I. GENETICALLY ENGINEERED RECOMBINANT AND CHIMERIC MEASLES VIRUSES.
II. SORTING OF MEMBRANE PROTEINS IN POLARIZED CELLS.
Hussein Y. Naim
Institut für Molekularbiologie
phone: +41-1-63 53112
Hussein Naim moved to the biotech company
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The long-term goal of my research focuses on: (i) construction of novel chimera and multivalent live vaccine vectors, based on the Edmonston B vaccine strain of measles virus. (ii) To understand the mechanisms of protein sorting in general, and specifically to understand assembly of measles virus in polarized epithelial cells and neurons.
I. GENETICALLY ENGINEERED RECOMBINANT AND CHIMERIC MEASLES VIRUSES.
Chimeric measles viruses combine the replication machinery
of one virus with the envelope of another. These viruses may be useful
tools for a variety of purposes. For basic research, these viruses could
be useful in studying virus structure and function and interactions with
the host, in particular with the immune system. In addition, they could
be used as models to develop novel monovalent live vaccines and gene delivery
After the establishment of the rescue of Mononegavirales (nonsegmented negative strand RNA viruses) from cDNA, we generated phenotypically mixed and chimeras based on MV, Edmonston B vaccine strain, cDNA. The Edmonston B strain of MV is known for decades as an inexpensive live human vaccine, proven both for its safety and efficacy, generally inducing life-long protection against MV in infants. MV has the potential of accommodating three additional transcription units in its genome or an additional coding sequence exceeding 3kb. The additional transgenes were stable after several rounds of replication implying that MV system is a potential candidate for developing recombinant and chimeric vaccines.
Since the expression of additional ORFs (LacZ and GFP reporter genes) is highly stable over many generations, the MV genome will be enriched with additional ORFs to express antigens of other viruses and pathogens. These viruses will be tested for MV-based multivalent vaccines.
For the purpose of therapeutic gene delivery, our system could be used for the construction of chimeric viruses containing engineered envelope proteins to redirect the virus to specific cells. For the purpose of generating novel monovalent vaccines, MV proteins could be genetically replaced by proteins of different viruses that are still lacking an efficient vaccine.
II. SORTING OF CELLULAR AND VIRAL PROTEINS IN POLARIZED CELLS.
Polarized cells, such as neurons and epithelial cells, maintain separate plasma membrane domains, each with a distinct protein and lipid composition, created through intracellular sorting mechanisms that recognize classes of proteins, and deliver them into separate vesicles for transport to the cognate surface. This "sorting" to the correct membrane is essential for the proteins to exhibit their biological functions, whereas missorting can often result in pathological conditions. The recognition events responsible for specifying transport to the basolateral surface of epithelial cells, or to cell bodies of neurons, have been found to mostly localize in the cytoplasmic domains of transmembrane glycoproteins. In addition to basolateral signals, many distinct types of signals for sorting proteins to the apical surface of epithelial cells or to axon of neurons, have been characterized. The glycolipid anchors, which direct proteins to the apical surface, apparently associate in the trans Golgi network (TGN) with detergent-insoluble membrane domains enriched in glycosphingolipids and cholesterol. Oligosaccharides on some secreted proteins specify apical transport although this mechanism does not apply to all secreted proteins. Thus, it appears that apical sorting involves multiple signals that are more variable than those involved in basolateral targeting.
Viruses follow specific sorting mechanisms in epithelial cells. Several studies have considered that the site of virus release is coupled to the sorting pattern of the envelope proteins. Measles virus (MV) infects epithelial cells from the apical domain and is mainly released from the same domain, irrespective of the envelope proteins sorting. We are interested to identify determinants that control polarized release of MV, and the implication of MV-proteins sorting in virus transmission and disease progression. Other cellular membrane glycoproteins are being used as internal markers to elucidate such a mechanism. For MV, we have evidence that viral proteins other than envelope glycoproteins determine the site of virus release. There are other cellular candidates that are involved in such a mechanism, which are currently under investigation.
Neumeister, C., Nanan, R., Cornu, T., Lüder, C., ter Mülen, V., Naim, H.Y., and Niewiesk, S. (2001). Measles virus and canine distemper virus target proteins into a ATP independent MHC class I-restricted antigen-processing pathway. J. Gen. Virol. 82, 441-447.
