A Cell-Based Examination of Modulators of Pre-Membrane Processing As a Target against Dengue Virus
Dengue Virus (DenV) is an arbovirus that represents a budding risk in the world. Every year, up to 100 million DenV infections manifest into Dengue Fever or in extreme cases Dengue Hemorrhagic Fever and Dengue Shock Syndrome. DenV research in vaccine development has proven to be a difficult feat due to the phenomenon of antibody dependent enhancement. Furthermore, there are currently no available antivirals to fight infection, viral protein processing or viral production. DenV lifecycle begins with its genomic release in the cytoplasm, where it is then translated as a single polypeptide embedded in the Endoplasmic Reticulum (ER) membrane. DenV, like so many other viruses, exploits a range of host enzymes in the Classical Secretory Pathway (CSP) for modifications. Among these important host enzymes are proteases such as the family of Proprotein Convertases (PCs), including furin. The modulation of the pre- membrane (pr-M) protein, most likely by PCs, is a critical step in the DenV lifecycle as an absence results in noninfectious progeny. Interestingly, the maturation of pr-M by the host enzymes during infection has been characterized as incomplete, thus rendering some viral particles noninfectious. Thus, the inhibition of pr-M cleavage presents an attractive target for potential antivirals. This assay is based on a fusion that contains an ER targeting signal sequence, the substrate of significance with flanking FLAG and HA epitopes, and a transmembrane (TM) domain. The assay in the context of the pr-M boundary has shown robust transportation with a wild phenotype in both transient and stable cell expression using retroviral technology. The original pr-M substrate included only 20 aa of the substrate boundary. Here, I have designed different substrate boundaries of pr-M to monitor important motifs in enzyme recognition and secretion, hypothesizing that by adapting to larger segments, we will have a powerful platform for the discovery of competitive inhibitors rather than inhibitors of the enzyme.
Aguiar, M., Stollenwerk, N., and Halstead, S. (2016). The impact of the newly licensed Dengue vaccine in endemic countries. PLOS Neglected Tropical Diseases, 10, 1–23. https://doi.org/10.1371/journal.pntd.0005179
Bardina, S. V, Bunduc, P., Tripathi, S., Duehr, J., Frere, J. J., Brown, J. A., Nachbagauer, R., Foster, G.A., Krysztof, D., and Tortorella, D. (2017). Enhancement of Zika virus pathogenesis by pre-existing anti-flavivirus immunity. Science, 356, 1-6. https://doi.org/10.1126/science.aal4365
Barlowe, C. K., and Miller, E. A. (2013). Secretory protein biogenesis and traffic in the early secretory pathway. Genetics, 193, 383–410. https://doi.org/10.1534/genetics.112.142810
Bonifacino, J. S., and Lippincott-Schwartz, J. (2003). Coat proteins: shaping membrane transport. Nature Reviews Molecular Cell Biology, 4, 409–414. https://doi.org/10.1038/nrm1099
Boonnak, K., Slike, B. M., Burgess, T. H., Mason, R. M., Wu, S.-J., Sun, P., Porter, K., Rudiman, I. F., Yuwono, D., Puthavathana, P., et al. (2008). Role of dendritic cells in antibody-dependent enhancement of Dengue virus infection. Journal of Virology, 82, 3939–3951. https://doi.org/10.1128/JVI.02484-07
Byk, L. A. , Iglesias, N. G., De Maio, F. A., Gebhard, L. G., Rossi, M., andGmarnik, A. V. (2016). Dengue virus genome uncoating requires ubiquitination. MBio, 7, 1–10. https://doi.org/10.1128/mBio.00804-16.Editor
Chauhan J. S., Rao, A., and Raghava G. P. S. (2013). In silico platform for prediction of N-, O- and C-Glycosites in eukaryotic protein sequences. PLoS ONE, 8, 1-10.
