all handling of the lysate components should be done at 4 °C or on ice.
      2. A ribonuclease-free environment is required during all of the operations:
        1. Add RNasin® Ribonuclease Inhibitor to all TNT® Lysate reactions to prevent RNA degradation.
        2. Microcentrifuge tubes and pipette tips should be RNase-free.
        3. Use a cleaning agent (such as RNaseZAPTM) to clean the bench and gloves to remove RNase.

  3. In vitro capsid assembly in RRL
    Our recent studies have shown that both HBc NTD and CTD are required for HBV capsid assembly at physiologically low HBc concentrations translated in RRL (Ludgate et al., 2016). CTD likely facilitates assembly under these conditions via RNA binding and protein-protein interactions. Moreover, CTD is subject to phosphorylation and dephosphorylation by endogenous host kinase(s) and phosphatase(s) in RRL, which further regulates capsid assembly.
    To confirm the roles of CTD in capsid assembly in RRL, especially its RNA binding activity and regulation of assembly by phosphorylation state, the following treatments, as shown in Figure 1, are applied to the HBc in vitro assembly assay in RRL.


    Figure 1. Conditions for in vitro capsid assembly in RRL. Mock, the reaction is incubated at 37 °C for 16 h without any treatment. CIAP, CIAP (10 U/µl) is added in the reaction during the assembly incubation. CIAP + RNase, RNase A (0.1 mg/ml) is added and incubated for another 1 h after the CIAP incubation. RNase + CIAP, the reaction is treated with RNase A before the CIAP incubation. PPI, PPI (1x) is added during the assembly incubation. PPI, a mixture of phosphatase inhibitors (see Recipes below), CIAP, calf intestinal alkaline phosphatase.

    1. Mock incubation
      1. Assemble the reaction components in a 1.5 ml microcentrifuge tube following the table below.

        Note: PI, a mixture of protease inhibitors (see Recipes below).
      2. Incubate the reaction at 37 °C for 16 h.
      3. Put the reactions on ice and proceed to the analysis steps or store the reactions at -80 °C for future use.
    2. CIAP incubation
      1. Assemble the reaction components in a 1.5 ml microcentrifuge tube following the table below.

      2. Incubate the reaction at 37 °C for 16 h.
      3. Put the reactions on ice and proceed to the analysis steps or store the reactions at -80 °C for future use.
    3. CIAP followed by RNase incubation
      1. Assemble the reaction components in a 1.5 ml microcentrifuge tube following the table below.

      2. Incubate the reaction at 37 °C for 16 h.
      3. Add 0.1 µl of RNase A (10 mg/ml) to the assembly reaction and incubate at 37 °C for another 1 h.
      4. Put the reactions on ice and proceed to the analysis steps or store the reactions at -80 °C for future use.
    4. RNase followed by CIAP incubation
      1. Assemble the reaction components in a 1.5 ml microcentrifuge tube following the table below.

      2. Incubate the reaction at 37 °C for 1 h.
      3. Add 1 µl of CIAP (10 U/µl) to the assembly reaction and incubate at 37 °C for another 16 h.
      4. Put the reactions on ice and proceed to the analysis steps or store the reactions at -80 °C for future use.
    5. PPI incubation
      1. Assemble the reaction components in a 1.5 ml microcentrifuge tube following the table below.

      2. Incubate the reaction at 37 °C for 1 h.
      3. Put the reactions on ice and proceed to the analysis steps or store the reactions at -80 °C for future use.

Notes

All work with [35S]methionine should be done with appropriate precautions and personal protective equipment like gloves and lab coat during all the operations. It is recommended to open the stock in a fume hood as [35S]methionine is volatile. Tools and equipment, such as PIPETMAN, centrifuge and incubators should be checked for contamination after using [35S]methionine.

Data analysis

The expression of HBc and capsid assembly in this cell-free system can be examined using SDS-PAGE, agarose gel electrophoresis, and Western blot, as shown in Figure 2 and Ludgate et al., 2016.


