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International audience; Background and aims: A single hepatitis B virus (HBV) particle is sufficient to establish chronic infection of the liver after intravenous injection, suggesting that the virus targets hepatocytes via a highly efficient transport pathway. We therefore investigated whether HBV utilizes a physiological liver-directed pathway that supports specific host-cell targeting in vivo.Methods: We established an ex vivo system of perfused human liver tissue that recapitulates the liver physiology to investigate HBV liver targeting. This model allowed us to investigate virus-host cell interactions in a cellular microenvironment mimicking the in vivo situation. Results: HBV was rapidly sequestered by liver macrophages within one hour after a virus pulse, but was detected in hepatocytes only after 16 hours. We found that HBV associates with lipoproteins. Electron- and immunofluorescence microscopy corroborated a co-localization in recycling endosomes within peripheral and liver macrophages. Importantly, recycling endosomes accumulated HBV and cholesterol, followed by transport of HBV back to the cell surface along the cholesterol efflux pathway. To reach hepatocytes as final target cells, HBV was able to utilise the hepatocyte-directed cholesterol transport machinery of macrophages.Conclusions: Our results propose that HBV by binding to liver targeted lipoproteins and utilizing the reverse cholesterol transport pathway of macrophages hijacks the physiological lipid transport pathways to the liver to most efficiently reach its target organ. This may involve trans-infection of liver macrophages and result in deposition of HBV in the perisinusoidal space from where HBV can bind its receptor on hepatocytes

Background & Aims Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism. Methods We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests. Results We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation. Conclusion Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition. Lay summary The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.

One of the best-studied cellular responses to toxic signals and pathogens is programmed cell death. Over the past years, it became apparent that the specific mechanisms of cell death have tremendous influence at both cellular and organismal level, highlighting the importance of sensors and pathways involved in this decision-making process. Central signalling molecules involved in a variety of cell death pathways are reactive oxygen species (ROS). However, the molecular mechanisms regulating differential responses and cellular fates to distinct ROS levels remain incompletely understood. Recently, we uncovered a caspase-independent cell-death pathway named 'oxeiptosis', which links the ROS sensing capacity of KEAP1 to a cell death pathway involving PGAM5 and AIFM1. Alike apoptosis, oxeiptosis is anti-inflammatory when activated by increased intracellular ROS levels and upon pathogens encounter. Here we discuss the potential impact of oxeiptosis in pathogens clearance and teratogenic cells.

In this issue of Cell Host & Microbe, Tripathi et al. (2015) report an in-depth meta-analysis of eight influenza virus siRNA screens combined with viral-host protein interactome data. The integration of the different omics datasets highlights candidate genes and pathways for further investigation and potential therapeutic targeting in the future.

Cytosolic viral RNA recognition by the helicases RIG-I and MDA5 is considered the major pathway for IFN-alpha/beta induction in response to RNA viruses. However, other cytoplasmic RNA sensors, including the double-stranded RNA-binding protein kinase R (PKR), have been implicated in IFN-alpha/beta production, although their relative contribution and mechanism have been unclear. Using cells expressing nonfunctional PKR or reduced levels of kinase, we show that PKR is required for production of IFN-alpha/beta proteins in response to a subset of RNA viruses including encephalomyocarditis, Theiler's murine encephalomyelitis, and Semliki Forest virus, but not influenza or Sendai virus. Surprisingly, although IFN-alpha/beta mRNA induction is largely normal in PKR-deficient cells, much of that mRNA lacks the poly(A) tail, indicating that its integrity is compromised. Our results suggest that PKR plays a nonredundant role in IFN-alpha/beta production in response to some but not all viruses, in part by regulating IFN-alpha/beta mRNA stability.

Virus infection elicits potent responses in all cells intended to contain virus spread before intervention by the adaptive immune system. Central to this process is the virus-elicited production of type I interferons (IFNs) and other cytokines. The sensors involved in coupling recognition of viruses to the induction of the type I IFN genes have only recently been uncovered and include endosomal and cytosolic receptors for RNA and DNA. Here, we review their properties and discuss how their ability to recognize the unusual presence of atypical nucleic acids in particular subcellular compartments is used by the body to detect virus presence.

Two synthetic approaches to the cytotoxic marine natural product known as mangicol A are described. The starting material common to both pathways is the cyclopentenonecarboxylate 11. The first tactic involves the 1,4-addition to 11 of the cuprate derivable from iodide 10, while the second proceeds via base-promoted conjugate addition of the regiospecifically generated enolate anion of 41. The first strategy proceeds by a series of efficient steps to tricyclic aldol 21 and subsequently to β-diketone 7. The latter proved to be totally unresponsive to schemes aimed at introduction of a butenyl group. The second approach involves earlier introduction of this substituent as realized in stereocontrolled fashion via transition state 42. While further passage to 44 proved uneventful, this advanced intermediate and analogs thereof proved remarkably recalcitrant to cyclization in the precedented fashion. In no instance was generation of a suitable product realized. These studies serve to underscore the extent to which steric considerations can complicate matters and the extent to which they must be skirted. Finally, a direct enantioselective route to the side chain aldehyde 2 is detailed.

