Abstract
Orally-transmitted viruses have evolved in a way to resist the extreme conditions of the host’s gastrointestinal environment, especially the proteolysis of their structural proteins. However, the mechanisms allowing these viruses to survive these harsh conditions remain unclear. Hepatitis E virus (HEV) is an orally-transmitted human pathogen. Its capsid protein contains three domains S, P1 and P2. The latter forms a homodimer protruding from the virus shell, making it the most exposed part. By combining biochemical and computational methods, we found the trypsin digestion sites to be highly conserved among the HEV strains. Furthermore, the constructs of the HEV capsid protein that contain an extended P2 domain were digested within the extensions leaving the P2 domain intact. The trypsinization seems to occur in three possible double cleavages at R451-R619, R460-R619 or R460-R631.The dimerization disrupts the trypsin action at three main sites in the P2 domain R542, K544 and K554. These sites are very exposed in the monomeric P2 domain constructs which makes the monomeric forms very susceptible to trypsin action. Therefore, we believe that dimerization is a structural feature that has been selected by the evolutionary forces to render the HEV capsid protein resistant to the host’s proteases; an evolutionary feature that could be common to some other (if not all) orally-transmitted viruses.
Highlights
Orally-transmitted viruses have evolved in a way to resist the extreme conditions of the host’s gastrointestinal environment, especially the proteolysis of their structural proteins
The different coding sequences were amplified from Hepatitis E virus (HEV) open reading frame 2 (ORF2), purified and separately inserted into pET-28a (+) vector
These results indicated that for p179, only when the proteolysis occurs in three sites (R451, R460 or R466) would lead to formation of fragments of molecular weights (17~19 kDa) similar to those observed in the trypsin digestion assay
Summary
Orally-transmitted viruses have evolved in a way to resist the extreme conditions of the host’s gastrointestinal environment, especially the proteolysis of their structural proteins. We believe that dimerization is a structural feature that has been selected by the evolutionary forces to render the HEV capsid protein resistant to the host’s proteases; an evolutionary feature that could be common to some other (if not all) orally-transmitted viruses. The first commercial hepatitis E vaccine (Hecolin) has been licensed in China in 2012 It was derived from HEV genotype 1 (aa 368–606 of ORF2) and produced in bacterial cells[9]. The HEV capsid is formed by capsomeres consisting of homodimers of a single structural capsid protein. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. We previously expressed, for the first time, in Pichia pastoris (P. pastoris) single-point mutated p179 proteins (aa 439–617, mutated at N562) that were naturally expressed as monomers and both were reactive against the anti-HEV neutralizing antibodies, indicating that the dimerization was inessential for p179 proteins interaction with the neutralizing antibodies[14]
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