Dahms, S. O., Arciniega, M., Steinmetzer, T., Huber, R., and Then, M. E. (2016). Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism. Proceedings of the National Academy of Sciences U.S.A., 113, 11196-11201. https://doi.org/10.1073/pnas.1613630113
Fluhrer, R., Steiner, H., and Haass, C. (2009). Intramembrane proteolysis by signal peptide peptidases: a comparative discussion of GXGD-type aspartyl proteases. Journal of Biological Chemistry, 284, 13975–13979. https://doi.org/10.1074/jbc.R800040200
Freire, J. M., Santos, N. C., Veiga, A. S., Da Poian, A. T., and Castanho, M. A. (2015).
Rethinking the capsid proteins of enveloped viruses: multifunctionality from genome packaging to genome transfection. FEBS J, 282, 2267–2278. https://doi.org/10.1111/febs.13274
Freire, J. M., Veiga, A. S., Conceição, T. M., Kowalczyk, W., Mohana-Borges, R., Andreu, D., Santos, N.C., Da Poian, A.T., and Castanho, M.A.R.B. (2013). Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein. PLoS ONE, 8, 1-10. https://doi.org/10.1371/journal.pone.0081450
Grief, C., Galler, R., Côrtes, L. M. C., and Barth, O. M. (1997). Intracellular localisation of Dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation. Archives of Virology, 142, 2347–2357. https://doi.org/10.1007/s007050050247
Gubler, D. J., and Kuno, G. (1998). Dengue and Dengue Hemorrhagic Fever. Clinical Microbiology Reviews, 11, 480–496.
Gyawali, N., Bradbury, R. S., and Taylor-Robinson, A. W. (2016). The epidemiology of dengue infection: harnessing past experience and current knowledge to support implementation of future control strategies. Journal of Vector Borne Diseases, 53, 293–304. http://www.ncbi.nlm.nih.gov/pubmed/28035105
Hsieh, S. C., Liu, I. J., King, C. C., Chang, G. J., and Wang, W. K. (2008). A strong endoplasmic reticulum retention signal in the stem-anchor region of envelope glycoprotein of Dengue virus type 2 affects the production of virus-like particles. Virology, 374, 338–350. https://doi.org/10.1016/j.virol.2007.12.041
Iglesias, N. G., Mondotte, J. A., Byk, L. A., De Maio, F. A., Samsa, M. M., Alvarez, C. and Gamarnik, A. V. (2015). Dengue virus uses a non-canonical function of the host GBF1-Arf-COPI system for capsid protein accumulation on lipid droplets. Traffic, 16, 962-977.
Kantanen, M. L., Leinikki, P., and Kuismanen, E. (1995). Endoproteolytic cleavage of HIV-1 gp160 envelope precursor occurs after exit from the trans-Golgi network (TGN).
Archives of Virology, 140, 1441–1449. https://doi.org/10.1007/BF01322670
Lee, M. C. S., Miller, E. A., Goldberg, J., Orci, L., and Schekman, R. (2004). Bi-directional protein transport between the ER and Golgi. Annual Review of Cell and Developmental Biology, 20, 87–123. https://doi.org/10.1146/annurev.cellbio.20.010403.105307
Li, L., Lok, S., Yu, I., Zhang, Y., Kuhn, R. J., Chen, J., and Rossmann, M. G. (2014). The
flavivirus precursor complex : Protein membrane-envelope structure and maturation,
Science, 319, 1830–1834. https://doi.org/10.1126/science.1153263
Liu, W. J., Sedlak, P. L., Kondratieva, N., and Khromykh, A. A. (2002). Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of NS3 in cis for virus assembly. Journal of Virology, 76, 10766–10775. https://doi.org/10.1128/JVI.76.21.10766-10775.2002
Lorenz, I. C., Kartenbeck, J., Mezzacasa, A., Allison, S. L., Heinz, F. X., and Helenius, A. (2003). Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus. Journal of Virology, 77, 4370–4382. https://doi.org/10.1128/JVI.77.7.4370-4382.2003
Mackenzie, J. (2005). Wrapping things up about virus RNA replication. Traffic 6, 967–977. https://doi.org/10.1111/j.1600-0854.2005.00339.x
Mackenzie, J.M., Jones, M. K., and Young, P. R. (1996). Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication.
