Uninfected Fibroblasts Research Articles

Target platelet antigen in homosexual men with immune thrombocytopenia.

A syndrome of isolated immune thrombocytopenic purpura (ITP) has recently been described in homosexual men. We have identified an antiplatelet antibody in the serum of 29 of 30 homosexual men with isolated ITP. The antibody binds to a platelet membrane antigen of 25,000 daltons, and binding is effected by the F(ab)2 portion of the immunoglobulin. Similar antibody activity was not detected in serum from 30 nonhomosexual patients with either ITP or nonimmune thrombocytopenia. The 25,000-dalton antigen was not found on other hematopoietic cells, and it was distinct from the core protein of the AIDS-associated retrovirus. In contrast, serum antibody reacted with a 25,000-dalton antigen associated with cultured herpes simplex virus Types I and II. In these experiments the antigen appeared to be derived from green-monkey kidney cells in which the herpes simplex viruses were grown. Identical antigenic activity was also demonstrated in uninfected human skin fibroblasts. We conclude that ITP in homosexual men is accompanied by a serum antibody directed against a platelet antigen of 25,000 daltons. The nature of the antigen and the relation of the serum antibody to ITP require further study.

Lysis of human cytomegalovirus infected fibroblasts by natural killer cells: demonstration of an interferon-independent component requiring expression of early viral proteins and characterization of effector cells.

We investigated the susceptibility of cells infected with human cytomegalovirus (HCMV) to lysis by human natural killer (NK) cells, examining in particular its relationship to sequential viral protein expression, interferon (IFN), and the nature of the effector cells. HCMV-infected fibroblasts were lysed by peripheral blood mononuclear cells from normal seronegative individuals. The effector cells were large granular lymphocytes of Leu-7+, Leu-11+, and to a lesser extent Leu-7- phenotype. Depletion studies suggested they were the same population of NK cells that lyse uninfected fibroblasts, but a subset of NK cells that lyse K562 cells. HCMV-infected cells treated with phosphonoformate and cells infected for 16 hr that only express the nonstructural HCMV immediate early and early proteins and not the late (structural) proteins were susceptible to lysis by IFN-pretreated effector cells, whereas cells expressing immediate early antigens alone were not. This enhanced susceptibility to lysis was associated with increased effector:target binding in target cell binding assays, and was competitively inhibited by uninfected fibroblasts in cold target competition assays. It was independent of IFN release from the infected target cells or effector cells. These results suggest that the increased susceptibility to lysis by NK cells produced by a human herpes virus HCMV i) is manifest when early viral proteins are expressed, ii) is related to enhanced expression of a target structure likely to be present on uninfected fibroblasts, and iii) has a major component that is independent of IFN.

Nucleoside pools of acyclovir-treated herpes simplex type 1 infected cells

Nucleoside pools of herpes simplex type 1 (HSV-1)-infected and uninfected African green monkey kidney (GMK) cells and human fetal lung fibroblasts (HL) have been analysed with high-performance liquid chromatography (HPLC). The only nucleosides found in measurable amounts were deoxythymidine (dThd) and adenosine (Ado). The dThd pool seemed to be greater in GMK cells than in HL cells. dThd was also the only nucleoside excreted into the medium. HSV-1 infection reduced the dThd concentration of GMK cells. Addition of acyclovir (ACV) to HSV-1-infected GMK cells inhibited virus replication. This resulted in a dThd concentration similar to that of uninfected GMK cells. dThd added to HSV-1-infected GMK and HL cells reduced the antiviral action of ACV but not that of phosphonoformic acid (PFA). ACV is known to be activated mainly by HSV-induced deoxythymidine kinase (dTK), an enzyme which utilizes dThd as a substrate, while the action of PFA is independent of dTK. The low antiviral activity of ACV in GMK cells as compared to HL cells may be explained by the presence of high amounts of dThd in GMK cells.

Natural killer cell activity in infants and children excreting cytomegalovirus.