Figure 2. HBV capsid assembly in RRL under different conditions. The WT HBc proteins were translated in RRL, and the translation reaction mixtures were resolved by agarose gel (1%) electrophoresis (A) or SDS-PAGE (12.5%) (B) without any further treatment (Input) (lane 1) or were treated with NEBuffer 3 alone overnight at 37 °C (Mock) (lane 2), with NEBuffer 3 plus CIAP overnight at 37 °C (CIAP) (lane 3), with NEBuffer 3 plus CIAP overnight at 37 °C followed by RNase treatment for one additional hour (CIAP + RNase) (lane 4), with RNase for 1 h followed by NEBuffer 3 plus CIAP overnight at 37 °C (RNase + CIAP) (lane 5), or with the mixture of phosphatase inhibitors overnight at 37 °C (PPI) (lane 6). All lanes contained 2 µl translation products. 35S-labeled HBc proteins were detected by autoradiography. C, HBc subunits; C-deP, dephosphorylated HBc subunits; Ca, capsids. (Adapted from Ludgate et al., 2016)

Recipes

  1. TE buffer
    10 mM Tris-HCl pH 8.0
    1 mM EDTA
  2. 10x phosphatase inhibitors (PPI)
    100 mM sodium fluoride
    500 mM β-glycerophosphate
    100 mM sodium pyrophosphate
    20 mM sodium orthovanadate
  3. 25x protease inhibitor
    1 tablet of cOmpleteTM EDTA-free Protease Inhibitor Cocktail dissolved in 2 ml of DNase/RNase free dH2O

Acknowledgments

This protocol is adapted from Ludgate et al., 2016. This work was supported by a Public Health Service grant (R01 AI043453 to J.H.) from the National Institutes of Health. K. L. is supported by a Scientific Research Staring Foundation (15042169-Y) from Zhejiang Sci-Tech University. The authors declare no conflicts of interest.

References

  1. Bartenschlager, R. and Schaller, H. (1992). Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J 11(9): 3413-3420.
  2. Birnbaum, F. and Nassal, M. (1990). Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol 64(7): 3319-3330.
  3. Daub, H., Blencke, S., Habenberger, P., Kurtenbach, A., Dennenmoser, J., Wissing, J., Ullrich, A. and Cotton, M. (2002). Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein. J Virol 76: 8124-8137.
  4. Gallina, A., Bonelli, F., Zentilin, L., Rindi, G., Muttini, M. and Milanesi, G. (1989). A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid particles but fails to bind nucleic acids. J Virol 63: 4645-4652.
  5. Hatton, T., Zhou, S. and Standring, D. (1992). RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their role in viral replication. J Virol 66: 5232-5241.
  6. Hu, J. (2016). In: Hepatitis B virus in human diseases. In: Liaw, Y. F. and Zoulim, F. (Eds.). Humana Press, Springer Chap. 1, pp: 1-34.
  7. Hu, J. and Lin, L. (2009). RNA-protein interactions in hepadnavirus reverse transcription. Front Biosci (Landmark Ed) 14: 1606-1618.
  8. Hu, J. and Seeger, C. (2015). Hepadnavirus genome replication and persistence. Cold Spring Harb Perspect Med 5(7): a021386.
  9. Kann, M. and Gerlich, W. H. (1994). Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus. J Virol 68: 7993-8000.
  10. Kann, M., Sodeik, B., Vlachou, A., Gerlich, W. H. and Helenius, A. (1999). Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J Cell Biol 145: 45-55.
  11. Lanford, R. E. and Notvall, L. (1990). Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity. Virology 176(1): 222-233.
  12. Liao, W. and Ou, J. H. (1995). Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs. J Virol 69: 1025-1029.
  13. Liu, K., Ludgate, L., Yuan, Z. and Hu, J. (2015). Regulation of multiple stages of hepadnavirus replication by the carboxyl-terminal domain of viral core protein in trans. J Virol 89(5): 2918-2930.
  14. Ludgate, L., Liu, K., Luckenbaugh, L., Streck, N., Eng, S., Voitenleitner, C., Delaney, W. E. 4th, and Hu, J. (2016). Cell-free hepatitis B virus capsid assembly dependent on the core protein C-terminal domain and regulated by phosphorylation. J Virol 90: 5830-5844.
  15. Ludgate, L., Ning, X., Nguyen, D. H., Adams, C., Mentzer, L. and Hu, J. (2012). Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids. J Virol 86: 12237-12250.
  16. Nassal, M. (1992). The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol 66: 4107-4116.
  17. Seeger, C., Zoulim, F., Mason, W. S. (2013). In: Fields virology. In: Knipe, D. M. and Howley, P. M., (Eds.). Lippincott, Williams & Wilkins, Philadelphia 2185-2221.
  18. Summers, J. and Mason, W. S. (1982). Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell 29(2): 403-415.
  19. Trepo, C., Chan, H. L. and Lok, A. (2014). Hepatitis B virus infection. Lancet 384(9959): 2053-2063.
  20. Wingfield, P. T., Stahl, S. J., Williams, R. W. and Steven, A. C. (1995). Hepatitis core antigen produced in Escherichia coli: subunit composition, conformational analysis, and in vitro capsid assembly. Biochemistry 34: 4919-4932.
  21. Yu, M. and Summers, J. (1994). Multiple functions of capsid protein phosphorylation in duck hepatitis B virus replication. J Virol 68: 4341-4348.

