In this study, we report the identification of three amino acid mutations in the HEV capsid protein that collectively contributed to virus attenuation in vivo
. The three amino acid mutations F51L, T59A, and S390L in the ORF2 capsid were identified in the genome of a genotype 3 HEV infectious clone (pSHEV-1) from a previous study (24
). ORF2 partially overlaps ORF3; however, the three mutations in the ORF2 capsid do not change the amino acid sequence of the ORF3 protein. In this study, three HEV mutants with a single amino acid change (rF51L, rT59A, and rS390L) were constructed, and all were shown to be replication competent and viable after transfection into a subclone of Huh7 human liver cells. No major difference was observed in the production of HEV ORF2 protein in cells transfected with the wild type and the mutants. The pig model system has been used to understand various aspects of HEV replication, pathogenesis, and cross-species infection (16
). Due to limited resources and restricted procedures for nonhuman primates, it is not possible to use a large number of nonhuman primates for HEV pathogenicity studies. Therefore, we conducted a pathogenicity study of these three single mutants using a large number of SPF pigs in each group (n
= 10). We showed that in the positive-control pigs inoculated with the wild-type pSHEV-3, fecal virus shedding preceded the onset of viremia, and the patterns of viremia and fecal virus shedding in pSHEV-3-infected pigs were consistent with those observed in previous studies (21
). An IgG anti-HEV response was detected 1 to 2 weeks after the onset of viremia and remained detectable until the end of the 8-week study. Peak viral loads in serum, liver, bile, and intestinal content were detected at 28 dpi, with a temporal decrease of viral genome titers concurrent with the increase of HEV antibodies. In pigs inoculated with the wild-type pSHEV-3, the virus was not fully cleared by the immune response by 56 dpi, as evidenced by continuous fecal shedding in all pigs and the presence of high viral loads in liver, bile, and intestinal content at the end of the 8-week study.
In a previous pilot study with only two pigs (24
), we demonstrated the viability of the triple mutant pSHEV-1 in vivo
. In this study, we confirmed that the triple mutant pSHEV-1 is infectious in vivo
, as HEV RNA was detected in serum and fecal samples as well as in liver, bile, and intestinal contents of inoculated pigs. However, the pSHEV-1 with the triple mutations in the capsid protein was clearly attenuated compared to the wild-type pSHEV-3, as fewer pigs were viremic or shed virus in feces. For those pigs that did have detectable viremia and fecal virus shedding, viremia was delayed by 5 weeks, serum viral loads were drastically lower, and fecal virus shedding was also delayed and had a shorter duration. In addition, none of the pigs inoculated with pSHEV-1 seroconverted by 56 dpi, which is consistent with our pilot study, in which the two pigs inoculated with pSHEV-1 did not seroconvert until 63 dpi (24
To determine which of the three amino acid mutations were responsible for the observed attenuation of the pSHEV-1 virus in pigs, we constructed three HEV single mutants (rF51L, rT59A, and rS390L), each containing a single amino acid mutation, and compared the in vivo pathogenicities of the three HEV single mutants in pigs to those of each other and the wild-type pSHEV-3. For the rF51L mutant, seroconversion and viremia were delayed 2 to 3 weeks compared to those with the wild-type pSHEV-3, and the incidence of viremia was lower. However, the serum viral loads were similar for the rF51L mutant and the wild-type pSHEV-3, and like the wild-type pSHEV-3, the mutant rF51L virus was also not fully cleared in pigs at the end of the 8-week study, as evidenced by viremia and fecal virus shedding at 56 dpi. Inefficient viral clearance for the rF51L mutant-inoculated pigs was also observed in liver, bile, and intestinal content, although viral loads were lower than those in the pigs inoculated with wild-type pSHEV-3. The results suggest that the F51L mutation in the capsid protein only partially contributes to virus attenuation.
