Jutta Seehafer

Synthesis of viral-specific polypeptides in Mengo virus-infected L cells: Evidence for asymmetric translation of the viral genome

Investigations of the synthesis of Mengo virus-specific polypeptides in L cells during the logarithmic phase of virus production have shown that, as is the case with other picornaviruses, the stable viral gene products are formed by posttranslational cleavage of larger primary proteins and that the pattern of cleavages by which they are produced is very similar to that proposed earlier for EMC virus (Butterworth et al., 1972b). However, analysis of the data suggests strongly that, on a molar basis, twice as much capsid as noncapsid viral protein is synthesized. This, in turn, suggests that translation of the viral genome is under some kind of control, the model most compatible with the data being one in which some viral protein(s) functions as termination factor(s).

PREPARATION OF FIBROBLAST-FREE CYTOTROPHOBLAST CULTURES UTILIZING DIFFERENTIAL EXPRESSION OF THE CD9 ANTIGEN

SummaryThe leukemia-associated antigen CD9 is present on a variety of normal cells, with apparent variable expression on normal human fibroblasts. In this study, we demonstrate by immunoperoxidase staining and direct binding studies that the CD9 antigen is uniformly expressed on normal human fibroblasts grown from first trimester and term placenta, embryonic fetal fibroblasts, and from human adult and fetal skin fibroblasts. Higher CD9 expression was present on fetal cells. CD9 antigen was not present on trophoblast. Over 99% of fibroblasts could be absorbed onto antibody to the CD9 antigen conjugated to magnetic beads. By applying this selective immunoadsorption of fibroblasts to term placental cytotrophoblast preparations, we demonstrated that fibroblast contamination could be nearly completely eliminated. This is a novel technique for purifying primary trophoblast cultures and may have wider applicability in cell culture of other cell types.

The functional cell surface glycoprotein CD9 is distinguished by being the major fatty acid acylated and a major iodinated cell-surface component of the human platelet

We showed that a 22 kDa protein (which comigrated with the leukocyte differentiation antigen CD9 as determined by immunoblotting with the platelet-activating mAb 50H.19) is a major iodinated component of the platelet surface. The iodinated protein was identified as CD9 by limited proteolysis analysis. The major acylated protein in platelets incubated with [3H]palmitic acid also had a mobility of 22 kDa. The radiolabelled fatty acid in CD9 appears to be ester bonded, as it is removed by treatment with hydroxylamine. Non-enzymatic ligation of the fatty acid is not involved. Since platelets lack protein synthetic capacity, the palmitolation of a surface protein indicates the existence of a plasma-membrane located transacylase which functions independently of protein synthesis. Limited proteolysis analysis of the palmitylated protein obtained by immunoprecipitation with mAb 50H.19 confirmed its identity as CD9. An additional novel minor component of 27 kDa was detected in platelets by immunoprecipitation of 125I-surface-labelled, or [3H]palmitic acid-labelled protein, and by immunoblotting with mAb 50H.19. The analogous cleavage patterns obtained by the limited proteolysis analysis of the 22, 24 and 27 kDa glycoproteins suggest that they may be differently modified variants of a single polypeptide.

The functional glycoprotein CD9 is variably acylated: localization of the variably acylated region to a membrane-associated peptide containing the binding site for the agonistic monoclonal antibody 50H.19

