Downloaded from www.microbiologyresearch.org by IP: 54.145.90.84 On: Mon, 07 Mar 2016 02:40:41 Journal of General Virology (2000), 81, 441–449. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Recombinant measles virus requiring an exogenous protease for activation of infectivity Andrea Maisner, 1 Branka Mrkic, 2 Georg Herrler, 1 † Markus Moll, 1 Martin A. Billeter, 2 Roberto Cattaneo 2 ‡ and Hans-Dieter Klenk 1 1 Institut fu  r Virologie, Philipps-Universita  t Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany 2 Institut fu  r Molekularbiologie, Universita  t Zu  rich, Winterthurerstraße 190, CH-8057 Zu  rich, Switzerland Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar tm -CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed. Introduction Measles virus (MV) is one of the most contagious human pathogens. Vaccination with attenuated live virus has greatly reduced the number of cases, but measles is still a major cause of serious disease and infant mortality in developing countries due to low vaccine coverage and the inability to vaccinate infants in the first 6 months of life. The virus is transmitted by the respiratory route. Entry and initial replication occur in the conjunctiva and the respiratory mucosa. Infection and inflam- mation of the lower respiratory tract and the lung follow. Viraemia and systemic infection inevitably occur before host defence mechanisms control virus replication and clear infected cells. Recently, a genetically modified mouse was established to study MV spread in an animal model. Mice expressing human CD46 and lacking the interferon receptor type I were Author for correspondence : Andrea Maisner. Fax 49 6421 286 8962. e-mail maisnermailer.uni-marburg.de † Present address : Institut fu  r Virologie, Tiera  rztliche Hochschule Hannover, Bu  nteweg 17, 30559 Hannover, Germany. ‡ Present address : Molecular Medicine Program, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. shown to be productively infected after intranasal and intracerebral inoculation (Mrkic et al., 1998). MV is the prototype member of the morbillivirus genus in the Paramyxoviridae family of negative-stranded RNA viruses. Virions have an envelope with two virus-encoded integral membrane glycoproteins, the viral attachment protein haemag- glutinin (H) and the fusion protein (F), which form spike-like projections on the outer surface. The H protein is responsible for binding to cellular receptors, such as CD46 (Do  rig et al., 1993 ; Naniche et al., 1993 ; Schneider-Schaulies et al., 1995), and is essential as a cofactor for fusion (Wild et al., 1991). The F protein is synthesized as an inactive precursor molecule F  , which is cleaved intracellularly by host proteases to generate two polypeptide subunits, F  and F  , held together by disulfide bonds. Infected cells exposing cleaved F protein on the surface fuse with adjacent cells at neutral pH, thereby causing syncytium formation. The multibasic cleavage site at which the F protein of MV is activated consists of five basic amino acids, R-R-H-K-R, at positions 108–112. Correct proteolytic cleavage after arginine 112 is essential, because changing this residue to leucine was shown to result in aberrant cleavage and loss of fusion ability (Alkathib et al., 1994). The major cellular protease responsible for correct cleavage of the F  precursor protein is furin, a subtilisin-like endo- 0001-6674  2000 SGM EEB