Natural killer cell (NKC) activity against uninfected and cytomegalovirus (CMV)-infected fibroblasts and K562 cells was assessed in 39 infants and children excreting CMV and 89 controls. NKC activity in cord blood specimens, healthy donors up to six months old, and donors older than six months showed significant increases (P less than .02) with age. NKC activities of CMV excreters correlated with acute disease characterized by interstitial pneumonitis (IP) and showed differential responses to the three target cells. CMV excreters younger than six months with IP showed greater mean percentage lysis (MPL; 36.6% +/- 10.5%) of CMV-infected cells than did those without pneumonitis (25.9% +/- 10%) or controls (22.1% +/- 8.0%). Excreters older than six months with IP showed activities comparable to controls; NKC activities of excreters without IP were depressed. Significantly elevated activity against uninfected fibroblasts characterized patients with IP regardless of age (MPL, 30.2% +/- 7.3% vs. 4.2% +/- 2.0% for healthy controls and 6.3% +/- 5.4% for excreters without IP). Increased cytolysis of uninfected fibroblasts was therefore associated with more active disease.

Human polymorphonuclear leukocyte-mediated cytotoxicity against varicella-zoster virus-infected fibroblasts.

Polymorphonuclear leukocytes (PMN) were studied for their ability to mediate cytotoxicity against varicella-zoster virus (VZV)-infected and uninfected human fibroblasts in 51Cr release assays. PMN were capable of mediating antibody-dependent cellular cytotoxicity (ADCC) against VZV-infected targets. Maximal ADCC was obtained with effector-to-target ratios of 100:1 and 18 h of incubation. Percent 51Cr release for 26 normal adults was 14.1 +/- 0.6 (mean +/- standard error) in the presence of pooled human seropositive sera (final dilution, 1:100) and 0.5 +/- 0.6 in the presence of pooled human seronegative sera. Addition of phorbol myristate acetate (PMA) enhanced PMN-mediated cytotoxicity against VZV-infected and uninfected targets. PMA-stimulated cytotoxicity was optimal with PMA concentrations of 200 ng/ml and effector-to-target ratios of 10:1, and antibody was not required; killing was detected as early as 3 h after incubation and was maximal after 18 h. Highly purified PMN were capable of mediating both ADCC and PMA-stimulated lysis. Catalase completely inhibited PMA-stimulated PMN cytotoxicity, but had no effect on PMN-mediated ADCC. PMN from patients with chronic granulomatous disease were capable of mediating ADCC, but not PMA-stimulated killing, against VZV-infected targets. Thus, PMN could kill VZV-infected targets by two different mechanisms: ADCC, which required antibody but not hydrogen peroxide (H2O2), and PMA-stimulated cytotoxicity, which required H2O2 but not antibody.

Expression of the chicken c-src gene in COS cells.

The cellular homolog of the Rous sarcoma virus transforming gene (v-src) was cloned into a plasmid containing the simian virus 40 origin of replication and transcriptional signals. This recombinant plasmid, designated pSVOHCS11 , directs the synthesis of relatively high levels of c-src mRNA and c-src protein ( pp60c -src), when the plasmid is studied 48 to 72 h after calcium phosphate-mediated DNA transfection of COS (monkey) cells. The level of c-src mRNA synthesis is 50-fold higher than the amount of c-src RNA produced in uninfected chicken embryo fibroblasts. Furthermore, the level of pp60c -src expressed in pSVOHCS11 -transfected COS cells is approximately the same as that of pp60v -src in Rous sarcoma virus-transformed cells. Using this recombinant plasmid, we demonstrated that c-src mRNA contains sequences which map 3' to the previously identified c-src-v-src regions of homology. In view of the small amount of c-src mRNA and protein that can be isolated from uninfected cells, this transient expression system offers a convenient source of material for further analyses of the c-src gene product.

Expression of the chicken c-src gene in COS cells.

The cellular homolog of the Rous sarcoma virus transforming gene (v-src) was cloned into a plasmid containing the simian virus 40 origin of replication and transcriptional signals. This recombinant plasmid, designated pSVOHCS11 , directs the synthesis of relatively high levels of c-src mRNA and c-src protein ( pp60c -src), when the plasmid is studied 48 to 72 h after calcium phosphate-mediated DNA transfection of COS (monkey) cells. The level of c-src mRNA synthesis is 50-fold higher than the amount of c-src RNA produced in uninfected chicken embryo fibroblasts. Furthermore, the level of pp60c -src expressed in pSVOHCS11 -transfected COS cells is approximately the same as that of pp60v -src in Rous sarcoma virus-transformed cells. Using this recombinant plasmid, we demonstrated that c-src mRNA contains sequences which map 3' to the previously identified c-src-v-src regions of homology. In view of the small amount of c-src mRNA and protein that can be isolated from uninfected cells, this transient expression system offers a convenient source of material for further analyses of the c-src gene product.