简介

乙型肝炎病毒(HBV)是一种重要的全球人类病原体,并且是肝炎,肝硬化和肝癌的主要原因。 HBV衣壳由单个病毒蛋白的多个拷贝组成,衣壳或核心蛋白(HBc)在病毒生命周期中起着多重作用,并且最近已经成为开发抗HBV病毒疗法的主要靶标。尽管已经开发了几种用于研究HBV衣壳组装的系统,包括异源过表达系统如细菌和昆虫细胞,使用纯化蛋白质和哺乳动物细胞培养系统进行体外组装,但对非生理浓度HBc和盐以及难以操纵装配的宿主调节物在生理相关条件下的衣壳装配的详细研究方面存在主要限制。我们最近开发了基于兔网织红细胞裂解物(RRL)的哺乳动物无细胞系统,其中HBc以生理浓度表达并在近生理条件下组装成衣壳。该系统已经揭示了HBc装配要求,这是以前装配系统所不能预料的。此外,该系统中的衣壳组装受可容易操作的内源宿主因子调控。在这里,我们提供了这种无细胞衣壳装配系统的详细协议,包括如何操纵调节装配的宿主因子的说明。

【背景】乙型肝炎病毒(HBV)是一种重要的全球人类病原体,其长期感染全世界数以亿计的人并且代表病毒性肝炎,肝硬化和肝癌的主要原因(Seeger等人, 2013; Trepo et。,2014)。 HBV通过逆转录RNA中间体(所谓的前基因组RNA(pgRNA))在核衣壳内(NC)复制其基因组DNA(一种宽松的环状部分双链DNA(RC DNA))(Summers和Mason,1982; Hu和Seeger,2015; Hu,2016),其将pgRNA的拷贝与病毒编码的逆转录酶(RT)蛋白一起包装(Bartenschlager和Schaller,1992; Hu和Lin,2009)。 RT将pgRNA转化为RC DNA是在NC内。

