Discovery of ultrapotent broadly neutralizing antibodies from SARS-CoV-2 elite neutralizers.

A fraction of COVID-19 convalescent individuals mount a potent antibody response to SARS-CoV-2 with cross-reactivity to SARS-CoV-1. To uncover their humoral response in detail, we performed single B cell analysis from 10 SARS-CoV-2 elite neutralizers. We isolated and analyzed 126 monoclonal antibodies, many of which were sarbecovirus cross-reactive, with some displaying merbecovirus- and embecovirus-reactivity. Several isolated broadly neutralizing antibodies were effective against B.1.1.7, B.1.351, B.1.429, B.1.617, and B.1.617.2 variants and 19 prominent potential escape sites. Furthermore, assembly of 716,806 SARS-CoV-2 sequences predicted emerging escape variants, which were also effectively neutralized. One of these broadly neutralizing potent antibodies, R40-1G8, is a IGHV3-53 RBD-class-1 antibody. Remarkably, cryo-EM analysis revealed that R40-1G8 has a flexible binding mode, targeting both "up" and "down" conformations of the RBD. Given the threat of emerging SARS-CoV-2 variants, we demonstrate that elite neutralizers are a valuable source for isolating ultrapotent antibody candidates to prevent and treat SARS-CoV-2 infection.

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.

Astrocyte Infection during Rabies Encephalitis Depends on the Virus Strain and Infection Route as Demonstrated by Novel Quantitative 3D Analysis of Cell Tropism.

Although conventional immunohistochemistry for neurotropic rabies virus (RABV) usually shows high preference for neurons, non-neuronal cells are also potential targets, and abortive astrocyte infection is considered a main trigger of innate immunity in the CNS. While in vitro studies indicated differences between field and less virulent lab-adapted RABVs, a systematic, quantitative comparison of astrocyte tropism in vivo is lacking. Here, solvent-based tissue clearing was used to measure RABV cell tropism in infected brains. Immunofluorescence analysis of 1 mm-thick tissue slices enabled 3D-segmentation and quantification of astrocyte and neuron infection frequencies. Comparison of three highly virulent field virus clones from fox, dog, and raccoon with three lab-adapted strains revealed remarkable differences in the ability to infect astrocytes in vivo. While all viruses and infection routes led to neuron infection frequencies between 7-19%, striking differences appeared for astrocytes. Whereas astrocyte infection by field viruses was detected independent of the inoculation route (8-27%), only one lab-adapted strain infected astrocytes route-dependently [0% after intramuscular (i.m.) and 13% after intracerebral (i.c.) inoculation]. Two lab-adapted vaccine viruses lacked astrocyte infection altogether (0%, i.c. and i.m.). This suggests a model in which the ability to establish productive astrocyte infection in vivo functionally distinguishes field and attenuated lab RABV strains.

Responsiveness of various reservoir species to oral rabies vaccination correlates with differences in vaccine uptake of mucosa associated lymphoid tissues.

Oral rabies vaccination (ORV) is highly effective in foxes and raccoon dogs, whereas for unknown reasons the efficacy of ORV in other reservoir species is less pronounced. To investigate possible variations in species-specific cell tropism and local replication of vaccine virus, different reservoir species including foxes, raccoon dogs, raccoons, mongooses, dogs and skunks were orally immunised with a highly attenuated, high-titred GFP-expressing rabies virus (RABV). Immunofluorescence and RT-qPCR screenings revealed clear differences among species suggesting host specific limitations to ORV. While for responsive species the palatine tonsils (tonsilla palatina) were identified as a main site of virus replication, less virus dissemination was observed in the tonsils of rather refractory species. While our comparison of vaccine virus tropism emphasizes the important role that the tonsilla palatina plays in eliciting an immune response to ORV, our data also indicate that other lymphoid tissues may have a more important role than originally anticipated. Overall, these data support a model in which the susceptibility to oral live RABV vaccine infection of lymphatic tissue is a major determinant in vaccination efficacy. The present results may help to direct future research for improving vaccine uptake and efficacy of oral rabies vaccines under field conditions.

