Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarrays

Ikeda, Keiko; Nakazawa, Hiroshi; Shimo-oka, Ai; Ishio, Kaoru; Miyata, Seiji; Hosokawa, Yoichiroh; Matsumura, Satoshi; Masuhara, Hiroshi; Belloncik, S.; Ahrends, Robert; Goshima, Naoki; Nomura, Nobuo; Morigaki, Kenichi; Kawai, Atsushi; Kuroita, Toshihiro; Kawakami, Bunsei; Endo, Yaeta; Mori, Hajime

doi:10.1002/pmic.200500022

Cited by 51 publications

(42 citation statements)

References 28 publications

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“…A follow-up biochemical study further suggested the existence of a "polyhedrin recognition signal" within amino acid residues 42 to 93 that is sufficient to immobilize foreign proteins tagged by this fragment (7,13). We took note of the fact that some proteins failed to be immobilized (9), suggesting that there could be alternative interpretations for the immobilization. Consistent with our ET data, the atomic model of TP (VP4) derived from an improved cryo-EM map at 3.1-Å resolution showed that the N-terminal segment is almost completely buried within the turret, making it unlikely that this segment binds polyhedrin molecules located in the exterior of the virus (17).…”

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“…TP is encoded by genome segment 4 and was formerly known as VP3 or V3 in previous literature, but it was renamed to VP4 (i.e., the protein encoded by genome segment 4) for clarity (5,20). In addition to making up the turret/B-spike, VP4, and specifically its N-terminal segment (residues 1 to 79), was shown to "immobilize" foreign proteins onto the polyhedron (8,9), suggesting it may bind polyhedrin within polyhedra. A follow-up biochemical study further suggested the existence of a "polyhedrin recognition signal" within amino acid residues 42 to 93 that is sufficient to immobilize foreign proteins tagged by this fragment (7,13).…”

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Electron Tomography Reveals Polyhedrin Binding and Existence of both Empty and Full Cytoplasmic Polyhedrosis Virus Particles inside Infectious Polyhedra

Chen

Sun

Atanasov

et al. 2011

J Virol

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Previous studies have described the structure of purified cytoplasmic polyhedrosis virus (CPV) and that of polyhedrin protein. However, how polyhedrin molecules embed CPV particles inside infectious polyhedra is not known. By using electron tomography, we show that CPV particles are occluded within the polyhedrin crystalline lattice with a random spatial distribution and interact with the polyhedrin protein through the A-spike rather than as previously thought through the B-spike. Furthermore, both full (with RNA) and empty (no RNA) capsids were found inside polyhedra, suggesting a spontaneous RNA encapsidating process for CPV assembly in vivo.Viruses in the Cypovirus genus of the Reoviridae, such as cytoplasmic polyhedrosis virus (CPV), are distinct architecturally in having only a single protein shell (reviewed in references 12 and 20). This is in stark contrast to viruses of all other genera of the Reoviridae, which have one or two additional shells surrounding a CPV-like inner core. Although its infection of silkworms causes a negative impact on the Asian economy, CPV is better recognized for its polyhedrin-binding property, which has tremendous potential as a nano-delivery tool and for its promise as an environmentally friendly pesticide for fruit and vegetable farming (11).In extracellular space, CPV particles are embedded inside a crystalline protein occlusion body, called the polyhedron (3, 11), which serves as an outer protective layer during viral spread across different hosts. The stability of the polyhedron in the environment and its sensitivity to the high pH of the insect midgut allows CPV to establish efficient infection through oral routes. In particular, the pH-sensitive release of embedded particles has great potential for drug delivery applications (2).Past studies have described the three-dimensional (3D) structures of both the polyhedron protein (2) and the isolated CPV virion (1,6,(16)(17)(18)(19)21) based on X-ray crystallography and cryoelectron microscopy (cryoEM), respectively. However, a 3D description documenting the packing pattern of CPV virions inside the polyhedron is lacking.In this study, we used electron tomography to resolve the packing of CPV within the polyhedron, as well as the interactions between the CPV capsid and the polyhedrin protein lattice. Our electron tomograms of stained sections of polyhedra revealed that the lattice of the polyhedron matches the polyhedrin arrangements shown by X-ray crystallography (2), thus validating our approach for resolution of molecular interactions. Our data suggest that, inside the polyhedron, CPV particles attach to the polyhedrin protein lattice via the Aspike of the capsid, not through the turret protein (TP; also known as the B-spike) as previously thought. In addition to full CPV capsids, empty CPV capsids containing no RNA material, as well as partially filled capsids, were found occluded in the polyhedra, supporting their physiological relevance in CPV assembly and spread.Polyhedron occlusion bodies are about 5 m in size, which ...

