Adenoviruses - from pathogens to therapeutics: a report on the 10th International Adenovirus Meeting

The study of adenovirus biology has led to seminal discoveries in mRNA splicing (Nobel Prize to Sharp and Roberts in 1993, Sharp, 1994), the eucaryotic first cell-free DNA replication system (Stillman, 1983), and fundamental insight into antigen presentation to T cells (Burgert and Kvist, 1985). These and many other advances in adenovirus molecular and cellular biology and epidemiology laid a strong foundation for cancer research, immunology, and genetic engineering. Notably, adenoviruses (AdVs), whose members include > 55 human types, are the most widely used vectors in clinical gene therapy fields. The types are classified into seven species, HAdV-A to G, mainly based on haemagglutination assays and genome sequences (Harrach et al., 2011). Together with other mammalian adenoviruses, these HAdVs form the genus Mastadenovirus, distinct from the aviadenoviruses infecting birds only. Two other genera include AdVs from more diverse hosts. Atadenoviruses infect reptiles predominantly, but occur also in birds and ruminants, whereas the host origin of the genus Siadenovirus is unclear as yet. The only known piscine AdV alone represents the most recently approved sixth genus Ichtadenovirus. A major quest in public health has been to find out how viruses cause disease. To solve this complex issue investigators classically employ reductionist's approaches and break the problem down into small units with specific solutions. The ultimate aim of this line of approach is to determine the atomic structure of the virus. This knowledge is primordial because it links major processes of propagation: entry, assembly, egress and spreading. The presentation from V. Reddy (The Scripps Research Institute, La Jolla, USA) gave new glimpses into the largest crystal structure of biomaterials solved so far, a human adenovirus type 5 from species HAdV-C (HAdV-5) containing the short fibre from HAdV-35 at 3.5 Å resolution (Reddy et al., 2010). This structural analysis extended earlier cryo-EM studies (Fabry et al., 2005; Liu et al., 2010), and showed that the virus has a T = 25 icosahedral symmetry, made up in large parts by the major virus protein hexon. The new study assigned the 4 helical bundle on the capsid exterior to a segment of IIIa protein, which was previously assigned to the C-termini of IX by the cryo-EM studies. While both IIIa and IX stabilize the hexon shell on the capsid exterior, VIII and yet-to-be-identified vertex proteins stabilize the hexon shell on the interior. The determination of the precise locations of the cementing proteins helps better understand how the virus is held together and how the double-stranded DNA genome is connected to the capsid. Many of the connections of the major capsid proteins to the ‘glue’ proteins are broken in a stepwise manner during virus entry (Greber et al., 1993). These connections determine if the viral DNA is deposited in the cell nucleus or degraded. A disassembly defective HAdV-2 mutant, ts1 (temperature-sensitive 1), unable to deliver DNA into the nucleus is non-infectious (Imelli et al., 2009). Ts1 lacks viral protease and contains immature non-processed ‘glue’ proteins. Remarkably, the outside structure of HAdV-2 and ts1 are identical as suggested by cryo-EM analyses at 10 Å resolution (Silvestry et al., 2009). The viruses interact with the same receptors, the coxsackievirus and adenovirus receptor (CAR) and alpha v integrins. G. Nemerow (The Scripps Research Institute, La Jolla, USA) presented atomic force microscopy (AFM) experiments showing that the fivefold axis of Ad5/35F has lower tensile strength than the twofold or threefold axis of the virus. Together with W. Roos and colleagues at the University of Utrecht (The Netherlands) they showed that alpha defensin HD5 binding to the fivefold axis stabilizes this region of the virus while binding of integrins to the same region loosens the fivefold structure. These studies provide a link between the mechanical properties of the virus and the disassembly process. Whether the physical properties of HAdV-2 and ts1 are different will be interesting to see in the future, as it might give an indication on the impact of host factors, such as integrins, defensins or blood factors on virus stability. It will also be interesting to see, how the flexibility of ts1 contributes to the resistance of this virus to the cellular uncoating cues that trigger the stepwise disassembly process of the wild-type virus. M. Suomalainen (University of Zurich, Switzerland) presented the identification of cellular cues, which promote the uncoating of wild-type HAdV-2 or 5. These cues are the drifting motions of CAR-bound virus on the cell surface, counteracted by the stationary motions of the alpha v integrin co-receptors (Burckhardt et al., 2011). The drifts are acto-myosin dependent. Together with integrins these motions trigger the shedding of the fibres and subsequently the exposure of the internal capsid protein VI, which appears to be the factor that disrupts endosomal membranes (Moyer et al., 2011). C. San Martin (Spanish National Center of Biotechnology, Madrid, Spain) presented data from cell-free HAdV-2 uncoating experiments and suggested that a unique vertex structure is opened before the virion eventually falls apart. It can be speculated that the compaction of the viral core into the capsid and subsequent maturation steps of the core by the viral protease would lead to an increase of internal capsid pressure, which then might facilitate uncoating steps of the virus (Perez-Berna et al., 2012). Whether such uncoating steps are modulated by posttranslational modifications of the HAdV-2/5 