The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion.

The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies.

Efficacy of the oral rabies virus vaccine strain SPBN GASGAS in foxes and raccoon dogs.

To test the immunogenicity and efficacy of a new oral rabies virus vaccine strain SPBN GASGAS in wildlife target species, one group of foxes and two groups of raccoon dogs were offered a bait containing 1.7 ml of the vaccine (106.6 FFU/ml; 106.8 FFU/dose) and subsequently challenged approximately 180 days later with a fox rabies virus isolate. One group of raccoon dogs (n=30) received the same challenge dose (100.7 MICLD50/ml) as the red foxes (n=29). The other group with raccoon dogs (n=28) together with 8 animals that received the vaccine dose by direct instillation into the oral cavity (DIOC) were infected with a 40-fold higher dose of the challenge virus (102.3 MICLD50/ml). All but one of the 29 vaccinated foxes survived the challenge infection; meanwhile all 12 control foxes succumbed to rabies. Twenty-eight of 30 vaccinated raccoon dogs challenged with the same dose survived the infection, however only six of 12 control animals succumbed. When the higher challenge dose was administered, all 12 control animals died from rabies and all 36 vaccinated animals (28 baited plus 8 DIOC) survived. Blood samples were collected at different time points post vaccination and examined by both RFFIT and ELISA. The kinetics of the measured immune response was similar for both species, although in RFFIT slightly higher values were observed in foxes than in raccoon dogs. However, the immune response as measured in ELISA was identical for both species. The oral rabies virus vaccine SPBN GASGAS meets the efficacy requirements for live rabies virus vaccines as laid down by the European Pharmacopoeia.

Oral vaccination of wildlife against rabies: Differences among host species in vaccine uptake efficiency.

Oral vaccination using attenuated and recombinant rabies vaccines has been proven a powerful tool to combat rabies in wildlife. However, clear differences have been observed in vaccine titers needed to induce a protective immune response against rabies after oral vaccination in different reservoir species. The mechanisms contributing to the observed resistance against oral rabies vaccination in some species are not completely understood. Hence, the immunogenicity of the vaccine virus strain, SPBN GASGAS, was investigated in a species considered to be susceptible to oral rabies vaccination (red fox) and a species refractory to this route of administration (striped skunk). Additionally, the dissemination of the vaccine virus in the oral cavity was analyzed for these two species. It was shown that the palatine tonsils play a critical role in vaccine virus uptake. Main differences could be observed in palatine tonsil infection between both species, revealing a locally restricted dissemination of infected cells in foxes. The absence of virus infected cells in palatine tonsils of skunks suggests a less efficient uptake of or infection by vaccine virus which may lead to a reduced response to oral vaccination. Understanding the mechanisms of oral resistance to rabies virus vaccine absorption and primary replication may lead to the development of novel strategies to enhance vaccine efficacy in problematic species like the striped skunk.

Comparative analysis of European bat lyssavirus 1 pathogenicity in the mouse model.

European bat lyssavirus 1 is responsible for most bat rabies cases in Europe. Although EBLV-1 isolates display a high degree of sequence identity, different sublineages exist. In individual isolates various insertions and deletions have been identified, with unknown impact on viral replication and pathogenicity. In order to assess whether different genetic features of EBLV-1 isolates correlate with phenotypic changes, different EBLV-1 variants were compared for pathogenicity in the mouse model. Groups of three mice were infected intracranially (i.c.) with 102 TCID50/ml and groups of six mice were infected intramuscularly (i.m.) with 105 TCID50/ml and 102 TCID50/ml as well as intranasally (i.n.) with 102 TCID50/ml. Significant differences in survival following i.m. inoculation with low doses as well as i.n. inoculation were observed. Also, striking variations in incubation periods following i.c. inoculation and i.m. inoculation with high doses were seen. Hereby, the clinical picture differed between general symptoms, spasms and aggressiveness depending on the inoculation route. Immunohistochemistry of mouse brains showed that the virus distribution in the brain depended on the inoculation route. In conclusion, different EBLV-1 isolates differ in pathogenicity indicating variation which is not reflected in studies of single isolates.