Virology, 220, 232–240. https://doi.org/10.1006/viro.1996.0307
Mackenzie, J. M., Khromykh, A. A., Jones, M. K., and Westaway, E. G. (1998). Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology, 245, 203–215. https://doi.org/10.1006/viro.1998.9156
Mandon, E. C., Trueman, S. F., and Gilmore, R. (2013). Protein translocation across the rough endoplasmic reticulum. Cold Spring Harbor Perspectives in Biology, 5, 1-14.
Nowak, T., and Wengler, G. (1987). Analysis of disulfides present in the membrane proteins of the West Nile flavivirus. Virology, 156, 127–137. https://doi.org/10.1016/0042- 6822(87)90443-0
Oliveira, E. R., Mohana-Borges, R., de Alencastro, R. B., and Horta, B. A. (2017). The flavivirus capsid protein: structure, function and perspectives towards drug design. Virus Research, 227, 115–123. https://doi.org/10.1016/j.virusres.2016.10.005
Orci, L., Stamnes, M., Ravazzola, M., Amherdt, M., Perrelet, A., Söllner, T. H., and Rothman, J. E. (1997). Bidirectional transport by distinct populations of COPI-coated vesicles. Cell, 90, 335–349. https://doi.org/10.1016/S0092-8674(00)80341-4
Perera, R., and Kuhn, R. J. (2008). Structural proteomics of Dengue virus. Current Opinion in Microbiology, 11, 369–377. https://doi.org/10.1016/j.mib.2008.06.004
Plevka, P., Battisti, A. J., Junjhon, J., Winkler, D.C., Holdaway, H. A, Keelapang, P. Sittisombut, N., Kuhn, R. J., Steven, A. C., and Rossman, M. G. (2011). Maturation
of flaviviruses starts from one or more icosahedrally independent nucleation centres.
EMBO Reports, 12, 602–606. https://doi.org/10.1038/embor.2011.75
Rodenhuis-Zybert, I. A., van der Schaar, H. M., da silva Voorham, J. M., van der Ende- Metselaar, H., Lei, H. Y., Wilschut, J., and Smit, J. M. (2010). Immature Dengue virus: A veiled pathogen? PLoS Pathogens, 6, 1-9. https://doi.org/10.1371/journal.ppat.1000718
Rodenhuis-Zybert, I. A., Wilschut, J., and Smit, J. M. (2010). Dengue virus life cycle: viral and host factors modulating infectivity. Cellular and Molecular Life Sciences, 67, 2773–2786. https://doi.org/10.1007/s00018-010-0357-z
Samsa, M. M., Mondotte, J. A., Caramelo, J. J., and Gamarnik, A. V. (2012). Uncoupling cis-acting RNA elements from coding sequences revealed a requirement of the N-
Terminal region of Dengue virus capsid protein in virus particle formation. Journal of Virology, 86, 1046–1058. https://doi.org/10.1128/JVI.05431-11
Samsa, M. M., Mondotte, J. A., Iglesias, N. G., Assunção-Miranda, I., Barbosa-Lima, G., Da Poian, A. T., Bozza, P. T., and Gamarnik, A. V. (2009). Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathogens, 5, 1-14. https://doi.org/10.1371/journal.ppat.1000632
Zhou, A., Webb, G., Zhu, X., and Steiner, D. F. (1999). Proteolytic processing in the secretory pathway. Journal of Biological Chemistry, 274, 20745–20748. https://doi.org/10.1074/jbc.274.30.20745
Zybert, I. A., van der Ende-Metselaar, H., Wilschut, J., and Smit, J. M. (2008). Functional importance of Dengue virus maturation: infectious properties of immature virions. The Journal of General Virology, 89, 3047–3051. https://doi.org/10.1099/vir.0.2008/002535-0
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