Viruses exploit cellular pathways for their own benefit, often achieved by providing high affinity binding sites on viral factors that recruit key-regulatory proteins from cellular pathways thereby outcompeting their physiological binding partner. This strategy allows viruses to infect cells and establish efficient replication with nothing else then the proteins associated with the entering viral genome and viral capsids. Besides transport of the genome to the replication sites, viruses also need to assure decondensation and transcriptional activation of the viral genome that in most cases has been packed and stored in the most economical way. Once the viral genome becomes transcriptionally activated new viral proteins are synthesized providing the virus with the capacity to reprogram the cell for viral replication.
Our scientific interest is to understand these immediate early steps during viral infection as they represent a decisive moment in the establishment of a productive infection of various pathogenic viruses. We investigate the role of viral core-associated factors being complexed with the incoming virus particle during the first phase of infection leading to the onset of gene expression during productive virus lifecycle. We found that such virion proteins share several features with their more potent non-structural counterparts, and may serve as surrogates to bridge the gap until viral gene expression is established. Based on our work, we suggest that such immediate early viral proteins are highly biologically active, posttranslationally modified and orchestrate immediate early cell activation and proliferation prior to viral gene expression. Further insights into the immediate early step of virus infections within the host will contribute to identify new therapeutic targets to limit or prevent human pathogenic virus-mediated diseases and mortality of patients.
1. Elucidating the bivalent role of PML nuclear bodies during Human Adenovirus (HAdV) infection
PML nuclear bodies (NBs) are matrix-bound nuclear structures that have been implicated in a variety of functions, including DNA repair, transcriptional regulation, protein degradation, and tumor suppression. These domains are also known to play an essential role in antiviral host-cell defense, most presumably mediated via accumulation of SUMO-dependent and interferon-induced antiviral host factors. This likely explains why they are targeted and subsequently manipulated by numerous viral regulatory proteins. Paradoxically, the genomes of numerous DNA viruses become associated with PML-NBs, and initial sites of viral transcription/replication are often juxtaposed to these domains. To date several HAdV regulatory proteins have shown association with PML-NBs, illustrating their crucial role during infection, although the functional consequences of this association are still largely elusive.
We recently reported that the viral transactivator protein E1A targets the PML-II isoform to efficiently activate viral and cellular transcription. In contrast, PML-associated proteins Daxx and ATRX, involved in cellular chromatin remodeling processes, are inhibited by early viral factors. Based on our data we hypothesize that this concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB associated proteins to actively establish a pro-viral environment in the host cell. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Specifically, we observed Ad-induced relocalization of the alternatively spliced Sp100 isoforms B, C and HMG from PML-NBs prior to the establishment of viral replication centers juxtaposed to these foci. In contrast, isoform Sp100A is retained at the virus-induced PML tracks that surround the newly formed viral replication centers, acting as designated sites of active transcription. The host restriction factors Sp100B, C and HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression. Our group wants to provide further evidence that Ad selectively counteracts antiviral responses, and at the same time benefits from proviral PML-NB-associated components by actively recruiting them to PML track-like structures, thereby creating a positive microenvironment for viral transcription and replication at these nuclear subdomains.
2. Functional analysis of covalent SUMO conjugation to virion-associated proteins during early HAdV infection
We plan to understand the biological functions of virion-associated proteins in detail to give perspectives for the development of novel virus intervention strategies. PML nuclear bodies represent hotspots for SUMOylation processes in the cell. Simultanously, initial HAdV replication/ transcription centers establish juxtaposed to these cellular domains. We therefore plan to study whether mainly virion-associated proteins are substrates of the SUMO machinery in the host and to investigate the functional consequences of virion-associated protein SUMOylation on viral replication.
3. Functional analyses of covalent ubiquitin conjugation to virion-associated proteins during early HAdV infection
Ubiquitin is the best-studied member of the UBL (ubiquitin-like) protein family. Covalent conjugation of ubiquitin to a substrate protein proceeds via a three-step cascade and involves the sequential enzymatic activity of E1, E2 and E3 proteins. PTM with ubiquitin is known to play a major role in protein stability, localization and numerous other protein functions. Based on these findings, we will investigate ubiquitinylation of virion-associated factors and study the role of this PTM during the course of early HAdV infection to understand these events and locate novel target structures for viral intervention strategies.
4. Role of virion-associated proteins in viral co-infections
Our data show that the HAdV virion-associated protein pVI severly impacts gene expression of non-related viruses, such as HCMV. Simultanously, activation of HAdV gene expression was also observed in presence of virion-associated proteins from other human pathogenic DNA viruses (HCMV, HPV). We want to challenge the question whether transcriptional activation by virion-associated proteins is a general mechanism of nuclear-replicating viruses. If we understand these generalities, we will be able to raise novel target structures for future virus intervention.
5. HAdV dependent modulation of chromatin-associated host cell determinants
For efficient infection of a cell, chromatin structure has to be modified and therefore the host-cellular DNA damage response (DDR) has to be manipulated. HAdV is known to interfere with the DDR in various ways, including the degradation of proteins involved in the DDR or pro-apoptotic processes like p53, Mre11, DNA ligase IV, Bloom Helicase (BLM) and Tip60. In this context, we recently showed that early viral proteins interfere with the cellular factors Daxx and X-linked α-thalassaemia retardation syndrome protein (ATRX) as well as with SPOC1, host determinants that are involved in transcriptional repression and chromatin compaction, in order to ensure proper virus replication. Our group wants to further investigate Ad dependent modulation of chromatin associated proteins and translate these findings into emerging fields such as AdV gene vector development and antiviral intervention strategies.