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Ms. Julia Hölper
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany

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0 herpesviruses
0 Viral infection
0 Crispr-Cas9
0 Cellular and Molecular Biology
0 Pseudorabies Virus

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Journal article
Published: 09 August 2021 in Viruses
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Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gDPass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gDPass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gDPass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gDPass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.

ACS Style

Julia Hölper; Finn Grey; John Baillie; Tim Regan; Nicholas Parkinson; Dirk Höper; Thiprampai Thamamongood; Martin Schwemmle; Katrin Pannhorst; Lisa Wendt; Thomas Mettenleiter; Barbara Klupp. A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gDPass. Viruses 2021, 13, 1574 .

AMA Style

Julia Hölper, Finn Grey, John Baillie, Tim Regan, Nicholas Parkinson, Dirk Höper, Thiprampai Thamamongood, Martin Schwemmle, Katrin Pannhorst, Lisa Wendt, Thomas Mettenleiter, Barbara Klupp. A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gDPass. Viruses. 2021; 13 (8):1574.

Chicago/Turabian Style

Julia Hölper; Finn Grey; John Baillie; Tim Regan; Nicholas Parkinson; Dirk Höper; Thiprampai Thamamongood; Martin Schwemmle; Katrin Pannhorst; Lisa Wendt; Thomas Mettenleiter; Barbara Klupp. 2021. "A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gDPass." Viruses 13, no. 8: 1574.

Journal article
Published: 10 June 2021 in Viruses
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The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.

ACS Style

Anna Dorsch; Julia Hölper; Kati Franzke; Luca Zaeck; Thomas Mettenleiter; Barbara Klupp. Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus. Viruses 2021, 13, 1117 .

AMA Style

Anna Dorsch, Julia Hölper, Kati Franzke, Luca Zaeck, Thomas Mettenleiter, Barbara Klupp. Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus. Viruses. 2021; 13 (6):1117.

Chicago/Turabian Style

Anna Dorsch; Julia Hölper; Kati Franzke; Luca Zaeck; Thomas Mettenleiter; Barbara Klupp. 2021. "Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus." Viruses 13, no. 6: 1117.

Research article
Published: 12 October 2020 in PLOS Pathogens
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Pathogenicity often differs dramatically among even closely related arenavirus species. For instance, Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever (AHF), is closely related to Tacaribe virus (TCRV), which is normally avirulent in humans. While little is known about how host cell pathways are regulated in response to arenavirus infection, or how this contributes to virulence, these two viruses have been found to differ markedly in their ability to induce apoptosis. However, details of the mechanism(s) governing the apoptotic response to arenavirus infections are unknown. Here we confirm that TCRV-induced apoptosis is mitochondria-regulated, with associated canonical hallmarks of the intrinsic apoptotic pathway, and go on to identify the pro- and anti-apoptotic Bcl-2 factors responsible for regulating this process. In particular, levels of the pro-apoptotic BH3-only proteins Noxa and Puma, as well as their canonical transcription factor p53, were strongly increased. Interestingly, TCRV infection also led to the accumulation of the inactive phosphorylated form of another pro-apoptotic BH3-only protein, Bad (i.e. as phospho-Bad). Knockout of Noxa or Puma suppressed apoptosis in response to TCRV infection, whereas silencing of Bad increased apoptosis, confirming that these factors are key regulators of apoptosis induction in response to TCRV infection. Further, we found that while the highly pathogenic JUNV does not induce caspase activation, it still activated upstream pro-apoptotic factors, consistent with current models suggesting that JUNV evades apoptosis by interfering with caspase activation through a nucleoprotein-mediated decoy function. This new mechanistic insight into the role that individual BH3-only proteins and their regulation play in controlling apoptotic fate in arenavirus-infected cells provides an important experimental framework for future studies aimed at dissecting differences in the apoptotic responses between arenaviruses, their connection to other cell signaling events and ultimately the relationship of these processes to pathogenesis. Arenaviruses are important zoonotic pathogens that present a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and/or neurological symptoms, other closely related species exhibit little or no pathogenicity. The basis for these dramatically different outcomes is insufficiently understood, but investigations of host cell responses have suggested that apoptosis, i.e. non-inflammatory programmed cell death, is regulated differently between pathogenic and apathogenic arenaviruses. However, many questions remain regarding how these viruses interact with cell death pathways upon infection. Here we demonstrate that apoptosis induced by the avirulent Tacaribe virus (TCRV), proceeds via the mitochondria (i.e. the intrinsic apoptotic signaling pathway), and is regulated by a combination of factors that appear to balance activation (i.e. Noxa and Puma) and inactivation (i.e. Bad-P) of this cascade. During TCRV infection, the balance of these pro- and anti-apoptotic signals shifts the equilibrium late in the infection towards cell death. Importantly, we also found that the highly pathogenic Junín virus (JUNV), which does not trigger caspase activation or apoptotic cell death, nonetheless induces pro-apoptotic factors, thus supporting the existence of a specific mechanism by which this virus is able to evade apoptosis at late stages in this process.