particle, for example phosphorylations as discussed by S. Lind (University of Uppsala, Sweden), remains to be demonstrated. In addition to providing insight into stepwise disassembly during entry, a high-resolution structure may also determine the arrangement of the hyper-variable (HV) loops of hexon. These HV loops are recognized by neutralizing antibodies and blood factors and influence the residence time of the cell-free virus in an organism, for example in the blood system of the mouse (Waddington et al., 2008). Work by A. Baker and colleagues (University of Glasgow, UK) showed that coagulation factor X (FX) is important for liver tropism of blood-exposed HAdV-5 in rodents. FX binds to the HAdV-5 hexon, not fibre, via an interaction between the FX Gla domain and HV loops of the hexon surface leading to heparansulfate–proteoglycan engagement. FX binding influences multiple HAdV types, but not canine type 2 (CAdV-2) (A. Bradshaw, University of Glasgow, UK). Baker suggested that liver gene transfer is mediated through a heparin-binding exo-site in the FX serine protease domain. A. Byrnes (Food and Drug Administration, Silver Spring, USA) showed that when the HV loops of hexon were genetically altered such that blood factors could no longer bind, these altered viruses were recognized by natural antibodies and the mouse complement system and subsequently degraded. It is presently unknown if adenovirus types, which use different receptors than CAR use the same cues to expose their protein VI as HAdV-2/5, or other mechanisms. Towards answering this question, receptor identification is a crucial step. U. Greber (University of Zurich, Switzerland) presented evidence that HAdV-3/7 (species HAdV-B) use the membrane cofactor CD46 as an attachment receptor, provided that CD46 is abundantly present to allow avidity binding to the fibre proteins of the virus (Trinh et al., 2012). A. Lieber (University of Washington, Seattle, USA) extended these studies and showed that HAdV-3 also uses human desmoglein 2 (DSG2) as a receptor (Wang et al., 2011b). DSG2 is localized on the basal and lateral membrane domains of polarized epithelial cells and involved in cell adhesion. A recombinant, self-dimerizing knob of the HAdV-3 fibre bound to DSG2 and inhibited infection (Wang et al., 2011a). Lieber also presented a transgenic mouse model expressing human DSG2, and suggested that the HAdV-3 fibre knob can be used to open the junctions between epithelial cells, and thereby enhance the therapeutic efficacy of adenovirus vectors in transducing epithelial cells, and possibly cancer targeting therapeutic antibodies (Wang et al., 2012). Besides CAR, CD46 and DSG2, there may be additional receptors for particular HAdV types, such as glycoproteins or glycolipids carrying sialic acid residues (Nilsson et al., 2011), as discussed by R. Storm (University of Umea, Sweden), or other soluble components that provide a link between the virus and the cells, as discussed by M. Nygren (University of Umea, Sweden). The biological relevance of these low affinity receptors remains to be determined. Efforts at the University of Umea are under way to develop sialic acid derivatives as anti-virals against adenoviruses causing epidemic keratoconjunctivitis, as pointed out by N. Arnberg (University of Umea, Sweden). Another central question has been how some viruses infect polarized cells, as these cells can be both gates and barriers to hosts. Paradoxically, many virus receptors are not present at the apical plasma membrane facing towards the outside. K. Excoffon (Wright State University, USA) using transfection and overexpression studies suggested that a low abundant splice variant of CAR is targeted to the apical domain of polarized canine MDCK cells, and this enhanced HAdV-5 infection from the apical side (Kolawole et al., 2012). Only a fraction of CAR is apically expressed, but this small fraction can be reduced by overexpression of MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and can be enhanced by MAGI-1 downregulation. It will be interesting to see if MAGI-1 is suppressed by cytokines, such as interleukin (IL)-8, which increase apical CAR levels in co-cultures of human epithelial cells and macrophages and boost apical infection with HAdV-2/5 (Lutschg et al., 2011). It will also be interesting to analyse the trafficking routes that direct CAR to the apical plasma membrane domain in epithelial cells. S. Salinas (Institut de Génétique Moléculaire de Montpellier, France) discussed CAR trafficking in neuronal cells. Her results suggested that CAR engagement by CAdV-2 or its fibre knob induced virus trafficking from Rab5-positive to neutrally charged lumens of Rab7-positive vesicles in axons (Salinas et al., 2009). This implies that virus-induced internalization of CAR can lead to CAR and virus cotransport. CAdV-2 appears to escape in the neuronal cell body while CAR is degraded. Work from C. Wiethoff (Loyola University, Maywood, USA) suggested that the rupture of endosomes by incoming HAdV-5 in epithelial cells elicits danger signals (Barlan et al., 2011). Wiethoff also suggested that the membrane lytic protein VI is required for the dissociation of the virus from broken endosomes, by virtue of its PPxY motive, and interactions with ubiquitin ligases. A mutant HAdV-5 lacking PPxY appeared to be cleared by autophagy. Wiethoff reported that the knock-down of the autophagy-related protein ATG5 fully restored the infectivity of HAdV-5 lacking the PPxY motive, suggesting that the PPxY motive had no additional functions for infection besides evasion of autophagy during cell entry. H. Wodrich (University of Bordeaux, France), however, suggested that the PPxY motive of the incoming protein VI was also