二十面体HBV衣壳由单个病毒蛋白的多个拷贝组成,HBV核心(衣壳)蛋白(HBc)。 HBc由N-末端结构域(NTD,aa 1-140)和C-末端结构域(取决于菌株的CTD,150-183或185)组成,其通过接头区(141-149)连接。在使用高浓度纯化的HBc和/或盐的包括细菌和昆虫细胞和体外组装反应的异源过表达系统中,无CTD的NTD单独足以用于衣壳装配;因此它也被称为装配域(Gallina等人,1989; Birnbaum和Nassal,1990; Lanford和Notvall,1990; Wingfield等人,1995)。尽管在这些系统中不需要衣壳装配,但高度碱性和富含精氨酸的CTD显示非特异性核酸结合活性(Hatton等,1992),并且在病毒RNA包装中起重要作用Nassal,1992),DNA合成(Nassal,1992; Yu和Summers,1994)和核衣壳的核输入(Liao和Ou,1995; Liu等人,2015),所有这些都是进一步受由宿主因子控制的CTD的动态磷酸化状态的调节(Kann和Gerlich,1994; Kann等人,1999; Daub等人,2002; Ludgate等人,等人,2012; Liu等人,,2015)。 CTD及其磷酸化状态是否在生理条件下的衣壳组装中发挥作用尚不清楚。

为了研究更多生理条件下的HBV衣壳组装,我们最近开发了基于常用的哺乳动物细胞提取物,兔网织红细胞裂解物(RRL)的哺乳动物无细胞装配系统,其中HBc以生理(低)浓度表达(25 -50nM,单体)并在接近生理条件下组装成衣壳(Ludgate等人,2016)。该系统使我们能够揭示CTD在衣壳组装中的出乎意料的作用,其进一步受到由内源宿主因子控制的CTD磷酸化状态的调节(Ludgate等人,2016)。该协议改编自Ludgate等人(2016),并且包括关于该无细胞衣壳组装系统的更详细信息,并且应用不同的处理来解决病毒和宿主因子的作用,例如作为CTD和宿主磷酸酶的RNA结合活性,在接近生理条件下的HBV衣壳组装中。该协议将有助于详细研究在生理条件下的衣壳装配和宿主调节以及鉴定靶向HBc的新型抗病毒药物。

关键字:乙型肝炎病毒, 无细胞衣壳组装系统, 兔网织红细胞裂解液, 磷酸化, RNA结合

材料和试剂

  1. 移液器吸头(Denville Scientific,产品目录号:P1096-FR,P1121,P1122,P1126)
  2. 1.5ml微量离心管(Denville Scientific,目录号:C2170)
  3. 手套和实验室外套(Denville Scientific,目录号:G4162; Medline Industries,目录号:83044QHW)
  4. 蛋白酶K,RNA级(Thermo Fisher Scientific,Invitrogen TM,目录号:25530049)
  5. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4509-1KG)
  6. 苯酚溶液(Sigma-Aldrich,目录号:P4557)
  7. 氯仿(Fisher Scientific,目录号:BP1145-1)
  8. 乙酸钠(Sigma-Aldrich,目录号:S2889-1KG)
  9. 乙醇(EtOH)(AmericanBio,目录号:AB00515-00100)
  10. UltraPure TM DNase / RNase-Free蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  11. 琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:15510027)
  12. TNT 偶联的兔网织红细胞裂解液(RRL)(Promega,目录号:L4610)
  13. EasyTag TM L - [35 S] - 甲硫氨酸(PerkinElmer,目录号:NEG709A001MC)
  14. RNasin Plus Plus核糖核酸酶抑制剂(Promega,目录号:N2611)
  15. RNaseZap TM RNase去污溶液(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9780)
  16. RNase A,DNase和无蛋白酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EN0531)
  17. NEBuffer 3(New England Biolabs,目录号:B7003S)
  18. 碱性磷酸酶,小牛肠(CIAP)(New England Biolabs,目录号:M0290S)
  19. Tris基地(Fisher Scientific,目录号:BP152-10)
  20. 乙二胺四乙酸,EDTA(Sigma-Aldrich,目录号:E5134)
  21. 氟化钠(Sigma-Aldrich,目录号:S7920)
  22. 焦磷酸四钠十水合物(Sigma-Aldrich,目录号:S6422)
  23. β-甘油磷酸酯(Sigma-Aldrich,目录号:G6251)
  24. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
  25. cOmplete TM TM,不含EDTA的蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693132001)
  26. TE缓冲液(见食谱)
  27. 10倍磷酸酶抑制剂(PPI)(见食谱)
  28. 25倍蛋白酶抑制剂(见食谱)

