Measles virus (MV) hemagglutinin: evidence that attachment sites for MV receptors SLAM and CD46 overlap on the globular head

Measles virus hemagglutinin (MVH) residues potentially responsible for attachment to the wild-type (wt) MV receptor SLAM (CD150) have been identified and localized on the MVH globular head by reference to a revised hypothetical structural model for MVH (www.pepscan.nl/downloads/measlesH.pdb). We show that the mutation of five charged MVH residues which are conserved among morbillivirus H proteins has major effects on both SLAM downregulation and SLAM-dependent fusion. In the three-dimensional surface representation of the structural model, three of these residues (D505, D507, and R533) align the rim on one side of the cavity on the top surface of the MVH globular head and form the basis of a single continuous site that overlaps with the 546-548-549 CD46 binding site. We show that the overlapping sites fall within the footprint of an anti-MVH monoclonal antibody that neutralizes both wt and laboratory-vaccine MV strains and whose epitope contains R533. Our study does not exclude the possibility that Y481 binds CD46 directly but suggests that the N481Y mutation of wt MVH could influence, at a distance, the conformation of the overlapping sites so that affinity to CD46 increases. The relevance of these results to present concepts of MV receptor usage is discussed, and an explanation is proposed as to why morbillivirus attachment proteins are H, whereas those from the other paramyxoviruses are HN (hemagglutinin-neuraminidase).     

Morbillivirus Downregulation of CD46

There is evidence that CD46 (membrane cofactor protein) is a cellular receptor for vaccine and laboratory-passaged strains of measles virus (MV). Following infection with these MV strains, CD46 is downregulated from the cell surface, and consequent complement-mediated lysis has been shown to occur upon infection of a human monocytic cell line. The MV hemagglutinin (H) protein alone is capable of inducing this downregulation. Some wild-type strains of MV fail to downregulate CD46, despite infection being prevented by anti-CD46 antibodies. In this study we show that CD46 is also downregulated to the same extent by wild-type, vaccine, and laboratory-passaged strains of rinderpest virus (RPV), although CD46 did not appear to be the receptor for RPV. Expression of the RPV H protein by a nonreplicating adenovirus vector was also found to cause this downregulation. A vaccine strain of peste des petits ruminants virus caused slight downregulation of CD46 in infected Vero cells, while wild-type and vaccine strains of canine distemper virus and a wild-type strain of dolphin morbillivirus failed to downregulate CD46. Downregulation of CD46 can, therefore, be a function independent of the use of this protein as a virus receptor.     

Multiple amino acid substitutions in hemagglutinin are necessary for wild-type measles virus to acquire the ability to use receptor CD46 efficiently

Measles virus (MV) possesses two envelope glycoproteins, namely, the receptor-binding hemagglutinin (H) and fusion proteins. Wild-type MV strains isolated in B-lymphoid cell lines use signaling lymphocyte activation molecule (SLAM), but not CD46, as a cellular receptor, whereas MV vaccine strains of the Edmonston lineage use both SLAM and CD46 as receptors. Studies have shown that the residue at position 481 of the H protein is critical in determining the use of CD46 as a receptor. However, the wild-type IC-B strain with a single N481Y substitution in the H protein utilizes CD46 rather inefficiently. In this study, a number of chimeric and mutant H proteins, and recombinant viruses harboring them, were generated to determine which residues of the Edmonston H protein are responsible for its efficient use of CD46. Our results show that three substitutions (N390I and E492G plus N416D or T446S), in addition to N481Y, are necessary for the IC-B H protein to use CD46 efficiently as a receptor. The N390I, N416D, and T446S substitutions are present in the H proteins of all strains of the Edmonston lineage, whereas the E492G substitution is found only in the H protein of the Edmonston tag strain generated from cDNAs. The T484N substitution, found in some of the Edmonston-lineage strains, resulted in a similar effect on the use of CD46 to that caused by the E492G substitution. Thus, multiple residues in the H protein that have not previously been implicated have important roles in the interaction with CD46.     

Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling

Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2-/- mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.     

Wild-type measles virus with the hemagglutinin protein of the edmonston vaccine strain retains wild-type tropism in macaques

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM(+) as well as CD46(+) cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM(+) cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM(+) lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo.     

