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Papers by Michael Nassal

Nature Communications

The discovery of nackednaviruses provided new insight into the evolutionary history of the hepati... more The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to ...

(A) Increased priming signals by co-expression of wild-type ε RNA and increased α-<su... more (A) Increased priming signals by co-expression of wild-type ε RNA and increased α-32P-dATP concentration. FLAG-tagged HBV P protein from cells transfected with only the P protein vector (lane P), or cotransfected with a wild-type ε RNA expression vector (lanes P + ε) was immobilized on anti-FLAG antibody beads. One third each of the immunoprecipitate was incubated with 1 µl or 2 µl of α-32P-dATP (3,000 Ci/mmol) as reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072798#pone.0072798-Jones1" target="_blank">[44]</a>; subsequently, the beads were boiled in SDS-PAGE sample buffer, and the released material was analyzed by SDS-PAGE and autoradiography. The remaining one third of the immunopellet was analyzed for FLAG-tagged P protein by Western blotting (panel anti-FLAG). (B) In vitro priming activities of selected ε RNA variants. FLAG-tagged P protein complexes with the indicated ε RNAs were expressed, affinity purified and subjected to in vitro priming conditions as in (A), using 2 µl α-32P-dATP. The molecular mass marker positions indicated on the left (in kDa) are approximations inferred from the respective marker protein positions on the SDS-PAGE gels used for the anti-FLAG immunoblots which were run in parallel under identical conditions. Numbers below the autoradiogram indicate mean signal intensities ± standard deviation from two independent experiments relative to that produced by the wild-type ε RNA complex which was set to 100.</p

(A) Enrichment of particulate HBc by Nycodenz gradient sedimentation.... more (A) Enrichment of particulate HBc by Nycodenz gradient sedimentation. Cytoplasmic lysate from HBV producing HepG2.117 cells was sedimented through a Nycodenz gradient. Fractions were analyzed by SDS-PAGE and CB staining (top panel; asterisks mark a 21 kDa band possibly representing HBc183; the complete gel is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.s014" target="_blank">S12A Fig</a>); by NAGE followed by immunoblotting with the anti-HBc assembly domain mAb 312 as PO conjugate (middle panel); and by hybridization with a 32P-labeled HBV probe. (B) Southern blot for capsid-borne HBV DNA in individual gradient fractions. M, DNA marker comprising HBV-specific fragments of the indicated sizes loaded in native ds form, or in heat-denatured ss form. (C) Phos-Tag SDS-PAGE immunoblot. Aliquots from the respective gradient fractions and recombinant HBc183 coexpressed with the indicated kinases, or not (ø), were separated by Phos-Tag SDS-PAGE and immuno-blotted with mAb 1D8. Short exposure showed one band with comparably strong retardation as SRPK1-phosphorylated HBc183. Longer exposure (left panel) revealed weak additional bands with mobilities similar to those of unmodified and PKC and PKA phosphorylated HBc183 (arrowheads). (D) Enveloped capsids contain phosphorylated HBc183. PEG-precipitated particles in supernatants from HepG2.117 cells, or from an HBc183 producing Huh7 line, H4-15 were analyzed by NAGE immunoblotting alongside E. coli HBc183 CLPs. The blot was sequentially probed with mAb T2212 (anti-phospho-CTD), mAb 312, and mAb 9H9 (anti-HBs). Note that T2212 detected only HBc from eukaryotic cells, including a low mobility species that comigrated with HBsAg and was absent from the H4-15 samples; it therefore represents enveloped capsids. Additional data employing mAb T2212 are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.s014" target="_blank">S12 Fig</a>.</p

(A) Selection of well-performing cell clones. Clones preselected for ... more (A) Selection of well-performing cell clones. Clones preselected for the presence of HBV DNA were grown in the absence (-) or presence (+) of DOX. Intracellular capsid-associated viral DNA was analyzed by Southern blotting, using a 32P labeled HBV specific probe. The wt-HBV producing Huh7.93 and HepG2.117 lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145746#pone.0145746.ref044" target="_blank">44</a>] served as reference. M, marker DNAs consisting of mixture of a 3.2 kb linear HBV genome in double-stranded (DL) and heat-denatured single-stranded (ss) form, plus a 3.2 kb plasmid (pla) including about 500 bp HBV sequence. RC, relaxed circular DNA. Virus variants and individual clone numbers are given on the top, an asterisk indicates the clone used further; sYC-PT is the double-mutant sY100C-P120T. Data for the other cell lines are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145746#pone.0145746.s001" target="_blank">S1 Fig</a>(B) Verification of DOX-regulation via capsid expression. Equal aliquots of cytoplasmic lysates from the selected cell clones grown without or with DOX were separated by native agarose gel electrophoresis (NAGE). Capsids were detected using the anti-core protein monoclonal antibody mAb312 conjugated to peroxidase plus chemiluminescent substrate. The mAb´s epitope around aa 80 of the core protein is conserved in WMHBV core protein. Note the virtual absence of capsids in cells grown with DOX.</p