设备

  1. 移液器(Gilson,P1000,P200,P20,P2)
  2. 通风橱(如,Protector Xstream Laboratory Hood,Labconco)
  3. 30°C / 37°C烤箱(SciGene,Robbins Scientific,型号:400型)
  4. 微量离心机(Fisher Scientific,Fisherbrand TM,型号:accuSpinTM Micro 17)
  5. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 1000)

程序

  1. 在兔网织红细胞裂解液中进行无RNA酶的体外翻译质粒
    1. 用蛋白酶K和SDS处理质粒DNA以去除质粒DNA中的任何污染性RNA。如下表所示,在50℃下用SDS(终浓度为0.6%w / v)和蛋白酶K(终浓度为0.4μg/μl)孵育HBc表达质粒30分钟。

      注意:
      1. 在加入蛋白酶K之前,首先将TE缓冲液中的20%SDS稀释。
      2. 任何含有可用于使用同源噬菌体RNA聚合酶进行体外转录的噬菌体启动子(以下实例中为T7)的质粒载体可用于在该系统中进行偶联转录和翻译。 br />
    2. 在消化反应中加入等量(200μl)的苯酚,充分混合并在室温下以最大速度(17,000×g克)旋转5分钟。将含水层(顶层)转移到新管中并重复此步骤。

    3. 如步骤A2所述,加入等体积的氯仿并萃取一次
    4. 测量水溶液并加入适量的3M乙酸钠(终浓度为0.3M)和100%乙醇(终浓度为70%),充分混合并在-20℃下孵育30分钟。在4℃下以最大速度(17,000×gg)离心20分钟,并用1ml 70%乙醇分别洗涤沉淀3次,以最大速度在4°下旋转10分钟C.
    5. 用30μl无RNase的水重悬DNA。用分光光度计(NanoDrop TM TM 1000)测量DNA浓度并将DNA浓度调节至1μg/μl,然后通过琼脂糖凝胶电泳验证DNA完整性。

  2. 体外翻译兔网织红细胞裂解物
    使用TNT 偶联的兔网织红细胞裂解物(RRL)体外翻译系统(Promega)按制造商推荐的方法表达HBc蛋白。
    1. 翻译工具包的准备
      1. 将TNT RNA聚合酶(T7)置于冰上。

      2. 通过手工加热迅速融化TNT 兔网织红细胞裂解液并立即置于冰上。
      3. 在RT(25°C)下解冻其他组分并置于冰上。
      注意:
      1. TNT ® 反应缓冲液可能在解冻后放置在冰上。通过在室温下涡旋30秒来重新溶解沉淀。
      2. 不要将裂解物冻融两次以上。
      3. 任何未使用的裂解物应在解冻后尽快在干冰/乙醇浴中重新冷冻,以尽量减少翻译活性的损失。

    2. 按照下表将反应组分装入1.5 ml离心管中。

      注意:小规模反应可以通过按比例减少所有组分来进行。

    3. 在30°C孵育反应90分钟
    4. 将反应置于冰上并进行组装步骤或将反应保存在-80°C以备将来使用。
      注意:
      1. 除了实际的翻译孵育之外,所有处理裂解物组分的操作都应在4°C或冰上进行。
      2. 在所有操作中都需要无核糖核酸酶的环境:
        1. 向所有TNT添加RNasin ® 核糖核酸酶抑制剂 ® 裂解反应以防止RNA降解。
        2. 微量离心管和移液管吸头应该不含RNase。
        3. 使用清洁剂(如RNaseZAPTM)清洁工作台和手套以去除RNase。

