Influenza viruses pose a global health threat, particularly to infants and the elderly. The viruses constantly change. As a consequence, vaccines have to be constantly adapted and therapeutics may cease to be effective. Therefore, we seek to develop novel influenza therapies. One focus of our work is on the host cell protease TMPRSS2 since we obtained evidence that TMPRSS2 depend on the protease for acquisition of infectivity and spread in the host. Moreover, we are investigating how defective interfering particles (DIPs) can be generated in the absence of infectious virus and how DIPs inhibit influenza virus infection.
Emerging viruses that are transmitted from animals to humans may cause severe disease. Outbreaks frequently occur abroad but the responsible viruses might be imported into Germany due to infected travelers. We are investigating how emerging viruses interact with host cells and cause disease. A recently started project focuses on lymphocytic choriomeningitis virus (LCMV). LCMV is related to the highly pathogenic Lassa virus, circulates globally and is responsible for outbreaks of lethal hepatitis in marmoset colonies. Moreover, LCMV may cause severe disease in immunosuppressed patients and can constitute a threat to pregnant woman and their unborn children. In addition to LCMV and Ebola virus we are also investigating MERS coronavirus. The aim of our research is to develop cell culture systems that allow predicting transmissibility and thus pandemic potential of novel MERS coronavirus variants.
Another focus of our research is on primate herpesviruses. The transmission of herpes B virus from macaques to humans as well as transmission of related viruses among non-human primates can cause serve disease. We are investigating which viral and host factors determine whether infection will result in severe disease. Moreover, we are developing diagnostics for herpesvirus infections of non-human primates. Finally, we are offering diagnostics for many other viral infections of non-human primates, including a chip-based antibody detection system useful for screening of non-human primate colonies.
Recent publications summarized in three sentences
Chloroquine does not inhibit SARS-CoV-2
Chloroquine blocks SARS-CoV-2 infection of the kidney cell line Vero and it thus used for COVID-19 therapy. Hoffmann and coworkers now show that chloroquine does not block SARS-CoV-2 infection of the lung cell line Calu-3 and that lack of inhibition is associated with SARS-CoV-2 activation by the cellular protease TMPRSS2. These results indicate that chloroquine and the closely related drug hydroxychloroquine should not be used for COVID-19 therapy.
Hoffmann et al, Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2. Nature. 2020, In press
Llama antibody blocks SARS-CoV-2 entry
Recombinant antibodies that neutralized SARS-CoV-2 could be used for COVID-19 therapy and prevention. Wrapp and colleagues report the identification of a llama single domain antibody that inhibits SARS-CoV-1 and SARS-CoV-2 entry into cells. This antibody or related ones could be developed to combat COVID-19.
Wrapp et al, Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies, Cell. 2020 May 28;181(5):1004-1015.e15.
Two-step activation of SARS-CoV-2
Cleavage of the SARS-CoV-2 surface protein spike by host cell proteases is required for viral infectivity. Hoffmann and colleagues demonstrate that spike protein cleavage by furin is required for viral infectivity. This finding and our previously published results demonstrate that spike protein cleavage by furin in infected cells is required for subsequent spike protein cleavage and activation by TMPRSS2, which occurs during viral entry and is essential for infection of lung cells.
Hoffmann et al, A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell. 2020 May 21;78(4):779-784.e5.
H2 influenza A viruses depend on TMPRSS2 for spread and pathogenesis
The cellular serine protease TMPRSS2 was shown to activate several influenza A viruses and coronaviruses. Lambertz and colleagues demonstrate that also H2 influenza A viruses depend on TMPRSS2 for spread and pathogenesis.
Lambertz et al, H2 influenza A virus is not pathogenic in Tmprss2 knock-out mice. Virol J. 2020 Apr 22;17(1):56.
Nafamostat mesylate inhibits SARS-CoV-2
Host cell entry of the novel coronavirus SARS-CoV-2 depends on activation of the viral spike protein by the cellular serine protease TMPRSS2. Hoffmann and colleagues show that Nafamostat mesylate, a serine protease inhibitors approved for treatment of pancreatitis in Japan, inhibits SARS-CoV-2 infection of lung cells.
Hoffmann et al, Nafamostat mesylate blocks activation of SARS-CoV-2: New treatment option for COVID-19. Antimicrob Agents Chemother. 2020 Apr 20. pii: AAC.00754-20.
Drug blocks SARS-CoV-2 infection of lung cells
Hoffmann and colleagues show that SARS-CoV-2 like SARS-CoV-1 uses the host cell proteins ACE2 and TMPRSS2 for entry into lung cells. TMPRSS2 is a serine protease that cleaves and thereby activates that viral spike protein – process that is disrupted by the clinically proven protease inhibitor camostat mesylate. Moreover, evidence is provided that sera from convalsecent SARS patients inhibit SARS-CoV-2 infection, although with low efficiency.