In vitro mouse cytomegalovirus infection of mouse tracheal epithelial cells requires the presence of other cell types.

Recently, methods have been developed to culture dissociated tracheal epithelial (TE) cells. Attempts were made to infect these epithelial cells with mouse cytomegalovirus (MCMV) to see if the dissociated epithelial cells share characteristics of infection with MCMV-infected tracheal organ cultures. When isolated TE cells were incubated with MCMV at multiplicities between 0.10 and 2.0, no infection or minimal infection resulted. Centrifugation of virus onto the TE cell sheets also resulted in only minimal infection. If MCMV-infected mouse embryo fibroblasts were added to cultures of TE cells, however, the TE cells subsequently became infected. TE cells could also be infected by incubating MCMV with mixed cultures of uninfected fibroblasts and TE cells. The epithelial nature of infected cells was confirmed by electron microscopy. Reconstruction experiments demonstrated that fibroblast-mediated infection of mouse TE cells with MCMV was not simply due to the large amounts of virus provided by the infected fibroblasts. It is suggested that cell-cell contact, or fusion with infected cells is required for productive infection of TE cells with MCMV.

Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses.

A cellular protein of 36,000 daltons becomes phosphorylated at tyrosine in chicken embryo fibroblasts transformed with avian sarcoma viruses. We have used cellular fractionation and immunofluorescence to locate the 36-kdalton protein in virus-transformed and uninfected chicken fibroblasts. The 36-kdalton protein in transformed cells fractionated mainly with high-speed particulate material, and in density gradient separations, the 36-kdalton protein was found in association with light density membranes together with most of the plasma membrane marker. Increasing the concentration of salt or adding ion chelators solubilized some of the 36-kdalton protein that otherwise was pelletable with high g forces. Based on these data, we conclude that this protein is peripherally or indirectly attached to light density membranes, including plasma membranes. Indirect immunofluorescent staining of the 36-kdalton protein in fixed cells revealed that it was located inside the cell in an extensive reticulum apposed to surface membranes. The same pattern of staining was found in both uninfected and virus-transformed cells. Pretreatment of cells with nonionic detergents before fixation altered or abolished 36-kdalton staining. The 36-kdalton protein appeared to be excluded from regions of the cells where actin cables were present. The pattern of staining observed with the anti-36-kdalton antibody was similar, but not identical, to that observed with antiserum against nonerythroid spectrin. Thus, the data obtained by biochemical fractionation and by immunofluorescent staining indicate that the 36-kdalton protein is found in a reticulum at the inner surface of the plasma membrane, possibly in association with cytoskeletal proteins.

Efficacy and selectivity of some nucleoside analogs as anti-human cytomegalovirus agents.

1-(2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluridine (FMAU), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouridine (FIAU), and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluridine (FEAU) were evaluated for antiviral activities against human cytomegalovirus (HCMV) and compared with 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and E-5-(2'-bromovinyl)-2'-deoxyuridine (BVDU). The relative anti-HCMV potencies of these compounds, as determined by calculating the dose of drug which inhibited 50% plaque formation, were in order of decreasing potency: FIAC greater than FIAU greater than FMAU greater than acyclovir greater than FEAU greater than BVDU. The antiviral activity of FIAC occurred at levels much lower than those that caused cytotoxic or cytostatic effects in uninfected fibroblasts. Neither thymidine nor deoxycytidine reversed the anti-HCMV activity of FIAC, indicating that this drug was not acting as an analog of the natural nucleosides. FIAC was not phosphorylated by cytosols of HCMV-infected cells to a greater extent that by those of uninfected cells, indicating that, unlike the antiviral activity against herpes simplex virus type 1, the selectivity of this drug is probably not based on a virus-specified pyrimidine kinase.