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Electron Tomography Reveals Polyhedrin Binding and Existence of both Empty and Full Cytoplasmic Polyhedrosis Virus Particles inside Infectious Polyhedra

Chen

Sun

Atanasov

et al. 2011

J Virol

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“…Immobilization of LIF using VP3 or H1 signals was accomplished by dual infection of the recombinant baculoviruses that express the LIF fusion proteins and a recombinant baculovirus AcCP-H expressing BmCPV polyhedrin. The resulting polyhedra were recovered and purified from Sf21 cells, and detected by western blotting as previous reported in Reference [16]. Polyhedra were stored in distilled water containing penicillin (100 U/mL) and streptomycin (100 mg/mL).…”

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confidence: 99%

“…We developed two targeting strategies for the incorporation of foreign proteins into polyhedra. Diverse foreign proteins can be immobilized into polyhedra by fusing a polyhedron-targeting tag sequence at the C-or N-terminus of the foreign protein [16].…”

Section: Introductionmentioning

confidence: 99%

The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells

Nishishita¹,

Ijiri²,

Takenaka³

et al. 2011

Biomaterials

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“…The CPVs are distinct from other members of this family because they exhibit a single rather than a double capsid layer and during replication they are commonly embedded within large proteinaceous inclusion bodies called polyhedra [1][2][3][4]. Recently, Coulibaly et al described the 2 Å crystal structures of both recombinant and infectious forms of the silkworm cypovirus polyhedra [5], which spurred the use of CPV polyhedra to immobilize foreign proteins within novel expression systems for analysis at the single-cell level [6][7][8].…”

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confidence: 99%

The complete nucleotide sequence of the type 5 Helicoverpa armigera cytoplasmic polyhedrosis virus genome

Tan

Zhang

et al. 2008

Virus Genes

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The S1-6, S8, and S9 segments of the type 5 Helicoverpa armigera cytoplasmic polyhedrosis virus (HaCPV-5, Chinese strain) were cloned and sequenced, completing the HaCPV-5 genome. We found that each HaCPV-5 segment exhibits the conserved terminal sequences AGUU and UUGC located at the 5' and 3' ends, respectively. We also analyzed the translation initiation codon of the HaCPV-5 genome and compared it with the available cypovirus sequences in GenBank. We postulated that the conserved purine at the -3 position in relation to the AUG codon is probably the most important nucleotide for efficient translation initiation in cypovirus. Although the nucleotide sequences of the HaCPV-5 segments S1-10 exhibit no significant similarity to other viruses, blast searches did reveal some similarities between predicted HaCPV-5 amino acid sequences and those of other viruses.

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Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarrays

Cited by 51 publications

References 28 publications

Electron Tomography Reveals Polyhedrin Binding and Existence of both Empty and Full Cytoplasmic Polyhedrosis Virus Particles inside Infectious Polyhedra

Electron Tomography Reveals Polyhedrin Binding and Existence of both Empty and Full Cytoplasmic Polyhedrosis Virus Particles inside Infectious Polyhedra

The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells

The complete nucleotide sequence of the type 5 Helicoverpa armigera cytoplasmic polyhedrosis virus genome

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