Rapid Reverse Genetics Systems for Rhabdoviruses: From Forward to Reverse and Back Again.

Methods to recover recombinant negative strand RNA viruses (rNSVs) from cloned cDNAs have been significantly improved in more than two decades of NSV reverse genetics . In particular, for non-segmented negative strand RNA viruses (NNSVs ) like rhabdoviruses , time-consuming generation of reverse genetics systems by stitching PCR subfragments of genomic rhabdovirus cDNAs using ligase-based conventional cloning approaches limited the number of available recombinant virus cDNA clones. As genetic variability is considered an intrinsic feature of RNA viruses, it is thus reasonable to conclude that reverse genetics approaches to investigate natural virus functions and pathogenesis require improved systems that reflect the complexity of naturally occurring wild-type viruses, and that largely exclude adaption to cell culture conditions.In order to allow rapid cloning of wild-type NSV genome populations into reverse genetics vector plasmids, we developed a system in which cDNA copies of complete rhabdovirus populations are inserted into a plasmid bank by linear-to-linear homologous RecE/T recombination (LLHR ). Limited requirements for sequence information a priori, high cloning efficiencies, and the possibility to directly generate recombinant viruses from individual cDNA clones now offer novel opportunities to combine forward genetic dissection of natural rhabdovirus populations and downstream reverse genetics approaches.

Cationic amphiphilic drugs enhance entry of lentiviral particles pseudotyped with rabies virus glycoprotein into non-neuronal cells.

Amiodarone and other cationic amphiphilic drugs (CADs) inhibit cell entry by diverse human pathogenic viruses including Filoviruses, Dengue virus and Japanese encephalitis virus. They are thus considered potential broad spectrum antiviral agents. Here we report the unexpected finding that amiodarone and other CADs markedly enhance rabies virus (RABV) glycoprotein- (GP-) mediated cell entry of pseudotyped lentiviruses into non-neuronal cells but not in neuronal cells. Increased cell entry can also be elicited when CADs are added several hours after pseudoviral attachment. Perturbing endosomal processing with phosphoinosite-3-kinase inhibitors wortmannin and LY294002 mimics the effects of CADs on RABV GP-mediated cell entry. Thus, CADs may enhance RABV GP-mediated cell entry of pseudotyped lentiviruses by promoting a late step of the pseudoviral cell entry process, possibly release from an endosomal compartment into the cytosol. In contrast to the pseudotyped lentiviruses, infection by fully infectious RABV was not enhanced by CADs, indicating, that the observed stimulation of RABV GP mediated lentivirus entry also depended on the used lentivirus vector backbone. In conclusion, we show that while CADs inhibit cell entry of diverse viruses they can also have a paradoxical enhancing effect on the ability of a viral glycoprotein to mediate cell entry depending on the cellular and viral context. Although, we show CAD-mediated enhancement of entry only for pseudoviruses, but not fully infectious RABV, the potential to unexpectedly enhance viral entry should be taken into account when considering use of CADs as antiviral agents.

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription.

UNLABELLED: Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors, and intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L), we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1 gene expression by both P and L. Finally, disruption of the motif in L resulted in a cell-type-specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step in virus replication. IMPORTANCE: Regulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown, and it is also not known whether the large enzymatic polymerase subunit L is involved. By fluorescence microscopy analysis of fluorescence-tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and reorganization, primary transcription regulation by DLC1, and regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L, our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well.

Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons.

UNLABELLED: Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 µm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 µm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 µm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCE: Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

Determination of sulfotransferase forms involved in the metabolic activation of the genotoxicant 1-hydroxymethylpyrene using bacterially expressed enzymes and genetically modified mouse models.

