Human CD46 enhances nitric oxide production in mouse macrophages in response to measles virus infection in the presence of gamma interferon: dependence on the CD46 cytoplasmic domains.

CD46 is a transmembrane complement regulatory protein widely expressed on nucleated human cells. Laboratory-adapted strains of measles virus (MV) bind to the extracellular domains of CD46 to enter human cells. The cytoplasmic portion of CD46 consists of a common juxtamembrane region and different distal sequences called Cyt1 and Cyt2. The biological functions of these cytoplasmic sequences are unknown. In this study, we show that expression of human CD46 with the Cyt1 cytoplasmic domain in mouse macrophages enhances production of nitric oxide (NO) in response to MV infection in the presence of gamma interferon (IFN-gamma). Human CD46 does not increase the basal levels of NO production in mouse macrophages and does not augment NO production induced by double-stranded polyribonucleotides. Replacing the cytoplasmic domain of human CD46 with Cyt2 reduces MV and IFN-gamma-induced NO production in mouse macrophages. Deleting the entire cytoplasmic domains of human CD46 does not prevent MV infection but markedly attenuates NO production in response to MV and IFN-gamma. Mouse macrophages expressing a tailless human CD46 mutant are more susceptible to MV infection and produce 2 to 3 orders of magnitude more infectious virus than mouse macrophages expressing human CD46 with intact cytoplasmic domains. These results reveal a novel function of CD46 dependent on the cytoplasmic domains (especially Cyt1), which augments NO production in macrophages. These findings may have significant implications for roles of CD46 in innate immunity and MV pathogenesis.

Expression of human complement regulatory protein CD46 restricts measles virus replication in mouse macrophages.

Measles virus (MV) can infect mouse macrophages to cause a prolonged non-cytopathic infection that produces low levels of infectious virus for days. We have generated RAW264.7 mouse macrophages expressing human CD46, a cell surface complement regulatory protein that serves as a receptor for laboratory-adapted strains of MV. Laboratory-adapted MV strains efficiently enter the CD46-positive mouse macrophages to cause a cytopathic infection with extensive multinucleated cells and pseudopodia-like extensions. However, MV infection of mouse macrophages through CD46 is self-limiting. Both viral protein synthesis and infectious virus production are abruptly terminated after the second day of infection. This novel virus-cell interaction is seen only in mouse macrophages but not in mouse or hamster fibroblasts expressing human CD46. The possible role of CD46 in macrophage antiviral response restricting MV replication is discussed.

The cytoplasmic domains of complement regulatory protein CD46 interact with multiple kinases in macrophages.

Membrane cofactor protein (CD46), which normally protects autologous cells from complement lysis, is the human cell receptor for measles virus (MV). Interaction between MV and CD46 on monocytes can lead to suppression of monocyte activation. We have investigated the interaction between the cytoplasmic sequences of CD46 and kinases in a mouse macrophage cell line. Glutathione-S-transferase (GST) fusion proteins bearing the Cyt1 or Cyt2 alternative cytoplasmic domain of CD46 associate with macrophage kinase activity, which phosphorylates multiple proteins co-purified with the GST fusion proteins. Association with the macrophage kinase activity correlates with tyrosine phosphorylation of the CD46 cytoplasmic domains. Removing the CD46 sequences or introducing a frame-shift mutation abrogates the association with macrophage kinase activity. Renaturation studies reveal multiple kinases with apparent molecular mass of 82, 79, 58, and 50/49 kDa, which associate specifically with both CD46 cytoplasmic domains. Alanine substitutions at a juxtamembrane Tyr-X-X-Leu motif in the Cyt1 domain completely abrogate the association with macrophage kinases and tyrosine phosphorylation of Cyt1; but similar substitutions in the Cyt2 domain only partially reduce the association with kinases and tyrosine phosphorylation of Cyt2. These results reveal a specific interaction between complement regulatory protein CD46 and macrophage kinases. These findings may provide an important clue for understanding immune modulation by MV.

Human cell receptor CD46 is down regulated through recognition of a membrane-proximal region of the cytoplasmic domain in persistent measles virus infection.