Wang, Z., Hangartner, L., Cornu, T., Martin, L., Zuniga, A., Billeter, M. A., and Naim, H. Y. (2001). Recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses. Vaccine 19, 2329-2336. Abstract
Ehrengruber, M.U., Hennou , S., Büeler, H-R., Naim,
H. Y., Déglon N., and
Naim, H.Y., Ehler, E, and Billeter, M.A (2000). Measles virus matrix protein specifies virus budding and envelope glycoprotein sorting in polarized epithelial cells. EMBO J. 19, 3576-3585. Abstract
Alfalah, M., Jacob, R., Preus, U., Zimmer, K.-P., Naim, H.Y., and Naim H.Y. (1999). O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts. Current Biology 9, 593-596. Abstract
Jakob, R., Preus, U., Panzer, P., Roth, M.G., Naim, Hassan Y., and Naim, H.Y. (1999). Hierarchy of sorting signals in chimeras of intestinal lactase-phlorizin hydrolase and the influenza virus hemagglutinin. J. Biol. Chem. 274, 8061-8067. Abstract
Roush, D.L., Gottardi, C.J., Naim, H.Y., Roth, M.G. and Caplan M.J. (1998). Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized MDCK and LLC-PK1 epithelial cells. J. Biol. Chem. 273, 26862-26869. Abstract
Fehr, T., Naim, H.Y., Bachmann, M.F. Ochsenbein, A., Spielhofer, P., Hangartner, H., Billeter, M.A., and Zingernagel, R.M. (1998). T-cell independent IgM and enduring protective IgG antibodies induced by chimeric measles viruses. Nature Medicine 4, 945-948. Abstract
Lin, S., Naim, H.Y., Rodriguez A.C., and Roth M.G. (1998). Mutations in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells. J.Cell Biol. 142, 51-57. Abstract
Spielhofer, P., Bächi, T., Fehr, T., Christiansen, G., Cattaneo, R., Kälin, K., Billeter, M., and Naim, H.Y. (1998). Chimeric measles viruses with a foreign envelope. J. Virol. 72, 2150-2159. Abstract
S. Lin, H.Y. Naim, and M.G. Roth. (1997). Tyrosine-dependent basolateral sorting signals are distinct from tyrosine-dependent internalization signals. J. Biol. Chem. 272, 26300-26305. Abstract
Jakob, R., Zimmer, K.-P., Naim, H., and Naim H.Y. (1997). The apical sorting of lactase-phlorizin hydrolase implicates sorting sequences found in the mature domain. Eur. J. Cell Biol. 72, 54-60. Abstract
Lazarovits, J., Naim, H.Y., Rodriguez A.C., Wang, R.-H., Fire, E., Bird, C., Henis, Y., and Roth, M.G. (1996). Endocytosis of Chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits J. Cell Biol. 134: 339-348. Abstract
Naim, H.Y., and Naim H. (1996). Dimerization of Pro-lactase-phlorizin hydrolase (pro-LPH) occurs in the endoplasmic reticulum, involves the membrane spanning domain and is required for an efficient transport of pro-LPH to the cell surface. Eur. J. Cell Biol. 70, 198-208. Abstract
Naim, H.Y., Dodds, D.T., Brewer C.B., and Roth, M.G. (1995). Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between hemagglutinin proteins with different internalization signals. J. Cell Biol. 129, 1241-1250. Abstract
Naim, H.Y. and Roth, M.G. (1994). SV40 expression vectors. In: Protein Expression in Animal Cells. Methods in Cell Biology, pp 113-136, Academic Press. Abstract
Naim, H.Y., Jacob, R., Naim, H., Sambrook, J., Gething, M.J. (1994). The Pro Region of Human Intestinal Lactase-Phlorizin Hydrolase. J. Biol. Chem. 269, 26933-26943. Abstract
Naim, H.Y. and Roth, M.G. (1994). Characteristics of the internalization signal in the Y543 influenza virus hemagglutinin suggest a model for recognition of internalization signals containing tyrosine. J. Biol. Chem. 269, 3928-3933. Abstract
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