Akiko Hirano

Human membrane cofactor protein (MCP, CD46): multiple isoforms and functions.

Human membrane cofactor protein (MCP, CD46) is a 45-70 kDa protein with genetic and tissue-specific heterogeneity, and is expressed on all nucleated cells. MCP consists from N-terminus of 4 short consensus repeats (SCRs), 1-3 serine/threonine-rich (ST) domains, a transmembrane domain (TM) and a cytoplasmic tail (CYT). More than 8 isoforms are generated secondary to alternative splicing due to combinations of various exons encoding the ST, TM and CYT domains. It serves as a cofactor of serine protease factor I for inactivation of complement C3b and C4b. Its primary role is to protect host cells from homologous complement attack by inactivating C3b/C4b deposited on the membrane. It also acts as receptors for measles virus (MV), some kinds of bacteria and for a putative ligand on oocytes. MV infection causes temporal host immune suppression, which may appear secondary to signaling events through MCP on macrophages and dendritic cells. These functional properties of human MCP may facilitate xenotransplantation and may be useful in the generation of animal models of measles by creating human MCP-expressing animals.

Functional Modulation of Human Macrophages Through CD46 (Measles Virus Receptor): Production of IL-12 p40 and Nitric Oxide in Association with Recruitment of Protein-Tyrosine Phosphatase SHP-1 to CD46

Human CD46, formerly membrane cofactor protein, binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic measles infection. Suppression of cell-mediated immunity, including down-regulation of IL-12 production, has been reported on macrophages (Mφ) by cross-linking their CD46. The intracellular events responsible for these immune responses, however, remain unknown. In this study, we found that 6- to 8-day GM-CSF-treated peripheral blood monocytes acquired the capacity to recruit protein-tyrosine phosphatase SHP-1 to their CD46 and concomitantly were able to produce IL-12 p40 and NO. These responses were induced by stimulation with mAbs F(ab′)2 against CD46 that block MV binding or by a wild-type MV strain Kohno MV strain (KO; UV treated or untreated) that was reported to induce early phase CD46 down-regulation. Direct ligation of CD46 by these reagents, but not intracellular MV replication, was required for these cellular responses. Interestingly, the KO strain failed to replicate in the 6- to 8-day GM-CSF-cultured Mφ, while other MV strains replicated to form syncytia under the same conditions. When stimulated with the KO strain, rapid and transient dissociation of SHP-1 from CD46 was observed. These and previous results provide strong evidence that CD46 serves as a signal modulatory molecule and that the properties of ligands determine suppression or activation of an innate immune system at a specific maturation stage of human Mφ.

Human receptor for measles virus (CD46) enhances nitric oxide production and restricts virus replication in mouse macrophages by modulating production of alpha/beta interferon.

ABSTRACT Complement regulatory protein CD46 is a human cell receptor for measles virus (MV). In this study, we investigated why mouse macrophages expressing human CD46 restricted MV replication and produced higher levels of nitric oxide (NO) in response to MV and gamma interferon (IFN-γ). Treatment of MV-infected CD46-expressing mouse macrophages with antibodies against IFN-α/β blocked NO production. Antibodies against IFN-α/β also inhibited the augmenting effect of MV on IFN-γ-induced NO production in CD46-expressing mouse macrophages. These antibodies did not affect NO production induced by IFN-γ alone. These data suggest that MV enhances NO production in CD46-expressing mouse macrophages through action of IFN-α/β. Mouse macrophages expressing a human CD46 mutant lacking the cytoplasmic domains were highly susceptible to MV. These cells produced much lower levels of NO and IFN-α/β upon infection by MV, suggesting the CD46 cytoplasmic domains enhanced IFN-α/β production. When mouse macrophages expressing tailless human CD46 were exposed to culture medium from MV-infected mouse macrophages expressing intact human CD46, viral protein synthesis and development of cytopathic effects were suppressed. Pretreating the added culture medium with antibodies against IFN-α/β abrogated these antiviral effects. Taken together, these findings suggest that expression of human CD46 in mouse macrophages enhances production of IFN-α/β in response to MV infection, and IFN-α/β synergizes with IFN-γ to enhance NO production and restrict viral protein synthesis and virus replication. This novel function of human CD46 in mouse macrophages requires the CD46 cytoplasmic domains.