1-Methylpyrene, a carcinogenic polycyclic aromatic hydrocarbon, forms benzylic DNA adducts, in particular N2-(1-methylpyrenyl)-2'-deoxyguanosine, in mice and rats. It is bioactivated via 1-hydroxymethylpyrene (1-HMP) to electrophilic 1-sulfooxymethylpyrene (1-SMP). In this study, we explored the role of individual mouse sulfotransferase (SULT) forms in this activation. First, we showed that all nine mouse SULTs tested were able to activate 1-HMP to a mutagen in the his- Salmonella typhimurium reversion test. Some activation was even observed with Sult2a3 and Sult5a1, orphan forms for which no substrates were identified hitherto. Subsequently, we used cytosolic preparations from tissues of four mouse lines (wild-type, Sult1a1-, Sult1d1-, and transgenic for human SULT1A1/2) for the activation of 1-HMP in the mutagenicity assay. The most prominent impacts of the genetic SULT status were 96% decrease in hepatic activation by Sult1a1 knockout, 99% decrease in renal activation by Sult1d1 knockout, and 100-fold increase in pulmonary activation by transgenic human SULT1A1/2. Finally, we treated the various mouse lines with 1-HMP (19.3 mg/kg, intraperitoneally), and then determined 1-SMP levels in plasma and DNA adducts in tissues. Transgenic human SULT1A1/2 strongly enhanced 1-SMP plasma levels and DNA adduct formation in the liver, lung, heart, and kidney but not in the colon. Sult1a1 and Sult1d1 knockout reduced plasma 1-SMP levels as well as DNA adduct formation in some tissues (strongest effects: 97% decrease in 1-SMP and 89% decrease in hepatic adducts in Sult1a1- mice). The adduct levels detected in various tissues did not accurately reflect the activation capacity of these tissues determined in vitro, probably due to the distribution of the reactive metabolite 1-SMP via the circulation. In conclusion, we demonstrated that many mouse SULT forms are able to activate 1-HMP. In vivo, we verified a prominent role of Sult1a1 in hepatic and renal adduct formation and a smaller but unambiguous role of Sult1d1, and demonstrated the strong impact of transgenic human SULT1A1/2.

Comparative studies on the genetic, antigenic and pathogenic characteristics of Bokeloh bat lyssavirus.

Bokeloh bat lyssavirus (BBLV), a novel lyssavirus, was isolated from a Natterer's bat (Myotis nattererii), a chiropteran species with a widespread and abundant distribution across Europe. As a novel lyssavirus, the risks of BBLV to animal and human health are unknown and as such characterization both in vitro and in vivo was required to assess pathogenicity and vaccine protection. Full genome sequence analysis and antigenic cartography demonstrated that the German BBLV isolates are most closely related to European bat lyssavirus type 2 (EBLV-2) and Khujand virus and can be characterized within phylogroup I. In vivo characterization demonstrated that BBLV was pathogenic in mice when inoculated peripherally causing clinical signs typical for rabies encephalitis, with higher pathogenicity observed in juvenile mice. A limited vaccination-challenge experiment in mice was conducted and suggested that current vaccines would afford some protection against BBLV although further studies are warranted to determine a serological cut-off for protection.

Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in embryonic stem cells.

Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.

Formation of hepatic DNA adducts by methyleugenol in mouse models: drastic decrease by Sult1a1 knockout and strong increase by transgenic human SULT1A1/2.

