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Methods Protoc. 2018, 1(2), 18; https://doi.org/10.3390/mps1020018

Function of the RNA Coliphage Qβ Proteins in Medical In Vitro Evolution

1
Department of Biological Sciences, College STEM, 1627 Hall Street, Montgomery, AL 36101, USA
2
Center for NanoBiotechnology Research, 1627 Harris Way, Montgomery, AL 36104, USA
*
Author to whom correspondence should be addressed.
Received: 27 March 2018 / Revised: 16 May 2018 / Accepted: 28 May 2018 / Published: 31 May 2018
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Abstract

Qβ is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qβ belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qβ is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (β) of a replicase, the maturation protein (A2) and the read-through or minor coat protein (A1), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qβ phage has attractive features for in vitro evolution. The A1 protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein’s UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A1 is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A1 protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qβ phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A1 proteins and exposed on the Qβ phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qβ system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qβ model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qβ phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses. View Full-Text
Keywords: Qβ; read-through protein A1; foot-and-mouth disease virus (FMDV); membrane proximal external region (MPER); human immunodeficiency virus (HIV); in vitro evolution; proofreading ; read-through protein A1; foot-and-mouth disease virus (FMDV); membrane proximal external region (MPER); human immunodeficiency virus (HIV); in vitro evolution; proofreading
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Singleton, R.L.; Sanders, C.A.; Jones, K.; Thorington, B.; Egbo, T.; Coats, M.T.; Waffo, A.B. Function of the RNA Coliphage Qβ Proteins in Medical In Vitro Evolution. Methods Protoc. 2018, 1, 18.

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