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. J. Leukoc. Biol. 62: 892–900; 1997.

Nucleoprotein phosphorylated on both serine and threonine is preferentially assembled into the nucleocapsids of measles virus.

The nucleoprotein (N) in the nucleocapsids of measles virus (MV) has different conformation and antigenicity than the free N-protein in MV-infected cells. These two forms of N-protein have identical methionine-containing tryptic peptides. The free N-protein contains 4 phosphorylated tryptic peptides. However, the nucleocapsid-associated N-protein has an additional phosphorylated peptide not found in the free N-protein. The free N-protein is phosphorylated only on serine residues, whereas the nucleocapsid-associated N-protein is phosphorylated on both serine and threonine residues. The MV N-protein expressed from a cloned gene in primate cells is also phosphorylated on both serine and threonine residues. These results suggest that cellular kinases phosphorylate the MV N-protein, and N-protein with phosphorylated serine and threonine is preferentially assembled into the viral nucleocapsids.

Expression and properties of the V protein in acute measles virus and subacute sclerosing panencephalitis virus strains

Measles virus (MV) inserts one guanosine (G) residue at a specific site in a subpopulation of the mRNA transcribed from the phosphoprotein (P) gene to produce V mRNA. Using an antiserum against the unique carboxyl-terminal region of the predicted V protein, we found that a phosphorylated V protein was expressed in two acute MV strains (Edmonston and Nagahata) and three SSPE virus strains (Biken, Yamagata, and Niigata). The V protein of Biken strain SSPE virus was electrophoretically and antigenically indistinguishable from the V protein of Nagahata strain acute MV, the likely progenitor of the Biken strain. The V protein of these two viruses was not present in the intracellular viral nucleocapsids, but was found only in the cytosolic free protein pool. Pulse-chase experiments failed to show transport of the V protein to the plasma membrane. The V protein was also absent in the extracellular virions. The P protein synthesized from the cloned gene associated with the MV nucleocapsids in vitro, but the V protein had no affinity to the MV nucleocapsids. These results suggest that expression and properties of the V protein are conserved in chronic MV infection.

Characteristics of avian sarcoma virus strain PRCIV and comparison with strain PRCII-p

Abstract Avian sarcoma virus strain Poultry Research Centre IV (PRCIV) ( J. G. Carr and J. G. Campbell (1958) Brit. J. Cancer 12 , 631–635) is highly oncogenic in chickens and transforms chicken embryo fibroblasts in culture. It is defective in replication, unable to code for complete functional products of the three essential viral genes gag , pol , or env . Nonproducing cells transformed by PRCIV lack the transforming protein of Rous sarcoma virus, pp6 src but synthesize a new transformation-specific protein of 170,000 MW. This P170 contains partial gag sequences, probably at its N-terminus and transformation-specific, cell-derived sequences, probably in the C-terminal portion of the molecule. In two-dimensional tryptic peptide maps P170 of PRCIV is indistinguishable from the transformation-specific protein P170 of the previously described PRCII-p ( M. L. Breitman, J. C. Neil, C. Moscovici, and P. K. Vogt (1981) Virology 108 , 1–12). Both P170 proteins also contain all but one tryptic peptide of the transformation-specific gag -linked protein P105 found in cells transformed by avian sarcoma virus PRCII. The P170 proteins of PRCIV and PRCII-p have an associated tyrosine-specific kinase activity similar to P105 of PRCII. All three agents, PRCII, PRCII-p, and PRCIV, belong to class II of avian sarcoma viruses. In cells transformed by PRCIV or PRCII-p secondary gag -linked transformation-specific proteins of less than 170,000 MW are sometimes seen, but limited in vivo and in vitro passage of the viruses shows the P170 to be a stable characteristic of PRCIV and PRCII-p transformation. The genomic RNA of PRCIV and PRCII-p sediments at 30 S corresponding to a size of about 6.1 kilobases.

