Previously, we found that among five rotavirus strains studied, RRV and SA11-FM had tropism for the biliary epithelial cell and could induce biliary atresia in the murine model; SA11-SM could be found in the hepatobiliary system but caused hepatitis instead of biliary obstruction; and EDIM and Wa caused no hepatobiliary disease (1
). To determine the molecular basis of RRV tropism for the biliary epithelial cell, we utilized the rotavirus property of reassortment to determine which RRV gene(s) governs its ability to induce biliary atresia in the murine model. Initially, we attempted to generate reassortants with the parental strains RRV and EDIM, but because EDIM does not replicate well within MA104 cells, the generation of new reassortants was challenging. To overcome this challenge, we tested other strains of rotavirus in the murine model of BA and identified the simian strain TUCH, which, after injection into newborn BALB/c pups, could be found within hepatobiliary tissue but did not cause BA. Because it replicated well in MA104 cells, we used it for the generation of reassortants. By using RRV and TUCH, we generated a complete set of 11 loss-of-function “knockout” single-gene reassortants and 11 reciprocal gain-of-function “knock-in” single-gene reassortants. The gain-of-function “knock-in” reassortants were important, confirming that the presence of a specific RRV gene was by itself capable of causing biliary atresia in the murine model. These rotavirus monoreassortants permit the identification of the RRV gene(s) that governs the ability to induce BA.
Using these single-gene reassortants, we identified RRV gene segments 3 and 4 as important determinants in the pathogenesis of murine BA. We found that when gene segment 4 of RRV was replaced by the corresponding gene derived from TUCH, the manifestations of biliary obstruction were totally abolished. In a reciprocal fashion, when newborn mice were infected with TR(VP4)
(the TUCH background clone with the VP4 gene derived from RRV), BA was induced. Supportive results were recently found using rotavirus strains RRV and UK (8
). Experiments performed by Feng et al. demonstrated the requirement of both genes VP4 and NSP1 from RRV for replication in the mouse biliary tract. RRV and TUCH share 91% amino acid homology in NSP1, which could explain why we did not observe a role for NSP1 in our experiments. Our in vitro
studies revealed that RRV gene segment 4 governed cholangiocyte binding and infectivity, establishing a mechanistic basis for the in vivo
results. The protein product of gene segment 4, VP4, has been found to be a major determinant of rotavirus pathogenicity in several systems. Using heterologous bovine × simian viral reassortants, Offit et al. linked gene segment 4 to tropism for the intestine in mice (27
). Two porcine rotavirus variants (4f and 4s) with different VP4 genes showed distinct pathogenicity profiles during serial passage in gnotobiotic piglets. Insertion of the pathogenic parental (4f) gene segment 4 into the nonpathogenic virus (4s) genome by reassortment caused the latter to develop tropism for the intestine and caused diarrhea in piglets (2
). VP4 is essential for early virus-cell interactions, because it participates in receptor binding and cell penetration (12
The early rotavirus-cell interactions constitute a multistep process (21
). In the multistep model, the initial contact of a neuraminidase-sensitive virus strain with the cell surface occurs through a sialic acid (SA)-containing cell receptor, using the VP8* subunit of VP4, which is positioned at the surfaces of rotavirus particles. The initial interaction of the virus with SA induces a subtle conformational change in VP4, which allows the virus to interact with a second cell receptor (currently proposed to be the α2β1 integrin) through a DGE-binding motif. After the second interaction, the virus interacts with integrins αXβ2, αVβ3, and hsc70. These interactions increase the permeability of the cell membrane, facilitating the penetration of the virus into the cell. Previously, we found that expression of the α2β1 integrin on the cell surface confers susceptibility to RRV infection on cholangiocytes (16
). The different mechanisms by which TUCH VP4 and RRV VP4 interact with the cell surface require further study. The VP4 proteins from these simian rotaviruses share 87% homology, so it is probable that the mechanistic basis for their differing abilities to infect cholangiocytes lies within the nonhomologous regions.