For the rT59A mutant, seroconversion in pigs was delayed 1 to 3 weeks. Viral RNA was detected in the liver, bile, intestinal content, and fecal materials at 21 to 35 dpi, but viremia was not detected in any of the rT59A-inoculated pigs until 56 dpi. Unlike the wild-type pSHEV-3 and mutant rF51L, the mutant rT59A virus was completely cleared from feces, liver, bile, and intestinal content at 56 dpi. Also, the viral loads in bile, liver, and intestinal contents collected at the necropsy at 28 dpi were lower than those of the wild-type pSHEV-3. Therefore, the T59A mutation in the capsid is important for virus attenuation in pigs.
For mutant rS390L, the inoculated pigs had a more drastic delay in seroconversion (3 to 4 weeks later than seroconversion with the wild-type pSHEV-3). Similarly, viremia and fecal virus shedding were also delayed compared to those with the wild-type pSHEV-3 virus. Among the three single mutants, mutant rS390L had the lowest viral loads in liver and bile of pigs necropsied at 21, 28, and 56 dpi. Pigs infected with mutant rS390L completely cleared the virus from serum and feces by 42 and 49 dpi, respectively, and the liver, bile, and intestinal content collected at the end of the 8-week study had no detectable virus. Therefore, the results indicate that the S390L mutation in the HEV capsid protein is critical for virus attenuation in pigs.
Mutations F51L and T59A are both located in the N-terminal region of the HEV capsid protein, which contains a signal peptide (aa 1 to 22) involved in the translocation of the protein across the endoplasmic reticulum membrane (72
), followed by an arginine-rich domain (aa 23 to 111) that may be involved in RNA encapsidation (59
). Therefore, it is possible that the F51L and T59A mutations contribute to virus attenuation by causing a defect in viral genomic RNA packaging, thus resulting in a lower level of infectious virus production in vivo
. The observed reduction of viral loads in bile, liver, and intestinal content and the lower incidence of viremia and fecal virus shedding with both mutants are consistent with this explanation. The T59A mutation appears to have a greater impact on viral replication than the F51L mutation, as no viremia was detected and viral loads and fecal virus shedding were greatly reduced. It is possible that the polarity change of the mutation T59A more drastically affects the function of the N-terminal domain. This would explain the observed difference in the level of attenuation between the two mutants, since the F51L mutation did not result in a polarity change.
The C terminus of the HEV capsid protein is the major structural domain responsible for virion assembly, immunogenicity, and host cell receptor binding (23
). Constructs (aa 112 to 607) lacking the N terminus have been shown to self-assemble into virus-like particles (VLPs) in a baculovirus expression system (34
), and a truncated C-terminal peptide, p239 (aa 368 to 606), of the HEV capsid protein induced protective immunity against HEV challenge in nonhuman primates (32
). This p239 peptide has also been reported to bind and penetrate cell lines that are susceptible to HEV, suggesting the presence of HEV receptor binding sites in this C-terminal region (23
). The crystal structure of genotype 3 HEV VLPs assembled from a truncated version of the ORF2 capsid protein (residues 129 to 606) was determined at 3.5-Å resolution (69
). Three structural domains have been defined within ORF2: S (residues 118 to 313), P1 (residues 314 to 453), and P2 (residues 454 to 606). P1 and S are closely associated and exposed on the surface of the capsid around the icosahedral 3-fold axis, and P1 and P2 are connected through a flexible proline-rich hinge that spans residues 445 to 467 (69
) (). The S domain forms a continuous capsid shell, whereas P2 is highly exposed and is largely responsible for antigenicity determination and virus neutralization (20
). Although there is no direct evidence regarding glycosylation of the HEV VLP capsid, consensus sites have been located within the S (Asn137-Leu-Ser and Asn130-Leu-Thr) and P2 (Asn562-Thr-Thr) domains (69
), with evidence pointing toward a direct role of Asn137 and Asn130 in virus infectivity and virion assembly (49
). Structural analysis of the P1 domain, an antiparallel β-barrel composed of six β-strands and four short helices (20
) (), reveals the presence of a putative sugar binding site (376
) that resembles the one site identified in the endosialidase of the K1F phage (PDB accession no. 1VOE
), which maps within a helix-turn-helix motif of P1 ( and C) and may be responsible for binding to cell receptors (20
). Amino acid position 390, which is conserved among all four major genotypes of mammalian HEV, is localized within this potential sialic acid binding site in the P1 linear domain. Although the HEV dimer shows a crossing topology of the C-terminal P2 domain versus the P1 and S domains, stability of the dimer is ensured by the interaction between the P2 domain of one monomer and the P2 and P1 domains of the other (69
