Summary In measles virus (MV)-infected cells the matrix (M)protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix-or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.

In order to analyze whether measles virus (MV) is transported via transmigrating leukocytes across endothelial barriers or whether virus spreads via infection of endothelial cells and basolateral release, we investigated the migratory behavior of infected human primary T lymphocytes across polarized cell layers of human brain microvascular endothelial cells. We found that the capacity of lymphocytes to migrate through filter pores was only slightly affected by wild-type MV infection, whereas their capacity to migrate through endothelial barriers was drastically reduced. MV infection stimulated the expression and activation of the leukocyte integrins LFA-1 and VLA-4, mediating a strong adherence to the surface of endothelial cells. Furthermore, the formation of engulfing membrane protrusions by endothelial cells, so-called transmigratory cups, was induced, but transmigration was impaired. As a consequence of this close cell-cell contact, MV infection was transmitted from lymphocytes to the endothelium. MV envelope proteins were expressed on the apical and basolateral surfaces of infected polarized endothelial cells, and virus was released from both sides. Wild-type MV infection did not induce the formation of syncytia, suggesting virus spread from cell to cell via cell processes and contacts. Our data indicate that transendothelial migration of infected T cells is strongly inhibited, whereas virus can cross endothelial barriers by productive infection of the endothelium and subsequent bipolar virus release.Leukocyte transmigration across vascular endothelium is a highly regulated process and is central to inflammation and the immune response (23,24). Underlying mechanisms such as interaction between adhesion molecules of both cell types and cytoskeletal rearrangements may be deregulated by viral infections. After transmission to the respiratory tract, measles virus (MV) is transported via dendritic cells or monocytes to draining lymph nodes, where a massive infection in CD150-positive activated dendritic cells, lymphocytes, and macrophages is initiated (10,13,22,38). The virus is then carried via infected leukocytes in the bloodstream to different organs, thereby establishing the systemic infection. To distribute infection into various organs, including the skin and the brain, MV must overcome endothelial cell barriers either within an infected leukocyte transmigrating through the barrier as a "Trojan horse" or by infection of endothelial cells and basolateral (abluminal) virus release or cell-to-cell spread. During the rash, MV-infected microvascular endothelial cells of the skin have been observed (18, 39); however, from these histological analyses it is not clear whether virus disseminates from there to underlying epithelial cell layers, leading to local cellular infiltrations, or whether the virus is first carried to epithelial cell layers via transmigrating leukocytes.MV is a highly immunosuppressive virus known to impair various functions of leukocytes, but it is not known if the infection affects transendo...

We previously demonstrated the presence of tyrosine-dependent motifs for specific sorting of two measles virus (MV) glycoproteins, H and F, to the basolateral surface in polarized epithelial cells. Targeted expression of the glycoproteins was found to be required for virus spread in epithelia via cell-to-cell fusion in vitro and in vivo. In the present study, recombinant MVs (rMVs) with substitutions of the critical tyrosines in the H and F cytoplasmic domains were used to determine whether the sorting signals also play a crucial role for MV replication and spread within lymphocytes, the main target cells of acute MV infection. Immunolocalization revealed that only standard glycoproteins are targeted specifically to the uropod of polarized lymphocytes and cluster on the surface of non-polarized lymphocytes. H and F proteins with tyrosine mutations did not accumulate in uropods, but were distributed homogeneously on the surface and did not colocalize markedly with the matrix (M) protein. Due to the defective interaction with the M protein, all mutant rMVs showed an enhanced fusion capacity, but only rMVs harbouring two mutated glycoproteins showed a marked decrease in virus release from infected lymphocytes. These results demonstrate clearly that the tyrosine-based targeting motifs in the MV glycoproteins are not only important in polarized epithelial cells, but are also active in lymphocytes, thus playing an important role in virus propagation in different key target cells during acute MV infection. INTRODUCTIONMeasles virus (MV) is still one of the leading causes of death among young children in developing countries, despite the availability of an effective vaccine for 40 years (WHO, 2007). During the course of an acute MV infection, many cell types, including polarized cells, are infected. As MV is transmitted via aerosols or droplets and replicates initially in the respiratory mucosa, polarized nasal or bronchial epithelial cells are among the first MV target cells. MV then enters local lymphatic tissues and spreads systemically through the lymphatic and blood systems. In the systemic phase of infection, monocytes and lymphocytes are the main target cells. Infected blood mononuclear cells are responsible for MV-induced transient immunosuppression and carry MV to various organs, such as skin, intestine, liver, lung and kidney, where different types of polarized cell are infected (Esolen et al., 1993;Osunkoya et al., 1990;Yanagi et al., 2006). Polarized cells differ from non-polarized cells in their ability to segregate proteins and lipids into distinct surface subdomains accompanied by morphological and functional asymmetry, as occurs with the apical and basolateral surfaces in polarized epithelia and the axonal and dendritic processes in neurons (Rodriguez-Boulan & Powell, 1992). Lymphocytes can also develop a polarized phenotype if they carry out certain functions, such as cell-cell interactions or migration (Bretscher, 1996;Sanchez-Madrid & del Pozo, 1999). In migrating T cells, polarization involves the f...

The spread of virus infection within an organism is partially dictated by the receptor usage of the virus and can be influenced by sorting signals present in the viral glycoproteins expressed in infected cells. In previous studies, we have shown that the haemagglutinin (H) and fusion protein (F) of the measles virus (MV) vaccine strain MV Edm harbour tyrosine-dependent sorting signals which influence virus spread in both lymphocytes and epithelial cells to a similar degree. In contrast with the vaccine strain, MV wild-type virus does not use CD46 but CD150/SLAM and a not clearly identified molecule on epithelial cells as receptors. To determine differences in viral spread between vaccine and wild-type virus, we generated recombinant MV expressing glycoproteins of both the wild-type strain WTFb and the corresponding tyrosine mutants. In contrast with observations based on vaccine virus glycoproteins, mutations in wild-type virus H and F differently influenced cell-to-cell fusion and replication in polarized epithelia and lymphocytes. For wild-type H, our data suggest a key role of the cytoplasmic tyrosine signal for virus dissemination in vivo. It seems to be important for efficient virus spread between lymphocytes, while the tyrosine signal in the F protein gains importance in epithelial cells as both signals have to be intact to allow efficient spread of infection within epithelia.