Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

Burwitz, Benjamin J.; Wettengel, Jochen M.; Mück-Häusl, Martin; Ringelhan, Marc; Ko, Chunkyu; Festag, Marvin M.; Hammond, Katherine B.; Northrup, Mina; Bimber, Benjamin N.; Jacob, Thomas; Reed, Jason S.; Norris, Reed; Park, Byung; Moller-Tank, Sven; Esser, Knud; Greene, Justin M.; Wu, Helen L.; Abdulhaqq, Shaheed; Webb, Gabriela M.; Sutton, William F.; Klug, Alex; Swanson, Tonya; Legasse, Alfred W.; Vu, Tania Q.; Asokan, Aravind; Haigwood, Nancy L.; Protzer, Ulrike; Sacha, Jonah B.

doi:10.1038/s41467-017-01953-y

Cited by 64 publications

(77 citation statements)

References 47 publications

(54 reference statements)

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“…HBV was purified and concentrated from HepAD38 cell culture supernatant using previously published protocols 27,62 . In brief, cells were cultivated in multi-layer flasks and supernatant collected every 3-4 days.…”

Section: Experimental Methodsmentioning

confidence: 99%

Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle

Chakraborty

Henning

et al. 2020

Preprint

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24Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) 25 DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA 26 that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and 27 host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium 28 taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral 29 life cycle. We employed a synchronized infection protocol to quantify HBV entry kinetics. HBV attachment to cells 30 at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation 31 at 12h post-infection. HBV uptake is clathrin-and dynamin dependent with actin and tubulin playing a role in the 32 first 6h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6h of infection and 33 cccDNA was first detected at 24h post-infection. Our studies show the majority (83%) of cell bound particles enter 34 HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting 35 this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell 36 culture systems and will inform the design and evaluation of physiologically relevant models that support efficient 37 HBV replication. 39Hepatitis B Virus (HBV) infects 257 million individuals worldwide and is a major driver of end-stage liver disease, 41 cirrhosis, and hepatocellular carcinoma (HCC). HBV is an enveloped DNA and prototypic member of the 42 hepadnaviridae that establishes its genome as an episomal, covalently closed circular DNA (cccDNA) in the nucleus 43 of infected hepatocytes. Current treatments suppress viral replication but are not curative, largely due to the 44 persistence of the cccDNA transcriptional template and failure to mount an effective anti-viral immune response 1 . 45In most cases, treatment is life-long and patients may still develop HCC 2 , highlighting a clinical need for new 46 curative therapies 3 . Despite its central role in the HBV life cycle our understanding of the host factors regulating 47 cccDNA genesis and half-life is limited 4 . 49Viral entry into a host cell represents the first step in the infectious life cycle and is mediated via specific 50 interactions between virus encoded proteins and cellular receptors that define internalization pathways 5 . The 51 discovery that sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV 6,7 enabled the 52 development of in vitro culture systems that support the complete HBV replication cycle. HBV encodes three 53 envelope glycoproteins, small (S), middle (M) and large(L) 8 . The preS1 domain of the L protein binds heparan 54 sulfate proteoglycan (HSPG) 9-11 that precedes high-affinity virus interaction with NTCP....

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Section: Experimental Methodsmentioning

confidence: 99%

Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle

Chakraborty

Henning

et al. 2020

Preprint

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“…To date, hepadnaviruses have been found in multiple Hominoids, including chimpanzees and orangutans (38). On the other hand, no HBV or hepadnavirus infections have been reported in Old-World monkeys, even though this family is the closest to the Hominoids (Figure 1) [Although a unique case of HBV genotype D infection was reported in Macaca fascicularis (cynomolgus macaques) from Mauritius (39), another recent paper showed that this macaques exhibited no evidence of current or prior HBV infection (29)]. Since Old-World monkeys are the closest species to human that are regarded to be non-susceptible to HBV or hepadnaviruses, we focused on these two lineages for the comparison among NTCP genes.…”

Section: Resultsmentioning

confidence: 99%

“…The sodium taurocholate cotransporting polypeptide [NTCP, also designated as solute carrier family 10A1 (SLC10A1)] was recently identified as a host factor that functions as an HBV entry receptor; NTCP, which originally was characterized as a hepatic transporter for the uptake of bile acids by hepatocytes, binds to the HBV envelope protein, notably to the preS1 region, thereby mediating viral entry into the host (26). NTCP has been suggested to be a key determinant of the species-specificity of HBV, as primary monkey hepatocytes can support the replication of intracellular HBV but not the entry of the virus into host cells (27), and complementation the monkey cells with human NTCP (hNTCP) permits HBV entry and thereby the whole infection cycle both in cell culture and in vivo (28, 29). These results indicate that the inability of monkey Ntcp to support HBV infection serves as the species barrier preventing HBV infection in monkey.…”

