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T C Wong

Publications and source records attributed to T C Wong.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of a cytoplasmic Tyr-X-X-Leu motif essential for down regulation of the human cell receptor CD46 in persistent measles virus infection.

To investigate the sequence requirements for measles virus (MV)-induced receptor down regulation, we transfected the human CD46 gene into simian cells persistently infected by the Biken strain of MV. Surface expression of CD46 is drastically reduced in these cells. Deletion analysis has shown that the juxtamembrane region of the CD46 cytoplasmic domain is essential for down regulation. Deleting a Tyr-Arg-Tyr-Leu sequence in this region or changing these residues to Ala prevents CD46 down regulation from the infected cell surface. Alanine-scanning mutagenesis has identified two amino acid residues, Tyr and Leu, forming a Tyr-X-X-Leu motif critical for CD46 down regulation. Mutations that prevent CD46 down regulation enhance syncytium formation. These results indicate that CD46 down regulation limits the cytopathic effects in a persistent MV infection and that CD46 down regulation requires a cytoplasmic Tyr-X-X-Leu sequence which resembles known motifs for membrane protein trafficking and receptor signalling.

Alanine↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1995

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.

Cells, Cultured↗
PubMed1995

Engineered serine protease inhibitor prevents furin-catalyzed activation of the fusion glycoprotein and production of infectious measles virus.

We have identified the major cellular endoprotease that activates the fusion (F) glycoprotein of measles virus (MV) and have engineered a serine protease inhibitor (serpin) to target the endoprotease and inhibit the production of infectious MV. The F-protein precursor of MV was not cleaved efficiently into the mature F protein in human colon carcinoma cells lacking functional furin, indicating that furin is the major enzyme responsible for activation of the MV F protein. A human serpin alpha 1-antitrypsin variant was engineered to specifically inhibit furin. When expressed from a recombinant vaccinia virus in primate cells infected by MV, the engineered serpin (alpha 1-PDX) specifically inhibited furin-catalyzed cleavage of the F-protein precursor without affecting synthesis of other MV proteins. We generated human glioma cells stably expressing alpha 1-PDX. MV infection in these cells did not result in syncytia. The infected cells produced all the MV proteins, but the F-protein precursor remained largely uncleaved. This did not prevent virus assembly. However, the released virions contained inactive F-protein precursor rather than mature F protein, and infectious-virus titers were reduced by 3 to 4 orders of magnitude. These results show that a mature F protein is not required for the assembly of MV but is crucial for virus infectivity. The engineered serpin may offer a novel molecular antiviral approach against MV.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Isolation and characterization of intranuclear ribonucleoprotein complexes associated with double-stranded RNA adenosine deaminase from brain cells: implications for RNA-editing and hypermutation of viral RNA in the CNS.

Double-stranded RNA adenosine deaminase (DsRAD), which converts adenosine in duplex RNA to inosine, has been implicated in editing of cellular mRNA and hypermutation of viral RNA in the central nervous system (CNS). We used subcellular fractionation to show that DsRAD in bovine brain tissues is associated with high-molecular-weight ribonucleoprotein (RNP) complexes in the nuclei. DsRAD-associated RNP complexes have apparent molecular mass of up to 500 kDa and buoyant density of 1.35 to 1.42 g cc-1 in CsCl solution. In human glioma cells, DsRAD is also found exclusively in intranuclear RNP complexes that co-sediment with the largest RNA species. These DsRAD-associated RNP complexes are dissociated by RNase A or high salt. The RNA component is not essential for DsRAD activity, and the protein component can be separated by dsRNA-affinity column, gel filtration column, and glycerol gradient into enzymatically active protein species with apparent molecular mass ranging from 120 kDa to 70 kDa in polyacrylamide gel. The bovine brain DsRAD has no apparent requirement for low-molecular-weight cofactors or metal ions. These results provide insight into the native state of DsRAD in brain cells and have interesting implications for its putative roles in RNA-editing and hypermutation of viral RNA in the CNS.

Adenosine Deaminase↗
PubMed1994

Solution conformation of a cyclic neurokinin antagonist: a NMR and molecular dynamics study.

