Stefan Seitz

Molecular Mechanism of Signal Perception and Integration by the Innate Immune Sensor Retinoic Acid-inducible Gene-I (RIG-I)

RIG-I is a major innate immune sensor for viral infection, triggering an interferon (IFN)-mediated antiviral response upon cytosolic detection of viral RNA. Double-strandedness and 5′-terminal triphosphates were identified as motifs required to elicit optimal immunological signaling. However, very little is known about the response dynamics of the RIG-I pathway, which is crucial for the ability of the cell to react to diverse classes of viral RNA while maintaining self-tolerance. In the present study, we addressed the molecular mechanism of RIG-I signal detection and its translation into pathway activation. By employing highly quantitative methods, we could establish the length of the double-stranded RNA (dsRNA) to be the most critical determinant of response strength. Size exclusion chromatography and direct visualization in scanning force microscopy suggested that this was due to cooperative oligomerization of RIG-I along dsRNA. The initiation efficiency of this oligomerization process critically depended on the presence of high affinity motifs, like a 5′-triphosphate. It is noteworthy that for dsRNA longer than 200 bp, internal initiation could effectively compensate for a lack of terminal triphosphates. In summary, our data demonstrate a very flexible response behavior of the RIG-I pathway, in which sensing and integration of at least two distinct signals, initiation efficiency and double strand length, allow the host cell to mount an antiviral response that is tightly adjusted to the type of the detected signal, such as viral genomes, replication intermediates, or small by-products.

Cryo‐electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions

Hepatitis B virus (HBV) is a major human pathogen causing about 750 000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two α‐helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing.

The Pre-S2 Domain of the Hepatitis B Virus Is Dispensable for Infectivity but Serves a Spacer Function for L-Protein-Connected Virus Assembly

ABSTRACT The envelope of the human hepatitis B virus (HBV) contains three membrane proteins (L, M, and S). They accomplish different functions in HBV infectivity and nucleocapsid envelopment. Infectivity determinants have been assigned to the N-terminal part of the pre-S1 domain of the L protein and the antigenic loop of the S domain in the L and/or S protein. Nucleocapsid envelopment requires a C-terminal sequence within pre-S1, including the five N-terminal amino acids of pre-S2 as part of the L protein. However, the role of the M protein and the pre-S2 domain of the L protein are not entirely understood. We addressed this question and analyzed assembly competence and infectivity of viruses that lack the M protein and, at the same time, carry alterations in the pre-S2 domain of L. These include deletions, in part frameshift mutations and a randomization of virtually the entire pre-S2 sequence. We found that the M protein is dispensable for HBV in vitro infectivity. Viruses that lack the M protein and contain a mostly randomized pre-S2 sequence assemble properly and are infectious in HepaRG cells and primary human hepatocytes. While deletions of 20 amino acids in the pre-S2 domain of L protein allowed the production of infectious virions, more extended deletions interfered with assembly. This indicates that the pre-S2 domain of the L protein serves an important role for virus assembly, presumably as a spacer that supports conformational changes of L protein but does not participate as part of the M protein or as a subdomain of the L protein in virus entry.

Deciphering the Origin and Evolution of Hepatitis B Viruses by Means of a Family of Non-enveloped Fish Viruses

Summary Hepatitis B viruses (HBVs), which are enveloped viruses with reverse-transcribed DNA genomes, constitute the family Hepadnaviridae. An outstanding feature of HBVs is their streamlined genome organization with extensive gene overlap. Remarkably, the ∼1,100 bp open reading frame (ORF) encoding the envelope proteins is fully nested within the ORF of the viral replicase P. Here, we report the discovery of a diversified family of fish viruses, designated nackednaviruses, which lack the envelope protein gene, but otherwise exhibit key characteristics of HBVs including genome replication via protein-primed reverse-transcription and utilization of structurally related capsids. Phylogenetic reconstruction indicates that these two virus families separated more than 400 million years ago before the rise of tetrapods. We show that HBVs are of ancient origin, descending from non-enveloped progenitors in fishes. Their envelope protein gene emerged de novo, leading to a major transition in viral lifestyle, followed by co-evolution with their hosts over geologic eras.

