AG Bogner - Replication mechanisms of herpesviruses

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Focus research

My lab aims at understanding what structure-function relationships are required for viral replication. One important component is the viral nanomotor. The viral nanomotor, which is involved in the cleavage-packaging process, is composed of the terminase and the portal. The packaging motor is a prerequisite because during insertion into the capsid the DNA is condensed to nearly crystalline structure.

Group members

Institute of Virology Charité: Janine Theiß
M.Sc. Janine Theiß

I am interested in the packaging mechanism of human cytomegalovirus (HCMV). Especially I am trying to characterize the functional motifs of the terminase subunit pUL89.

Projects

Mechanism of DNA packaging

DNA packaging of human cytomegalovirus (HCMV) is the key step in maturation of herpesviruses. This projects aims at understanding what structure-function relationships are required for viral replication. One important component is the viral nanomotor. The viral nanomotor, which is involved in the cleavage-packaging process, is composed of the terminase and the portal protein, pUL104. Terminases catalyze the ATP-dependent translocation of genomic DNA into the procapsids, bind and cleave concatenated DNA. The terminase subunit pUL56, which has ATPase activity, directly interacts with the portal protein thus leading to the formation of most effective biochemical nanomotor. The subunit pUL89 seems to be mainly required for cleavage of concatemeric DNA into unit-length genomes. It is most likely that several additional viral proteins are required for the complex DNA packaging process One aspect of our research address the identification and characterization of additional DNA packaging proteins. One candidate is the protein UL77 (pUL77), a conserved core gene of HCMV. We identified this essential capsid-associated protein and demonstrated that pUL77 has the ability to form a homodimer. In silico analyses revealed the presence a coil-coiled motif (CCM) in pUL77. Experiments with mutant pUL77 lacking the CCM demonstrated that this motif is crucial for oligomerization. In addition, according to its proposed function in DNA packaging it was shown that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein (MCP). Furthermore, the ability of pUL77 to bind dsDNA was shown by an in vitro assay. The DNA binding was lost if the CCM was deleted, thus indicating that oligomerization is a prerequisite for DNA binding of pUL77 (Meissner et al., 2011). Our results implicated a potential function of pUL77 during packaging. One possible explanation could be that HCMV pUL77 plays a role in anchoring the encapsidated DNA during the late stage of packaging (Meissner et al., 2011; Köppen-Rung et al., 2016).

Identification of new antiviral targets

Effect of DSTP-27 on HCMV entry. Prechilled HELF cells were treated with 10 µM DSTP-27 or 10µg/ml heparin or left untreated (w/o) for 30 min at 4°C prior to infection with prechilled HCMV-GFP (MOI 0.25) for 2 h at 4°C. After 48, 72 h or 7 d of infection at 37°C the cells were subjected to GFP fluorescence.

Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults. All current available drugs against HCMV target the viral DNA polymerase. They have limited effects, suffer from long-term toxicity and cause additional complications including drug resistance. Considering that present therapeutic approaches are limited new strategies are needed that may result from a better understanding of viral molecular biology. This, in turn, would necessitate the characterization of new targets, which are non-nucleosidic and have a different mechanism of action. This project deals with the development of compounds that interfere with the cleavage and packaging mechanisms of the viral DNA as well as entry inhibitors.

a)    Viral targets

A new class of replication inhibitors, benzimidazole-D-ribonucleosides, target HCMV DNA packaging (Gentry et al., 2019). Recently we identified new derivatives with an inhibitory effect on DNA cleavage, Cl4RB and BTCRB (Hwang et al., 2007). Interestingly the tetrahalogenates have an antiviral activity against HCMV clinical isolates (GCV-sensitive; GCV resistant), HSV-1, MCMV, RCMV and VZV (Hwang et al., 2009). Their antiviral activity was quantified by measurement of viral plaque formation (plaque reduction) as well as viral growth kinetics. Furthermore, by using pulsed-field gel electrophoresis we demonstrated that both compounds prevent cleavage of concatenated DNA into unit-length genomes. In addition, we could demonstrate that both compounds are effective against rat cytomegalovirus strain England (RCMV-E). Furthermore, yield assays showed that the tetrahalogenated compounds have a high efficacy in three-dimensional environments (Dittmer et al., 2017).

b)    Cellular targets

This part of the project deals with the characterization of the dispirotripiperazine derivative DSTP-27 and the effect on HCMV replication. Cells pre-treated with DSTP-27 showed a growth defect up to 3 log. Interestingly, we provide evidence that DSTP-27 exhibited strong antiviral activity against two laboratory HCMV strains with different cell tropism as well as a GCV-sensitive and GCV-resistant clinical isolate. Furthermore, neither infectious nor non-infectious viral particles were observed by electron microscopy. Taken together, pre-treatment of infected cells with DSTP-27 prevents (i) penetration of HCMV but not attachment (Paeschke et al., 2014). It is discussed that DSTP-27 prevents virus infection by binding to cell surface heparan sulfate glycosaminoglycans (HS). In summary, the stability of DSTP-27 together with its strong inhibitory activity will be a powerful tool for antiviral therapy against HCMV reactivation. This would be a promising therapy for the increasing number of transplant patients suffering from HCMV infection or reactivation

Structure-function relationships of DNA packaging enzymes

Surface-rendered presentation of the pUL56 structure as determined by 3D reconstruction from single particles. two connected rings. Each ring measures about 9 nm in diameter and 2.5 nm in height and the central protein deficit is approximately 3.5 by 2.5 nm across. pUL56 is viewed as face-on(a) and side on (b, giving rise to the square-shaped projection; and d, the u-shaped projection) projection. The presentation in C is obtained when tilting the ring-like view by 45°.