Modulation of plasminogen activator activity in human skin fibroblasts infected with mycoplasmas.

Secreted and cell-associated plasminogen activator was measured in human skin fibroblasts infected with mycoplasmas. The infected fibroblasts exhibited significantly higher levels of secreted and cell-associated plasminogen activator activity compared to uninfected skin fibroblasts. Subconfluent infected fibroblasts had increased plasminogen activator activity compared to confluent infected fibroblasts. Plasminogen activator may be important in tissue destruction in disease states in which mycoplasma infection has been implicated.

Transformation-defective Rous sarcoma virus mutants with altered p19 of the gag gene and their inhibitory effect on host cell growth.

Mutants (PH2010, PH2011, PH2012) of Rous sarcoma virus which have a growth-inhibitory effect on chicken embryo fibroblasts were isolated from a temperature-sensitive mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (tsNY68). The growth rate of fibroblasts infected with these viruses was about 50 to 60% of that of uninfected fibroblasts. A morphological difference between mutant-infected and uninfected fibroblasts was observed at logarithmic phase but not at stationary phase. Neither the protein p60src nor its associated protein kinase activity was significantly detected by an immunoprecipitation assay in the cells infected with these mutants. Analysis of the unintegrated DNA of the mutant PH2010 showed that a sequence of about 1.4 kilobase pairs at the src gene region is deleted. Further examination of the viral structural proteins in infected cells as well as in virions by immunoprecipitation and peptide mapping revealed that the molecular size of the Pr76 gag protein of the mutant RSV is smaller than that of the mutant tsNY68 because of partial deletion at the p19 gag gene. The peptide maps suggest that the deleted region of the altered p19 of the mutant is near the carboxy terminal of p19. The amount of Prgp92env synthesized in the mutant-infected cells was about fivefold more than that in tsNY68-infected cells.

Heterogeneity of human NK cells: comparison of effectors that lyse HSV-1-infected fibroblasts and K562 erythroleukemia targets.

In this study, we compared the natural killer (NK) cells that lyse HSV-1-infected NK(HSV-1) or uninfected [NK-(FS)] fibroblasts to those that lyse K562 erythroleukemia cells [NK(K562)]. Activity against all three targets was found in Percoll gradient fractions enriched for large granular lymphocytes, which suggests that these effector cells have a common morphology. In competition studies between 51Cr-labeled targets and unlabeled targets, both the infected and the uninfected fibroblasts competed for lysis of NK(HSV-1) and NK(FS) activity, whereas K562 cells competed poorly. In contrast, when 51Cr-labeled K562 cells were used, the unlabeled K562 cells competed well, but HSV-1-infected and uninfected fibroblasts competed poorly. Panning studies and complement elimination experiments using monoclonal antibodies were performed to describe cell surface markers on the NK cell populations. Treatment with an antibody to an la framework antigen reduced NK(HSV-1) but not NK(K562) activity. In contrast, the majority of NK(K562) effectors were recognized by antibodies to the E-rosette receptor (Lyt-3 and OKT11A), whereas NK(HSV-1) activity was much less sensitive to this antibody. OKM1, OKT10, and Leu-7 (HNK-1) markers were found on a portion, but not all, of the cells that lysed both the HSV-FS and K562 targets, while treatment with HLA plus complement totally abrogated both NK activities. Taken together, these data are consistent with the concept that human NK cells are heterogeneous and that we are dealing with at least three subpopulations of effector cells--one that kills the infected or uninfected fibroblasts; one that kills K562 cells; and a third population that may be able to kill all three targets. Patient studies provide additional evidence for heterogeneity within the NK cells that lyse the fibroblasts and K562 cells. We have studied a number of individuals who have normal NK activity with one target (HSV-FS or K562) but have low or no activity against the other. These patients provide strong evidence not only that NK cells are heterogeneous but also that these NK subpopulations can be regulated independently of each other in vivo.

Transformation-sensitive protein associated with the cell substratum of chicken embryo fibroblasts.