), with the sugar binding site exposed in the opposite interface of P1 (). Interestingly, He et al. showed that various monoclonal antibodies directed against conformational and linear epitopes in P1 and P2 were able to block the binding of a truncated peptide (p239, residues 368 to 606) of ORF2, which occurs as 23-nm particles, to various cell lines (23
). Largely based on selective blocking by monoclonal antibodies, He et al. concluded that the region comprising residues 368 to 606 should contain at least two distinct sites, located separately in the monomeric and dimeric domains, involved in binding. Remarkably, two of the monoclonal antibodies against linear epitopes in P1 (residues 423 to 438 [antibody 12A10] and 423 to 443 [antibody Ab 16D7]) that neutralize the binding of p239 particles to Huh7 cells map on the same interface as Ser390 (23
) (), suggesting a potential site of contact within that region. Therefore, the mutation from a polar residue (Ser) to a nonpolar residue (Leu) at position 390 may prevent the rS390L mutant from efficiently interacting with the host cell receptor. Crystal structure studies have shown that this putative sugar binding motif may be responsible for destabilization of HEV capsid trimers during uncoating (20
). Defects in receptor binding and uncoating would both result in a lower level of virus replication in the host and thus would explain why mutant rS390L appeared to contribute the most to virus attenuation among the three single HEV mutants.
It has been well documented that one or more amino acid changes in capsid or envelope proteins can contribute to the attenuation phenotype of viruses (8
). Single point mutations in the capsids of poliovirus (65
), murine norovirus (4
), infectious bursal disease virus (64
), and adeno-associated viruses (67
) have all been demonstrated to have significant effects on both in vitro
and in vivo
growth characteristics of the viruses. Reversion of an attenuated phenotype to a pathogenic wild type has also been reported for many animal viruses, such as poliovirus (35
), porcine reproductive and respiratory syndrome virus (48
), and infectious bursal disease virus (54
). In the current study, the F51L and T59A mutants are genetically stable in pigs, as the viruses recovered from the inoculated pigs over the course of infection all retained these two mutations. However, in pigs inoculated with the mutant rS390L, a mixture of both the wild-type and attenuated mutant phenotype viruses was recovered from feces, serum, bile, and intestinal content as early as 21 dpi. As the course of infection progressed, the wild-type phenotype virus population increasingly became predominant, and by 42 dpi, the rS390L mutant virus was no longer detectable in some infected pigs. The fact that the mutant rS390L had mutated back to the wild-type sequence at later stages of virus infection indicates a strong biological importance of this amino acid residue in the functionality of the HEV capsid protein. It took at least 21 days of replication in pigs before the wild-type phenotype population reached a level similar to that of the mutant virus population in infected pigs (data not shown). The lower populations of the wild-type virus, during the first few weeks of infection contribute to the observed attenuation in pigs inoculated with the mutant rS390L. However, in our previous pilot study with only two pigs (24
), the three nonsilent mutations F51L, T59A, and S390L in the capsid gene of the triple mutant pSHEV-1 were retained in the genome for up to 70 dpi. Therefore, reversion to the wild-type phenotype appears to be greater for the single mutant (such as rS390L) than the triple mutant pSHEV-1, since only one nucleotide change is required to restore the original sequence. All three mutants (F51L, T59A, and S390L) are created by a single nucleotide change, and therefore it will be interesting to see in a future animal study if the attenuation phenotype of mutant S390L can be stabilized by changing two nucleotides of codon 390.
Taken together, the results from this study indicated that the three amino acid mutations in the HEV capsid protein collectively contribute to virus attenuation. The F51L mutation resulted in only partial attenuation, whereas the T59A and S390L mutations attenuated the virus more drastically. The significant attenuation for the triple mutant pSHEV-1 was likely due to the additive effect of the three individual amino acid mutations. Additional phenotypic markers of HEV attenuation need to be identified and characterized for the development of a modified live-attenuated vaccine when HEV can be efficiently propagated in vitro.