Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. IMPORTANCERecurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. P orcine epidemic diarrhea virus (PEDV) belongs to the genusAlphacoronavirus in the family Coronaviridae and is the causative agent of porcine epidemic diarrhea (1). The virus is prevalent in East Asia, inflicting severe economic damage due to high mortality rates in young piglets, and recently made its first appearance on the North American subcontinent (2-4). PEDV infects the epithelia of the small intestine, an environment rich in proteases, and causes villous atrophy, resulting in diarrhea and dehydration. Intriguingly, in vitro propagation of PEDV isolates requires supplementation of trypsin to the cell culture supernatant (5). It has been hypothesized that trypsin mediates activation of virions for membrane fusion by cleaving the spike (S) glycoprotein (5, 6). Trimeric S proteins decorate the virion envelope and mediate receptor binding and membrane fusion. The S protein has been recognized as a class I fusion protein by its molecular features (7,8).Class I fusion proteins are generated in a locked conformation to prevent premature triggering of the fusion mechanism and are subsequently prepared for action by proteolytic processing, a step called priming (reviewed in reference 9). This cleavage is separating two functionally distinct protein domains, a soluble head domain responsible for receptor binding and a membrane bound subunit comprising the fusion machinery. A characteristic feature of the cleaved, fusion-ready subunit is an N-terminal fusion peptide. Proteolytic priming can occur in the virus-producing cell, in the extracellular environment, or after contact with the target cell membrane. Priming of the PEDV S protein is ...

Silk–elastin block copolymers have such physical and biological properties that make them attractive biomaterials for applications ranging from tissue regeneration to drug delivery. Silk–elastin block copolymers that only assemble into fibrils at high concentrations can be used for a template-induced fibril assembly. This can be achieved by additionally including template-binding blocks that promote high local concentrations of polymers on the template, leading to a template-induced fibril assembly. We hypothesize that template-inducible silk-fibril formation, and hence high critical concentrations for fibril formation, requires careful tuning of the block lengths, to be close to a critical set of block lengths that separates fibril forming from nonfibril forming polymer architectures. Therefore, we explore herein the impact of tuning block lengths for silk–elastin diblock polypeptides on fibril formation. For silk–elastin diblocks E S m –S Q n , in which the elastin pentamer repeat is E S = GSGVP and the crystallizable silk octamer repeat is S Q = GAGAGAGQ, we find that no fibril formation occurs for n = 6 but that the n = 10 and 14 diblocks do show concentration-dependent fibril formation. For n = 14 diblocks, no effect is observed of the length m (with m = 40, 60, 80) of the amorphous block on the lengths of the fibrils. In contrast, for the n = 10 diblocks that are closest to the critical boundary for fibril formation, we find that long amorphous blocks ( m = 80) oppose the growth of fibrils at low concentrations, making them suitable for engineering template-inducible fibril formation.

Consensus motifs for sequences of both crystallizable and amorphous blocks in silks and natural structural analogues of silks vary widely. To design novel silklike polypeptides, an important question is therefore how the nature of either the crystallizable or the amorphous block affects the self-assembly and resulting physical properties of silklike polypeptides. We address herein the influence of the amorphous block on the self-assembly of a silklike polypeptide that was previously designed to encapsulate single DNA molecules into rod-shaped viruslike particles. The polypeptide has a triblock architecture, with a long N-terminal amorphous block, a crystallizable midblock, and a C-terminal DNA-binding block. We compare the self-assembly behavior of a triblock with a very hydrophilic collagen-like amorphous block (GXaaYaa)132 to that of a triblock with a less hydrophilic elastin-like amorphous block (GSGVP)80. The amorphous blocks have similar lengths and both adopt a random coil structure in solution. Nevertheless, atomic force microscopy revealed significant differences in the self-assembly behavior of the triblocks. If collagen-like amorphous blocks are used, there is a clear distinction between very short polypeptide-only fibrils and much longer fibrils with encapsulated DNA. If elastin-like amorphous blocks are used, DNA is still encapsulated, but the polypeptide-only fibrils are now much longer and their size distribution partially overlaps with that of the encapsulated DNA fibrils. We attribute the difference to the more hydrophilic nature of the collagen-like amorphous block, which more strongly opposes the growth of polypeptide-only fibrils than the elastin-like amorphous blocks. Our work illustrates that differences in the chemical nature of amorphous blocks can strongly influence the self-assembly and hence the functionality of engineered silklike polypeptides.

By using recombinant DNA technology, many groups have designed protein-based block copolymers that combine silk-like blocks with either elastin-like or collagenlike blocks. Materials made out of such protein-based block copolymers combine the properties of the blocks from which they are made. For example, silk-elastinlike polypeptides (SELPs) combine the strength of the crystallisable blocks of B. mori silk with the flexibility of elastin, resulting in a tough biomaterial 115. The properties of the biomaterial can be precisely tuned by changing for example the relative lengths of the silk-and elastin-like blocks 116 , the type of guest residue X Design rules for protein-based synthetic viruses To implement requirements that are not yet covered by the current design of our artificial viral capsid protein or to optimise the design for other applications, various design rules should be taken into account. Design rules for viral and non-viral