Frontiers in Molecular Biosciences, 2021

We here establish the phosphorylation sites in the human hepatitis B virus (HBV) large envelope p... more We here establish the phosphorylation sites in the human hepatitis B virus (HBV) large envelope protein (L). L is involved in several functionally important interactions in the viral life cycle, including with the HBV cellular receptor, HBV capsid, Hsc70 chaperone, and cellular membranes during fusion. We have recently shown that cell-free synthesis of the homologous L protein of duck HBV in wheat germ extract results in very similar phosphorylation events to those previously observed in animal cells. Here, we used mass spectrometry and NMR to establish the phosphorylation patterns of human HBV L protein produced by both in vitro cell-free synthesis and in E. coli with the co-expression of the human MAPK14 kinase. While in the avian virus the phosphorylation of L has been shown to be dispensable for infectivity, the identified locations in the human virus protein, both in the PreS1 and PreS2 domains, raise the intriguing possibility that they might play a functional role, since they...

Journal of Hepatology, 2019

BACKGROUND AND AIM Noncytolytic curing of hepatitis B virus (HBV) infected hepatocytes by cytokin... more BACKGROUND AND AIM Noncytolytic curing of hepatitis B virus (HBV) infected hepatocytes by cytokines including type I interferons (IFNs) is of importance for resolving acute and chronic infection. However, as IFNs stimulate hundreds of genes those most relevant for HBV suppression remain largely unknown. Amongst them are the large Mx GTPases. Human MX1 (or MxA) is active against many RNA viruses while MX2 (or MxB) was recently found to restrict human immunodeficiency virus 1, hepatitis C virus, and herpesviruses. Here we investigated the anti-HBV activity of MX2. METHODS Potential anti-HBV activity of MX2 and functional variants was assessed in transfected and HBV infected hepatoma cells and primary human hepatocytes, employing multiple assays to determine HBV nucleic acids as well as their synthesis and decay. The specific roles of MX2 in IFN-α inhibition of HBV transcription and replication were addressed by MX2-specific shRNA interference (RNAi). RESULTS MX2 alone as well as IFN-α substantially inhibited HBV replication, due to significant deceleration of the synthesis and slight acceleration of the turnover of viral RNA. RNAi knock-down of MX2 significantly reduced the inhibitory effects of IFN-α. Strikingly, MX2 inhibited HBV infection by reducing covalently closed circular DNA (cccDNA), most likely by indirectly impairing relaxed circular DNA to cccDNA conversion rather than destabilizing existing cccDNA. Various mutations affecting the GTPase activity and oligomerization status reduced MX2's anti-HBV activity. CONCLUSION MX2 is an important IFN-α inducible effector that decreases HBV RNA levels but can also potently inhibit HBV infection by indirectly impairing cccDNA formation. MX2 likely has the potential of therapeutic application aimed at curing HBV infection by eliminating cccDNA.

Journal of Viral Hepatitis, 2015

Nucleic Acids Research, 1993

Journal of Biomolecular NMR, 2021

Progress in NMR in general and in biomolecular applications in particular is driven by increasing... more Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