required to release host-mediated repression of adenoviral early gene expression (Schreiner et al., 2012). This was in some disagreement with the presentation of M. Suomalainen who reported that incoming protein VI was degraded within 2–3 h post infection, that is, before or at the onset of early viral gene expression. The latter was also in agreement with earlier reports (Greber et al., 1993; Burckhardt et al., 2011). Based on protein VI overexpression studies Wodrich et al. also suggested that protein VI displaces Daxx (death domain-associated protein) from PML-NBs, and that this was dependent on the PPxY motive, which facilitated interaction with Nedd4 ubiquitin ligases (Schreiner et al., 2012). Surprisingly, the repression of Daxx by protein VI and activation of adenoviral early promoters could be mimicked by structural proteins from unrelated human viruses, such as human cytomegalovirus (HCMV) or papillomavirus, and protein VI overexpression activated the early HCMV promoter. Wodrich suggested that capsid proteins contain trans-activating properties for viral gene expression. Whether these properties function during entry or replication, or if they work at high or low copy numbers of viral genomes remains to be determined. Regardless of the mechanism, further studies will be needed to explore how the trafficking of viral genomes relates to the way the genomes are detected by the cellular surveillance systems. An important mechanism of host interference with viral infection is transcriptional repression, as emphasized in the presentation of S. Schreiner (Heinrich Pette Institute, Hamburg, Germany). The Daxx protein located in promyelocytic leukaemia nuclear bodies (PML-NBs) may be a negative regulator of adenovirus gene expression at low multiplicity of infection (Schreiner et al., 2010). Schreiner et al. suggested that Daxx/ATRX (X-linked α-thalassemia mental retardation syndrome protein) and SWI/SNF (SWItch/Sucrose NonFermentable) chromatin remodelling complexes play a role in regulating viral gene expression. ATRX acts as the core ATPase subunit, while Daxx recruits histone deacetylase, and leads to transcriptional repression of viral promoters (Schreiner et al., 2012). The early viral E1B-55K protein inhibits Daxx functions, and targets ATRX for proteasome degradation depending on the cullin E3-ubiquitin ligase, thereby counteracting host cell chromatin remodelling functions (Schreiner et al., 2011). It is currently unknown if the impact of E1B-55K on the transcriptome profile is comparable to the large-scale epigenetic impact of the immediate early transcriptional regulator E1A (Ferrari et al., 2008; Horwitz et al., 2008), and how E1B-55K affects viral replication. Adenoviral replication is a high fidelity process, in part due to the proof-reading features of the viral DNA polymerase. R. Hoeben et al. (Leiden University Medical Center, The Netherlands) presented an elegant series of experiments to make the viral DNA more by some > and to some in the binding and the domain of the and the in a cell viruses were and for such as virus with enhanced in the splice the adenovirus protein, and induced which was as A clinical could be better Another important of genome is G. (University of Uppsala, showed data to that the viral splice protein which splicing of the viral is by protein kinase and protein kinase A et al., 2012). by inhibits while by This mechanism suggested new for and in Two are currently in the a capsid model a ATPase the viral DNA into a capsid and a model by the properties of capsid proteins. The work presented by W. National USA) the that of the viral protease the AdV genome the protease to the capsid and thereby the capsid and core proteins. a process of the protease within the which is in DNA and It will be important to determine how the protease the viral or if the protease on the viruses the cell by a lytic process. and experiments were presented by A. (University of Zurich, Switzerland) to that of expressing HAdV-2 by transfer of viruses which and transfer et al., 2012). This implies that the of lytic cell-free to viral infection between cells can be by or of the virus in and viral can be in this were mainly reported on virus interactions with cells, viral host and genetic analyses of host factors in M. (University of at USA) presented a analysis of the interaction of HAdV-2 with the major complex for antigen degradation of proteins by the proteasome or proteins by and present these to T cells at the cell discussed how HAdV-2 antigen presentation to T cells. et al. (Institut de Génétique Moléculaire de Montpellier, France) the question how adenovirus complexes the of cells in particular the maturation of immature It was that mediated knock-down of acid receptors reduced while knock-down of the enhanced knock-down of receptor or gene protein reduced factor alpha This suggested that HAdV complexes maturation by with and thereby link and to (Loyola University and USA) reported interesting of A function of is to of macrophages and It is known that cells develop which is more to than the appeared to from for example which is of nuclear factor suggested that clinical of (species which syndrome et al., protein than and to dependent experiments further showed that the on the viral early protein which is a of the appears to on and these may macrophages of the cell biology of are to more of the of AdV It will also be interesting to see if the of (University of UK) a form of an protein of from the (species appears to be unique to species and binds to cells of the but not or epithelial cells. studies suggested that of cells, and may to A mouse approach to host involved in AdV was by K. 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