  3. RRL
    体外衣壳装配 我们最近的研究表明HBc NTD和CTD都是HBV R衣壳装配所需的生理学低HBc浓度翻译成RRL(Ludgate等人,2016)。 CTD可能通过RNA结合和蛋白质 - 蛋白质相互作用在这些条件下促进装配。此外,CTD受RRL中内源性宿主激酶和磷酸酶的磷酸化和去磷酸化作用,其进一步调节衣壳组装。
    为了证实CTD在RRL中的衣壳组装中的作用,特别是其RNA结合活性和通过磷酸化状态对组装的调节,如图1所示的以下处理应用于HBc体外组装在RRL中检测。


    图1. RRL中体外衣壳装配的条件模拟,将反应物在37℃孵育16小时而不进行任何处理。 CIAP,CIAP(10 U /μl)在组装培养过程中加入到反应中。加入CIAP + RNase,RNA酶A(0.1mg / ml)并在CIAP温育后再温育1小时。 RNase + CIAP,反应在CIAP孵育前用RNA酶A处理。 PPI,PPI(1x)在组装孵化过程中添加。 PPI,磷酸酶抑制剂的混合物(见下面的食谱),CIAP,小牛肠碱性磷酸酶。

    1. 模拟孵化

      1. 按照下表将反应组分装入1.5 ml离心管中。

        注意:PI,蛋白酶抑制剂的混合物(见下面的食谱)。

      2. 在37°C孵育反应16小时。
      3. 将反应置于冰上并进行分析步骤或将反应保存在-80°C以备将来使用。
    2. CIAP孵化

      1. 按照下表将反应组分装入1.5 ml离心管中。


      2. 在37°C孵育反应16小时。
      3. 将反应置于冰上并进行分析步骤或将反应保存在-80°C以备将来使用。
    3. CIAP之后是RNase孵化

      1. 按照下表将反应组分装入1.5 ml离心管中。


      2. 在37°C孵育反应16小时。
      3. 向装配反应中加入0.1μlRNA酶A(10 mg / ml),并在37°C下再孵育1 h。
      4. 将反应置于冰上并进行分析步骤或将反应保存在-80°C以备将来使用。
    4. RNase随后进行CIAP培养

      1. 按照下表将反应组分装入1.5 ml离心管中。


      2. 在37°C孵育反应1小时。
      3. 向装配反应中加入1μlCIAP(10 U /μl),并在37°C下再孵育16 h。
      4. 将反应置于冰上并进行分析步骤或将反应保存在-80°C以备将来使用。
    5. PPI孵化

      1. 按照下表将反应组分装入1.5 ml离心管中。


      2. 在37°C孵育反应1小时。
      3. 将反应置于冰上并进行分析步骤或将反应保存在-80°C以备将来使用。

笔记

所有使用[35S]甲硫氨酸的工作应在所有操作过程中采取适当的预防措施和个人防护装备,如手套和实验室涂层。建议在通风橱中打开库存,因为[35S]甲硫氨酸是挥发性的。在使用[35S]甲硫氨酸后,应检查工具和设备,如PIPETMAN,离心机和培养箱的污染情况。

数据分析

如图2和Ludgate等人2016所示,可以使用SDS-PAGE,琼脂糖凝胶电泳和Western印迹检查HBc和衣壳组装体在该无细胞系统中的表达。 br />

图2.在不同条件下RRL中的HBV衣壳装配。 WT HBc蛋白质在RRL中翻译,并且翻译反应混合物通过琼脂糖凝胶(1%)电泳(A)或SDS-PAGE(12.5%)(B)解析而无需任何进一步处理(输入)( (第1泳道)或用NEBuffer 3在37℃过夜(模拟)(泳道2),NEBuffer 3加CIAP在37℃过夜(泳道3),NEBuffer 3加CIAP在37°过夜(RNase + RNAP)(第4道),RNase处理1小时,然后用NEBuffer 3加CIAP在37°C过夜(RNase + CIAP)(泳道5),或用磷酸酶抑制剂在37°C过夜(PPI)(泳道6)。所有泳道都含有2μl翻译产物。通过放射自显影检测35 S标记的HBc蛋白。 C,HBc亚基; C-deP,去磷酸化的HBc亚基;钙,衣壳。 (改编自Ludgate等人,2016年)

