* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Redox rescues virus from eR trap - Institute of Molecular Life Sciences
Survey
Document related concepts
Cell nucleus wikipedia , lookup
Extracellular matrix wikipedia , lookup
Cell membrane wikipedia , lookup
Cell culture wikipedia , lookup
Cell growth wikipedia , lookup
Cellular differentiation wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Cytokinesis wikipedia , lookup
Endomembrane system wikipedia , lookup
Transcript
news and views The authors have investigated the epigenetic memory of an active state and propose a role for H3.3 in this process. How the epigenetic memory of silent chromatin is handled will be the next item on the agenda, and is likely to be equally important for reprogramming efficiency. Does memory of silent states occur as a default mechanism in the absence of H3.3 simply because no transcription priming event can occur? It may thus be important to further analyse the links between replication and transcription reprogramming. In this context, it would be interesting to examine whether the high replication reprogramming promoted by mitotic extracts12 is paralleled by a higher incorporation of replicative H3. With the cellular systems now available13,14,15, analysis of the dynamics of histone H3 variants will surely be important to fully understand the network of interactions and find ways to manipulate the system. Although the study by Ng and Gurdon raises several questions, it reinforces the importance of histones as a possible means of conveying epigenetic information. These advances, combined with other reprogramming techniques such as cellular fusions13 or recently developed cellular models in which a limited set of factors is expressed in somatic cells14,15, should extend our basic understanding of epigenetics and open avenues for the treatment of diseases such as cancer, and for the development of cell replacement therapies. 1. Ng, R. K. & Gurdon, J. B. Nature Cell Biol. 10, 102– 109 (2008). 2. Tsunoda, Y. & Kato, Y. Differentiation 69, 158–161 (2002). 3. Gurdon, J. B. J Embryol. ������������� Exp. Morphol. 10, 622–640 (1962). 4. Santos, F. et al. Curr Biol.13, 1116–1121 (2003). 5. Ng, R. K. & Gurdon, J. B. Proc. Natl Acad. Sci. USA 102, 1957–1962 (2005). 6. Sarma, K. & Reinberg, D. Nature Rev. Mol. ����������� Cell. Biol. 6, 139–149 (2005). 7. Loyola, A. & Almouzni, G. Trends Biochem. Sci. ���� 32, 425–433 (2007). 8. van der Heijden, G. W. et al. Mech. Dev. 122, 1008– 1022 (2005). 9. Yang, X. et al. Nature Genet. 39, 295–302 (2007). 10.Klose, R. J. & Zhang, Y. Nature Rev. Mol. Cell. Biol. 8, 307–318 (2007). 11.Wysocka, J. et al. Cell 121, 859–872 (2005). 12.Lemaitre, J. M., Danis, E., Pasero, P., Vassetzky, Y. & Mechali, M. Cell 123, 787–801 (2005). 13.Blau, H. M., Chiu, C. P. & Webster, C. Cell 32, 1171– 1180 (1983). 14.Meissner, A., Wernig, M. & Jaenisch, R. Nature Biotechnol. 25, 1177–1181 (2007). 15.Takahashi, K. et al. Cell 131, 861–872 (2007). Redox rescues virus from ER trap Christoph J. Burckhardt and Urs F. Greber How viruses manage to resist physical and chemical stress and yet open their protective coats during cell infection has been a longstanding, fundamental question. A study with the DNA tumour virus SV40 now shows that protein folding and quality-control factors of the endoplasmic reticulum reshuffle disulfide bonds within the viral capsid, providing a molecular mechanism for the exit of infectious virions from the endoplasmic reticulum. The concept of a viral replication cycle is exceedingly simple, yet its mechanisms hold many secrets. Upon engulfment of a virus by the host cell, uncoating of the viral genome from the protective capsid is required for the activation of viral genes, using the transcriptional machinery of the host cell to drive synthesis of viral progeny. Newly formed particles exit the cell, infect new cells, and the cycle starts again. On their way into a cell, animal viruses pass through cellular compartments where they encounter triggers that initiate viral coat destabilization. Examples of destabilization events include virus–receptor interactions, shedding of minor capsid proteins, or the low pH in the digestive tract or in endosomal vesicles activating viral or cellular proteases that catalyse limited proteolysis1. In the past, therapeutic Christoph J. Burckhardt & Urs F. Greber are at the Institute of Zoology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. email: [email protected] inhibition of uncoating has been a prominent strategy in the development of anti-viral drugs, particularly for non-enveloped viruses, such as picornaviruses2. Although in principle successful, the strategy has been largely abandoned due to the rapid appearance