Hoffmann et al, SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020 181(2):271-28
Polymorphisms in DPP4 gene might impact MERS-CoV infection
MERS-coronavirus uses the host cell protein DPP4/CD26 for entry into target cells. Kleine-Weber and colleagues show that polymorphisms in the DPP4 gene can reduce DPP4 binding and viral entry. It will thus be interesting to determine whether such polymorphisms are present in the Saudia Arabian population and whether they impact the course of MERS-coronavirus infection.
Kleine-Weber et al, Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus. Emerg Microbes Infect. 2020 Dec;9(1):155-168
Plasmid system for generation of Cercopithecine Alphaherpesvirus 2
Macacine alphaherpesvirus 1 (McHV-1) but not the closely related Cercopithecine Alphaherpesvirus 2 (CeHV-2) can cause fatal disease in humans. It is unknown why these viruses exhibit differential virulence in humans and this question is in the focus of current research efforts of the Infection Biology Unit. We now report the generation of a plasmid system which allows generation and analysis of CeHV-2 in cell culture.
Chukhno et al, A Fosmid-Based System for the Generation of Recombinant Cercopithecine Alphaherpesvirus 2 Encoding Reporter Genes. Viruses. 2019 Nov 5
No evidence for a role of IFITM3(2) in SIV infection of macaques
IIFITM proteins are known to inhibit viral entry into target cells but the role of IFITM3(2) in simian immunodeficiency virus (SIV) infection has been unknown. Winkler and colleagues show that although IFITM3(2) blocks SIV entry into a cell line with moderate effciency, polymorphisms in IFITM3(2) do not impact the course of SIV infection in macaques. These results, jointly with previously published data, argue against an important role of IFITM proteins in SIV infection of macaques.
Winkler et al, Role of rhesus macaque IFITM3(2) in simian immunodeficiency virus infection of macaques. PLoS One. 2019 Nov 4
Ebola virus resistance against cathepsin B/L inhibitors
The Ebola virus glycoprotein (EBOV-GP) mediates viral entry into traget cells, which depends on cleavage of GP by the host cell proteases cathepsin B and L. The study by Hoffmann and colleagues shows that GP rapidly acquires resistance mutations upon viral spread in the presence of cathepsin B/L inhibitors. If these inhibitors are used for antiviral therapy, rapid resistance development is to be expected.
Hoffmann et al, Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins. Pathogens. 2019 Oct 15;8(4).
Influenza B virus hemagglutinin: Perfect key for viral entry into human lung cells
Influenza B viruses (IBV) employ their hemagglutinin protein to enter target cells. This manuscript shows that IBV hemagglutinin can use a very broad spectrum of host cell proteases with high efficiency for activation and that its temperature requirments and pH sensitivity are optimal for expression and function, respectively, in human lung cells. These findings might reflect extensive adaptation of IBV to infection of humans.
Laporte et al, Hemagglutinin cleavability, acid-stability and temperature dependence optimize influenza B virus for replication in human airways. J Virol. 2019 Oct 9.
Novel technique to analyze interactions between IFITM proteins
Interferon-induced transmembrane proteins (IFITM) are cellular proteins that can block infection by diverse viruses. It has been suggested that IFITM-IFITM interactions are required for antiviral activity but quantifying these interactions is technically challenging. This manuscript reports a fluorescence resonance energy transfer (FRET)-based system that allows quantification of IFITM-IFITM interactions in cells via fluorescence-activated cell scanning (FACS).
Winkler et al, Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay. Int J Mol Sci., 2019, 8;20(16). pii: E3859.
MERS-coronaviruses from African dromedary camels can efficiently enter human cells
The MERS-coronavirus is endemic in the Middle East and is transmitted from camels, the natural reservoir, to humans, who may develop severe disease. Camels in Africa are also infected by the virus but seem to harbor viral variants with reduced capacity to spread in human cells, which might account for the absence of documented MERS cases in Africa. The present study provides evidence that viruses from African camels might enter human cells with high efficiency, indicating that a step in viral replication other than entry is limiting viral spread in human cells.
Kleine-Weber et al, Spike proteins of novel MERS-coronavirus isolates from North- and West-African dromedary camels mediate robust viral entry into human target cells. Virology. 2019 Jul 19;535:261-265.
