• Characterization of Endogenous SERINC5 Protein as anti-HIV-1 Factor.

      Passos, Vânia; Zillinger, Thomas; Casartelli, Nicoletta; Wachs, Amelie S; Xu, Shuting; Malassa, Angelina; Steppich, Katja; Schilling, Hildegard; Franz, Sergej; Todt, Daniel; et al. (American Society of Microbiology, 2019-10-09)
      When expressed in virus-producing cells, the cellular multipass-transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed HA epitope (Jurkat SERINC5(iHA-knock-in) T-cells). This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. IFN-α treatment enhanced cell surface levels of SERINC5 in a Ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA-knock-in) T-cells shared the ability to produce infectious wildtype HIV-1, but not HIV-1 Δnef SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. Association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35 kDa species, as opposed to heterologous SERINC5 that presented as 51 kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5 and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface.IMPORTANCE SERINC5 is the long-searched antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.
    • Yellow Fever: Integrating Current Knowledge with Technological Innovations to Identify Strategies for Controlling a Re-Emerging Virus.

      Kleinert, Robin D V; Montoya-Diaz, Eduardo; Khera, Tanvi; Welsch, Kathrin; Tegtmeyer, Birthe; Hoehl, Sebastian; Ciesek, Sandra; Brown, Richard J P; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MPDI, 2019-10-17)
      Yellow fever virus (YFV) represents a re-emerging zoonotic pathogen, transmitted by mosquito vectors to humans from primate reservoirs. Sporadic outbreaks of YFV occur in endemic tropical regions, causing a viral hemorrhagic fever (VHF) associated with high mortality rates. Despite a highly effective vaccine, no antiviral treatments currently exist. Therefore, YFV represents a neglected tropical disease and is chronically understudied, with many aspects of YFV biology incompletely defined including host range, host-virus interactions and correlates of host immunity and pathogenicity. In this article, we review the current state of YFV research, focusing on the viral lifecycle, host responses to infection, species tropism and the success and associated limitations of the YFV-17D vaccine. In addition, we highlight the current lack of available treatments and use publicly available sequence and structural data to assess global patterns of YFV sequence diversity and identify potential drug targets. Finally, we discuss how technological advances, including real-time epidemiological monitoring of outbreaks using next-generation sequencing and CRISPR/Cas9 modification of vector species, could be utilized in future battles against this re-emerging pathogen which continues to cause devastating disease
    • Characterization of Equine Parvovirus in Thoroughbred Breeding Horses from Germany.

      Meister, Toni Luise; Tegtmeyer, Birthe; Brüggemann, Yannick; Sieme, Harald; Feige, Karsten; Todt, Daniel; Stang, Alexander; Cavalleri, Jessika-M V; Steinmann, Eike; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MPDI, 2019-10-18)
    • Quantitative proteomics of Uukuniemi virus - host cell interactions reveals GBF1 as proviral host factor for phleboviruses.

      Uckeley, Zina M; Moeller, Rebecca; Kühn, Lars I; Nilsson, Emma; Robens, Claudia; Lasswitz, Lisa; Lindqvist, Richard; Lenman, Annasara; Passos, Vania; Voss, Yannik; et al. (American Society for Biochemistry and Molecular Biology, 2019-09-30)
      Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immuno-precipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both occurs in the nucleus. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.
    • Synthesis of 4′/5′-Spirocyclopropanated Uridine and d -Xylouridine Derivatives and Their Activity against the Human Respiratory Syncytial Virus

      Köllmann, Christoph; Wiechert, Svenja M.; Jones, Peter G.; Pietschmann, Thomas; Werz, Daniel B.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (American Chemical Society, 2019-09-06)
    • Gamma secretase dependent release of the CD44 cytoplasmic tail upregulates IFI16 in cd44 <sup>-/-</sup> tumor cells, MEFs and macrophages

      Schultz, Kristin; Grieger, Christina; Li, Yong; Urbánek, Pavel; Ruschel, Anne; Minnich, Kerstin; Bruder, Dunja; Gereke, Marcus; Sechi, Antonio; Herrlich, Peter; et al. (PLOS, 2018-12-01)
    • Chronic Hepatitis E Virus Infection during Lymphoplasmacytic Lymphoma and Ibrutinib Treatment.