食谱

  1. TE缓冲区
    10 mM Tris-HCl pH 8.0
    1 mM EDTA
  2. 10倍磷酸酶抑制剂(PPI)
    100 mM氟化钠
    500 mMβ-甘油磷酸酯
    100 mM焦磷酸钠
    20 mM原钒酸钠
  3. 25倍蛋白酶抑制剂
    1片cOmplete TM不含EDTA的蛋白酶抑制剂混合物溶于2ml不含DNase / RNase的dH 2 O 2中。

致谢

该协议改编自Ludgate等人,2016年。这项工作得到了国立卫生研究院的公共卫生服务资助(R01 AI043453致J.H.)的支持。 K. L.由浙江理工大学科学研究基金会(15042169-Y)资助。作者宣称没有利益冲突。

参考

  1. Bartenschlager,R.和Schaller,H。(1992)。 嗜肝DNA病毒装配通过聚合酶与病毒RNA基因组中的衣壳蛋白信号结合而启动。 EMBO J 11(9):3413-3420。
  2. Birnbaum,F.和Nassal,M.(1990)。 乙型肝炎病毒核衣壳装配:核心蛋白中的一级结构要求 J Virol 64(7):3319-3330。
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  4. Gallina,A.,Bonelli,F.,Zentilin,L.,Rindi,G.,Muttini,M.和Milanesi,G。(1989)。 具有鱼精蛋白样结构域缺失的重组乙型肝炎核心抗原多肽自组装成衣壳颗粒,但不能结合核酸。 J Virol 63:4645-4652。
  5. Hatton,T.,Zhou,S.and Standring,D。(1992)。 乙型肝炎病毒衣壳蛋白中的RNA和DNA结合活性:它们的模型在病毒复制中的作用。 J Virol 66:5232-5241。
  6. Hu,J。(2016)。在:乙型肝炎病毒在人类疾病中在:Liaw,YF和Zoulim,F. (编辑)。 Humana Press ,Springer Chap。 1,pp:1-34。
  7. Hu,J。和Lin,L。(2009)。 嗜肝DNA病毒逆转录中的RNA-蛋白质相互作用 Front Biosci(Landmark Ed ) 14:1606-1618。
  8. Hu,J。和Seeger,C。(2015)。 嗜肝DNA病毒基因组复制和持久性 冷泉港Harb Perspect Med 5(7):a021386。
  9. Kann,M.和Gerlich,W.H。(1994)。 蛋白激酶C对肝炎核心颗粒内核糖核酸蛋白磷酸化的影响B病毒。 J Virol 68:7993-8000。
  10. Kann,M.,Sodeik,B.,Vlachou,A.,Gerlich,W.H。和Helenius,A。(1999)。 乙型肝炎病毒核心颗粒与核孔复合体的磷酸化依赖性结合。
  11. Lanford,R.E和Notvall,L。(1990)。 乙型肝炎病毒核心和前核抗原在昆虫细胞中的表达和核心相关激酶活性的表征。 病毒学 176(1):222-233。
  12. Liao,W.和Ou,J.H。(1995)。 乙型肝炎病毒核心蛋白的磷酸化和核定位:三种重复中丝氨酸的意义SPRRR基元。 J Virol 69:1025-1029。
  13. Liu,K.,Ludgate,L.,Yuan,Z.和Hu,J。(2015)。 通过病毒核心蛋白的羧基末端结构域反式调节多个阶段的肝炎病毒复制J Virol 89(5):2918-2930。
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引用:Liu, K. and Hu, J. (2018). Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System. Bio-protocol 8(8): e2813. DOI: 10.21769/BioProtoc.2813.
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