The initial step of production of new virions is the packaging of newly synthesized, concatenated viral DNA into procapsids. We provide evidence that the HCMV terminase, consisting of the subunits pUL56 and pUL89, catalyze the generation of unit length-genomes during DNA-packaging. Although we have identified and characterized the terminase, the structure requirements are not completely understood.

The first important aspect of this research is the identification of DNA-binding domains of the  terminase subunits. According to the results achieved the identified DNA binding domains will be verified by generation of different HCMV deletion mutants. Another part of the project will focus on the interaction domain in pUL89 with pUL56. Recombinant viruses with a deletion of this domain were generated. The mutant viruses were characterized concerning their growth kinetics and viral particle formation. A majority of the recombinant viruses were able to infect cells but lost the ability for virus spread. This viral mutants are essential for HCMV maturation (Theiß et al., in preparation).   

Despite the importance of these proteins and intensive research into biochemical aspects, there is still a lack of structural information. Structural analysis of recombinant proteins pUL56 (Fig.1; Savva et al., 2004) and pUL89 (Theiß et al., in preparation) was achieved by using electron microscopy in conjunction with single particle analysis. These results will lead to a consistent model of HCMV DNA replication and will be important for the development of highly specific antivirals targeting the essential domains of the terminase subunits

Collaborators

  • Prof. John C. Drach, Department of Biologic and Material Sciences, University of Ann Arbor, Michigan, USA
  • Prof. Andreas Holzenburg, Division of Research, Innovation and Economic Development, Department of Biomedical Sciences, School of Medicine, The University of Texas Rio Grande Valley, Brownsville-Edinburg-Harlingen, USA
  • Dr. Michael Laue, Robert-Koch-Institut, Berlin, Germany
  • Prof. Vadim Makarov, A. N. Bakh Institute of Biochemistry RAS, Moscow, Russia
  • Prof. Michaela Schmidtke, Jena University Hospital, Section Experimental Virology, Jena, Germany
  • Prof. Leroy B. Townsend, College of Pharmacy, University of Ann Arbor, Michigan, USA
  • Dr. Lüder Wiebusch, Charité-Universitätsmedizin Berlin, Klinik für Pädiatrie m.S. Onkologie und Hämatologie.
  • Prof. Dr. Gabriele Pecher, Charité-Universitätsmedizin Berlin, Medizinische Klinik m.S. Onkologie und Hämatologie CCM

Significant publications

A current list of all publications can be found in the pubmed-Liste.

Key publications:   

  • Dittmer, A., I. Woskobojnik, R. Adfeldt, J.C. Drach, L.B. Townsend, S. Voigt and E. Bogner. (2017). Tetrahalogenated benzimidazole D-ribonucleosides are active against rat cytomegalovirus. Antiviral Res. 137: 102-107
  • Paeschke, R., I. Woskobojnik, V. Makarov, M. Schmidtke, and E. Bogner. (2014). DSTP-27 prevents entry of human cytomegalovirus Antimicrob. Agents Chemother. 58, 1963-1971
  • Meissner, C.S., S. Suffner, M. Schauflinger, J. von Einem and E. Bogner. (2012) A leucine zipper motif of a tegument protein triggers final envelopment of human cytomegalovirus. J. Virol. 86, 3370-3382
  • Hwang, J.-S., O. Kregler, R. Schilf, N. Bannert, J. C. Drach, L.B. Townsend and E. Bogner. (2007) Identification of acetylated, tetrahalogenated benzimidazole D-ribonucleotides with enhanced activity against human cytomegalovirus. J. Virol. 81, 11604-11611
  • Thoma, C., Borst, E., Messerele, M., Rieger, M., Hwang, J.-S. and Bogner, E. (2006). Identifcation of the the interaction domain of the small terminase subunit pUL89 with the large subunit pUL56 of human cytomegalovirus. Biochemistry, 45, 8855-8863
  • Sava, C.G.W., Holzenburg, A. andf Bogner, E., (2004). Three dimensional structure of the terminase subunit pUL56 of human cytomegalovirus. FEBS Letters, 563, 135-140
  • Scholz, B., Rechter, S., Drach, J.C.,  Townsend, L.B., & Bogner, E. (2003). Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovirus. Nucl. Acids Res. 31: 1426-1433.
  • Bogner, E. (2002) Human cytomegalovirus terminase as a target for antiviral therapy. Rev. Med. Virol. 12:115-127.
  • Scheffczik, H., Savva, C.G.W., Holzenburg, A., Kolesnikova, L., & Bogner, E. (2002). The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA metabolizing proteins with toroidal structure. Nucl. Acids Res. 30: 1695-1703.