A plan for enantioselective construction of the mangicol A framework by means of intramolecular Diels–Alder cycloaddition is outlined. First to be assembled is the enantiopure cyclopentenecarboxylic acid 16 . Of the several approaches targeting the 1,3-diene component 56 , only that involving palladium-catalyzed enyne cyclization proved successful. Following the coupling of 16 to 56 , we were unable to bring about any detectable level of (4π+2π) cycloaddition. Activation of the diene by incorporation of an OSiEt 3 substituent on a terminal sp 2 -hybridized center likewise proved unsuccessful. Further facilitation was sought in the form of cyclopentenonecarboxylate 66 . However, thermal activation, Lewis acid catalysis, and high-pressure conditions proved ineffective and did not lead to C–C bond formation. These studies serve to underscore the extent to which steric complications can complicate matters and the extent to which they must be skirted to arrive at the title compound.

Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus termed SARS-CoV. No antiviral treatment has been established so far. Interferons are cytokines which induce the synthesis of several antivirally active proteins in the cell. In this study, we demonstrated that multiplication of SARS-CoV in cell culture can be strongly inhibited by pretreatment with interferon-beta. Interferon-alpha and interferon-gamma, by contrast, were less effective. The human MxA protein is one of the most prominent proteins induced by interferon-beta. Nevertheless, no interference with SARS-CoV replication was observed in Vero cells stably expressing MxA. Therefore, other interferon-induced proteins must be responsible for the strong inhibitory effect of interferon-beta against SARS-CoV.

An eight-step synthesis of an orthogonally protected 3,8-diazabicyclo[3.2.1]octane from commercially available pyroglutamic acid was developed. The target compound can be used as a versatile building block for combinatorial synthesis of pharmacologically useful compounds and features a hidden α-, β-, δ- and e-amino acid, which can also be seen as an example of a new class of cocaine analogues.

Phleboviruses are a group of emerging viruses with a wide spectrum of virulence: For example, Rift Valley Fever virus (RVFV) is highly pathogenic, whereas Sandfly fever Sicilian virus (SFSV) causes intermediate pathogenicity. The major virulence factor is the non-structural protein NSs, a suppressor of the type I interferon (IFN) system. While the NSs protein of highly pathogenic RVFV inhibits global host cell transcription via sequestration and degradation of the TFIIH subunits p44 and p62, respectively, the mode of action of the NSs protein of intermediately pathogenic SFSV has remained elusive. We therefore aimed to characterize the IFN-inhibitory function employed by the NSs of SFSV. Using mass spectrometry, our group has identified multiple candidate host interactors, among them interferon regulatory factor 3 (IRF3). Indeed, co-immunoprecipitation confirmed the interaction of SFSV NSs with IRF3, but not with other IRF family members like IRF2, IRF5 and IRF9. Furthermore, SFSV NSs specifically abrogated IRF3 activation and thus IFN- β promoter activity, but not general host cell transcription. Hence, SFSV encodes a NSs protein that efficiently inhibits the induction of type I IFN. Different to the highly pathogenic RVFV, which induces a general host transcription shutoff, however, SFSV specifically targets IRF3-dependent IFN promoter activation. Although RVFV and SFSV are highly related, their NSs proteins display remarkably diverse strategies of counteracting the type I IFN system. Possibly, the quality of NSs-host factor interactions correlates with the virulence levels of phleboviruses.

The Pyrin and HIN200 domain-containing (PYHIN) protein family consists of AIM2, IFI16, MNDA, PYHIN1 and POP3 in humans and at least 13 proteins in mouse. Mouse AIM2 and p204 have been previously described to be intracellular dsDNA sensors leading to caspase-1 activation and STING-dependent IFN- β induction respectively. However the function of most mouse PYHIN proteins remains unknown. Here we characterized one such mouse PYHIN protein, p207. P207 contains an N-terminal Pyrin and a C-terminal HINB domain that share strong sequence similarity with the corresponding domains of p204 (85% and 92% respectively). P207 is highly expressed in BMDMs and BMDCs and its expression is further enhanced after dsDNA stimulation of cells in an IFN-dependent manner. In contrast to p204, reduction of p207 expression by shRNA had no effect on DNA-induced IFN- β . Rather, p207 shRNA studies in macrophage cell lines showed an impairment in TNF mRNA induction following stimulation with dsDNA, transfected poly(I:C) or LPS as well as after infection with HSV. Induction of other LPS-dependent genes including CCL5, IL-1, IL-12 or IL-6 was not p207-dependent. Further, ectopic expression of p207 induced a murine TNF promoter reporter, while p207 shRNA inhibited LPS-stimulated RNA polymerase II recruitment to the TNF promoter. Furthermore, p207 interactome analysis by mass spectrometry revealed a list of potential interaction partners involved in transcription. These candidates and their involvement in p207-mediated effects on TNF induction are being currently pursued. These results demonstrate the existence of a novel PYHIN-dependent mechanism of regulation of TNF induction during pattern recognition receptor responses. Thus mouse p207 is another PYHIN protein involved in mediating innate immune responses. Further, the requirement for a PYHIN protein for maximal TNF responses may be conserved in humans.

Tick-borne encephalitis virus (TBEV) is a very important virus medically, causing mild or severe encephalitis often with long-lasting sequelae. Treatment of tick-borne encephalitis is limited to su ...