(A) Visualization of RNA content in HBc183_F97L CLPs by NAGE and RNA vs. protei... more (A) Visualization of RNA content in HBc183_F97L CLPs by NAGE and RNA vs. protein staining. HBc183_F97L CLPs expressed in the absence (-) or presence (+) of NHisSRPK1ΔNS1 were enriched to sucrose gradient sedimentation. The indicated fractions were separated by NAGE in the presence of ethidium bromide (EB); subsequently proteins were stained by Coomassie Blue (CB). Note the high vs. low EB to CB signal ratios in the -SRPK1 vs. the +SRPK1 samples but their nearly identical mobility. Negative staining EM (EM) did not reveal differences between the two HBc183_F97L samples, nor between the respective wild-type HBc183 samples (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.s005" target="_blank">S3B Fig</a>). (B) Impact of NHisSRPK1ΔNS1 coexpression vs. CTD truncations on RNA content. CLPs from the indicated truncated HBc variants were subjected to NAGE in EB-free gels, then stained with Sybr Green 2 (SG2) for RNA; after documentation the gels were counterstained with Sypro Ruby (SR) for protein. Green and red signals were semiquantitatively evaluated by laser scanning at the indicated conditions (grey-scale panels). The ratios of SG2 to SR fluorescence in each sample are given as percent of the respective value for HBc183 CLPs expressed without kinase. (C) Similarly strong reduction in absolute CLP RNA content by NHisSRPK1ΔNS1 coexpression as by CTD deletion. RNA contents of the indicated HBc CLPs (in nt per HBc protein monomer (N/P)) were calculated from UV/VIS spectra [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.ref073" target="_blank">73</a>]. Black bars show the mean N/P values ± SD (n≥3). For comparison, the relative values derived from (B) are shown as grey bars; the scale was set such that the 100% value (HBc183) matched the mean N/P value (~16) of the same sample. In either assay, the RNA content of HBc183 and HBc183_F97L CLPs coexpressed with SRPK1 was as low as that of HBc140 CLPs.</p

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a new class of anti-HBV antiv... more Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a new class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking. We show that solid-state NMR can provide such information, including for wild-type full-length Cp183, and we find that in these assemblies, the asymmetric unit comprises a single Cp molecule rather than the four quasi-equivalent conformers typical for the icosahedral T=4 symmetry of the normal HBV capsids. Furthermore, while in contrast to truncated Cp149, full-length Cp183 assemblies appear, on the mesoscopic level, unaffected by CAM-A, NMR reveals that on the molecular level, Cp183 assemblies are equally aberrant. Finally, we use a ...

(A) Position of S/T>A mutations, dominant m/z peak observed and calculated M... more (A) Position of S/T>A mutations, dominant m/z peak observed and calculated MH+ mass of best-fitting phospho-species. Note that S181A is the only single-site variant with a best-fit m/z for seven-fold phosphorylation; all other single-site mutants showed the best match to six phosphoryl groups. (B) Impact of number and position of phospho-sites on Phos-tag SDS-PAGE mobility. Following Phos-tag SDS-PAGE HBc proteins were analyzed by immunoblotting using the HBc assembly-domain specific mAb 1D8. All SRPK1-coexpressed proteins contained seven (HBc183, S181A) or six phosphoryl groups (all others) but variants S170A (*) and especially T160A and S162A (**) were less retarded, even though much more than three-fold PKAcd phosphorylated HB183. The impact of the mutations on recognition by the phospho-CTD specific mAb T2212 is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.s014" target="_blank">S12 Fig</a>. (C) Impact on CLP RNA content. CLPs from the indicated HBc proteins were analyzed by NAGE and sequential SG2 vs. SR staining using an improved, background-reducing protocol (Materials and Methods). SG2: SR ratios are indicated in percent of that in non-phosphorylated HBc183 CLPs. Bars show the mean of ≥3 determinations; error bars represent standard deviation (SD). Original NAGE fluorograms plus data for additional variants are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.s010" target="_blank">S8 Fig</a>. Note the exceptionally low SG2 to SR ratio for S181A and the higher ratio for S170A vs. all other single S/T>A variants (red arrows). The minor impact of PKA and PKC coexpression on RNA content (blue bars) correlated with the predominance of only three-fold phosphorylated species (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.s009" target="_blank">S7 Fig</a>) and their less pronounced retardation in Phos-tag SDS-PAGE (Fig 5B and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007488#ppat.1007488.g009" target="_blank">Fig 9C</a>).</p

Progress in NMR in general and in biomolecular applications in particular is driven by increasing... more Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins and viral capsids and others. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

