The functional significance of the HEV PPR remains unknown. The data obtained in the present study suggest that the substantial sequence variability of PPR plays an important role in viral adaptation. Although the exact role of the PPR in HEV adaptation is not known, several findings indicate that the PPR may be involved in determination of host range. The HEV lineages of genotypes 1 and 2 are anthropotropic, while the genotype 3 and 4 HEV strains infect not only humans but also several animal species 
, suggesting their zoonotic origin. Consistent with the broad host range of zoonotic HEV lineages, PPR in HEV genotypes 3 and 4 is ~2-fold more heterogeneous than in HEV genotype 1 (). The limited number of available sequences did not allow for assessing the degree of PPR heterogeneity in genotype 2.
Analysis of distribution of highly homoplastic sites along ORF1 was especially informative (). It was found that the PPR had the 3-fold greater HD than any other region within ORF1 of HEV genotypes 3 and 4, although no difference in HD was observed along ORF1 in genotype 1. Moreover, Shannon entropy in ORF1 from genotypes 3 and 4 was >2 times greater than in genotype 1 in the PPR (). The presence of many highly homoplastic sites indicates the operation of recurrent selection pressures on ORF1 and especially the PPR in the zoonotic genotypes 3 and 4. This finding suggests convergent evolution of the PPR, which is probably related to shuttling HEV infection among various susceptible species of hosts.
The PPR is the only region in ORF1 that contains sites under positive selection (). All 3 HEV genotypes, for which the sufficient number of sequences was available for analysis, contain 4–10 codons with dN/dS>1. Since both anthropotropic and zoonotic genotypes experienced detectable positive selection, the PPR is not only involved in adaptation to the host range but expresses other adaptive traits shared by all genotypes.
The PPR also has a low content of bulky hydrophobic aas (Ile, Met, Phe, Trp and Tyr) and a high proportion of polar and charged aas (Ala, Gly, Pro and Ser) () 
. The fractional content of aa in the PPR is maintained across all four HEV genotypes (). The confluence of findings showing high Pro density, high average Shannon entropy (), positive selection () and reduction in ordered secondary structure () strongly suggests that the HEV PPR is an IDR. Analysis carried out on the PPR 3D-model for HEV genotype 3 also confirmed that the PPR lacks regular structure, is highly polarized, negatively charged, largely solvent accessible and flexible, all characteristics of IDRs () 
. All HEV sequences studied have this IDR, including sequences from avians and rats. As HEV belongs to rubi-like viruses, other members of this family were tested for IDR; only rubivirus and CTV have a region of high polyproline density. Rubivirus possesses two IDR-like domains: one domain associated with its PPR and the other domain located between the macro domain and the endopeptidase (pfam05407) (). It should be noted that the latter domain in rubivirus does not have the high Pro density or positive dN/dS
. Thus, the PPR is the only region under positive selection in HEV, rubivirus and CTV ().
Positive selection and high homoplastic density in the PPR of HEV genotype 3 and 4 should lead to a highly diverse population of sequences within the PPR. Indeed, the PPR has a high degree of divergence () and accordingly, has been called the hypervariable region 
. All these factors taken together with reduction in aa composition complexity should substantially scramble phylogenetic relationships of the PPR from different HEV strains because of the small contribution of many sites to homology in this region. However, an examination of the PPR shows that it has approximately the same degree of phylogenetic signal as seen in most other regions of similar size in ORF1 (). Application of this region to phylogenetic analysis of HEV genotypes and subtypes seems to be as accurate as the use of any other genomic regions 
. This observation indicates that the observed homoplasy and positive selection are specific to HEV genotypes and subtypes, and occur within the boundaries of the major HEV lineages. Thus, both homoplasy and positive selection most probably reflect recurrent adaptation events experienced by each HEV lineage and are specific to these lineages. These considerations explain the reduced contribution of these factors to the phylogenetic noise at the level of genotypes and subgenotypes.
The PPR does not appear to be required for the replication of rubivirus 
or HEV 
. Immediately upstream from the PPR is a region with no known function 
. Tzeng et al.
showed that a deletion of this region in rubella virus created a mutant that was unable to replicate. However, Pudupakam et al.
found that deletions in the PPR did not abolish infectivity of HEV in vivo
or in vitro
, although near-complete deletion of the PPR yielded evidence of attenuation. Moreover, Nguyen et al.
isolated naturally occurring variants isolated in serum and feces from a patient chronically infected with HEV that had deletions in the PPR and macro domain. These observations suggest that the PPR plays a regulatory role in HEV replication, which may be related to proper positioning of structured protein domains 
. The folding of such hinges is known to regulate the self-assembly of large multiprotein complexes 
and the PPR may interact with viral and host factors 
The recently observed association between deletions in the PPR and variation in levels of viral RNA replication is consistent with the PPR being able to modulate the rate of HEV replication 
. Ropp et al.
observed that after extended incubations of 24–36 hours, the HEV ORF1 polyprotein, expressed as a vaccinia recombinant, was cleaved to yield two products in vivo
. Mutagenesis of Cys in the active site of the putative HEV papain-like protease failed to abolish cleavage, indicating that this protease is not involved in the observed ORF1-protein processing. However, Koonin et al.
had noted earlier that the papain-like protease motifs found in HEV were atypical 
, which may also explain the result of the mutagenesis experiment. The location of the potential cleavage site 
appears to be situated near the two putative LM protease cleavage sites identified here (). This finding may explain, at least in part, the results reported by Pudupakam et al.
Sequence analyses of the ORF1 of all HEV genotypes identified the PPR as an IDR (). The identified propensity of the HEV PPR for the disorder-to-order transitions upon interaction with a protein ligand () is an important IDR property. The PPR region capable of such transitions is most negatively charged () and most prone to protein-ligand binding. As an IDR, the PPR may be involved in regulating viral transcription and translation 
. IDRs are known to affect protein folding and to bind to large numbers of proteins due to the intrinsically disordered nature of these regions 
. An examination of LMs found seven putative linear motifs located within the IDR. These include two protease-cleavage sites, three ligand binding sites and two kinase phosphorylation sites ().
Additionally, the PPR 3D-model was used to predict the occurrence of molecular functional roles using GO annotations. This analysis revealed several sites potentially involved in interactions with many protein ligands ( and ). The motifs include putative peptide cleavage sites, sites modified by enzymes and sites that bind to proteins, nucleotides and metal ions. Such interactions have been shown to contribute to regulation of cellular signal transduction, protein phosphorylation as well as transcription and translation 
. Thus, these findings suggest that the PPR is involved in protein-protein interactions associated with the regulation of HEV replication. Of further interest are recent reports of isolations of virus/host recombinants found in patients chronically infected with HEV 
. In both instances a fragment from a human ribosomal protein was inserted in-frame into the PPR, in the first case as a 174-nt (58-aa) insertion from S17 
and in the second a 117-nt (39-aa) insertion from S19 
. The finding of these insertions provides an additional support to the hypothesis that the PPR has regulatory functions essential for viral adaptation rather than functions critical for viral replication.
In conclusion, the data shown here strongly suggest the role of the PPR in HEV adaptation, including the host-specific adaptation. Being an IDR, the PPR is likely involved in fine tuning of viral replication through protein-protein interactions. Delineation of these interactions will lead to a better understanding of the HEV life cycle and to development of novel anti-viral drugs.