Sorting signals in the measles virus wild-type glycoproteins differently influence virus spread in polarized epithelia and lymphocytes.

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.

Measles virus-induced block of transendothelial migration of T lymphocytes and infection-mediated virus spread across endothelial cell barriers.

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.

Glycoprotein targeting signals influence the distribution of measles virus envelope proteins and virus spread in lymphocytes.

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.

Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein.

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.