• Cellular Antiviral Factors that Target Particle Infectivity of HIV-1.

      Goffinet, Christine; TWINCORE, Zentrum für Experimentelle und Klinische Infektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2016)
      In the past decade, the identification and characterization of antiviral genes with the ability to interfere with virus replication has established cell-intrinsic innate immunity as a third line of antiviral defense in addition to adaptive and classical innate immunity. Understanding how cellular factors have evolved to inhibit HIV-1 reveals particularly vulnerable points of the viral replication cycle. Many, but not all, antiviral proteins share type I interferon-upregulated expression and sensitivity to viral counteraction or evasion measures. Whereas well-established restriction factors interfere with early post-entry steps and release of HIV-1, recent research has revealed a diverse set of proteins that reduce the infectious quality of released particles using individual, to date poorly understood modes of action. These include induction of paucity of mature glycoproteins in nascent virions or self-incorporation into the virus particle, resulting in poor infectiousness of the virion and impaired spread of the infection.
    • [cGAS, an antiviral weapon: role in the recognition of HIV-1 transmitted from cell to cell].

      Ducroux, Aurélie; Goffinet, Christine; TwinCore, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2017-10-10)
    • 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.
    • Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

      Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; et al. (2017-05-31)
      Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC.
    • 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.
    • Susceptibility of Chikungunya Virus to Inactivation by Heat and Commercially and World Health Organization-Recommended Biocides.

      Franz, Sergej; Friesland, Martina; Passos, Vânia; Todt, Daniel; Simmons, Graham; Goffinet, Christine; Steinmann, Eike; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Oxford University Press, 2018-09-22)
      Despite increasing clinical relevance of Chikungunya virus (CHIKV) infection, caused by a rapidly emerging pathogen, recommended guidelines for its inactivation do not exist. In this study, we investigated the susceptibility of CHIKV to inactivation by heat and commercially available hand, surface, and World Health Organization-recommended disinfectants to define CHIKV prevention protocols for healthcare systems.