A Slow Maturation Process Renders Hepatitis B Virus Infectious

Hepatitis B virus (HBV) replication is strictly limited to the liver. Virions attach to hepatocytes through interactions of the viral PreS envelope protein domain with heparan sulfate proteoglycans (HSPGs). However, HSPG is ubiquitously present on many cell types, suggesting that HBV employs mechanisms to avoid attachment at extrahepatic sites. We demonstrate that HBV particles are released from cells in an inactive form with PreS hidden in the interior. These HSPG-non-binding (N-type) particles develop receptor binding competence by translocating PreS across the envelope onto their surface. Conversion into HSPG-binding (B-type) particles occurs spontaneously and renders HBV infectious. Low-dose inoculation of mice with human liver xenografts demonstrates superiority of N-type particles in establishing infections, while mature B-type virions, generated via N-type conversion, are profoundly impaired, correlating with non-selective accumulation in extrahepatic tissues. This dynamic topology switch represents a maturation process utilized by HBV to most likely avoid non-productive docking outside the liver.

HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is sensitive to interferon (IFN)-based therapy, whereas hepatitis B virus (HBV) infection is not. It is unclear whether HBV escapes detection by the IFN-mediated immune response or actively suppresses it. Moreover, little is known on how HBV and HCV influence each other in coinfected cells. We investigated interactions between HBV and the IFN-mediated immune response using HepaRG cells and primary human hepatocytes (PHHs). We analyzed the effects of HBV on HCV replication, and vice versa, at the single-cell level. METHODS PHHs were isolated from liver resection tissues from HBV-, HCV-, and human immunodeficiency virus-negative patients. Differentiated HepaRG cells overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (dHepaRGNTCP) and PHHs were infected with HBV. Huh7.5 cells were transfected with circular HBV DNA genomes resembling viral covalently closed circular DNA (cccDNA), and subsequently infected with HCV; this served as a model of HBV and HCV coinfection. Cells were incubated with IFN inducers, or IFNs, and antiviral response and viral replication were analyzed by immune fluorescence, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assays, and flow cytometry. RESULTS HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or up-regulation of IFN-stimulated genes. In coinfected cells, HBV did not prevent IFN-induced suppression of HCV replication. CONCLUSIONS In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response.

Novel non-heteroarylpyrimidine (HAP) capsid assembly modifiers have a different mode of action from HAPs in vitro

Abstract One of the most promising viral targets in current hepatitis B virus (HBV) drug development is the core protein due to its multiple roles in the viral life cycle. Here we investigated the differences in the mode of action and antiviral activity of representatives of six different capsid assembly modifier (CAM) scaffolds: three from the well‐characterized scaffolds heteroarylpyrimidine (HAP), sulfamoylbenzamide (SBA), and phenylpropenamide (PPA), and three from novel scaffolds glyoxamide‐pyrrolamide (GPA), pyrazolyl‐thiazole (PT), and dibenzo‐thiazepin‐2‐one (DBT). The target activity and antiviral efficacy of the different CAMs were tested in biochemical and cellular assays. Analytical size exclusion chromatography and transmission electron microscopy showed that only the HAP compound induced formation of aberrant non‐capsid structures (class II mode of action), while the remaining CAMs did not affect capsid gross morphology (class I mode of action). Intracellular lysates from the HepAD38 cell line, inducibly replicating HBV, showed no reduction in the quantities of intracellular core protein or capsid after treatment with SBA, PPA, GPA, PT, or DBT compounds; however HAP‐treatment led to a profound decrease in both. Additionally, immunofluorescence staining of compound‐treated HepAD38 cells showed that all non‐HAP CAMs led to a shift in the equilibrium of HBV core antigen (HBcAg) towards complete cytoplasmic staining, while the HAP induced accumulation of HBcAg aggregates in the nucleus. Our study demonstrates that the novel scaffolds GPA, PT, and DBT exhibit class I modes of action, alike SBA and PPA, whereas HAP remains the only scaffold belonging to class II inhibitors. HighlightsThis is the first comprehensive comparison of 6 different CAM scaffolds, including 3 unclassified ones (GPA, PT and DBT).Our data suggest the three unclassified scaffolds have a class I mode of action.Currently, the HAP remains the only CAM scaffold that exhibits a class II mode of action.