      Schlevogt, Bernhard; Kinast, Volker; Reusch, Julia; Kerkhoff, Andrea; Praditya, Dimas; Todt, Daniel; Schmidt, Hartmut H; Steinmann, Eike; Behrendt, Patrick; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MPDI, 2019-08-22)
      Hepatitis E virus (HEV) is an increasingly recognised pathogen, affecting several hundred thousand individuals in western countries each year. Importantly, the majority of immunocompromised individuals are not able to clear HEV but develop a chronic course of infection. In the case of lymphoma, which is an inherent immunosuppressive disease per se, chemotherapy can even further exacerbate the immunosuppressive status. As the mechanism of HEV chronification is barely understood, it is important to gain knowledge about the influence of chemotherapeutic drugs on the HEV replication cycle to guide rational clinical management of HEV infection in such patients. In this case report, a 70 year old man was diagnosed with lymphoplasmacytic lymphoma. As we observed the occurrence of chronic HEV after treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib in vivo, we investigated the influence of BTK signaling and ibrutinib treatment in the HEV replication cycle in vitro. First, we detected an HEV-induced mobilisation of BTK in human liver cells during HEV replication. A moderate antiviral effect against HEV replicating isolates including genotypes 1 and 3 was observed, suggesting that ibrutinib did not support HEV replication in a direct manner. Combinatory treatments of ibrutinib with ribavirin indicated that ibrutinib did not influence the antiviral effect of ribavirin. Taken together, chemotherapy targeting cellular factors for the treatment of lymphomas may be a neglected risk factor for the chronification of HEV. For ibrutinib, despite the upregulation of its target BTK during HEV replication, we observed neither a proviral effect on HEV replication nor an influence on the antiviral effect of ribavirin, suggesting that the chronification of HEV may be favoured by its immunosuppressive effect.
    • Chronic equine hepacivirus infection in an adult gelding with severe hepatopathy.

      Tegtmeyer, Birthe; Echelmeyer, Julia; Pfankuche, Vanessa M; Puff, Christina; Todt, Daniel; Fischer, Nicole; Durham, Andy; Feige, Karsten; Baumgärtner, Wolfgang; Steinmann, Eike; et al. (Wiley Open Access, 2019-01-01)
      Background: Equine hepacivirus (EqHV) in equids represents the closest homologue to hepatitis C virus(HCV) infecting humans. A majority of HCV infected patients develop a chronic course of infection leading toliver fibrosis, cirrhosis and liver failure. However, in horses mostly transient mild subclinical infections arereported for EqHV to date. Objectives: EqHV can be involved in chronic liver diseases of horses. Methods:Biochemical parameters in serum samples were measured. Viral load was determined using qPCR. Next gener-ation sequencing (NGS) of serum was performed. Liver tissue was stained with haematoxylin and eosin andanalysed for viral RNA with fluorescentin situ-hybridization. Results: The horse showed symptoms of severehepatopathy and was chronically infected with EqHV. Viral RNA was detectable in the liver during disease.To rule out other infectious agents NGS was performed and showed the highest abundance for EqHV. Theidentified virus sequence was similar to other circulating equine hepaciviruses. Conclusions: EqHV can be asso-ciated with liver disease in horses. Whether it causes the disease or contributes in a multifactorial mannerneeds further investigation
    • In vivo Neutralization of Pro-inflammatory Cytokines During Secondary Streptococcus pneumoniae Infection Post Influenza A Virus Infection

      Sharma-Chawla, Niharika; Stegemann-Koniszewski, Sabine; Christen, Henrike; Boehme, Julia D.; Kershaw, Olivia; Schreiber, Jens; Guzmán, Carlos A.; Bruder, Dunja; Hernandez-Vargas, Esteban A.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers Media SA, 2019-08-14)
      An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the Streptococcus pneumoniae strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies.
    • Blocking IL-10 receptor signaling ameliorates Mycobacterium tuberculosis infection during influenza-induced exacerbation

