A Single Adaptive Mutation in Sodium Taurocholate Cotransporting Polypeptide Induced by Hepadnaviruses Determines Virus Species Specificity

Abstract

Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. However, there are no reports providing direct molecular evidence for such a coevolutionary “arms race” between hepadnaviruses and their hosts. Here, we present evidence suggesting that the adaptive evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals showed that most NTCP residues are highly conserved among species, exhibiting evolution under negative selection (dN/dS ratio [ratio of nonsynonymous to synonymous evolutionary changes] of <1); this observation implies that the evolution of NTCP is restricted by maintaining its original protein function. However, 0.7% of NTCP amino acid residues exhibit rapid evolution under positive selection (dN/dS ratio of >1). Notably, a substitution at amino acid (aa) 158, a positively selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested a close association of the aa 158 positive selection with the pressure by virus infection. Moreover, the aa 158 sequence determined attachment of the HBV envelope protein to the host cell, demonstrating the mechanism whereby HBV infection would create positive selection at this NTCP residue. In summary, we provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing adaptive mutation in host receptor. IMPORTANCE HBV and its hepadnavirus relatives infect a wide range of vertebrates, with a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive mutations in hosts to escape from infection while simultaneously allowing adaptive mutations in viruses to overcome host barriers. However, there is no published molecular evidence for such a coevolutionary arms race between hepadnaviruses and hosts. In the present study, we performed coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays for investigating the biological significance of NTCP sequence variation. Our data provide the first molecular evidence supporting that HBV-related hepadnaviruses drive adaptive evolution in the NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with their hosts, permitting the acquisition of strong species specificity.

Circadian control of hepatitis B virus replication

“…The HDV supernatant was concentrated (x50) using an Amicon centrifugal filter (MWCO: 100 kDa, Merck, Germany) dHepaRG cells were infected with HDV at a MOI of 25 genome equivalents per cell in the presence of 5% polyethylene glycol (PEG) 8000 for 24 h. The cells were washed three times with medium to remove viral inoculum and cultured for an additional 6 days. HDV infection was evaluated by detecting HDAg by immunofluorescence assay and by quantifying HDV RNA by qRT-PCR as previously described 76 .…”

Diverse Effects of the NTCP p.Ser267Phe Variant on Disease Progression During Chronic HBV Infection and on HBV preS1 Variability

“…HBV has been evolving together with Homo sapiens over millennia and many studies have reported the evolutionary “arms race” between the HBV and the host immune system (Kramvis et al, 2018; Lumley et al, 2018). Recently, two studies reported that HBV-related hepadnaviruses drive the evolutionary history of their mammalian host receptor and induce signatures of adaptive selection in the cellular receptor NTCP (Jacquet et al, 2018; Takeuchi et al, 2018). Host genes also influence the evolution of the virus.…”

Animal Models of Hepatitis B Virus Infection–Success, Challenges, and Future Directions

“…On the other hand, hNTCP expression in cynomolgus macaque, rhesus macaque, and pig hepatocytes enables productive HBV infection, indicating that the barriers experienced in mice are not an issue in macaques [ 29 ]. Notably, amino acid residue 158 in hNTCP determines species specificity towards HBV infection in Old World Monkeys (OWM); substitution of this residue to that of cynomolgus macaque NTCP impedes HBV infection [ 42 ].…”