Recent studies have shown that [3H]palmitic acid strongly labels both glycosylated forms (gp22 and gp24) of the signal-initiating cell surface glycoprotein CD9. We performed a two-dimensional limited proteolysis analysis with Staphylococcus aureus V8 proteinase in order to localize the palmitylation sites to final peptides on both glycosylated forms of CD9. Analysis of [3H]leucine- and [3H]amino acid mixture-labeled gp22 delineated 4 final peptides of 11, 8, 7 and 4 kDa. gp24 produced a similar pattern with the exception that the 11 kDa peptide was replaced by an N-glycosylated 13 kDa peptide. Since all four final peptides (total molecular mass of 30/32 kDa) could not be accommodated by a parent molecule of 22/24 kDa, it is likely that one of the final peptide coexists in two differently modified states. Palmitic acid labeled the 11 kDa/13 kDa final peptides, and the 7 kDa final peptide, with equal intensity, but was not incorporated into the 4 kDa final peptide, demonstrating that fatty acid is ligated in two distinct regions of the molecule. The 8 kDa final peptide was strongly labeled by [3H]palmitic acid, but only weakly by [3H]leucine. We present evidence that this peptide is derived by further acylation of the region defined by the 7 kDa peptide, and that this occurs in only 15% of the molecules. Palmitic acid is turned over faster at these additional sites, indicating that they may be more accessible to membrane transacylases. Proteolysis of CD9 on the intact cell with papain enabled the highly acylated region to be localized to a membrane-associated fragment which contains the binding site for the agonistic monoclonal antibody 50H.19. The co-localization of a functional domain with a region of variable acylation suggests that acylation events may play a role in the transduction of the signal initiated by interaction of the antibody with CD9.

A comparative study of BK and polyoma viruses

Abstract A comparative study of BK and polyma (Py) viruses has shown that the molecular anatomy of the two virions is strikingly similar. The capsids of both are composed of 72 capsomeres arranged in a T = 7d icosahedral lattice. Analysis of the DNAs of the two viruses suggest that the full-length DNA of BK virus may be slightly smaller than that of Py virus. The two virions contain the same number of structural polypeptides, but the molecular mass of BK-VP1 is about 4000 daltons less, and those of BK-VP2 and 3 about 4000 daltons more than those of the corresponding Py polypeptides. The observation that the major capsid polypeptide, BK-VP1, is smaller than Py-VP1, is compatible with the observed difference in the diameters of the two virions (BK = 405 ± 10A; Py = 430 ± 15A). The two viruses differ sharply in the relative efficiencies with which they hemagglutinate guinea pig and human erythrocytes. BK virus, unlike Py virus, replicates only in certain human or monkey cells; and of the cells examined in this study, human fetal kidney cells are the most satisfactory for the propagation of this agent.

Isolation and characterization of BK virus-transformed hamster cells.

Abstract BK virus (BKV) was used to transform baby hamster kidney (HK) and hamster embryo fibroblast (HE) cells in culture. Six clones of each of the BKV-transformed HK and HE cells were isolated and characterized with respect to a number of biological properties. None of the cloned lines was found to produce infectious BKV, and all 12 lines were shown to contain BKVT antigen, to have a lower serum dependency for growth and to grow to higher saturation densities than the corresponding untransformed cells, to have higher plating efficiencies than control cells, to have acquired the ability to produce colonies in soft agar, and to produce progressively growing tumors when injected subcutaneously into weanling hamsters. Differences were found to exist among the cell lines of each group with respect to these parameters, and, in general, transformed HK cells have a lower serum dependency, grow to higher saturation densities, and have higher plating efficiencies and a greater capacity to produce colonies in soft agar than do the transformed HE cells. Infection of secondary cultures of HE cells with various input multiplicities of BKV showed that transformation is a multiplicity-dependent phenomenon and can be achieved with an input multiplicity as low as 3 PFU/cell.

Observations on the growth and plaque assay of BK virus in cultured human and monkey cells.

Although human embryo kidney (HEK), muscle (HEM) and lung (HEL) cells are capable of supporting the replication of BK virus (BKV) through passage levels 9, 12 and 12 respectively, only third, fourth and fifth passage level HEK cells were found to be satisfactory for the plaque assay of the virus. BSC-I and VERO cells can also be used for the plaque assay of BKV. However, in HEK cells plaques can be visualized in 20 days (compared to 28 days in BSC-I cells), and since in VERO cells the plaques are poorly defined and the titre about I log10 lower than in either HEK or BSC-I cells, HEK cells were the ones chosen for use.