Monkey cells persistently infected by measles virus (MV) Biken strain (Biken-CV-1 cells) showed no cytopathic effects and lacked surface expression of a homolog of human cell receptor, membrane cofactor protein CD46. Transfection of a human CD46 gene into these cells induced extensive cell fusion, indicating that down regulation of the endogenous CD46 homolog was essential for the maintenance of a noncytopathic mode of infection. Surface expression of the exogenously introduced human CD46 was also drastically down regulated in the persistently infected cells compared with uninfected cells. The down regulation was specific for CD46 and did not affect surface expression of exogenously introduced CD4. Exogenous human CD46 was synthesized efficiently in the persistently infected cells, but it did not accumulate on the cell surface. Fusion of Biken-CV-1 cells required the extracellular hemagglutinin (H-protein)-binding domain but not the cytoplasmic domain. Replacing the transmembrane and cytoplasmic domains of CD46 with a glycosylphosphatidylinositol anchor did not prevent cell fusion but completely alleviated down regulation of the glycosylphosphatidylinositol-anchored CD46 in Biken-CV-1 cells. Deletion analyses revealed that the membrane-distal sequences of the CD46 cytoplasmic domain were not only unnecessary but also inhibitory for CD46 down regulation. By contrast, the six amino acid residues proximal to the membrane contained a sequence required for CD46 down regulation in the persistently infected cells. These results indicate that CD46 is down regulated in the persistently infected cells by a mechanism that recognizes a membrane-proximal sequence in the CD46 cytoplasmic domain.

Mapping human telomere regions with YAC and P1 clones: chromosome-specific markers for 27 telomeres including 149 STSs and 24 polymorphisms for 14 proterminal regions.

A YAC library enriched for telomere clones was constructed and screened for the human telomere-specific repeat sequence (TTAGGG). Altogether 196 TYAC library clones were studied: 189 new TYAC clones were isolated, 149 STSs were developed for 132 different TY-ACs, and 39 P1 clones were identified using 19 STSs from 16 of the TYACs. A combination of mapping methods including fluorescence in situ hybridization, somatic cell hybrid panels, clamped homogeneous electric fields, meiotic linkage, and BLASTN sequence analysis was utilized to characterize the resource. Forty-five of the TYACs map to 31 specific telomere regions. Twenty-four linkage markers were developed and mapped within 14 proterminal regions (12 telomeres and 2 terminal bands). The polymorphic markers include 12 microsatellites for 10 telomeres (1q, 2p, 6q, 7q, 10p, 10q, 13q, 14q, 18p, 22q) and the terminal bands of 11q and 12p. Twelve RFLP markers were identified and meiotically mapped to the telomeres of 2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs. More than 30,000 nucleotides derived from the TYAC vector-insert junction regions or from regions flanking TYAC microsatellites were compared to reported sequences using BLASTN. In addition to identifying homology with previously reported telomere sequences and human repeat elements, gene sequences and a number of ESTs were found to be highly homologous to the TYAC sequences. These genes include human coagulation factor V (F5), Weel protein tyrosine kinase (WEE1), neurotropic protein tyrosine kinase type 2 (NTRE2), glutathione S-transferase (GST1), and beta tubulin (TUBB). The TYAC/P1 resource, derivative STSs, and polymorphisms constitute an enabling resource to further studies of telomere structure and function and a means for physical and genetic map integration and closure.

Isolation of YAC clones from the pericentromeric region of chromosome 10 and development of new genetic markers linked to the multiple endocrine neoplasia type 2A gene.

Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology form the basis of our strategy to clone and define the genomic structure of the pericentromeric region of chromosome 10 containing the multiple endocrine neoplasia type 2A gene. Thus far YAC walks have been initiated from five chromosome 10 pericentromeric loci including RBP3, D10S94, RET, D10Z1, and FNRB. Long range pulsed-field gel electrophoresis maps are constructed from the YACs isolated to define clone overlaps and to identify putative CpG islands. Bidirectional YAC walks are continued by rescreening the YAC library with sequence-tagged site assays developed from end-clones. Several new restriction fragment length polymorphisms and simple sequence repeat polymorphism markers have been identified from the YAC clones. In particular, two highly informative (CA)n dinucleotide repeat markers, sTCL-1 from proximal chromosome 10p (16 alleles, PIC = 0.68) and sJRH-1 from the RBP3 locus (18 alleles, PIC = 0.88), provide useful reagents for a polymerase chain reaction-based predictive genetic test that can be performed rapidly from small amounts of DNA.