Production and Biomedical Application of Flavivirus-like Particles Ewelina Krol, Gabriela Brzuska, Boguslaw Szewczyk  Trends in Biotechnology  DOI: 10.1016/j.tibtech.2019.03.013 Copyright © 2019 The Authors Terms and Conditions

Figure 1 The Assembly of Flavivirus Virions. The genome encodes three structural proteins (C, prM/M, E) and seven nonstructural proteins, which are initially translated into a single polyprotein and then further proteolytically cleaved. During the late steps of the replication cycle in a host cell, the assembly of virions occurs in the rough endoplasmic reticulum, where the cleavage of C protein from prM protein via the viral protease NS2B/NS3 complex is performed. This process is required for subsequent cleavage of prM and E protein by host cell signal peptidase, resulting in the assembly of immature particles composed of heterodimers of prM-E proteins that are organized into 60 trimers, which gives the particle a spiky structure. In the next step, virions are transported to the trans-Golgi network and subsequently packed into vesicles for secretion. The pr fragment of prM protein in immature state of virions covers the fusion loop of E protein, protecting it from premature fusion with membranes during egression through the acidic environment of the trans-Golgi network. Immediately before the release of virions from the host cell, the pr fragment is cleaved by cellular furin protease causing the secretion of mature virus particles. Cleavage of the pr fragment changes the conformation of E protein, forming mature particles. Differences in the expression levels of these proteases in various cells that are permissive to flaviviruses give rise to a heterogeneous population of particles: mature and immature virus particles and particles that can be defined as partially mature. ER, Endoplasmic reticulum; TGN, trans-Golgi network; VLP, virus-like particle. Trends in Biotechnology DOI: (10.1016/j.tibtech.2019.03.013) Copyright © 2019 The Authors Terms and Conditions

Figure 2 Schematic Representation of Mature, Immature, and Partially Mature Virus-like Particles (A) and Virions (B). During the natural replication cycle of flaviviruses, heterogeneous population of virus-like particles (VLPs) and virions are released from the host cell. Maturation state of VLPs and virions depends greatly on the conformation of E protein (dimeric for mature particles/trimers for immature particles) and the presence of mature M protein released following cleavage from the precursor prM by furin or immature, uncleaved prM. Moreover, at pH <6 both VLPs and virions can undergo irreversible change of structure from dimeric to trimeric form of E protein, resulting in the exposure of the fusion loop on the particle surface (C). Trends in Biotechnology DOI: (10.1016/j.tibtech.2019.03.013) Copyright © 2019 The Authors Terms and Conditions

Figure 3 General Strategies for Flavivirus Virus-like Particle (VLP) Construction. Three structural proteins (C-prM-E) expressed as a single cassette can form VLPs composed of prM/M and E proteins only when coexpressed with NS2B/NS3 viral protease required for the cleavage of C protein. VLPs can also be generated by coexpression of prM and E proteins with the inclusion of respective signal sequences (ss) (cleaved by host signal peptidase I complex) in both cis and trans conformation. Another approach, studied mostly for diagnostic purposes, includes coexpression of Flavivirus proteins and reporter protein from separate vectors; one vector encodes structural proteins (prM-E) while the second vector contains C protein, reporter protein: GFP/ luciferase (luc) gene and nonstructural viral proteins genes (NS1-5). In these strategies maturation of VLPs greatly depends on the presence of furin protease required for the cleavage of the pr fragment. Trends in Biotechnology DOI: (10.1016/j.tibtech.2019.03.013) Copyright © 2019 The Authors Terms and Conditions