of viral escape mutants in patients treated with such antiviral drugs. Development of therapeutics directed against the host may be an interesting alternative. A recent paper by Schelhaas et al.3 reveals new host functions required for the infectious entry of the polyoma virus Simian Virus 40 (SV40). Polyoma viruses are non-enveloped DNA viruses that cause cancer in rodents, and traces of their genome can be found in human tumours. Notably, SV40 was accidentally inoculated into millions of humans during vaccinations with chemically inactivated poliovirus in the 1950s when the vaccine virus was produced in SV40-infected monkey cells. The pas���� sage of SV40, mouse polyomavirus (mPy) and human BK virus into cells is intriguing, given that these viruses use glycolipids as their cell nature cell biology volume 10 | number 1 | JANUARY 2008 © 2008 Nature Publishing Group attachment sites4. Polyoma viruses are assembled in the nucleus under reducing conditions, and their capsids become oxidized outside the cell. The spatial separation of viral assembly and disassembly explains in part why a virus can be stably assembled in an infected cell and disassembled on entry into a naïve cell5. The work by Schelhaas et al.3 shows how a virus uses the protein folding and quality-control apparatus for uncoating and membrane translocation. It provides a molecular explanation for two long-standing observations, namely that the incoming SV40 is found in large amounts inside the ER6 and that infectious particles are present in the cytosol7. The SV40 capsid has icosahedral symmetry and is composed of 72 homopentamers of the major capsid protein VP1. Twelve pentamers are five-coordinated, the others are six-coordinated. It is unusually structured because the VP1 proteins are linked together by a network of interchain Cys9–Cys9 and Cys104–Cys104 disulfide bonds (Fig. 1).The carboxy-terminal news and views a ERp57 Caveolar endocytosis ERAD PDI Derlin-1 Sel1L Ca2+ high ER b Cys 104 Cys9 Cys9 Cys 104 Cys104 ERp57 Cys104 Cytosol Ca2+ low Cys104 Cys9 Cys9 Cys104 Figure 1 ERp57 and low calcium are involved in SV40 pentamer dissociation. (a) Infectious entry of SV40 into human and monkey cells occurs via caveolin- and lipid raft-dependent endocytosis on binding to the GM1 ganglioside receptors at the plasma membrane13,14. From the caveosome, the viruses are transported inside vesicles along microtubules to the ER where they accumulate 15. A cryo-electron microscopy structure of intact SV40 capsids is shown in blue; reduced and calciumdepleted particles that released the pentavalent pentamers of the major capsid protein VP1 are shown in yellow3. The ER thiol-disulfide oxidoreductase ERp57 isomerises SV40 interchain disulfide bonds. PDI, Derlin-1 and Sel1L — three proteins involved in ER-associated degradation (ERAD) — are then thought to retrotranslocate particles to the cytosol, where the low calcium concentration leads to pentamer dissociation. Cytosolic particles then enter the nucleus via the nuclear pore complex, where viral transcription and replication take place. (b) Model of SV40 disulfide isomerisation mediated by ERp57. Individual monomers in a five-coordinated pentamer (blue) of the major capsid protein VP1 are connected to neighbouring six-coordinated pentamers (orange and green) by interchain Cys9–Cys9 and Cys104–Cys104 disulfide bonds (possible bonds are displayed by dashed lines). During disulfide bond isomerisation, intrachain Cys9–Cys104 disulfide bonds are formed in the five-coordinated pentamer of the VP1 molecule whereas the six-coordinated pentamers are connected by Cys104– Cys104 disulfide bonds. This isomerisation reaction uncouples the five-coordinated pentamer from the disulfide bond network of the virus capsid, which may provide a signal for recognition of the particle by the ERAD machinery for translocation to the cytosol. peptide of VP1 extends to binding sites on VP1 molecules of neighbouring pentamers that are stabilized by calcium ions8. In the life cycle of polyoma viruses, progeny capsids are assembled in the nucleus under reducing conditions and oxidized outside the cell. They do not come in contact with the enzymatic redox system in the ER; the site of viral uncoating. The initial trigger for the research by Schelhaas et al. was the observation that in SDS polyacrylamide gels, treatment of nonreduced virus with alkylating agents yields VP1 multimers indicative of intersubunit disulfide bonds. These are not observed in the absence of alkylation, where free cysteines are available for reshuffling the interchain disulfides of VP1. Moreover, alkylation of virus blocked 10 infection and alkylated virus did not release VP1, indicative of a requirement for disulfide