Indian Journal of Experimental Biology Vol. 46 November 2008, pp. 793-796
Assembly of recombinant coat protein of sugarcane streak mosaic virus into potyvirus-like particles M Hema1*, Ch V Subba Reddy1, H S Savithri2 & P Sreenivasulu1 # 1
Department of Virology, College of Biological and Earth Sciences, Sri Venkateswara University, Tirupati 517 502, India 2
Department of Biochemistry, Indian Institute of Science, Bangalore 526 012, India Received 11 March 2008; revised 22 August 2008
Coat protein (CP) gene of sugarcane streak mosaic virus-AP isolate (SCSMV-AP) was expressed in E. coli and recombinant CP (SCSMV-AP rCP) was purified by linear sucrose density gradient centrifugation. Observation of purified SCSMV-AP rCP under electron microscope revealed the presence of potyvirus-like particles (PVLPs). The assembled particles were shown to encapsidate CP gene transcripts by slot-blot hybridization. Keywords: Potyvirus-like particles, Recombinant coat protein, Sugarcane streak mosaic virus
Expression of viral coat proteins (CPs) in microbial system and their assembly into virus-like particles provide useful ways to identify CP regions necessary for particle assembly by site-directed mutagenesis1. Foreign peptides can be expressed on coat protein surface of viruses by either deletion or substitution2. The system also facilitates large scale production of coat protein in vitro which represents pure antigen free from the contaminating plant proteins and can be used as an antigen to produce polyclonal antiserum3-6. The reassembly of rCPs expressed in bacteria or in plants into virus-like particles has been studied in detail earlier for several potyviruses and it has been proposed as a means to study the molecular assembly of capsids2, 7, 9-12. Translational fusion of small foreign peptides is used as efficient system to display foreign epitopes or viral antigenic peptides on the viral particles to use as vaccine12-14. Sugarcane streak mosaic virus (SCSMV) causes mosaic disease of sugarcane in India and several other Asian countries like Pakistan, Bangladesh, Sri Lanka, Thailand and Vietnam and suggested that it probably belongs to an unassigned new genus of Potyviridae family15. The assembly of SCSMV-AP rCP into potyvirus-like particles (PVLPs) in Escherichia coli —————— # Correspondent author Phone: +91 9849102267 E-mail:
[email protected] *Present address: Department of Biotechnology, Sri Padmavathi Women’s University, Tirupati 517 502, India
has been investigated in this study. Purification of SCSMV-AP rCP from E. coli – Cloning of coat protein (CP) gene and expression of recombinant CP was done as reported by Hema et al.3. A plasmid construct (pR-SC CP) was transformed into the E. coli BL21 (DE3) pLys S strain. Purification of SCSMV-AP rCP was done by following the method as described by Mira et al.16 with modifications. A single colony was inoculated into 25 ml of LB medium containing 50 μg / ml of ampicillin and 34 mg / ml of chloramphenicol and incubated for overnight at 37oC. The culture (4%) was inoculated into 500 ml of terrific broth containing 50 μg / ml of ampicillin and 34 mg / ml of chloramphenicol. After 3 hr of growth, cells were induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG). Cells were harvested after 4 hr, suspended in 20 mM HEPES buffer (pH 8.0) and sonicated for 30 min at 50% duty cycle with an output control limit of 8 in an ice bath. The supernatant was subjected to 8% (w/v) polyethylene glycol (PEG) precipitation for 2 h and centrifuged at 12,000 rpm for 15 min. Pellet was resuspended in 20 mM HEPES buffer (pH 7.2, resuspension buffer) and centrifuged as above. The supernatant was subjected to high speed centrifugation at 35,000 rpm in a T-865 (Sorvall Combiplus) rotor for 2 hr. Pellet was resuspended in resuspension buffer and loaded onto 20-50% (w/v) linear sucrose density gradients and centrifuged at
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26,000 rpm for 3 hr in AH-629 rotor (Sorvall Combiplus). Light scattering zones seen upon illumination of gradients were collected, diluted with resuspension buffer (1:3) and pelleted by centrifugation at 35,000 rpm for 3 hr at 4oC. Pellet was resuspended in resuspension buffer and stored at 4oC. Western blot analysis – Crude and purified SCSMV-AP rCP were analysed by 12% of SDSPAGE17. The remaining steps were performed as per Burgermeister and Koenig 18. The resolved proteins from gel were blotted on to nitrocellulose membrane (Millipore, USA). The membrane was transferred into blocking solution, washed with TBS-T and probed with polyclonal antiserum raised against SCSMV-AP purified virus19. Then the membrane was washed and probed with HRP-conjugated goat anti-rabbit antibodies (Genei, Bangalore). Finally, HRP specific diamino benzidine (DAB) substrate (Genei, Bangalore) was added to detect SCSMV-AP rCP. Electron microscopy – Carbon shadowed formavar coated grids applied with purified SCSMV-AP rCP were negatively stained with 2% phosphotungstic acid (w/v) in water and observed under electron microscope (JOEL, NIMHANS, Bangalore) at a magnification of 40,000x. Slot-blot hybridization – CP gene