Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry.

Following endocytosis, enveloped viruses employ the changing environment of maturing endosomes as cues to promote endosomal escape, a process often mediated by viral glycoproteins. We previously showed that both high [K+] and low pH promote entry of Bunyamwera virus (BUNV), the prototypical bunyavirus. Here, we use sub-tomogram averaging and AlphaFold, to generate a pseudo-atomic model of the whole BUNV glycoprotein envelope. We unambiguously locate the Gc fusion domain and its chaperone Gn within the floor domain of the spike. Furthermore, viral incubation at low pH and high [K+], reminiscent of endocytic conditions, results in a dramatic rearrangement of the BUNV envelope. Structural and biochemical assays indicate that pH 6.3/K+ in the absence of a target membrane elicits a fusion-capable triggered intermediate state of BUNV GPs; but the same conditions induce fusion when target membranes are present. Taken together, we provide mechanistic understanding of the requirements for bunyavirus entry.

Protease-Independent Production of Poliovirus Virus-like Particles in Pichia pastoris: Implications for Efficient Vaccine Development and Insights into Capsid Assembly.

The production of enterovirus virus-like particles (VLPs) that lack the viral genome have great potential as vaccines for a number of diseases, such as poliomyelitis and hand, foot, and mouth disease. These VLPs can mimic empty capsids, which are antigenically indistinguishable from mature virions, produced naturally during viral infection. Both in infection and in vitro, capsids and VLPs are generated by the cleavage of the P1 precursor protein by a viral protease. Here, using a stabilized poliovirus 1 (PV-1) P1 sequence as an exemplar, we show the production of PV-1 VLPs in Pichia pastoris in the absence of the potentially cytotoxic protease, 3CD, instead using the porcine teschovirus 2A (P2A) peptide sequence to terminate translation between individual capsid proteins. We compare this to protease-dependent production of PV-1 VLPs. Analysis of all permutations of the order of the capsid protein sequences revealed that only VP3 could be tagged with P2A and maintain native antigenicity. Transmission electron microscopy of these VLPs reveals the classic picornaviral icosahedral structure. Furthermore, these particles were thermostable above 37°C, demonstrating their potential as next generation vaccine candidates for PV. Finally, we believe the demonstration that native antigenic VLPs can be produced using protease-independent methods opens the possibility for future enteroviral vaccines to take advantage of recent vaccine technological advances, such as adenovirus-vectored vaccines and mRNA vaccines, circumventing the potential problems of cytotoxicity associated with 3CD, allowing for the production of immunogenic enterovirus VLPs in vivo. IMPORTANCE The widespread use of vaccines has dramatically reduced global incidence of poliovirus infections over a period of several decades and now the wild-type virus is only endemic in Pakistan and Afghanistan. However, current vaccines require the culture of large quantities of replication-competent virus for their manufacture, thus presenting a potential risk of reintroduction into the environment. It is now widely accepted that vaccination will need to be extended posteradication into the foreseeable future to prevent the potentially catastrophic reintroduction of poliovirus into an immunologically naive population. It is, therefore, imperative that novel vaccines are developed which are not dependent on the growth of live virus for their manufacture. We have expressed stabilized virus-like particles in yeast, from constructs that do not require coexpression of the protease. This is an important step in the development of environmentally safe and commercially viable vaccines against polio, which also provides some intriguing insights into the viral assembly process.

Production and Characterisation of Stabilised PV-3 Virus-like Particles Using Pichia pastoris.

Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and Pakistan. However, the continued use of these vaccines poses potential risks to the eradication of PV. The production of recombinant PV virus-like particles (VLPs), which lack the viral genome offer great potential as next-generation vaccines for the post-polio world. We have previously reported production of PV VLPs using Pichia pastoris, however, these VLPs were in the non-native conformation (C Ag), which would not produce effective protection against PV. Here, we build on this work and show that it is possible to produce wt PV-3 and thermally stabilised PV-3 (referred to as PV-3 SC8) VLPs in the native conformation (D Ag) using Pichia pastoris. We show that the PV-3 SC8 VLPs provide a much-improved D:C antigen ratio as compared to wt PV-3, whilst exhibiting greater thermostability than the current IPV vaccine. Finally, we determine the cryo-EM structure of the yeast-derived PV-3 SC8 VLPs and compare this to previously published PV-3 D Ag structures, highlighting the similarities between these recombinantly expressed VLPs and the infectious virus, further emphasising their potential as a next-generation vaccine candidate for PV.