Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii.

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

Oral delivery of a functional algal-expressed TGF-ß mimic halts colitis in a murine DSS model.

Inflammatory bowel disease (IBD) is a set of immunological disorders which can generate chronic pain and fatigue associated with the inflammatory symptoms. The treatment of IBD remains a significant hurdle with current therapies being only partially effective or having significant side effects, suggesting that new therapies that elicit different modes of action and delivery strategies are required. TGM1 is a TGF-ß mimic that was discovered from the intestinal helminth parasite Heligmosomoides polygyrus and is thought to be produced by the parasite to suppress the intestinal inflammation response to help evade host immunity, making it an ideal candidate to be developed as a novel anti-inflammatory bio-therapeutic. Here we utilized the expression system of the edible green algae Chlamydomonas reinhardtii in order to recombinantly produce active TGM1 in a form that could be ingested. C. reinhardtii robustly expressed TGM1, and the resultant recombinant protein is biologically active as measured by regulatory T cell induction. When delivered orally to mice, the algal expressed TGM1 is able to ameliorate weight loss, lymphadenopathy, and disease symptoms in a mouse model of DSS-induced colitis, demonstrating the potential of this biologic as a novel treatment of IBD.

Modification of Three Amino Acids in Sodium Taurocholate Cotransporting Polypeptide Renders Mice Susceptible to Infection with Hepatitis D Virus In Vivo.

UNLABELLED: Sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for hepatitis D virus (HDV) and its helper hepatitis B virus (HBV). In cultured cell lines, HDV infection through mouse NTCP is restricted by residues 84 to 87 of the receptor. This study shows that mice with these three amino acids altered their corresponding human residues (H84R, T86K, and S87N) in endogenous mouse NTCP support de novo HDV infection in vivo HDV infection was documented by the presence of replicative forms of HDV RNA and HDV proteins in liver cells at day 6 after viral inoculation. Monoclonal antibody specifically binding to the motif centered on K86 in NTCP partially inhibited HDV infection. These studies demonstrated specific interaction between the receptor and the viral envelopes in vivo and established a novel mouse model with minimal genetic manipulation for studying HDV infection. The model will also be useful for evaluating entry inhibitors against HDV and its helper HBV. IMPORTANCE: NTCP was identified as a functional receptor for both HDV and HBV in cell cultures. We recently showed that neonatal C57BL/6 transgenic (Tg) mice exogenously expressing human NTCP (hNTCP-Tg) in liver support transient HDV infection. In this study, we introduced alterations of three amino acids in the endogenous NTCP of FVB mice through genome editing. The mice with the humanized NTCP residues (H84R, T86K, and S87N) are susceptible to HDV infection, and the infection can be established in both neonatal and adult mice with this editing. We also developed a monoclonal antibody specifically targeting the region of NTCP centered on lysine residue 86, and it can differentiate the modified mouse NTCP from that of the wild type and partially inhibited HDV infection. These studies shed new light on NTCP-mediated HDV infection in vivo, and the NTCP-modified mice provide a useful animal model for studying HDV infection and evaluating antivirals against the infection.

Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide.

Hepatitis D virus (HDV) is the smallest virus known to infect human. About 15 million people worldwide are infected by HDV among those 240 million infected by its helper hepatitis B virus (HBV). Viral hepatitis D is considered as one of the most severe forms of human viral hepatitis. No specific antivirals are currently available to treat HDV infection and antivirals against HBV do not ameliorate hepatitis D. Liver sodium taurocholate co-transporting polypeptide (NTCP) was recently identified as a common entry receptor for HDV and HBV in cell cultures. Here we show HDV can infect mice expressing human NTCP (hNTCP-Tg). Antibodies against critical regions of HBV envelope proteins blocked HDV infection in the hNTCP-Tg mice. The infection was acute yet HDV genome replication occurred efficiently, evident by the presence of antigenome RNA and edited RNA species specifying large delta antigen in the livers of infected mice. The resolution of HDV infection appears not dependent on adaptive immune response, but might be facilitated by innate immunity. Liver RNA-seq analyses of HDV infected hNTCP-Tg and type I interferon receptor 1 (IFNα/ßR1) null hNTCP-Tg mice indicated that in addition to induction of type I IFN response, HDV infection was also associated with up-regulation of novel cellular genes that may modulate HDV infection. Our work has thus proved the concept that NTCP is a functional receptor for HDV infection in vivo and established a convenient small animal model for investigation of HDV pathogenesis and evaluation of antiviral therapeutics against the early steps of infection for this important human pathogen.

Viral entry of hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.

UNLABELLED: The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population, renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs. IMPORTANCE: Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), are important human pathogens. Available therapeutics against HBV are limited, and there is no drug that is clinically available for HDV infection. A liver bile acids transporter (sodium taurocholate cotransporting polypeptide [NTCP]) critical for maintaining homeostasis of bile acids serves as a functional receptor for HBV and HDV. We report here that the NTCP-binding lipopeptide that originates from the first 47 amino acids of the pre-S1 domain of the HBV L protein blocks taurocholate transport. Some bile salts dose dependently inhibit HBV and HDV infection mediated by NTCP; molecular determinants of NTCP critical for HBV and HDV entry overlap with that for bile acids transport. This work advances our understanding of NTCP-mediated HBV and HDV infection in relation to NTCP's physiological function. Our results also suggest that bile acids or their derivatives hold potential for development into novel drugs against HBV and HDV infection.

Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide.

Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), primarily infect humans, chimpanzees, or tree shrews (Tupaia belangeri). Viral infections in other species are known to be mainly restricted at the entry level since viral replication can be achieved in the cells by transfection of the viral genome. Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for HBV and HDV, and amino acids 157 to 165 of NTCP are critical for viral entry and likely limit viral infection of macaques. However, the molecular determinants for viral entry restriction in mouse NTCP (mNTCP) remain unclear. In this study, mNTCP was found to be unable to support either HBV or HDV infection, although it can bind to pre-S1 of HBV L protein and is functional in transporting substrate taurocholate; comprehensive swapping and point mutations of human NTCP (hNTCP) and mNTCP revealed molecular determinants restricting mNTCP for viral entry of HBV and HDV. Remarkably, when mNTCP residues 84 to 87 were substituted by human counterparts, mNTCP can effectively support viral infections. In addition, a number of cell lines, regardless of their species or tissue origin, supported HDV infection when transfected with hNTCP or mNTCP with residues 84 to 87 replaced by human counterparts, highlighting the central role of NTCP for viral infections mediated by HBV envelope proteins. These studies advance our understanding of NTCP-mediated viral entry of HBV and HDV and have important implications for developing the mouse model for their infections.

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.DOI:http://dx.doi.org/10.7554/eLife.00049.001.

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.