fragment was released from pR-SC CP3 with BamHI and HindIII digestion and labeled by random primer method as described by Sambrook and Russel20. SCSMV was purified from mechanically inoculated Sorghum bicolor cv Rio seedlings19. RNA was isolated from 1 mg of purified SCSMV-AP rCP and purified virus as described by Zaitlin21, serially diluted in 1x SSC buffer (pH 7.0) and spotted onto Nylon membrane (Millipore, USA) using Hybrislot-manifold (GIBCOBRL). The membrane was baked for 2 hr at 80oC. Prehybridisation was carried out in 50% (w/v), formamide; 10% (w/v), dextran sulfate; 1% (w/v), SDS; 0.9 M, NaCl; and 20 μg / ml, salmon sperm DNA for 6 hr at 42oC. Hybridization was carried out at 42oC for 16 hr using 32P-labeled SCSMV-AP coat protein gene as a probe. Membrane was washed with 2x SSC + SDS (0.1%) followed by 1x, SSC; 0.5x, SSC, and 0.1x, SSC at 60oC and exposed for autoradiography 20. Authenticity of sucrose density gradient purified SCSMV-AP rCP was confirmed by western blot analysis using polyclonal antibodies raised against native SCSMV-AP isolate (Fig. 1). Electron
microscopic observation of purified SCSMV-AP rCP showed potyvirus-like particles (PVLPs, Fig. 2) which were heterogeneous in length and scanty in contrast to purified SCSMV-AP. Expression of CP genes in bacteria or in plants can give rise to viruslike particles, as shown for several other potyviruses as different as Pepper vein banding virus (PVBV)10, Johnsongrass mosaic virus (JGMV)11, Potato virus Y (PVY)11, Zucchini yellow mosaic virus2 and Papaya ring spot virus (PRSV)12. In the present study, it was observed that SCSMV
Fig. 1—(A) SDS-PAGE analysis of the crude and purified rCP of SCSMV-AP expressed in E. coli. [M. Protein Mr marker (Genei, Bangalore); (1)-Crude extract of E. coli expressing SCSMV-AP rCP; (2)-Purified SCSMV-AP rCP]; (B) Western blot of crude and purified rCP with SCSMV-AP antibodies
Fig. 2—Electron micrograph of sucrose density gradient purified SCSMV-AP rCP showing assembled PVLPs. (Stained with 1%, phosphotungstic acid; 40,000 x)
NOTES
rCP got self assembled into potyvirus-like particles in E. coli and additional 36 amino acids at N-terminus of rCP of SCSMV due to cloning strategy did not influence the assembly. It has been shown earlier that conserved core, but not the N or C terminus of potyviral CP is required for virus assembly2, 22, 23. A novel approach with potential biological application involves the translational fusion of small foreign peptides to viral proteins24, 25. Arazi et al. 2 have reported expression of 16 amino acid (aa) peptide from the human c-Myc and 16-aa FMDV CP immunogenic epitope on Zucchini yellow mosaic potyvirus (ZYMV) CP. Saini and Vrati14 have reported that presentation of Japanese encephalitis virus peptide on JGMV-like particles induces protection in mice against lethal challenge. When JGMV CP is expressed in E. coli and Saccharomyces cerevisiae, expressed protein self-assembles into potyvirus-like particles (PVLPs) and chimeric PVLPs are used as vaccine carriers14. Foot-and-mouth disease virus non-structural protein epitopes are expressed on tymovirus-like particles and used for detection of FMDV-NSP antibodies26. Encapsidation of viral RNA is one of the main functions of potyviral CP. In slot blot nucleic acid hybridization, RNA isolated from SCSMV PVLPs reacted with 32P-labeled SCSMV CP gene probe and showed that assembled PVLPs encapsidated CP mRNA (Fig. 3). Earlier, it has been demonstrated that PVY CP can encapsidate RNA and assemble into short virus-like particles (VLPs)7, 9. In case of rodshaped viruses, assembled PVBV recombinant CP particles have been shown to encapsidate CP mRNA10. In this study, SCSMV rCP assembled into PVLPs and encapsidation of CP mRNA indicated that the
assembly might not require the presence of 5′terminal sequence of the virus RNA. This further suggested that if there was an ‘origin of assembly’ motif or sequence, it might lie within the CP gene as encapsidation occurred even in the absence of 5’ end sequences of the genome. However, further experimental evidence is required to clearly know the encapsidation process of SCSMV-AP RNA. Thus, the assembly of recombinant CP of SCSMV-AP into potyvirus-like particles provided a model system to study the molecular details of potyvirus particle assembly. This system can be used to investigate the importance of crucial residues involved in vector transmission, transport or cell-to-cell movement and also the interaction of CP with genomic RNA. Further, this system facilitates the possibility of using SCSMV-AP for the presentation of heterologous peptides. The author (MH) acknowledges the financial assistance from the CSIR, New Delhi, India. References 1
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Fig. 3—Slot-blot hybridization of the RNA isolated from SCSMV-AP PVLPs using 32P- labeled SCSMV-AP CP gene as probe [Lane (A) RNA isolated from SCSMV rCP PVLPs; (B) RNA isolated from purified native SCSMV-AP. (1) 1:10; (2) 1: 100; (3) 1:1,000; (4) 1: 2,000; (5) 1:4,000; (6) 1:8,000; (7) 1: 16,000 dilutions of RNAs; (8) –ve control]
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