ACS Style

Julia Holzerland; Lucie Fénéant; Logan Banadyga; Julia E. Hölper; Michael R. Knittler; Allison Groseth. BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis. PLOS Pathogens 2020, 16, e1008948 .

AMA Style

Julia Holzerland, Lucie Fénéant, Logan Banadyga, Julia E. Hölper, Michael R. Knittler, Allison Groseth. BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis. PLOS Pathogens. 2020; 16 (10):e1008948.

Chicago/Turabian Style

Julia Holzerland; Lucie Fénéant; Logan Banadyga; Julia E. Hölper; Michael R. Knittler; Allison Groseth. 2020. "BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis." PLOS Pathogens 16, no. 10: e1008948.

Journal article
Published: 17 July 2020 in Virus Research
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During herpesvirus replication, newly synthesized nucleocapsids exit the nucleus by a vesicle-mediated transport, which requires the nuclear egress complex (NEC), composed of the conserved viral proteins designated as pUL31 and pUL34 in the alphaherpesviruses pseudorabies virus (PrV) and herpes simplex viruses. Oligomerization of the heterodimeric NEC at the inner nuclear membrane (INM) results in membrane bending and budding of virus particles into the perinuclear space. The INM-derived primary envelope then fuses with the outer nuclear membrane to release nucleocapsids into the cytoplasm. The two NEC components are necessary and sufficient for induction of vesicle budding and scission as shown after co-expression in eukaryotic cells or in synthetic membranes. However, where and when the NEC is formed, how membrane curvature is mediated and how it is regulated, remains unclear. While monospecific antisera raised against the different components of the PrV NEC aided in the characterization and intracellular localization of the individual proteins, no NEC specific tools have been described yet for any herpesvirus. To gain more insight into vesicle budding and scission, we aimed at generating NEC specific monoclonal antibodies (mAbs). To this end, mice were immunized with bacterially expressed soluble PrV NEC, which was previously used for structure determination. Besides pUL31- and pUL34-specific mAbs, we also identified mAbs, which reacted only in the presence of both proteins indicating specificity for the complex. Confocal microscopy with those NEC-specific mAbs revealed small puncta (approx. 0.064 μm2) along the nuclear rim in PrV wild type infected cells. In contrast, ca. 5-fold larger speckles (approx. 0.35 μm2) were detectable in cells infected with a PrV mutant lacking the viral protein kinase pUS3, which is known to accumulate primary enveloped virions in the PNS within large invaginations of the INM, or in cells co-expressing pUL31 and pUL34. Kinetic experiments showed that while the individual proteins were detectable already between 2–4 hours after infection, the NEC-specific mAbs produced significant staining only after 4–6 hours in accordance with timing of nuclear egress. Taken together, the data indicate that these mAbs specifically label the PrV NEC.

ACS Style

Julia Hölper; Sven Reiche; Kati Franzke; Thomas C. Mettenleiter; Barbara G. Klupp. Generation and characterization of monoclonal antibodies specific for the Pseudorabies Virus nuclear egress complex. Virus Research 2020, 287, 198096 .

AMA Style

Julia Hölper, Sven Reiche, Kati Franzke, Thomas C. Mettenleiter, Barbara G. Klupp. Generation and characterization of monoclonal antibodies specific for the Pseudorabies Virus nuclear egress complex. Virus Research. 2020; 287 ():198096.

Chicago/Turabian Style

Julia Hölper; Sven Reiche; Kati Franzke; Thomas C. Mettenleiter; Barbara G. Klupp. 2020. "Generation and characterization of monoclonal antibodies specific for the Pseudorabies Virus nuclear egress complex." Virus Research 287, no. : 198096.

Journal article
Published: 31 March 2020 in Journal of Virology
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Herpesviruses encode an exceptional vesicle formation and scission machinery, which operates at the inner nuclear membrane, translocating the viral nucleocapsid from the nucleus into the perinuclear space. The conserved herpesviral nuclear egress complex (NEC) orchestrates this process. High-resolution imaging approaches as well as the recently solved crystal structures of the NEC provided deep insight into the molecular details of vesicle formation and scission. Nevertheless, the molecular mechanism of nucleocapsid incorporation remained unclear. In accordance with structure-based predictions, a basic amino acid could be pinpointed in the most membrane-distal domain of the NEC (pUL31-K242), indicating that capsid incorporation might depend on a direct electrostatic interaction. Our follow-up study, described here, however, shows that the positive charge is not relevant but that the overall structure matters.