isomerisation rather than disulfide reduction for productive infection. Depletion of the two ER thiol–disulfide oxidoreductases, ERp57 and the closely related protein disulfide isomerase (PDI), by small-interfering RNA (siRNA) led to a significant reduction of infection, but only the depletion of ERp57 inhibited VP1 release in vivo, indicating that only ERp57 resolves the interchain disulfide bonds. ERp57 is an essential soluble protein of the ER, where it catalyses disulfide reduction, isomerization and dithiol oxidation in substrate proteins. These findings are complemented by an in vitro assay where both purified ERp57 and PDI were able to induce the loss of VP1 on isolated viruses. To determine whether ERp57 and PDI serve as isomerases or reductases, alkylated (non-isomerizing) virus was used. Following treatment with ERp57, alkylated SV40 capsids did not release VP1 monomers although PDI was able to release VP1, suggesting that ERp57 functions as an isomerase and PDI as a reductase. However, native SV40 released VP1 following ERp57 treatment, supporting the siRNA knockdown data. The disulfide isomerization activity of ERp57 is needed to resolve interchain disulfide bonds of the pentamers, yielding the intrachain disulfide bond Cys9–Cys104 (Fig. 1). Nevertheless, isomerization of disulfide bonds is not sufficient for the dissociation of the pentamers; the loss of the VP1-associated Ca2+ ions is also required. Cryo-electron microscopy of partially uncoated viruses revealed that it was the five-coordinated pentamers that were released, suggesting that they are less tightly associated than the six-coordinated pentamers. Interestingly, the Cys9–Cys104 disulfide bond is already in place in native mouse polyoma virus VP1, which has no interchain disulfide bonds and hence does not require the Erp57 activity for infection9. The conformational changes around the disulfides of SV40 VP1 weaken the capsid such that the myristoylated amino-terminal region of VP2 may swing out, and potentially positions the capsid to the lumenal face of the ER membrane. Electron micrographs suggest that SV40 is proximal to the lumenal ER membrane6. A similar principle of conformational change is used by mPy, where in vitro experiments have shown that the thioredoxin-like ERp29 is required for membrane association of the virion9. But how is a nucleo-protein complex of 50 nm in diameter translocated through the ER membrane? The observation that proteasome inhibitors block SV40 entry suggests that ER-associated degradation (ERAD) may be involved 3. ERAD requires chaperones that recognize misfolded proteins and the retrotranslocation machinery10. Cholera toxin, for example, requires reduced PDI for unfolding and presentation to the ERAD machinery. Accordingly, knockdown of PDI, Derlin-1 and Sel1L reduced SV40 infection, suggesting a role of ERAD in SV40 retrotranslocation. Derlin-1 is a putative pore-forming component, whereas Sel1L may be required for substrate recognition. The sensor that recognizes the disulfidereshuffled virus in the ER has not been identified, but may involve PDI or the signal nature cell biology volume 10 | number 1 | JANUARY 2008 © 2008 Nature Publishing Group news and views peptide peptidase, perhaps recognizing VP2 (ref. 10). This sensor may target the virus to the retrotranslocation complex. The rather low efficiency of SV40 release from the ER into the cytosol (estimated to be 1%) may explain why it remains unclear whether the translocated virus particles are ubiquitinated, a modification that normally occurs on proteins that are targeted to ERAD. Nevertheless, the translocated virus with isomerized disulfides sheds the VP1 pentamers in the low-calcium conditions of the cytosol. This poises the particle for the release of infectious DNA into the nucleus, as shown for other DNA viruses11. One can speculate that the co-option of the ERAD machinery by SV40 occurs in conjunction with biosynthetic functions of the ER membranes, such as lipid synthesis or lipid droplet formation12. Viral escape from the ER is, however, reminiscent of viral gene products that co-opt the ERAD machinery to induce rapid degradation of host receptors or of immune surveillance molecules, such as major histocompatibility complex (MHC) class I proteins10. The ER redox system contains more than fifteen different disulfide isomerases; other viruses, such as the papilloma viruses or picornaviruses, that visit the ER or interfere with ER functions, may use a similar pathway as SV40, perhaps by engaging different isomerases. Future studies will uncover additional host factors, and these may be useful targets for anti-viral therapies as they are not subject to viral resistance mutations. 