Novel herpesvirus identified in Colobus monkey suffering from primary effusion lymphoma
This manuscript reports the identification and genome structure of a new Kaposi Sarcoma Herpesvirus (KSHV)-related virus found in Colobus monkeys, which was termed Colobine gammaherpesvirus 1 (CbGHV-1). The animal examined (housed at a zoological garden) suffered from disease similar to primary effusion lymphoma (PEL), a cancer found in KSHV infected humans, and was not coinfected with an immunosuppressive virus. This finding and the results of a parallel study summarized below suggest that experimental CbGHV-1 infection of Colobus monkeys might serve as new animal model for KSHV infection of humans.
Dhingra et al, Novel Virus Related to Kaposi's Sarcoma-Associated Herpesvirus from Colobus Monkey. Emerg Infect Dis. 2019, 25(8):1548-1551
New herpesvirus identified in Guereza monkeys with Kaposi sarcoma
The Kaposi sarcoma herpesvirus (KSHV) infects humans and causes cancer, including the Kaposi sarcoma (KS). KSHV-related viruses have been identified in non-human primates and were found to only induce disease in the context of immunosuppression. This paper reports the identification of a KSHV-related virus in Colobus monkeys, Colobine gammaherpesvirus 1 (CbGHV-1), and shows that a naturally CbGHV-1 infected animal developed a KS-like disease without overt signs for immunosuppression.
Seroprevalence of viral infections in macaques at DPZ
Viral infections threaten th health of macaques and humans in contact with these animals. The Infection Biology Unit employed a novel CHIP-based system to detect antibodies against viruses in macaques housed at DPZ. The data show that the animals do not have antibodies against herpes B virus, which can cause severe disease in humans, but frequently have antibodies against other herpesviruses that usually threaten neither animal nor human health.
Kaul et al, Seroprevalence of viral infections in captive rhesus and cynomolgus macaques.Primate Biology, 2019 May 26
H10 influenza viruses depend on the host cell factor TMPRSS2 for disease induction
The hemagglutinin of influena viruses mediates host cell entry and depends on activation by host cell proteases to transit into an active form. TMPRSS2 is a host cell protease that can activate influenza hemagglutinin proteins in cell culture. The study by Lambertz et al from the laboratory of Prof. Schughart, Helmholtz Center for Infection Research, Braunschweig, shows that TMPRSS2 can cleave the hemagglutinin of the H10 subtype and that TMPRSS2 is essential for disease induction upon H10 influenza virus infection.
Lambertz et al, Tmprss2 knock-out mice are resistant to H10 influenza A virus pathogenesis. Journal of General Virology, 17 May 2019
Guanylate-Binding Proteins 2 and 5 block virus activation
Viral glycoproteins mediated host cell entry viruses and depend on activation by host cell proteases to transit into an active form. The interferon response is an important component of innate immunity and can inhibit virus infection. The present study from the laboratory of Prof. Sauter, University Ulm, shows that the cellular protein Guanylate-Binding Proteins 2 and 5, which are known to interferon-induced, block activation of viral glycoproteins.
Braun et al. Guanylate-Binding Proteins 2 and 5 Exert Broad Antiviral Activity by Inhibiting Furin-Mediated Processing of Viral Envelope Proteins. Cell Rep. 2019 May 14
IFITM proteins promote immune evasion of hepatitis C virus
Interferon induced transmembrane proteins (IFITM) block host cell entry of hepatitis C virus (HCV), a globally circulating causative agent of liver cancer. The present paper from the laboratory of Prof. Baumert, Strasbourg, France, reports findings of Florian Wrensch, an alumni of the Infection Biology Unit, and colleagues that show that IFITM proteins augment antibody-mediated neutralization of HCV. This effect forces the virus to change and to thereby evade control by the antibody response.
Wrensch et al. Interferon-Induced Transmembrane Proteins Mediate Viral Evasion in Acute and Chronic Hepatitis C Virus Infection. Hepatology. 2019 May 7
Inhibitors of signal peptide peptidase and subtilisin/kexin-isoenzym 1 block host cell entry of Ebola virus
Host cell proteases that activate viruses are potential targets for antiviral intervention. However, many protease inhibitors have a broad specificity and may interfere with virus infection in several ways. This study shows that inhibitors of the cellular proteases signal peptide peptidase and subtilisin/kexin-isoenzym 1 block host cell entry of Ebola viruses by interfering with activity and cellular localization, respectively, of the protease cathepsin L.
Plegge et al. Inhibitors of signal peptide peptidase and subtilisin/kexin-isozyme 1 inhibit Ebola virus glycoprotein-driven cell entry by interfering with activity and cellular localization of endosomal cathepsins. PLoS One. 2019 Apr 11
tRNA modulates HIV Gag/Gag-Pol frameshifting
Production of infectious human immunodeficiency virus (HIV) depends on a frameshift that allows synthesis of the Gag-Pol protein. Which factors impact frameshifting efficiency has been unclear. This study shows that a tRNA that binds to the frameshifting site (slippery side) modulates efficiency and type of frameshifting and that the virus can use an alternative site for frameshifting after having acquired mutations that confer resistance to antiretroviral therapy.