Efficiency of measles virus entry and dissemination through different receptors

The efficiency with which different measles virus (MV) strains enter cells through the immune cell-specific protein SLAM (CD150) or other receptors, including the ubiquitous protein CD46, may influence their pathogenicity. We compared the cell entry efficiency of recombinant MV differing only in their attachment protein hemagglutinin (H). We constructed these viruses with an additional gene expressing an autofluorescent reporter protein to allow direct detection of every infected cell. A virus with a wild-type H protein entered cells through SLAM two to three times more efficiently than a virus with the H protein of the attenuated strain Edmonston, whereas cell entry efficiency through CD46 was lower. However, these subtle differences were amplified at the cell fusion stage because the wild-type H protein failed to fuse CD46-expressing cells. We also proved formally that a mutation in H protein residue 481 (asparagine to tyrosine) results in improved CD46-specific entry. To define the selective pressure exerted on that codon, we monitored its evolution in different H protein backgrounds and found that several passages in CD46-expressing Vero cells were necessary to shift it in the majority of the MV RNA. To verify the importance of these observations for human infections, we examined MV entry into peripheral blood mononuclear cells and observed that viruses with asparagine 481 H proteins infect these cells more efficiently.     

Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion,Hemadsorption, Virus Growth,and Penetration Rate

Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.Measles virus (MV), which belongs to the genus Morbillivirus in the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome encodes six structural proteins: the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene also encodes two other accessory proteins, the C and V proteins. The C protein is translated from an alternative translational initiation site leading a different reading frame, and the V protein is synthesized from an edited mRNA. MV has two envelope glycoproteins, the F and H proteins. The former is responsible for envelope fusion, and the latter is responsible for receptor binding (12).Wild-type MV strains isolated in B95a cells and laboratory-adapted MV strains have distinct phenotypes (18). Wild-type MV strains can grow in B95a cells but not in Vero cells, while laboratory-adapted MV strains can grow in both B95a and Vero cells. Wild-type MV strains do not cause hemadsorption (HAd) in African green monkey red blood cells (AGM-RBC), while most of laboratory-adapted MV strains cause HAd. Importantly, wild-type MV strains are pathogenic and induce clinical signs that resemble human measles in experimentally infected monkeys while laboratory-adapted MV strains do not.One approach to identify amino acid substitutions responsible for these phenotypic differences is the comparison of a wild-type MV strain with a standard laboratory-adapted MV strain such as the Edmonston strain. With regard to the H protein, amino acid substitutions important for HAd activity and cell-cell fusion in tissue culture cells were identified by expressing the H proteins in mammalian cells (15, 21). Recently, Tahara et al. revealed that the M, H, and L proteins are responsible for efficient growth in Vero cells by constructing a series of recombinant viruses in which part of the genome of the wild-type MV was replaced with the corresponding sequences of the Edmonston strain (45, 46, 47).Another approach is the comparison of wild-type MV strains with their Vero cell-adapted MV strains. It was reported that Vero cell-adapted MV strains could be obtained by successive blind passages of wild-type MV strains in Vero cells (18, 24, 30, 43). Interestingly, in vivo and in vitro phenotypes of Vero cell-adapted MV strains were similar to those of laboratory-adapted standard MV strains (18, 19, 24, 30, 43). Comparison of the complete nucleotide sequences of the genomes of wild-type MV strains with those of Vero cell-adapted wild-type MV strains revealed amino acid substitutions in the P, C, V, M, H, and L proteins (27, 42, 48, 53).At present, these phenotypic differences are explained mainly by the receptor usage of MV. Wild-type MV strains can use signaling lymphocyte activation molecule (SLAM; also called CD150) but not CD46 as a cellular receptor, whereas laboratory-adapted MV strains can use both SLAM and CD46 as cellular receptors (7, 10, 16, 29, 56, 60).However, receptor usage per se cannot explain all of the phenotypic differences (20, 25, 48, 53). For example, recombinant Edmonston strains expressing wild-type H proteins can grow in Vero cells to some extent (17, 54). Several reports suggested the presence of the third MV receptor on Vero cells (14, 44, 54, 60). Other reports indicated the contribution of the M protein on cell-cell fusion and growth of MV in Vero cells (4, 27, 47). Recently, the unidentified epithelial cell receptor for MV was predicted in primary culture of human cells (1, 55) and several epithelial cell lines (23, 51). However, the identity of the third receptor on Vero cells and the unidentified epithelial cell receptor is not clear yet. Thus, the mechanism of Vero cell adaptation of wild-type MV is not completely understood.In order to understand the molecular mechanism of these phenotypic changes of wild-type MV strains during adaptation in Vero cells, we determined the complete nucleotide sequences of the genomes of the wild-type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strains (43) and examined the effect of individual amino acid substitutions using a mammalian cell expression system and reverse genetics. We show here that previously unrecognized new amino acid substitutions in the H and F proteins are important for MV adaptation and HAd activity.     