Altered translation of the matrix genes in Niigata and Yamagata neurovirulent measles virus strains

Niigata and Yamagata strains measles virus were isolated from subacute sclerosing panencephalitis patients. These viruses were defective in virion production and expression of matrix (M) protein. The Niigata M protein-coding frame was interrupted by an in-frame termination codon, whereas the Yamagata M gene lacked the normal translational initiation codon. These mutations prevented translation of a normal M protein. However, RNA derived from the cloned Niigata and Yamagata M genes was translatable in vitro into low levels of aberrant proteins that reacted with M-specific antiserum. These proteins were also translated from poly(A)+ RNA from cells infected by Niigata and Yamagata virus strains. The aberrant M protein of Niigata virus was initiated at a downstream AUG codon created by a second mutation. The Yamagata M gene produced two aberrant proteins: one initiated mainly in vitro at an ACG codon, and a second species initiated at a downstream site both in vitro and in vivo. These results define the abnormal translational functions of the Niigata and Yamagata M genes, and further implicate the involvement of M protein defects in chronic central nervous system infections by measles virus.

Delayed activation of altered fusion glycoprotein in a chronic measles virus variant that causes subacute sclerosing panencephalitis

We compared the intracellular processing of the fusion (F) glycoproteins of an acute measles virus (MV) Nagahata strain and its relative Biken strain that caused subacute sclerosing panencephalitis (SSPE), Nagahata strain synthesizes a precursor F0 which acquires three asparagine (N)-linked oligosaccharide chains sequentially in 1 h. One oligosaccharide chain on the partially glycosylated F0 is less accessible to endo-beta-N-acetylglucosaminidase H (endo-H) but becomes accessible as the protein becomes fully glycosylated, suggesting a protein conformational change. Biken strain SSPE virus synthesizes a similarly glycosylated F0. However, one oligosaccharide chain on the Biken F0 remains less accessible to endo-H even after the protein is fully glycosylated. The Nagahata F0 is cleaved into the F1 and F2 subunits with a half life of 1 h. The Biken F0 is cleaved with a half life of 4 h. We cloned the F genes of Nagahata and Biken strains and showed by transfection that the defect causing delayed cleavage of F0 resides in the Biken F gene. Sequence analysis predicts a mutation in the cleavage recognition sequence, a truncated carboxyl-terminus, and multiple mutations in F1 of the Biken F protein. Expression of chimeric F genes showed the mutated cleavage recognition sequence and the carboxyl-terminal truncation do not delay cleavage of F0. Instead, delayed F0 cleavage is due to multiple mutations in the extracellular domain of F1, and four amino acid substitutions near the transmembrane region impair endo-H access to the oligosaccharide chain. These results provide detailed information on the normal maturation process of the F protein of MV and additional clues to the mechanisms of MV persistence in the CNS.

Avian sarcoma virus PRCII: Conditional mutants temperature sensitive in the maintenance of fibroblast transformation

Abstract Avian sarcoma virus PRCII was mutagenized with 5-azacytidine, and from single foci of transformed cells induced by this virus preparation three conditional mutants were isolated that are temperature sensitive for oncogenic transformation of fibroblasts. Two of these, LA 42 and LA 47, are not affected in their replication potential at the nonpermissive temperature (41.5°) nor are the virions heat labile. The third mutant, LA 46, is coordinately temperature sensitive in replication and transformation. A shift of the incubation temperature from 36° to 41.5° at any time after infection causes transformed cells by any one of the three mutants to revert to normal morphology.