      Ring, Sarah; Eggers, Lars; Behrends, Jochen; Wutkowski, Adam; Schwudke, Dominik; Kröger, Andrea; Hierweger, Alexandra Maximiliane; Hölscher, Christoph; Gabriel, Gülsah; Schneider, Bianca E.; et al. (American Society for Clinical Investigation, 2019-01-01)
      Epidemiological findings indicate that coinfection with influenza viruses is associated with an increased risk of death in patients suffering from tuberculosis but the underlying pathomechanisms are not well understood. In this study, we demonstrate that influenza A virus (IAV) coinfection rapidly impairs control of Mycobacterium tuberculosis (Mtb) in C57BL/6 mice. IAV coinfection was associated with significantly increased bacterial loads, reduced survival and a substantial modulation of innate and adaptive immune defenses including an impaired onset and development of Mtb-specific CD4+ T cell responses and the accumulation of macrophages with increased arginase-1 production in the lungs. Our findings strongly indicate that IAV coinfection compromises the host's ability to control Mtb infection via the production of IL-10 which was rapidly induced upon viral infection. The blockade of IL-10 receptor signaling reduced the bacterial load in coinfected mice to a level comparable with that in Mtb-only-infected animals. Taken together, our data suggest that IL-10 signaling constitutes a major pathway that enhances susceptibility to Mtb during concurrent IAV infection
    • The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.

      Volckmar, Julia; Knop, Laura; Stegemann-Koniszewski, Sabine; Schulze, Kai; Ebensen, Thomas; Guzmán, Carlos A; Bruder, Dunja; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-08-14)
      Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8+ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.
    • Anti-infective Properties of the Golden Spice Curcumin.

      Praditya, Dimas; Kirchhoff, Lisa; Brüning, Janina; Rachmawati, Heni; Steinmann, Joerg; Steinmann, Eike; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Frontiers, 2019-01-01)
      The search for novel anti-infectives is one of the most important challenges in natural product research, as diseases caused by bacteria, viruses, and fungi are influencing the human society all over the world. Natural compounds are a continuing source of novel anti-infectives. Accordingly, curcumin, has been used for centuries in Asian traditional medicine to treat various disorders. Numerous studies have shown that curcumin possesses a wide spectrum of biological and pharmacological properties, acting, for example, as anti-inflammatory, anti-angiogenic and anti-neoplastic, while no toxicity is associated with the compound. Recently, curcumin's antiviral and antibacterial activity was investigated, and it was shown to act against various important human pathogens like the influenza virus, hepatitis C virus, HIV and strains of Staphylococcus, Streptococcus, and Pseudomonas. Despite the potency, curcumin has not yet been approved as a therapeutic antiviral agent. This review summarizes the current knowledge and future perspectives of the antiviral, antibacterial, and antifungal effects of curcumin.
    • Identification of Keratin 23 as a Hepatitis C Virus-Induced Host Factor in the Human Liver.

      Kinast, Volker; Leber, Stefan L; Brown, Richard J P; Vieyres, Gabrielle; Behrendt, Patrick; Eßbach, Constanze; Strnad, Pavel; Vondran, Florian W R; Cornberg, Markus; Wex, Cora; et al. (MPDI, 2019-06-18)
      Keratin proteins form intermediate filaments, which provide structural support for many tissues. Multiple keratin family members are reported to be associated with the progression of liver disease of multiple etiologies. For example, keratin 23 (KRT23) was reported as a stress-inducible protein, whose expression levels correlate with the severity of liver disease. Hepatitis C virus (HCV) is a human pathogen that causes chronic liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. However, a link between KRT23 and hepatitis C virus (HCV) infection has not been reported previously. In this study, we investigated KRT23 mRNA levels in datasets from liver biopsies of chronic hepatitis C (CHC) patients and in primary human hepatocytes experimentally infected with HCV, in addition to hepatoma cells. Interestingly, in each of these specimens, we observed an HCV-dependent increase of mRNA levels. Importantly, the KRT23 protein levels in patient plasma decreased upon viral clearance. Ectopic expression of KRT23 enhanced HCV infection; however, CRIPSPR/Cas9-mediated knockout did not show altered replication efficiency. Taken together, our study identifies KRT23 as a novel, virus-induced host-factor for hepatitis C virus.
    • Equine Parvovirus-Hepatitis Frequently Detectable in Commercial Equine Serum Pools.