Isolation and characterization of BK virus-transformed rat and mouse cells.

The isolation and characterization of four groups of BK virus (BKV)-transformed rat embryo fibroblast (RE) and mouse kidney (MK) cells are described. They consist of (1) seven RE lines transformed with a BKV pool containing a high proportion of defective virions, and (2) 16 RE, (3) 14 Balb/c-MK and (4) 2 Swiss ICR-MK lines, all transformed, at different input multiplicities, with a pool of BKV free of defective virions. None of the lines produces BKV, all contain BKV T antigen and all grow to higher saturation densities and have higher plating efficiencies than do the corresponding control cells. Cells of the RE lines, transformed with the BKV pool containing defective virions, form colonies in soft agar and produce tumours in irradiated weanling rats, while those of the RE lines transformed with the defective virion-free pool do neither. Cells of the Balb/c-MK, but not of the ICR-MK lines are tumorigenic, although cells of both groups form colonies in soft agar. In general, those lines transformed at higher multiplicities express the biological properties associated with transformation more strongly than do those transformed at lower multiplicities.

Myristic acid is incorporates into the two acylatable domains of the functional glycoprotein CD9 in ester, but not in amide bonds

CD9 is a signal-initiating glycoprotein of uncertain membrane insertion which contains more than one locus of acylation and is distinguished by being the major acylatable platelet protein. The N-terminus of CD9 is blocked to Edman degradation. We investigated whether [3H]myristic acid could be incorporated into CD9, whether that incorporation occurred via an amide linkage, and whether myristate and palmitate were differentially incorporated into the two domains. Pulse-labeling studies, performed on the human osteogenic sarcoma cell line SKOSC which expresses 22 and 24 kDa variants of CD9 demonstrated that the respective precursors of 20.5 and 23 kDa were not radiolabeled by either [3H]myristic acid or [3H]palmitic acid, but that both fatty acids could be ligated to CD9 during the later stages of protein maturation. The failure to incorporate myristic acid cotranslationally suggest that CD9 does not contain amino-terminal amide-bonded myristic acid. Incorporation of radiolabel from both fatty acids proceeded very rapidly and could be visualized after a 10 s pulse. Although myristic acid was partially metabolized into palmitic acid, incorporation of authentic [3H]myristate into CD9 could be demonstrated. The myristic acid bonds were shown to be as sensitive to hydroxylamine treatment as those linking palmitate. Both fatty acids were also incorporated into CD9 in hydroxylamine-sensitive bonds in the presence of cycloheximide, reaching 30-40% of the levels in untreated controls. The sensitivity of myristate ligands to hydroxylamine demonstrates that this fatty acid is not linked via amide, but rather via ester bonds. The sensitivity of [3H]myristate and [3H]palmitate bonds to 2-mercaptoethanol further suggests that either fatty acid is linked via thioester rather than hydroxyester bonds to each domain on CD9. Limited proteolysis analysis with Staphylococcus aureus V8 proteinase of CD9, labeled in the absence or presence of cycloheximide, showed that [3H]myristic acid and [3H]palmitic acid labeled identical peptides, and to the same extent, suggesting that myristate is an alternative substrate for the transacylase(s) involved.

Evidence for the expression of TSTA in BKV-transformed cells: Cross-reaction with SV40 TSTA

Abstract BALB/c mice which had received two intraperitoneal injections of BK virus-transformed mouse, rat, and hamster cells (killed by exposure to γ irradiation) were shown to be resistant to subsequent challenge with cells of the syngeneic, SV40-transformed line, mKSA-ASC. The degree of resistance acquired by the mice differed sharply depending on the line used for immunization, with resistance indices ranging from 0 to >6000. A low but significant level of resistance was induced by the injection of BKV, but none was induced by the injection of either polyoma virus-transformed cells or untransformed mouse, rat, or hamster cells. The data show that BKV-transformed cells contain a tumor-specific transplantation antigen (TSTA) which is closely related immunologically to the SV40 TSTA.