Korniy et al, Modulation of HIV-1 Gag/Gag-Pol frameshifting by tRNA abundance. Nucleic Acids Res. 2019 Apr 10.
Link to the publication For further information please click here
Calu-3 cells are largely resistant to cell entry of Ebola virus
The highly pathogenic Ebola virus can enter most cell types. Which cellular factors might limit cell entry of Ebola virus is largely unknown. Our study shows that the lung-derived cell line Calu-3 is largely resistant to Ebola virus entry and that entry effciency is increased upon directed expression of the protease cathepsin L and the lectin DC-SIGN.
González-Hernández et al, Calu-3 cells are largely resistant to entry driven by filovirus glycoproteins and the entry defect can be rescued by directed expression of DC-SIGN or cathepsin L. Virology. 2019 Apr 3;532:22-29.
Identification of a cell line that is not susceptible to Ebola virus entry
The highly pathogenic Ebola virus (EBOV) exhibits an extremely broad cell tropism. Zapatero-Belinchón and colleagues demonstrate that the cell line SH-SY5Y is not susceptible to EBOV entry due to lack of factors that promote viral attachment. Therefore, SH-SY5Y cells can by used as tools to identify novel EBOV attachment factors and to characterize known ones.
Zapatero-Belinchón et al, Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors. Viruses. 2019 Mar 19;11(3).
A system for production of defective interfering particles
Defective interfering particles (DIP) inhibit influenza virus infection and are being developed for influenza therapy. However, so far, DIPs can only be produced joinlty with infectious virus, which is associated with safety concerns. Here, we report that a cell line stably expressing the viral protein PB2 is suitable for production of segment 1-based DIPs in the absence of infectious virus.
Bdeir et al. A system for production of defective interfering particles in the absence of infectious influenza A virus. PLoS One. 2019 Mar 1;14(3):e0212757.
Link to the publication For further information please click here.
Virosomes interfere with Ebola virus control by the immune system
The Ebola virus glycoprotein is release from cells within non-infectious particles, termed virosomes. Virosomes function as antibody decoys and interfere with macrophage function. Release of virosomes is blocked by the interferon-induced antiviral host cell protein tetherin.
Nehls et a.l Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner. Cell Reports. 2019, 26, 1841–1853
Focal epithelial hyperplasia and papillomavirus infection in a bonobo
In a biopsie of a bonobo with focal epithelial hyperplasia (FEH) the Pan paniscus papillomavirus 1 (PpPV1) was detected. The animal was part of a bonoboa cohort in which FEH and PpPV1 infection were already detected in the 1980s and comparison of viral genome sequences revealed that the present genomic sequence different in 23 positions for the sequence detected in the 19180s. This finding indicates that either the animal was infected twice with different variants of PpPV1 or that the PpPV1 sequence changed more profound during the course of infection than expected from published data.
Hoffmann et al Disease Manifestation and Viral Sequences in a Bonobo More Than 30 Years after Papillomavirus Infection. Pathogens. 2019 Jan 26;8(1). pii: E13. doi: 10.3390/pathogens8010013.
Tetherin inhibits spread of Nipah virus in fruit bat cells
Ebola and Nipah virus cause severe disease in humans but not fruit bats and it is unclear whether the antiviral host cell factor tetherin contributes to control of viral infection in fruit bat cells. Our study shows that tetherin efficiently blocks Nipah virus spread in fruit bat cells, suggesting that it may contribute to viral control in infected animals. In contrast, inhibition of Ebola virus spread by tetherin was modest and it is at present unclear whether this is due to tetherin counteraction by the viral glycoprotein.
Hoffmann et al Tetherin inhibits Nipah virus but not Ebola virus replication in fruit bat cells. J Virol. 2018 Nov 14. pii: JVI.01821-18.
A mutation protects MERS coronavirus against antibodies
The MERS coronavirus causes severe disease and there is concern that the virus might mutate and spread globally. Our work shows that a MERS coornavirus variant emerged during a large outbreak in South Korea that was partially protected against antibody responses raised in infected patients. Our results show that the virus can adapt to more efficient spread in infected patients and suggest that usage of single antíbodies for MERS therapie mgiht not be effective.
Kleine Weber et al. Mutations in the spike protein of MERS-CoV transmitted in Korea increase resistance towards antibody-mediated neutralization. J Virol. 2018 Nov 7. pii: JVI.01381-18.