ACS Style

Sebastian Rönfeldt; Kati Franzke; Julia E. Hölper; Barbara G. Klupp; Thomas C. Mettenleiter. Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction. Journal of Virology 2020, 94, 1 .

AMA Style

Sebastian Rönfeldt, Kati Franzke, Julia E. Hölper, Barbara G. Klupp, Thomas C. Mettenleiter. Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction. Journal of Virology. 2020; 94 (8):1.

Chicago/Turabian Style

Sebastian Rönfeldt; Kati Franzke; Julia E. Hölper; Barbara G. Klupp; Thomas C. Mettenleiter. 2020. "Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction." Journal of Virology 94, no. 8: 1.

Research article
Published: 30 March 2020 in PLOS Pathogens
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Herpesviral encephalitis caused by Herpes Simplex Virus 1 (HSV-1) is one of the most devastating diseases in humans. Patients present with fever, mental status changes or seizures and when untreated, sequelae can be fatal. Herpes Simplex Encephalitis (HSE) is characterized by mainly unilateral necrotizing inflammation effacing the frontal and mesiotemporal lobes with rare involvement of the brainstem. HSV-1 is hypothesized to invade the CNS via the trigeminal or olfactory nerve, but viral tropism and the exact route of infection remain unclear. Several mouse models for HSE have been developed, but they mimic natural infection only inadequately. The porcine alphaherpesvirus Pseudorabies virus (PrV) is closely related to HSV-1 and Varicella Zoster Virus (VZV). While pigs can control productive infection, it is lethal in other susceptible animals associated with severe pruritus leading to automutilation. Here, we describe the first mutant PrV establishing productive infection in mice that the animals are able to control. After intranasal inoculation with a PrV mutant lacking tegument protein pUL21 and pUS3 kinase activity (PrV-ΔUL21/US3Δkin), nearly all mice survived despite extensive infection of the central nervous system. Neuroinvasion mainly occurred along the trigeminal pathway. Whereas trigeminal first and second order neurons and autonomic ganglia were positive early after intranasal infection, PrV-specific antigen was mainly detectable in the frontal, mesiotemporal and parietal lobes at later times, accompanied by a long lasting lymphohistiocytic meningoencephalitis. Despite this extensive infection, mice showed only mild to moderate clinical signs, developed alopecic skin lesions, or remained asymptomatic. Interestingly, most mice exhibited abnormalities in behavior and activity levels including slow movements, akinesia and stargazing. In summary, clinical signs, distribution of viral antigen and inflammatory pattern show striking analogies to human encephalitis caused by HSV-1 or VZV not observed in other animal models of disease.

ACS Style

Julia Sehl; Julia E. Hölper; Barbara G. Klupp; Christina Baumbach; Jens P. Teifke; Thomas C. Mettenleiter. An improved animal model for herpesvirus encephalitis in humans. PLOS Pathogens 2020, 16, e1008445 .

AMA Style

Julia Sehl, Julia E. Hölper, Barbara G. Klupp, Christina Baumbach, Jens P. Teifke, Thomas C. Mettenleiter. An improved animal model for herpesvirus encephalitis in humans. PLOS Pathogens. 2020; 16 (3):e1008445.

Chicago/Turabian Style

Julia Sehl; Julia E. Hölper; Barbara G. Klupp; Christina Baumbach; Jens P. Teifke; Thomas C. Mettenleiter. 2020. "An improved animal model for herpesvirus encephalitis in humans." PLOS Pathogens 16, no. 3: e1008445.

Journal article
Published: 17 March 2020 in Cells
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Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

ACS Style

Julia E. Hölper; Barbara G. Klupp; G. W. Gant Luxton; Kati Franzke; Thomas C. Mettenleiter. Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress. Cells 2020, 9, 738 .

AMA Style

Julia E. Hölper, Barbara G. Klupp, G. W. Gant Luxton, Kati Franzke, Thomas C. Mettenleiter. Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress. Cells. 2020; 9 (3):738.

Chicago/Turabian Style

Julia E. Hölper; Barbara G. Klupp; G. W. Gant Luxton; Kati Franzke; Thomas C. Mettenleiter. 2020. "Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress." Cells 9, no. 3: 738.