Interestingly, VP7, the other major constituent of the outer protein layer, did not, under these conditions, govern hepatobiliary tropism. As an outer capsid protein, VP7 plays a role in viral entry; however, reassortants RT(VP7)
behaved similarly to their parents. The function of VP7 is to facilitate cell entry and infection by interacting with αXβ2 and αVβ3 (12
) in a postbinding step. Because the VP7 proteins of RRV and TUCH were both G3 types and had 90.5% homology, it is likely that manipulation of this gene segment did not change the protein structure of VP7 and thus did not affect cell entry/replication.
The mechanism by which gene segment 3 and its translated protein VP3 govern the induction of murine BA is unknown. At least one function of VP3 is to act as a guanylyltransferase and methyltransferase, enabling capping of the 5′ end of the mRNA synthesized in viroplasms by the RNA-dependent RNA polymerase, VP1 (5
). The cap stabilizes viral mRNA, potentially protecting it from degradation by nucleases (6
), and enables its translation by interaction with cellular translation protein complexes. Association of VP3 with three other genes (VP4, VP7, NSP4) in rotavirus virulence and host range restriction and attenuation has been suggested in reassortant studies (27
). In these studies, piglets failed to develop diarrhea when challenged with a single-gene reassortant that derived its 3rd gene from an avirulent background, while diarrhea was induced only when virulence-associated genes encoding VP3, VP4, VP7, and NSP4 were present together on an avirulent-rotavirus background. Our results are somewhat different from these observations in that reassortant RT(VP3)
did not abolish biliary injury; rather, it elicited an attenuated phenotype compared with that of RRV. Reassortant TR(VP3)
caused a significant increase in the proportion of animals with biliary injury symptoms, similar to that with RRV, but mortality was minimal. We speculate that gene segment 3 might affect viral replication rates, thereby decreasing mortality, but this was not observed in infectivity assays. The basis for the intermediate phenotype induced by gene segment 3 is currently unclear, but it might result from the interactions of multiple factors, including the host immune response.
It was noteworthy that TUCH reassortants containing the RRV VP6, NSP2, NSP3, NSP4, or NSP5 gene increased symptoms of hepatobiliary injury without eliciting bile duct obstruction or mortality. The reverse effect was not seen in the reciprocal RRV reassortants containing the same TUCH genes. The TUCH reassortants produced larger amounts of replication-competent virus than did the TUCH parent both in vivo and in vitro. The increase in symptoms encountered following infection with these reassortants could be a consequence of viral loads causing hepatobiliary inflammation rather than obstruction. Further study will be required to determine how these genes contribute to pathogenesis.
The basis for the contributions of these gene segments to pathogenesis may extend beyond the mechanisms discussed. Several recent investigations have established both a cell- and an antibody-mediated immunological role in biliary obstruction. A recent study by Mack and colleagues showed that a component of VP4 results in the generation of an antibody that recognizes α-enolase expressed on cholangiocytes (22
). This mimicry may contribute to BA pathogenesis. Interestingly, within the segment of interest identified in that study lie several amino acid differences between RRV and TUCH that may cause conformational changes in the peptide structure which could contribute to antibody recognition. Site-directed mutagenesis will be necessary to confirm these findings.
In summary, a set of 22 single-gene reassortants was generated that allowed the identification of RRV gene segments 3 and 4 as important determinants of murine BA. RRV gene segment 4, through its translated protein VP4, determined RRV tropism for the biliary system. The mechanism of binding to receptors on the surfaces of cholangiocytes remains to be characterized. Replacement of the 3rd gene segment of RRV did not affect viral infectivity in vitro
; however, it caused an intermediate phenotype in vivo
. Although rotavirus has been refractory to direct genetic manipulation, a reverse-genetics system has recently been developed which permits the rescue of a viral RNA segment produced from cDNA in vitro
into replication-competent progeny virus (18
). In the future, this approach will be utilized to determine the precise molecular mechanism by which RRV gene segments 3 and 4 contribute to the pathogenesis of BA.