Methyleugenol--a natural constituent of herbs and spices--is hepatocarcinogenic in rodent models. It can form DNA adducts after side-chain hydroxylation and sulfation. We previously demonstrated that human sulfotransferases (SULTs) 1A1 and 1A2 as well as mouse Sult1a1, expressed in Salmonella target strains, are able to activate 1'-hydroxymethyleugenol (1'-OH-ME) and 3'-hydroxymethylisoeugenol (3'-OH-MIE) to mutagens. Now we investigated the role of these enzymes in the formation of hepatic DNA adducts by methyleugenol in the mouse in vivo. We used FVB/N mice [wild-type (wt)] and genetically modified strains in this background: Sult1a1 knockout (ko), transgenic for human SULT1A1/2 (tg) and the combination of both modifications (ko-tg). Methyleugenol (50mg/kg body mass) formed 23, 735, 3770 and 4500 N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adducts per 10(8) 2'-deoxyribonucleosides (dN) in ko, wt, ko-tg and tg mice, respectively. The corresponding values for an equimolar dose of 1'-OH-ME were 12, 1490, 12 400 and 13 300 per 10(8) dN. Similar relative levels were observed for the minor adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine. Thus, the adduct formation by both compounds was nearly completely dependent on the presence of SULT1A enzymes, with human SULT1A1/2 producing stronger effects than mouse Sult1a1. Moreover, a dose of 0.05 mg/kg methyleugenol (one-fourth of the estimated average daily exposure of humans) was sufficient to form detectable adducts in humanized (ko-tg) mice. Although 3'-OH-MIE was equally mutagenic to 1'-OH-ME in Salmonella strains expressing human SULT1A1 or 1A2, it only formed 0.14% of hepatic adducts in ko-tg mice compared with an equimolar dose of 1'-OH-ME, suggesting an important role of detoxifying pathways for this isomer in vivo.

Conventional knockout of Tbc1d1 in mice impairs insulin- and AICAR-stimulated glucose uptake in skeletal muscle.

In the obesity-resistant SJL mouse strain, we previously identified a naturally occurring loss-of-function mutation in the gene for Tbc1d1. Characterization of recombinant inbred mice that carried the Tbc1d1(SJL) allele on a C57BL/6J background indicated that loss of TBC1D1 protects from obesity, presumably by increasing the use of fat as energy source. To provide direct functional evidence for an involvement of TBC1D1 in energy substrate metabolism, we generated and characterized conventional Tbc1d1 knockout mice. TBC1D1-deficient mice showed moderately reduced body weight, decreased respiratory quotient, and an elevated resting metabolic rate. Ex vivo analysis of intact isolated skeletal muscle revealed a severe impairment in insulin- and AICAR-stimulated glucose uptake in glycolytic extensor digitorum longus muscle and a substantially increased rate of fatty acid oxidation in oxidative soleus muscle. Our results provide direct evidence that TBC1D1 plays a major role in glucose and lipid utilization, and energy substrate preference in skeletal muscle.

Chimeric rabies viruses for trans-species comparison of lyssavirus glycoprotein ectodomain functions in virus replication and pathogenesis.

The glycoprotein G of lyssaviruses is the major determinant of virus pathogenicity and serves as a target for immunological responses to virus infections. However, assessment of the exact contribution of lyssavirus G proteins to observed differences in the pathogenicity of lyssavirus species is challenging, since the direct comparison of natural lyssaviruses does not allow specific ascription to individual virus proteins or domains. Here we describe the generation and characterization of recombinant rabies viruses (RABV) that express chimeric G proteins comprising of a RABV cytoplasma domain fused to transmembrane and ectodomain G sequences of a virulent RABV (challenge virus standard; CVS-11) or two European bat lyssaviruses (EBLV- and EBLV-2). These "envelope-switched" recombinant viruses were recovered from cDNAs. Similar growth kinetics and protein expression in neuroblastoma cell cultures and successful targeting of primary neurons showed that the chimeric G proteins were able to replace the authentic G protein in a RABV based virus vector. Inoculation of six week old CD-1 mice by the intracranial (i. c.) route of infection further demonstrated that all recombinant viruses were able to spread in the brain and to induce disease. The "envelope-switched" RABV therefore represent an important tool to further investigate the influence of lyssavirus ectodomains on virus tropism, and pathogenicity.

Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.

Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1ß- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro.