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M Oie

Publications and source records attributed to M Oie.

32 records · Page 2Linked to original sources
PubMed1990

The function of the vaccinia hemagglutinin in the proteolytic activation of infectivity.

The vaccinia virus hemagglutinin (HA) has specific affinity for the structural protein, VP37K. The nature of this affinity and its relationship to the function of the HA were analyzed using HA mutants. The VP37K reactive site of the HA molecule is located in its transmembrane region, and the vaccinia virus HA associates with the viral particle via the VP37K-HA affinity. The viruses possessing an HA with fusion inhibitor activity were largely of the low infectivity form, whereas the viruses that associated mutant HAs defective in the activity were of the high infectivity form. D1 mutant virus does not produce HA. When it was incubated with the HA of the IHD-J strain, the HA associated with the virus particle. The HA-loaded D1 mutant virus acquired a high affinity not only for chick erythrocytes but also for KB and Vero cells. At the same time, the infectivity for Vero cells was decreased. The original high infectivity was recovered by treatment with trypsin. The virion-associated vaccinia HA has two functions; the HA protects the infectivity of the virus by the fusion inhibitor activity and exhibits affinity against host cells. Vaccinia virus first adsorbs to the cell via HA, and then proteolysis of the HA activates the second adsorption site which seems to be the fusogenic site of the virus. Proteolytic activation represents removal of the fusion inhibitor activity of the HA.

Adsorption↗
PubMed1990

Hemadsorption and fusion inhibition activities of hemagglutinin analyzed by vaccinia virus mutants.

Vaccinia virus IHD-J strain induces hemagglutinin (HA) on the surface membrane of infected cells and does not elicit cell-cell fusion (F-). We isolated 21 independent hemadsorption-negative (HAD-) mutant viruses from IHD-J and five HAD+ revertants from one of these mutants. Of the 21 mutants, 19 that synthesized either no or little HA at the cell surface caused cell-cell fusion (F+), whereas none of the five revertants that synthesized HA at the cell surface induced cell-cell fusion. Furthermore, anti-HA monoclonal antibody B2D10 induced extensive polykaryocytosis of IHD-J-infected cells and suppressed the ability of the IHD-J-infected cell extract to inhibit the polykaryocytosis induced by IHD-W. The other 2 of the 21 HAD- mutants, B1 and A2, which induced HAs at the cell surface, showed F- and F+ phenotype, respectively. The HA molecule of mutant B1 had a single amino acid substitution of Lys for Glu-121 in its extracellular domain, whereas that of mutant A2 had a single substitution mutation of Tyr for Cys-103. We conclude that the vaccinia HA is a fusion inhibition protein, that the active sites for the two activities reside separately in its extracellular domain, and that cysteine-103 is important in forming the proper tertiary structure of the protein to exert both activities.

Animals↗
PubMed1988

Epitope mosaic on the surface proteins of orthopoxviruses.

Epitopes on the surface components of orthopoxviruses were analyzed with monoclonal antibodies (MAbs) against monkeypox and vaccinia viruses by enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), radioimmunoprecipitation (RIP), and competitive binding inhibition assay (CBIA). When compared by ELISA, three vaccinia virus strains exhibited a similar reactivity to 99 tested MAbs despite their remote passage history. All five isolates of monkeypox virus closely resembled one another, irrespective of the host species (human, monkey, squirrel) from which they were isolated. Taterapox virus reacted similar to vaccinia virus against 97 of the 99 tested MAbs, and reacted with 2 MAbs which were cross-reactive with monkeypox and mousepox. Mousepox and cowpox viruses reacted with these MAbs in a species-specific manner: MAbs reactive to cowpox virus distinctly differ from those reactive to mousepox virus. Of the 99 tested MAbs, 32 reacted with all the 11 tested orthopoxviruses, indicating that the corresponding epitopes existed in all the viruses. Fifty-four MAbs reacted with two or more virus species and were classified as partially common MAbs. Eight MAbs were apparently type-specific for monkeypox, and five were specific for vaccinia and taterapox viruses. No strain-specific epitope was detected. Sera of monkeypox-infected patients, when analyzed by CBIA, interfered with the binding of monkeypox-specific MAb H12C1 but not of vaccinia-specific MAb G6C6. Sera of monkeypox-infected patients who had been vaccinated competed against both MAbs, demonstrating the original antigenic sin phenomenon. The two MAbs could distinguish between the sera of monkeypox patients and those of vaccinated persons. However, the serum of a smallpox patient was competitive against these apparently vaccinia- or monkeypox-specific MAbs. Three of the eight monkeypox-specific epitopes were recognized by the above CBIA test, which suggests that they also exist in smallpox virus. The mosaic-like combination of common epitopes and the small number of type-specific epitopes manifested the antigenic characteristics of orthopox viruses. The species boundary was obscured due to the partially common epitopes, but the total composition of epitopes was stable enough to maintain the antigenic species-specificity. The mutual relationship of the orthopoxviruses was visualized in a three-dimensional network.