Identification of a second major site for CD46 binding in the hemagglutinin protein from a laboratory strain of measles virus (MV): potential consequences for wild-type MV infection

Natural or wild-type (wt) measles virus (MV) infection in vivo which is restricted to humans and certain monkeys represents an enigma in terms of receptor usage. Although wt MV is known to use the protein SLAM (CD150) as a cell receptor, many human tissues, including respiratory epithelium in which the infection initiates, are SLAM negative. These tissues are CD46 positive, but wt MV strains, unlike vaccinal and laboratory MV strains, are not thought to use CD46 as a receptor. We have identified a novel CD46 binding site at residues S548 and F549, in the hemagglutinin (H) protein from a laboratory MV strain, which is also present in wt H proteins. Our results suggest that although wt MV interacts with SLAM with high affinity, it also possesses the capacity to interact with CD46 with low affinity.     

Interaction of measles virus (Hallé strain) with CD46: evidence that a common binding site on CD46 facilitates both CD46 downregulation and MV infection.

CD46 acts as a cellular receptor for vaccine strains of measles virus (MV). The MV/CD46 interaction-mediated by the MV attachment glycoprotein, the hemagglutinin (H)-not only facilitates infection but also induces CD46 downregulation. A conflict of opinion exists as to whether a single MVH binding site on CD46, or two separate sites, facilitates the two phenomena. To investigate this conundrum we first tested and compared a panel of CD46-specific monoclonal antibodies (mAbs) for their capacity to block both processes. One (mAb 13/42) abrogated both MV fusion and CD46 downregulation. Mutation of an amino acid (arg59 in the SCR1 of CD46) essential for the epitope of mAb 13/42 resulted in the abrogation of both CD46 downregulation and viral fusion. This strongly suggests that the same MV binding site on CD46 is responsible for both CD46 downregulation and MV infection.     

Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake.

CD46, which serves as a receptor for measles virus (MV; strain Edmonston), is rapidly downregulated from the cell surface after contact with viral particles or infected cells. We show here that the same two CD46 complement control protein (CCP) domains responsible for primary MV attachment mediate its downregulation. Optimal downregulation efficiency was obtained with CD46 recombinants containing CCP domains 1 and 2, whereas CCP 1, alone and duplicated, induced a slight downregulation. Using persistently infected monocytic/promyelocytic U937 cells which release very small amounts of infectious virus, and uninfected HeLa cells as contact partners, we then showed that during contact the formation of CD46-containing patches and caps precedes CD46 internalization. Nevertheless, neither substances inhibiting capping nor the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly-OH (FIP) blocked CD46 downregulation. Thus, CD46 downregulation can be uncoupled from fusion and subsequent virus uptake. Interestingly, in that system cell-cell contacts lead to a remarkably efficient infection of the target cells which is only partially inhibited by FIP. The finding that the contact of an infected with uninfected cells results in transfer of infectious viral material without significant (complete) fusion of the donor with the recipient cell suggests that microfusion events and/or FIP-independent mechanisms may mediate the transfer of MV infectivity from cell to cell.     

Is mRNA and protein level of CD46 altered in measles virus vaccine strain S191-infected cells?

Previous research showed that the expression of measles virus receptor CD46 was downregulated after expression of measles virus hemagglutinin protein on the surface of the virus infected cell or triggered by infected cell-to-cell contact. We reported here that the mRNA level of CD46 in MV infected cells was not changed which was tested by real-time quantitative PCR. To further analyse the surface expression alteration of CD46 after MV infection, flow cytometric analysis and indirect immunofluorescence were used to detect the protein level of CD46. Altogether, our results provided a demonstration that the expression of CD46 was not downregulated by the infection of MV strain S191 both on mRNA level and cellular surface protein level. Previous results reported that the "downregulation" of CD46 expression on the cell surface may take place because H protein masks the antibody recognition site on CD46 which results in "downregulation" of the expression of CD46.     

A human lung carcinoma cell line supports efficient measles virus growth and syncytium formation via a SLAM- and CD46-independent mechanism

Measles virus (MV) propagates mainly in lymphoid organs throughout the body and produces syncytia by using signaling lymphocyte activation molecule (SLAM) as a receptor. MV also spreads in SLAM-negative epithelial tissues by unknown mechanisms. Ubiquitously expressed CD46 functions as another receptor for vaccine strains of MV but not for wild-type strains. We here show that MV grows and produces syncytia efficiently in a human lung adenocarcinoma cell line via a SLAM- and CD46-independent mechanism using a novel receptor-binding site on the hemagglutinin protein. This infection model could advance our understanding of MV infection of SLAM-negative epithelial cells and tissues.     