The solution structure of a hexapeptide, cyclo (Gln-Trp-Phe-Gly-Leu-Met), which is a selective NK-2 antagonist, has been studied by a combination of two-dimensional nmr and molecular dynamics (MD) techniques. The simulation based on nmr and MD data resulted in the convergence to a family of structures. Free molecular dynamics for 50 ps in the presence of DMSO solvent molecules shows that the structure is energetically stable. One intramolecular hydrogen bond between the amide proton of Gln and the carbonyl oxygen of Gly was revealed. This result is consistent with the results from the measurement of the temperature coefficient of the amide protons. The extent of intermolecular hydrogen bonding between the amide protons of the peptide and DMSO was also revealed by the free MD simulation. The resulting structure of the cyclic peptide contains a variation type I' beta-turn in the Gly-Leu-Met-Gln segment. Comparison of the structure of this peptide with that of other NK-2 antagonist cyclic hexapeptides was made, and the activity of cyclic antagonists appears to be inversely related to the conformational rigidity of the cyclic peptides.

Amino Acid Sequence↗
PubMed1994

1H- and 13C-nmr assignments and structural determination of a novel glycoalkaloid from Solanum platanifolium.

Four steroidal glycoalkaloids, solasonine [2], solamargine [3], khasianine [4], and ravifoline [5], with solasodine [1] as the aglycone moiety, have been isolated from Solanum platanifolium. The present study involved the characterization of the novel compound 5 by one- and two-dimensional nmr techniques and has also led to the unambiguous and total 13C- and 1H-nmr assignments of all of these steroidal glycoalkaloids and the direct elucidation of their glycosidic linkages.

Carbohydrate Sequence↗
PubMed1993

Solution conformation study of substance P methyl ester and [Nle10]-neurokinin A (4-10) by NMR spectroscopy.

High-resolution proton spectra at 500 MHz of two tachykinin peptides, substance P methyl ester (SPOMe) and [Nle10]-neurokinin A (4-10), have been obtained in dimethylsulfoxide (DMSO), and for SPOMe, also in 2,2,2-trifluoroethanol (TFE)/water mixtures. Complete chemical shift assignments for these peptides were made based on two-dimensional (2D) nmr techniques, correlated spectroscopy and total COSY. J coupling measurement and nuclear Overhauser effect spectroscopy (NOESY) were then used to determine the conformation of these peptides in the various solvents. Based on the J coupling, NOE correlations, and temperature coefficients of the NH resonances, it is concluded that these two peptides exist in DMSO at room temperature as a mixture of conformers that are primarily extended. For SPOMe in TFE/water with high TFE content, however, helical structures are found to be present, and they become quite clear at temperatures between 270 and 280 K. The variation of the 13C chemical shifts of the C alpha (the secondary shift) with TFE contents corroborates this conclusion. The NOE and C alpha shifts show that the main helical region for SPOMe lies between 4P and 9G. The C-terminus segment L-M-NH2 is found to be quite flexible, which appears to be quite common for neurokinin-1 selective peptides.

Amino Acid Sequence↗
PubMed1993

Conformational study of two substance P hexapeptides by two-dimensional NMR.

The conformation of two substance P (SP) related hexapeptides. Glp-Phe-Phe-(L-Pro)-Leu-Met.NH2 (I) and Glp-Phe-Phe-(D-Pro)-Leu-Met.NH2 (II), in two solvents, chloroform-d and trifluoroethanol(TFE)-d3/H2O, was studied by two-dimensional NMR methods, including COSY, TOCSY, ROESY and HMQC. The study shows that these two peptides exist predominantly in the extended form in TFE/H2O, but in general exhibit a reverse-turn structure in chloroform. I is clearly less ordered than II in both solvents. Furthermore, extensive Phe3-Pro4 cis<==>trans isomerization was found in I but not in II. The differences in the conformational behavior of these two peptides, which are selective agonists for neurokinin NK1 and NK2 receptors, respectively, are discussed.

Amino Acid Sequence↗
PubMed1993

Functional analysis of matrix proteins expressed from cloned genes of measles virus variants that cause subacute sclerosing panencephalitis reveals a common defect in nucleocapsid binding.