Section: Introductionmentioning

confidence: 99%

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

Takeuchi

Fukano

Iwamoto

et al. 2018

Preprint

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24Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates from 25 fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. 26However, there are no reports providing direct molecular evidence for such a coevolutionary "arms 27 race" between hepadnaviruses and their hosts. Here, we present evidence suggesting the adaptive 28 evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been 29 influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals 30 showed that most NTCP residues are highly conserved among species, exhibiting evolution under 31 negative selection (dN/dS < 1); this observation implies that the evolution of NTCP is restricted by 32 maintaining its original protein function. However, 0.7 % of NTCP amino acid (aa) residues exhibit 33 rapid evolution under positive selection (dN/dS > 1). Notably, a substitution at aa 158, a positively 34 selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to 35 support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the 36 human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested 37 that positive selection at aa 158 was induced by virus infection. Moreover, the aa 158 sequence 38 determined attachment of the HBV envelope protein to host cell, demonstrating the mechanism 39 whereby HBV infection would create positive selection at this residue in NTCP. In summary, we 40 provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing an 41 adaptive mutation in host receptor. 42 3 Importance 43 Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, with 44 a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive 45 mutations in hosts to escape from infection, while simultaneously allowing adaptive mutations in 46 viruses to overcome host barriers. However, there is no published molecular evidence for such a 47 coevolutionary "arms race" between hepadnaviruses and hosts. In the present study, we performed 48 coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate 49 cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays 50 for investigating the biological significance of NTCP sequence variation. Our data provide the first 51 molecular evidences supporting that HBV-related hepadnaviruses drive adaptive evolution in the 52 NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral 53 susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with 54 their hosts, permitting the acquisition of strong species-specificity. 55 56 57 4

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“…(11) However, attempts to replicate those findings were unsuccessful. (12) When we initially isolated WMHBV, we examined spider monkeys as surrogate hosts, since woolly monkeys are endangered. In that study, maximum viral titers of 10 5 GE/mL were obtained with viremia that lasted only six weeks.…”

Section: Discussionmentioning

confidence: 99%

“…However, a recent attempt to replicate infection of Mauritian cynomolgus macaques with that strain of HBV was unsuccessful. (12) In 1998, a hepatitis B-like virus was isolated from zoo-housed woolly monkeys suffering from fulminant hepatitis (WMHBV), (13) and an infectious clone of the virus was reported in 2003. (14) WMHBV is a hepadnavirus with the same genetic organization as human HBV.…”

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confidence: 99%

Woolly Monkey–HBV Infection in Squirrel Monkeys as a Surrogate Nonhuman Primate Model of HBV Infection

et al. 2020

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Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high‐sequence homology of the HBV receptor, Na+/taurocholate co‐transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah−/−, NOD, Rag1−/−, Il2Rgnull (FNRG) mice engrafted with human or squirrel monkey hepatocytes were challenged with HBV or Woolly Monkey HBV (WMHBV). In addition, adult squirrel monkeys were inoculated with HBV, WMHBV, adeno‐associated virus containing an infectious genome of HBV (AAV‐HBV), and AAV‐WMHBV. Finally, neonate squirrel monkeys were assessed for the potential of chronic infection with WMHBV. PreS1 peptide efficiently bound to human and squirrel monkey NTCP but not to mouse or capuchin NTCP. FNRG mice engrafted with squirrel monkey hepatocytes were susceptible to infection by WMHBV but not human HBV. Similarly, adult squirrel monkeys could be infected with WMHBV but not human HBV, whereas chimeric mice engrafted with human hepatocytes were susceptible to HBV but not WMHBV. Infection of squirrel monkeys with AAV‐WMHBV yielded maximum viremia of 108 genomes/mL with detectable virus for up to 8 months. Notably, covalently closed circular DNA was detected in the liver of these animals. Infection of neonates with WMHBV led to detectable viremia for up to 6 months. Conclusions: Adult and neonate squirrel monkeys exhibited prolonged WMHBV viremia lasting 6‐8 months. This is greater than twice the duration of viremia achieved in other nonhuman primates and suggests that squirrel monkeys may be a suitable model for testing HBV therapeutics.

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Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

Cited by 64 publications

References 47 publications

Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle

Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle

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

Woolly Monkey–HBV Infection in Squirrel Monkeys as a Surrogate Nonhuman Primate Model of HBV Infection

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