The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein

AbstractGlobal vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.

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Conjugation of an antibody Fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles

Biochemical Journal ◽

10.1042/bj3560867 ◽

2001 ◽

Vol 356 (3) ◽

pp. 867-873 ◽

Cited By ~ 20

Author(s):

Kay STUBENRAUCH ◽

Stefan GLEITER ◽

Ulrich BRINKMANN ◽

Rainer RUDOLPH ◽

Hauke LILIE

Keyword(s):

Coat Protein ◽

Mammalian Cells ◽

Recombinant Antibody ◽

Specific Antigen ◽

Antibody Fragment ◽

Fusion Peptides ◽

Virus Like Particles ◽

Vector Systems ◽

Virus Coat

The development of cell-type-specific delivery systems is highly desirable for gene-therapeutic applications. Current virus-based vector systems show broad cell specificity, which results in the need to restrict the natural tropism of these viral systems. Here we demonstrate that tumour-cell-specific virus-like particles can be functionally assembled in vitro from recombinant viral coat protein expressed in Escherichia coli. The insertion of a negatively charged peptide in the HI loop of polyoma VP1 interferes with the binding of VP1 to the natural recognition site on mammalian cells and also serves as an adapter for the coupling of antibody fragments that contain complementary charged fusion peptides. A recombinant antibody fragment of the tumour-specific anti-(Lewis Y) antibody B3 could be coupled to the mutant VP1 by engineered polyionic peptides and an additional disulphide bond. With this system an entirely recombinant cell-specific delivery system assembled in vitro could be generated that transfers genes preferentially to cells presenting the tumour-specific antigen on the cell surface.

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The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus

Virology ◽

10.1016/j.virol.2004.10.039 ◽

2005 ◽

Vol 331 (2) ◽

pp. 316-324 ◽

Cited By ~ 46

Author(s):

Tao Ren ◽

Feng Qu ◽

T. Jack Morris

Keyword(s):

Transcription Factor ◽

Coat Protein ◽

Nuclear Localization ◽

Turnip Crinkle Virus

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Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Journal of Virology ◽

10.1128/jvi.02364-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1169-1177 ◽

Cited By ~ 14

Author(s):

Jason Porta ◽

Vidya Mangala Prasad ◽

Cheng-I Wang ◽

Wataru Akahata ◽

Lisa F. P. Ng ◽

...

Keyword(s):

Chikungunya Virus ◽

Neutralizing Antibody ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virus Like Particles ◽

Fab Fragments ◽

Cell Transmission ◽

Viral Surface

ABSTRACTChikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur.IMPORTANCEAlphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to theAlphavirusgenus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments.

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Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.02034-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2294-2303 ◽

Cited By ~ 38

Author(s):

Yuliang Liu ◽

Luis Cocka ◽

Atsushi Okumura ◽

Yong-An Zhang ◽

J. Oriol Sunyer ◽

...

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Ebola Virus ◽

Intracellular Localization ◽

Gel Filtration ◽

Point Mutations ◽

Marburg Virus ◽

Structure Stability ◽

Wild Type ◽

Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

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Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya

Journal of General Virology ◽

10.1099/0022-1317-82-11-2827 ◽

2001 ◽

Vol 82 (11) ◽

pp. 2827-2836 ◽

Cited By ~ 32

Author(s):

Chu-Hui Chiang ◽

Ju-Jung Wang ◽

Fuh-Jyh Jan ◽

Shyi-Dong Yeh ◽

Dennis Gonsalves

Keyword(s):

Coat Protein ◽

Sequence Similarity ◽

Transcriptional Gene Silencing ◽

Papaya Ringspot Virus ◽

Wild Type ◽

Coding Region ◽

Transgenic Papaya ◽

Cp Gene ◽

Post Transcriptional Gene Silencing

Transgenic papaya cultivars SunUp and Rainbow express the coat protein (CP) gene of the mild mutant of papaya ringspot virus (PRSV) HA. Both cultivars are resistant to PRSV HA and other Hawaii isolates through homology-dependent resistance via post-transcriptional gene silencing. However, Rainbow, which is hemizygous for the CP gene, is susceptible to PRSV isolates from outside Hawaii, while the CP-homozygous SunUp is resistant to most isolates but susceptible to the YK isolate from Taiwan. To investigate the role of CP sequence similarity in overcoming the resistance of Rainbow, PRSV HA recombinants with various CP segments of the YK isolate were constructed and evaluated on Rainbow, SunUp and non-transgenic papaya. Non-transgenic papaya were severely infected by all recombinants, but Rainbow plants developed a variety of symptoms. On Rainbow, a recombinant with the entire CP gene of YK caused severe symptoms, while recombinants with only partial YK CP sequences produced a range of milder symptoms. Interestingly, a recombinant with a YK segment from the 5′ region of the CP gene caused very mild, transient symptoms, whereas recombinants with YK segments from the middle and 3′ parts of the CP gene caused prominent and lasting symptoms. SunUp was resistant to all but two recombinants, which contained the entire CP gene or the central and 3′-end regions of the CP gene and the 3′ non-coding region of YK, and the resulting symptoms were mild. It is concluded that the position of the heterologous sequences in the recombinants influences their pathogenicity on Rainbow.

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