Ablation of Nectin4 Binding Compromises CD46 Usage by a Hybrid Vesicular Stomatitis Virus/Measles Virus

Measles virus (MV) immunosuppression is due to infection of SLAM-positive immune cells, whereas respiratory shedding and virus transmission are due to infection of nectin4-positive airway epithelial cells. The vaccine lineage MV strain Edmonston (MV-Edm) acquired an additional tropism for CD46 which is the basis of its oncolytic specificity. VSVFH is a vesicular stomatitis virus (VSV) encoding the MV-Edm F and H entry proteins in place of G. The virus spreads faster than MV-Edm and is highly fusogenic and a potent oncolytic. To determine whether ablating nectin4 tropism from VSVFH might prevent shedding, increasing its safety profile as an oncolytic, or might have any effect on CD46 binding, we generated VSVFH viruses with H mutations that disrupt attachment to SLAM and/or nectin4. Disruption of nectin4 binding reduced release of VSVFH from the basolateral side of differentiated airway epithelia composed of Calu-3 cells. However, because nectin4 and CD46 have substantially overlapping receptor binding surfaces on H, disruption of nectin4 binding compromised CD46 binding and greatly diminished the oncolytic potency of these viruses on human cancer cells. Thus, our results support continued preclinical development of VSVFH without ablation of nectin4 binding.     

The Measles Virus Hemagglutinin β-Propeller Head β4-β5 Hydrophobic Groove Governs Functional Interactions with Nectin-4 and CD46 but Not Those with the Signaling Lymphocytic Activation Molecule

Wild-type measles virus (MV) strains use the signaling lymphocytic activation molecule (SLAM; CD150) and the adherens junction protein nectin-4 (poliovirus receptor-like 4 [PVRL4]) as receptors. Vaccine MV strains have adapted to use ubiquitous membrane cofactor protein (MCP; CD46) in addition. Recently solved cocrystal structures of the MV attachment protein (hemagglutinin [H]) with each receptor indicate that all three bind close to a hydrophobic groove located between blades 4 and 5 (β4-β5 groove) of the H protein β-propeller head. We used this structural information to focus our analysis of the functional footprints of the three receptors on vaccine MV H. We mutagenized this protein and tested the ability of individual mutants to support cell fusion through each receptor. The results highlighted a strong overlap between the functional footprints of nectin-4 and CD46 but not those of SLAM. A soluble form of nectin-4 abolished vaccine MV entry in nectin-4- and CD46-expressing cells but only reduced entry through SLAM. Analyses of the binding kinetics of an H mutant with the three receptors revealed that a single substitution in the β4-β5 groove drastically reduced nectin-4 and CD46 binding while minimally altering SLAM binding. We also generated recombinant viruses and analyzed their infections in cells expressing individual receptors. Introduction of a single substitution into the hydrophobic pocket affected entry through both nectin-4 and CD46 but not through SLAM. Thus, while nectin-4 and CD46 interact functionally with the H protein β4-β5 hydrophobic groove, SLAM merely covers it. This has implications for vaccine and antiviral strategies.     

Susceptibility of human dendritic cells (DCs) to measles virus (MV) depends on their activation stages in conjunction with the level of CDw150: role of Toll stimulators in DC maturation and MV amplification

Human CD46 (membrane cofactor protein, or MCP) and CDw150 (signaling lymphocyte activation molecule, or SLAM) serve as receptors for measles virus (MV), which induces marked host immune suppression. Although monocytes express CD46, they are considerably resistant to MV. Once monocytes differentiate into immature myeloid dendritic cells (iDCs) (GM-CSF + IL-4-treated), the cells become susceptible to MV. Therefore, we have identified CD46-adapted and CDw150-adapted strains of MV, and the dynamics of CD46 and CDw150 during monocyte-iDC conversion were examined in conjunction with MV susceptibility. Strikingly, CDw150 was not detected in monocytes and moderately induced in iDCs, while CD46 was constantly expressed in monocyte-to-iDC differentiation. Thus, iDCs were found to become highly permissive to CDw150-adapted MV strains via expression of CDw150. In fact, polyclonal and monoclonal antibodies that specifically blocked the MV receptor function of CD46 or CDw150 cancelled MV replication in iDCs according to the preferential usage of either CD46 or CDw150 in each strain of MV. Next, we showed that DCs that matured via stimulation of their Toll-like receptors (TLRs) 2 and/or 4 exhibited an approximately fivefold increase in CDw150 at the protein level, and concomitantly, higher levels of MV amplification were observed in mixed culture of lymphocytes than in iDCs without TLR2/4 stimuli. Hence, amplification of CDw150-dependent MV strains was augmented in DCs parallel with the levels of CDw150 in the presence of lymphocytes possessing CDw150. TLR-mediated functional potential of DCs may affect the degree of MV amplification through distinct MV strain-specific receptor usage of CDw150 or CD46.     

Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins

The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.     

Receptor (CD46) modulation and complement-mediated lysis of uninfected cells after contact with measles virus-infected cells.

Recently, it has been observed that the infection of human target cells with certain measles virus (MV) strains leads to the downregulation of the major MV receptor CD46. Here we report that CD46 downregulation can be rapidly induced in uninfected cells after surface contact with MV particles or MV-infected cells. Receptor modulation is detectable after 30 min of cocultivation of uninfected cells with MV-infected cells and is complete after 2 to 4 h, a time after which newly synthesized MV hemagglutinin (MV-H) cannot be detected in freshly infected target cells. This contact-mediated receptor modulation is also induced by recombinant MV-H expressed by vaccinia virus and is inhibitable with antibodies against CD46 and MV-H. By titrating the effect with MV Edmonston strain-infected cells, a significant contact-mediated CD46 modulation was detectable up to a ratio of 1 infected to 64 uninfected cells. As a result of CD46 downregulation, an increased susceptibility of uninfected cells for complement-mediated lysis was observed. This phenomenon, however, is MV strain dependent, as observed for the downregulation of CD46 after MV infection. These data suggest that in acute measles or following measles vaccination, uninfected cells might also be destroyed by complement after contacting an MV-infected cell.     

Single-chain antibody displayed on a recombinant measles virus confers entry through the tumor-associated carcinoembryonic antigen

To redirect the tropism of the vaccine strain of measles virus (MV), Edmonston B, to a targeted cell population, we displayed on the viral hemagglutinin (H) a single-chain antibody (scAb) specific for the tumor-associated carcinoembryonic antigen (CEA). We generated H fusion proteins with three forms of the scAb appended, differing in the lengths of the linkers separating the VH and VL domains and thus in the oligomerization states of the scAbs. All proteins were stable, appeared properly folded, and were transported to the cell surface, but only H displaying the long-linker form of scAb was functional in supporting cell-cell fusion. This protein induced extensive syncytia in cells expressing the normal virus receptor CD46 and also in CD46-negative cells expressing the targeted receptor, human CEA. Replication-competent MV with H replaced by H displaying the long-linker form of scAb was recovered and replicated efficiently in both CD46-positive and CD46-negative, CEA-positive cells. Thus, MV not only tolerates the addition of a scAb on its H protein but also infects cells via a novel interaction between the scAb and its targeted receptor.     

Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion

Clinical isolates of measles virus (MV) use signaling lymphocyte activation molecule (SLAM) as a cellular receptor, whereas vaccine and laboratory strains may utilize the ubiquitously expressed CD46 as an additional receptor. MVs also infect, albeit inefficiently, SLAM(-) cells, via a SLAM- and CD46-independent pathway. Our previous study with recombinant chimeric viruses revealed that not only the receptor-binding hemagglutinin (H) but also the matrix (M) protein of the Edmonston vaccine strain can confer on an MV clinical isolate the ability to grow well in SLAM(-) Vero cells. Two substitutions (P64S and E89K) in the M protein which are present in many vaccine strains were found to be responsible for the efficient growth of recombinant virus in Vero cells. Here we show that the P64S and E89K substitutions allow a strong interaction of the M protein with the cytoplasmic tail of the H protein, thereby enhancing the assembly of infectious particles in Vero cells. These substitutions, however, are not necessarily advantageous for MVs, as they inhibit SLAM-dependent cell-cell fusion, thus reducing virus growth in SLAM(+) B-lymphoblastoid B95a cells. When the cytoplasmic tail of the H protein is deleted, a virus with an M protein possessing the P64S and E89K substitutions no longer grows well in Vero cells yet causes cell-cell fusion and replicates efficiently in B95a cells. These results reveal a novel mechanism of adaptation and attenuation of MV in which the altered interaction of the M protein with the cytoplasmic tail of the H protein modulates MV growth in different cell types.     

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.