Antibodies, Monoclonal↗
PubMed1987

Modification of vaccinia virus penetration proteins analyzed by monoclonal antibodies.

Modifications induced in structural vaccinia virus proteins that elicit the high infectious state by virus activating treatments involving trypsin and phosphatidylserine were analyzed using antivaccinia monoclonal antibodies (MABs). MABs reactive against each of the five outer layer proteins (VP54K, 34K, 32K, 29K, and 17K-25K) neutralized infectivity. VP54K possesses at least two neutralizing epitopes. Treatment with trypsin or with isolated plasma membrane cleaved VP54K into TVP41K carrying epitope A and removed a fragment containing epitope B from the virus. MABs against either of the epitopes could neutralize the virus. The exposure of epitope A concomitantly activated virus infectivity, and it was an essential step of penetration. MABs against VP17K-25K reacted more efficiently with trypsin-treated virus than with untreated virus, but the size of VP17K-25K was not affected by trypsin; this finding indicated that trypsin treatment rendered the VP17K-25K epitopes more accessible to antibody and hence to neutralization. MABs against VP32K and VP29K neutralized infectivity to the same extent irrespective of the state of activation. Virus treated with phosphatidylserine (PS) was neutralized more efficiently by MAB against VP34K than untreated virus, but the amount of antibody that reacted with the virus was the same before and after treatment with PS. Phosphatidylserine did not modify epitope structure itself, but it activated the function of VP34K. It was concluded that blocking of the functions attributed to any of the five proteins resulted in neutralization of virus infectivity, and treatment with trypsin and phosphatidylserine activates infectivity of vaccinia virus by modifying three of them (VP54K, VP34K, VP17K-25K) with characteristic behavior for each protein.

Antibodies, Monoclonal↗
PubMed1985

Reversible inactivation and reactivation of vaccinia virus by manipulation of viral lipid composition.

The role of phospholipids in vaccinia virus was investigated by substituting viral lipids with specific phospholipids. Treatment of virus with sodium dodecyl sulfate, sodium deoxycholate, or Nonidet-P40 (NP-40) resulted in almost complete removal of viral lipid and led to inactivation of the virus. The inactivation induced by the former two was irreversible, but NP-40-treated virus was reactivated upon reassociation with phospholipids. Individual phospholipids, including phosphatidylserine (PS), phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, lysolecithin, sphingomyelin, and acyl bis(monoacylglycero)phosphate (ABMP), were tested for ability to reactivate NP-40-treated virus. Reactivation was induced only by PS. The infectivity of virus that had been treated with NP-40 and then with PS was unstable; the reactivated virus was inactivated within a short period. It was also very sensitive to trypsin. Treatment of NP-40-treated virus with mixtures of PS and ABMP yielded virus that was more resistant to spontaneous and trypsin-induced inactivation. Thus, PS appears to be an essential for infectivity and ABMP appears to play a supplementary role for maintenance of infectivity, perhaps by protecting against inactivating factors.

Deoxycholic Acid↗
PubMed1984

Location of DNA-binding proteins and disulfide-linked proteins in vaccinia virus structural elements.