We have developed an in vitro nucleocapsid-binding assay for studying the function of the matrix (M) protein of measles virus (MV) (A. Hirano, A. H. Wang, A. F. Gombart, and T. C. Wong, Proc. Natl. Acad. Sci. USA, 89:8745-8749, 1992). In this communication we show that the M proteins of three MV strains that cause acute infection (Nagahata, Edmonston, and YN) bind efficiently to the viral nucleocapsids whereas the M proteins of four MV strains isolated from patients with subacute sclerosing panencephalitis (SSPE) (Biken, IP-3, Niigata, and Yamagata) fail to bind to the viral nucleocapsids. MV Biken (an SSPE-related virus) produces variant M sequences which encode two antigenically distinct forms of M protein. A serine-versus-leucine difference is responsible for the antigenic variation. MV IP-3 (an SSPE-related virus) also produces variant M sequences, some of which have been postulated to encode a functional M protein responsible for the production of an infectious revertant virus. However, the variant M proteins of Biken and IP-3 strains show no nucleocapsid-binding activity. These results demonstrate that the nucleocapsid-binding function is conserved in the M proteins of MV strains that cause acute infection and that the M proteins of MV strains that cause SSPE exhibit a common defect in this function. Analysis of chimeric M proteins indicates that mutations in the amino-terminal, carboxy-proximal, or carboxy-terminal region of the M protein all abrogate nucleocapsid binding, suggesting that the M protein conformation is important for interaction with the viral nucleocapsid.

Antigens, Viral↗
PubMed1993

Conformational maturation of measles virus nucleocapsid protein.

We have obtained a polyclonal antiserum, N-BE, against the denatured, amino-terminal half of the measles virus (MV) nucleocapsid (N) protein and a monoclonal antibody (MAb), N46, which recognizes a conformation-dependent epitope in the same region. Amino acid residues 23 to 239 were required and sufficient for the formation of the conformational epitope. Using these antibodies, we show that the N protein of MV is synthesized as a relatively unfolded protein which first appears in the free-protein pool. This nascent N protein undergoes a conformational change into a more folded mature form. This change does not require the participation of other viral proteins or genomic RNA. The mature N protein does not accumulate in the free-protein pool but is quickly and selectively incorporated into the viral nucleocapsids. The mature N protein is a target for interaction with the phosphoprotein (P protein) of MV. This interaction interferes with the recognition of the N protein by the N46 MAb. This suggests that the association with the P protein may mask the binding site for the N46 MAb or that it induces a conformational change in the N protein.

Antibodies, Monoclonal↗
PubMed1992

The matrix proteins of neurovirulent subacute sclerosing panencephalitis virus and its acute measles virus progenitor are functionally different.

Persistence of measles virus in the brains of patients with subacute sclerosing panencephalitis (SSPE) is accompanied by changes in the viral matrix (M) protein. To understand the significance of these changes, cell culture and cell-free assays were developed to compare the functions of the M proteins of an SSPE virus Biken strain and its acute measles virus progenitor Nagahata strain. The Nagahata viral M protein is associated with the intracellular viral nucleocapsids and the plasma membrane, whereas the Biken viral M protein is localized mainly in the cytosol. The lack of M protein in the Biken viral nucleocapsids is due to a failure of the Biken M protein to bind to the viral nucleocapsids. The Biken M protein also fails to bind to the Nagahata viral nucleocapsids. Conversely, the Nagahata M protein can bind to the Biken viral nucleocapsids, although this association is not as stable at physiological salt concentration. These results offer concrete evidence that the M protein of an SSPE virus is functionally different from that of its progenitor acute measles virus.

Capsid↗
PubMed1992

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.

Animals↗
PubMed1992

Irreversible modification of measles virus RNA in vitro by nuclear RNA-unwinding activity in human neuroblastoma cells.

The matrix (M) gene of a measles virus (MV) variant passaged in IMR-32 human neuroblastoma cells displays numerous uridine-to-cytosine transitions called biased hypermutation. Using an in vitro assay, we show that IMR-32 cells contain high levels of an activity which unwinds and irreversibly alters the base pairing of double-stranded RNA synthesized from the M gene of MV. This activity is found exclusively in the cellular nucleus and is present at a lower level in African green monkey kidney Vero cells. Experiments with mixed cell extracts suggest that the low activity in Vero cells is not due to inhibitory factors. These findings support the hypothesis that this RNA-modifying and -unwinding activity is responsible for biased hypermutation of MV strains that infect the central nervous system. Possible functions of this neural cell activity and implications for central nervous system disorders are discussed.

Animals↗
PubMed1991

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.

Animals↗