      Meister, Toni Luise; Tegtmeyer, Birthe; Postel, Alexander; Cavalleri, Jessika-M V; Todt, Daniel; Stang, Alexander; Steinmann, Eike; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MDPI, 2019-05-21)
      An equine parvovirus-hepatitis (EqPV-H) has been recently identified in association with equine serum hepatitis, also known as Theiler's disease. This disease was first described by Arnold Theiler in 1918 and is often observed after applications with blood products in equines. So far, the virus has only been described in the USA and China. In this study, we evaluated the presence of EqPV-H in several commercial serum samples to assess the potential risk of virus transmission by equine serum-based products for medical and research applications. In 11 out of 18 commercial serum samples, EqPV-H DNA was detectable with a viral load up to 105 copies/mL. The same serum batches as well as three additional samples were also positive for antibodies against the EqPV-H VP1 protein. The countries of origin with detectable viral genomes included the USA, Canada, New Zealand, Italy, and Germany, suggesting a worldwide distribution of EqPV-H. Phylogenetic analysis of the EqPV-H NS1 sequence in commercial serum samples revealed high similarities in viral sequences from different geographical areas. As horse sera are commonly used for the production of anti-sera, which are included in human and veterinary medical products, these results implicate the requirement for diagnostic tests to prevent EqPV-H transmission.
    • A central hydrophobic E1 region controls the pH range of hepatitis C virus membrane fusion and susceptibility to fusion inhibitors.

      Banda, Dominic H; Perin, Paula M; Brown, Richard J P; Todt, Daniel; Solodenko, Wladimir; Hoffmeyer, Patrick; Kumar Sahu, Kamlesh; Houghton, Michael; Meuleman, Philip; Müller, Rolf; et al. (Elsevier, 2019-06-01)
    • Essential role of IκB for in vivo CD4 T cell activation, proliferation and Th1 cell differentiation during Listeria monocytogenes infection in mice.

      Frentzel, Sarah; Katsoulis-Dimitriou, Konstantinos; Jeron, Andreas; Schmitz, Ingo; Bruder, Dunja; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2019-05-03)
      Acquisition of effector functions in T cells is guided by transcription factors including NF-κB that itself is tightly controlled by inhibitory proteins. The atypical NF-κB inhibitor IκBNS is involved in the development of Th1, Th17 and Treg cells. However, it remained unclear to which extend IκBNS contributes to the acquisition of effector function in T cells specifically responding to a pathogen during in vivo infection. Tracking of adoptively transferred T cells in Listeria monocytogenes infected mice uncovered antigen-specific activation of CD4+ T cells following in vivo pathogen encounter to strongly rely on IκBNS . While IκBNS was largely dispensable for the acquisition of cytotoxic effector function in CD8+ T cells, IκBNS -deficient Th1 effector cells exhibited significantly reduced proliferation, marked changes in the pattern of activation marker expression and reduced production of the Th1-cell cytokines IFNγ, IL2 and TNFα. Complementary in vitro analyses using cells from novel reporter and inducible knockout mice revealed that IκBNS predominantly affects the early phase of Th1-cell differentiation while its function in terminally differentiated cells appears to be negligible. Our data suggest IκBNS as a potential target to modulate specifically CD4+ T-cell responses. This article is protected by copyright. All rights reserved.
    • Antiviral Meroterpenoid Rhodatin and Sesquiterpenoids Rhodocoranes A-E from the Wrinkled Peach Mushroom, Rhodotus palmatus.

      Sandargo, Birthe; Michehl, Maira; Praditya, Dimas; Steinmann, Eike; Stadler, Marc; Surup, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (American Chemical Society, 2019-05-03)
      Rhodatin (1), a meroterpenoid featuring a unique pentacyclic scaffold with both spiro and spiroketal centers, and five unusual acorane-type sesquiterpenoids, named rhodocoranes A-E (2-6, respectively), are the first natural products isolated from the basidiomycete Rhodotus palmatus. Their structures were elucidated by two-dimensional NMR experiments and HRESIMS, while the absolute configuration of the substance family was determined by Mosher's method utilizing 2. Rhodatin strongly inhibited hepatitis C virus, whereas 4 displayed cytotoxicity and selective antifungal activity.
    • HCV Pit Stop at the Lipid Droplet: Refuel Lipids and Put on a Lipoprotein Coat before Exit.