PLOS ONE, 2015

(A-C) Interference with the Salp15-OspC interaction. (A) Competitive ELISA sche... more (A-C) Interference with the Salp15-OspC interaction. (A) Competitive ELISA scheme. In the usual sequential procedure (left) Salp15 can (1) bind via its interaction region (red) to immobilized OspC. Bound Salp15 can then be detected (2) using anti-Salp15 mabs 19/7.4 and 18/12.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136180#pone.0136180.ref010" target="_blank">10</a>]. As the mab epitopes are outside the OspC binding region, preincubation (premix) of Salp15 with the mabs should not interfere with OspC binding (left part). If the vaccination induced polyclonal IS contain antibodies to the OspC binding region (in red), the sequential format should still allow Salp15—OspC binding; however, in the premix format such antibodies would compete with OspC binding. (B) Assay validation using non-competing mabs. DsbA-fused Salp15, Iric-1 or non-OspC binding SaΔN59 protein as negative control were premixed with excess mab 19/7.4 or 18/12.1, then the mixtures were added to plate-immobilized B. burgdorferi OspCa or OspCb. Bound mab complexes were detected using a secondary anti-mouse-PO conjugate. As expected, no interference was observed. (C) Antibodies in vaccination-induced IS compete with OspC binding. In the sequential format, the polyclonal IS to wt-Iric-1 (pc α-wt-Iric1) and pooled IS to CLPs presenting Cys-free Salp15 and Iric-1 (pc α-Salp/Iric Cys- CLP) gave strong signals upon OspCa-mediated binding of DsbA-fused wt-Salp15 and wt-Iric-1. In contrast, signals were markedly reduced in the premix format. Comparable results were obtained for OspCb, and B. afzelii OspC A3 and YU (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136180#pone.0136180.s006" target="_blank">S6 Fig</a>). (D) Anti-Salp15 antibodies in the presence of Salp15 enhance killing of borreliae by human complement. B313 borreliae were preincubated in the absence or presence of recombinant H6-Salp15 with either anti-Salp15-specific mouse IS (Salp-IS) or non-immune mouse serum (niMS), and thereafter in 25% (v/v) normal human serum (NHS). Cell counts were determined in triplicate by a fluorescence-based survival assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136180#pone.0136180.ref038" target="_blank">38</a>] and normalized to the mean value obtained in NHS without any additives (set to 100%); error bars denote SEM. Note that the simultaneous presence of both Salp15 and anti-Salp15 antibodies (Salp-IS + Salp) caused a further, significant drop in viability (p = 0.031) compared to niMS ± Salp. Comparable results with factor B depleted NHS (NHS-B) confirmed the antibody-dependence of the effect. See text for further controls and experimental details.</p

(A) N terminally His6 tagged tHRF (H6-tHRF).Left: ... more (A) N terminally His6 tagged tHRF (H6-tHRF).Left: SDS-PAGE analysis of native lysate of transformed BL21*CP cells. T, total unfractionated lysate; P and S, insoluble pellet and soluble supernatant after centrifugation. The gel was stained with Coomassie Blue. Middle: Ni2+ IMAC of the soluble lysate fraction. Equal aliquots from the IMAC fractions eluted by increasing imidazole concentration were analyzed by SDS-PAGE. Right: Size exclusion chromatography (SEC). Peak fractions from IMAC were subjected to SEC on a Superdex S75 16/60 column; elution volumes are indicated on the top. By comparison with the elution volumes of marker proteins the peak of tHRF eluted at a volume corresponding to a molecular mass of ~27 kDa (arrowhead). This was confirmed for a non-tagged version of recombinant tHRF (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136180#pone.0136180.s001" target="_blank">S1 Fig</a>). (B) HBc149_c/e1-tHRF.Left: Cleared native lysate of bacteria expressing the contiguous chain fusion protein were subjected to sucrose gradient sedimentation. Equal aliquots from fractions 1 to 13 (out of 14 total) were analyzed by SDS-PAGE. Middle: Immunoblot after native agarose gel electrophoresis (NAGE). Material from fraction 8 of the gradients shown in (B) and (C) was subjected to NAGE, blotted and detected using the HBc particle-specific mab mc275. Right: Negative staining EM. (B) HBc183_c/e1-tHRF. Sedimentation and EM analysis were performed as in (B).</p

Nature Communications, 2020

Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure... more Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure are lacking, and the knowledge of virus-host interactions is still limited. Here, we perform a genome-wide gain-of-function screen using a poorly permissive hepatoma cell line to uncover host factors enhancing HBV infection. Validation studies in primary human hepatocytes identified CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in highly permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, we identify a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and ...

Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is establ... more Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is established

PLOS Pathogens, 2022

Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny... more Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P—ε...

Nature Communications

Frontiers in Molecular Biosciences, 2021

Journal of Hepatology, 2019

Journal of Viral Hepatitis, 2015

Nucleic Acids Research, 1993

Journal of Biomolecular NMR, 2021

Progress in NMR in general and in biomolecular applications in particular is driven by increasing... more Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins and viral capsids and others. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

PLOS ONE, 2015

Nature Communications, 2020

PLOS Pathogens, 2022