1. Guglielmi, K. M., Johnson, E. M., Stehle, T. & Dermody, T. S. Curr. Top. ���������������� Microbiol. Immunol. �������� 309, 1–38 (2006). 2. Li, C. et al. Curr. Med. Chem. 14, 847–856 (2007). 3. Schelhaas, M. et al. Cell 131, 516–529 (2007). 4. White, M. K. & Khalili, K. Virology 324, 1–16 (2004). 5. Greber, U. F., Singh, I. & Helenius, A. Trends Microbiol. 2, 52–56 (1994). 6. Kartenbeck, J., Stukenbrok, H. & Helenius, A. J. Cell Biol. 109, 2721–2729 (1989). 7. Greber, U. F. & Kasamatsu, H. Trends Cell Biol. 6, 189–195 (1996). 8. Stehle, T., Gamblin, S. J., Yan, Y. & Harrison, S. C. Structure 4, 165–182 (1996). 9. Magnuson, B. et al. Mol. Cell 20, 289–300 (2005). 10.Loureiro, J. & Ploegh, H. L. Adv. Immunol. 92, 225– 305 (2006). 11.Greber, U. F. & Way, M. Cell 124, 741–754 (2006). 12.Ploegh, H. L. Nature 448, 435–438 (2007). 13.Tsai, B. et al. Embo J. 22, 4346–4355 (2003). 14.Smith, A. E., Lilie, H. & Helenius, A. FEBS Lett. 555, 199–203 (2003). 15.Pelkmans, L., Kartenbeck, J. & Helenius, A. Nature Cell Biol. 3, 473–483 (2001). Cilia put a brake on Wnt signalling Xi He Two studies suggest that the primary cilium, a microtubule-based structure protruding from the surface of most vertebrate cells, has a role in restraining Wnt/β-catenin signalling. These findings have implications for the pathogenesis of a plethora of diseases associated with abnormal cilia; however, the mechanism linking Wnt signalling and cilia remains a mystery. Signalling by the Wnt family of secreted growth factors is important in cell proliferation and differentiation during development1,2. Wnt proteins engage multiple receptors and activate a number of distinct signalling pathways3,4. These include the canonical Wnt/β-catenin pathway, which controls stability of the transcription coactivator β-catenin (and therefore gene expression)1–3, and the non-canonical Wnt/planar cell polarity (PCP) pathway, which primarily governs cytoskeletal architectures associated with cell polarity and movement4. Defects in Wnt signalling pathways are associated with human cancers and other diseases1,2. The primary cilium consists of a microtubule-based axoneme enclosed by a specialized plasma membrane5,6. The cilium protrudes into the extracellular space and is tethered to the cell by the basal body, a centriolar-based Xi He is at the F. M. Kirby Neurobiology Center, Children’s Hospital Boston, Harvard Medical School, Enders 461.2, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. e-mail: [email protected] structure that forms the interface between the cilium and the cytoplasm5,6 (Fig. 1). The formation and function of the cilium requires intraflagellar transport (IFT), which shuttles molecules in and out of the cilium via IFT proteins and microtubule-based motor complexes, including Kinesin-2 and Dynein5,6. The cilium proteome consists of hundreds to a thousand polypeptides5, reflecting diverse cilia functions that include cell motility, chemo- and mechano-sensing, and signal transduction5,6. Many human diseases, collectively referred to as ciliopathies, are associated with defective cilia formation and/or function, such as polycystic kidney disease (PKD), nephronophthisis, primary cilia dyskinesia, situs inversus (reversal of left-right asymmetry), and Bardet-Biedl Syndrome (BBS)5,6. A critical role of the cilium in cell signalling was discovered in the study of neural patterning by Sonic Hedgehog in mice7. The role of cilia in Hedgehog signalling is unique to vertebrates. Cilia are present in most vertebrate cells, whereas in Drosophila Melanogaster, nature cell biology volume 10 | number 1 | JANUARY 2008 © 2008 Nature Publishing Group only sperm and sensory neurons exhibit cilia. Hedgehog signalling therefore occurs independently of cilia in flies, supporting the idea that Hedgehog signalling uses both common and distinct mechanisms in Drosophila and vertebrates7. By comparison, the Wnt signalling pathway is highly conserved between flies and vertebrates1–4. Given that cilia cannot have a widespread role in Drosophila, the findings by Katsanis and colleagues and Reiter and colleagues that cilia restrain Wnt/β-catenin signalling in vertebrates is surprising8, 9. Earlier studies have implicated ciliary proteins in Wnt and PCP signalling10,11. Inversin (Inv), mutations of which cause nephronophthisis characterized by renal cysts and situs inversus, was found to inhibit Wnt/β-catenin signalling10. However, Inv protein localization to other cellular compartments in addition to cilia complicates the interpretation that its ciliary role is directly implicated in the control of Wnt signalling. Inv is also required for Xenopus gastrulation10, a process governed by Wnt/PCP signalling4. Inv binds to and promotes the degradation of cytosolic, 11