      Vieyres, Gabrielle; Pietschmann, Thomas; TWINCORE, Zentrum für Experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (MPDI, 2019-03-12)
      The replication cycle of the liver-tropic hepatitis C virus (HCV) is tightly connected to the host lipid metabolism, during the virus entry, replication, assembly and egress stages, but also while the virus circulates in the bloodstream. This interplay coins viral particle properties, governs viral cell tropism, and facilitates immune evasion. This review summarizes our knowledge of these interactions focusing on the late steps of the virus replication cycle. It builds on our understanding of the cell biology of lipid droplets and the biosynthesis of liver lipoproteins and attempts to explain how HCV hijacks these organelles and pathways to assemble its lipo-viro-particles. In particular, this review describes (i) the mechanisms of viral protein translocation to and from the lipid droplet surface and the orchestration of an interface between replication and assembly complexes, (ii) the importance of the triglyceride mobilization from the lipid droplets for HCV assembly, (iii) the interplay between HCV and the lipoprotein synthesis pathway including the role played by apolipoproteins in virion assembly, and finally (iv) the consequences of these complex virus–host interactions on the virion composition and its biophysical properties. The wealth of data accumulated in the past years on the role of the lipid metabolism in HCV assembly and its imprint on the virion properties will guide vaccine design efforts and reinforce our understanding of the hepatic lipid metabolism in health and disease.
    • ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

      Büssow, Konrad; Themann, Philipp; Luu, Sabine; Pentrowski, Paul; Harting, Claudia; Majewski, Mira; Vollmer, Veith; Köster, Mario; Grashoff, Martina; Zawatzky, Rainer; et al. (Plos, 2019-01-01)
      Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
    • Hepatitis C Virus Strain-Dependent Usage of Apolipoprotein E Modulates Assembly Efficiency and Specific Infectivity of Secreted Virions.

      Weller, Romy; Hueging, Kathrin; Brown, Richard J P; Todt, Daniel; Joecks, Sebastian; Vondran, Florian W R; Pietschmann, Thomas; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen-Str.7,30625 Hannover, Germany. (American Society for Microbiology, 2017-09-15)
      Hepatitis C virus (HCV) is extraordinarily diverse and uses entry factors in a strain-specific manner. Virus particles associate with lipoproteins, and apolipoprotein E (ApoE) is critical for HCV assembly and infectivity. However, whether ApoE dependency is common to all HCV genotypes remains unknown. Therefore, we compared the roles of ApoE utilizing 10 virus strains from genotypes 1 through 7. ApoA and ApoC also support HCV assembly, so they may contribute to virus production in a strain-dependent fashion. Transcriptome sequencing (RNA-seq) revealed abundant coexpression of ApoE, ApoB, ApoA1, ApoA2, ApoC1, ApoC2, and ApoC3 in primary hepatocytes and in Huh-7.5 cells. Virus production was examined in Huh-7.5 cells with and without ApoE expression and in 293T cells where individual apolipoproteins (ApoE1, -E2, -E3, -A1, -A2, -C1, and -C3) were provided in trans All strains were strictly ApoE dependent. However, ApoE involvement in virus production was strain and cell type specific, because some HCV strains poorly produced infectious virus in ApoE-expressing 293T cells and because ApoE knockout differentially affected virus production of HCV strains in Huh-7.5 cells. ApoE allelic isoforms (ApoE2, -E3, and -E4) complemented virus production of HCV strains to comparable degrees. All tested strains assembled infectious progeny with ApoE in preference to other exchangeable apolipoproteins (ApoA1, -A2, -C1, and -C3). The specific infectivity of HCV particles was similar for 293T- and Huh-7.5-derived particles for most strains; however, it differed by more than 100-fold in some viruses. Collectively, this study reveals strain-dependent and host cell-dependent use of ApoE during HCV assembly. These differences relate to the efficacy of virus production and also to the properties of released virus particles and therefore govern viral fitness at the level of assembly and cell entry.IMPORTANCE Chronic HCV infections are a major cause of liver disease. HCV is highly variable, and strain-specific determinants modulate the response to antiviral therapy, the natural course of infection, and cell entry factor usage. Here we explored whether host factor dependency of HCV in particle assembly is modulated by strain-dependent viral properties. We showed that all examined HCV strains, which represent all seven known genotypes, rely on ApoE expression for assembly of infectious progeny. However, the degree of ApoE dependence is modulated in a strain-specific and cell type-dependent manner. This indicates that HCV strains differ in their assembly properties and host factor usage during assembly of infectious progeny. Importantly, these differences relate not only to the efficiency of virus production and release but also to the infectiousness of virus particles. Thus, strain-dependent features of HCV modulate ApoE usage, with implications for virus fitness at the level of assembly and cell entry.