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

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 (A 2 ) and the read-through or minor coat protein (A 1 ), 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 A 1 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, A 1 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 A 1 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 A 1 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.

Toward innovative veterinary nanoparticle vaccines

“…Qβ is a positive single-stranded RNA bacteriophage that infects E. coli (Singleton, et al 2018). The capsid of Qβ is composed of 180 monomeric proteins that self-assemble into an icosahedral VLP with a diameter of 28 nm (Golmohammadi et al 1996;Machida and Imataka 2015).…”

RNA Phage VLP-Based Vaccine Platforms

“…In some cases, it has been possible to exploit the Qß readthrough phenomenon to actually display peptides and proteins on intact, infectious recombinant bacteriophages [ 32 , 33 ]. Apparently, some portions of the readthrough protein are dispensable and can be interrupted with foreign sequences.…”

“…Apparently, some portions of the readthrough protein are dispensable and can be interrupted with foreign sequences. The importance of readthrough protein for infectivity and the existence of the virion’s variable upper limit on extension copy number imposes additional constraints on display density that may restrict the utility of infectious Qß, but the ability to produce the particle by growing it as a self-replicating viral entity introduces the possibility of using its relatively low replication fidelity to spontaneously mutagenize and then affinity-optimize displayed peptides and proteins [ 32 , 33 ]. So far, Qß has been little used for this purpose, but it deserves further effort.…”

Advancements in protein nanoparticle vaccine platforms to combat infectious disease

“…Qβ is a bacteriophage that infects E . coli through the F‐pilus protein, which is not present on mammalian cells, and therefore the virus is noninfectious to humans (Singleton et al, 2018). Qβ is found in abundance in sewage and animal feces.…”