The determinants of HAV replication and pathogenesis are poorly understood. Experimentation with HAV is difficult because it grows poorly in cell culture. Since primates are the only animal models for this virus, development of a small-animal model to study the pathogenesis of HAV is highly desirable. A mouse model for HAV would be ideal, but unfortunately, HAV does not replicate efficiently in cells of murine origin.
To circumvent this problem, we attempted to adapt HAV to grow efficiently in MMH-D3 cells, a nontransformed mouse hepatocyte cell line (2
). To maintain optimal proliferation and retain their hepatocyte-like characteristics, the MMH-D3 cells were cultivated in the presence of insulin, EGF, and IGF II. Our results showed that HAV can grow in MMH-D3 cells transfected with virion RNA but not in MMH-D3 cells infected with HAV particles, which implies a problem at the cell entry level. The cell entry block(s) was overcome by withdrawal of EGF from the culture medium of the MMH-D3 cells. This acquired susceptibility to HAV infection could be due to several reasons, for instance, (i) the expression of an endogenous mouse gene(s) capable of serving as a cellular receptor for HAV, (ii) the activation of transcriptional or posttranscriptional events that allowed normally expressed genes to function as HAV cellular receptors, or (iii) the repression of a mouse gene(s) that prevented cell entry of HAV. More research will be required to elucidate the molecular basis for the gain of susceptibility to HAV infection after withdrawal of EGF from the cell culture medium of the MMH-D3 cells.
Interestingly, hepatitis A (HA) is an age-dependent disease. Children 6 years old or younger usually develop subclinical forms of HA whereas older individuals develop more-severe manifestations of the disease (13
). Because aging reduces the response to EGF treatment and the phosphorylation of the EGF receptor (18
; reference 31
and references therein), it is tantalizing to postulate an inverse relationship between the severity of HA and the response to EGF. The response to EGF may also play a role in the severity of HA. Further research will be required to determine whether EGF could also influence the susceptibility of human hepatocytes to HAV infection and to determine whether EGF plays a role in the severity of HA.
It has previously been reported that HAV can grow efficiently in cells of primate origin and some cells of nonprimate origin but not in mouse cells. Takeda et al. (35
) detected negative-strand HAV RNA in mouse L929 and NIH/3T3 cells after infection with HAV. However, low levels of HAV (101.8
s) were produced after transfection of viral RNA, suggesting that a very limited infection occurred in these cells. We have recently shown that mouse Ltk- cells can support HAV growth after transfection with HAV infectious cDNA (19
). It should be pointed out that Ltk- cells were not susceptible to HAV infection and that the cDNA-transfected Ltk- cells that supported efficient levels of HAV growth had to be selected by several rounds of single-cell cloning. This paper is the first report of mouse cells that are susceptible to HAV infection and support the efficient growth of this virus. We showed that MMH-D3 cells grown under suboptimal conditions gained susceptibility to HAV infection and produced approximately 105
of virus/ml. Additionally, serial passages of HAV in HP-MMH cells, which were derived from MMH-D3 cells cultured under suboptimal conditions, resulted in the selection of viral variants that acquired the capability of infecting hepatocyte-like MMH-D3 cells cultured under optimal conditions. These mouse-adapted HAV-MMH variants contained four amino acid substitutions in the capsid region. Mutations N1237D and D2132G were present in the six clones that we analyzed, which suggested that these two substitutions were responsible for the adaptation of HAV to mouse hepatocyte-like cells. We are currently constructing HAV mutants to determine whether these two changes are indeed responsible for the adaptation of HAV to mouse hepatocyte-like cells. Although the structure of HAV has not yet been resolved, a model of the HAV capsid predicted that both mutations are located at the viral surface (20
). Interestingly, N1237D is adjacent to Q1232, a residue that is within the major antigenic site of HAV (26
). In addition, Y1236 has been shown to be accessible to iodination in highly purified virions (30
), which also suggests that this residue is exposed at the virion surface. Therefore, it is possible that amino acid residues 237 of VP1 and 132 of VP2 are indeed located at the surface of the HAV particle and that mutations N1237D and D2132G allowed the interaction of HAV-MMH with a mouse receptor that mediated cell entry. Further research and the elucidation of the structure of HAV will be required to validate this hypothesis.
A multigene family of mouse orthologs of hhavcr-1 has recently been identified (24
). Two members of this multigene family, Tim1 and Tim2, share a high degree of homology with hhavcr-1. It is presently unknown whether members of the mouse Tim family have HAV receptor function. However, preliminary results (D. Feigelstock and G. Kaplan, unpublished data) indicated that withdrawal of EGF from the cell culture medium of MMH-D3 cells did not affect the levels of Tim1 and Tim2 expression. Further work will be required to determine whether members of the Tim family have HAV receptor function.
In this paper, we showed for the first time that mouse cells code for all the cellular factors required for the efficient growth of HAV. We also showed that MMH-D3 cells gained susceptibility to HAV infection upon withdrawal of EGF from the cell culture medium. Our data also suggest that mutations at the viral surface allowed the entry of HAV into mouse hepatocyte-like cells. The experimental system reported here will contribute to the identification of cellular factors required for HAV growth and possibly to the development of a mouse model for study of the pathogenesis of HAV.