Treatment with sodium dodecyl sulfate (SDS) converted the vaccinia virus strain IHD-J into particles of two types: (i) ghosts which possessed a thin-membrane vesicle derived from basement part of the virus membrane with attached lateral bodies and a membranous structure derived from the core wall and (ii) aggregates of a DNA-nucleoprotein eluted from the core. These particles lacked lipids, and all the viral phospholipids were detected in the SDS-soluble fraction. The viral membrane was composed of an SDS-soluble coat layer and the basement membrane, and the basement membrane was maintained by a mechanism other than the lipid bilayer. By comparisons of protein species in morphologically distinct subviral particles prepared by several solubilizing methods, protein compositions of viral structural elements were suggested as follows: 25,000-molecular-weight viral protein-17,000-molecular-weight viral protein ( VP25K - VP17K ), viral basement membrane; VP13 . 8K , major component of the lateral body; VP70K , VP69K , VP66K , and VP64K , minor components of the lateral body; VP61K , outer layer of core wall; VP57K - VP22K , inner layer of core wall; and VP27K - VP13K , nucleoprotein. These structural elements found in the SDS-insoluble particles dissolved in the same SDS solution under reducing conditions, indicating that the disulfide linkages seem to have a principal role in maintaining their morphological integrity. VP57K , VP27K , VP13 . 8K , and VP13K were revealed to possess affinity for DNA. Denatured calf thymus DNA and viral DNA in double- or single-stranded form associated equally well with these proteins, but RNA did not bind. Therefore, it was strongly suggested that disulfide-linked VP27K - VP13K represented the nucleoproteins of vaccinia virus. A structural model of vaccinia virus is proposed and discussed.

Carcinoma↗
PubMed1983

The activation of vaccinia virus infectivity by the transfer of phosphatidylserine from the plasma membrane.

Purified vaccinia virus usually contains a large proportion of noninfectious virus which can be converted to infectious virus by incubation with purified plasma membrane. This activating reaction which is mediated by a heat stable component of the membrane has been studied. A suspension of liposomes containing the lipids extracted from plasma membrane of either KB cells or mouse RBCs activated the noninfectious virus in the same manner as heated plasma membrane. The phospholipid fraction of the KB cell lipids had the activating ability, but neither neutral lipid nor glycolipid fraction activated the virus. Liposomes containing phosphatidylserine activated the virus, whereas other tested phospholipids, including phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and sphingomyelin had no effect on virus infectivity. Lysolecithin reduced the infectivity. Treatment with isolated plasma membrane or liposomes increased hydrophobicity of the virus slightly, but did not change its density. Analysis of activated and then purified virus showed that all phospholipid species in the coincubated plasma membranes and liposome samples were transferred to the virus. The transfer was not a phospholipid exchange reaction but a one-way net transfer, and took place rapidly at 37 degrees to reach saturation within 1 hr of coincubation. Neither activation of virus nor transfer of phospholipid occurred when the mixture was incubated at a temperature below 8 degrees. The virus has great ability to extract phospholipids from coincubated lipid bilayer membranes, and association with phosphatidylserine gives the virus high infectivity.

Animals↗
PubMed1981

Target antigen of vaccinia-infected cells recognized by virus-specific cytotoxic T lymphocytes.

A vaccinia-specific target antigen for recognition of anti-vaccinia cytotoxic T lymphocytes (CTL) was found to be formed on the surface of infected cells through two distinct processes. In the first phase, the expression of the target antigen was dependent on the dose of inoculated virus, without specific protein synthesis. The target antigen seems to be produced by virus-cell fusion. In the second phase, the expression of the target antigen was accompanied by synthesis of an early protein. In spite of the difference in their mode of expression, the first-phase and the second-phase target antigens were cross-reactive in cytotoxicity inhibition assays. Cowpox virus, CPR Cl strain, brought about a lower response than vaccinia virus, IHD-J strain, in both sensitization of CTL and formation of CTL-susceptible cells at both the first and the second phase. The cross-reactive, but inefficient, recognition of anti-vaccinia CTL for cowpox-infected cells suggested a slight difference in the target antigens of the two viruses. Attempts to identify the target antigen were then made by comparing the polypeptide composition of vaccinia virus, cowpox virus, and their recombinants. SDS-PAGE analysis of trypsin-activated viruses revealed 44K (cowpox)/45K (vaccinia) polypeptides which corresponded to the difference in target cell formation. Trypsinization of the viruses also increased the ability of the virus to induce the production of CTL-susceptible target cells.

Animals↗