The interaction of cultured cells with their growth substrata has been studied as a function of oncogenic transformation by using chicken embryo fibroblasts infected with the temperature-sensitive mutant of Rous sarcoma virus, LA24, and grown in plastic culture dishes. In comparison to total cell fractions, substratum-associated material (SAM), prepared by EGTA release of transforming cells from culture dishes, is enriched in a 21-kilodalton (kDal) protein. Synthesis and deposition of this protein in SAM are stimulated within hours of transfer of cells to the permissive temperature (35 degrees C), peak around 8 hr, and decline to levels 1.3-fold higher than those of controls at 41 degrees C by 20 hr after the temperature shift. In contrast, incorporation of (3)H-labeled amino acids into newly synthesized fibronectin in SAM is not significantly influenced by the transformation process during this time. Furthermore, although the presence of fibronectin in SAM is influenced by cell density, the 21-kDal protein is increased in SAM of transforming cells at all densities examined. The 21-kDal protein is not present in increased amounts in SAM from normal, uninfected chicken embryo fibroblasts grown at 41 degrees C and 35 degrees C or from cells infected with the wild-type Rous sarcoma virus (Prague A), which are fully transformed. It is not a mannose-containing glycoprotein and does not appear to be phosphorylated. Furthermore, it is not a product of normal cell protein degradation induced by transformation but results from de novo protein synthesis after shift of LA24-infected cells to the permissive temperature. Finally, turnover of the 21-kDal protein is slower at 35 degrees C than at 41 degrees C. This amplifies the effect of increased synthesis and results in a net accumulation in SAM during the early stages of transformation.

The cytoskeleton of murine leukemia virus (MuLV)-infected mouse fibroblasts as observed under varying conditions of formaldehyde fixation.

Mouse fibroblasts chronically infected with Moloney murine leukemia virus (MuLV) were fixed using variable amounts of formaldehyde, then examined by indirect immunofluorescence light microscopy. Several antisera were employed to detect both external and internal antigens associated with the cells, eg, MuLV gp70, tubulin, vimentin, and actin. Our results indicate that the cell membranes could be partially permeabilized to IgG molecules directed against the three cytoskeletal antigens only after 3.7%, but not 1%, formaldehyde treatment. Complete permeabilization was achieved by subsequent acetone treatment of cells after 3.7% formaldehyde fixation. In such cells, normal-appearing cytoskeletal networks of microtubules and intermediate filaments were observed. Stress fibers were also seen; however, they appeared less numerous and thinner than those of uninfected mouse fibroblasts. Further, a significant amounts of F-actin fluorescence was localized in granules in the cytoplasm of infected cells. Similar observations were made using JLS-V9 mouse cells chronically infected with 334C virus, another MuLV. These results taken together suggest that subtle differences exist in the organization of actin within MuLV-infected and uninfected mouse fibroblasts.

Inhibition of human cytomegalovirus by trifluorothymidine.

The antiviral activity of trifluorothymidine (TFT) singly and in combination with other antiviral agents against human cytomegalovirus (HCMV) was evaluated by using an infectious center plaque reduction assay. The 50% inhibitory dose of TFT against six different patient HCMV strains was 0.57 (+/- 0.24, standard deviation) microM and ranged from 0.32 to 0.97 microM. The 50% inhibitory dose for the laboratory-adapted HCMV strain, AD-169, was 2.1 microM. When TFT (0.17 microM) was combined with human fibroblast interferon (25 U/ml), the combination was additive against all four HCMV isolates evaluated. Synergism was observed when TFT (0.17 microM) was combined with phosphonoformic acid (25 microM) for all strains studied or with acyclovir (20 microM) for three of the four clinical HCMV strains tested. Each of the three antiviral agents, when combined with TFT, exhibited additive effects against strain AD-169. TFT at concentrations of 0.5, 1.7, and 3.5 microM had an increasing inhibitory effect on uninfected human embryonic lung fibroblast (HEL) cell growth over 72 h, with 16% growth inhibition at 3.5 microM after 3 days. There was no increased toxicity to growing HEL cells when the paired antiviral agent combinations were evaluated. These findings suggest that TFT may be useful singly or in combination with other antiviral agents in treating HCMV infections.

Altered Cell Spreading in Cytochalasin B: a Possible Role for Intermediate Filaments

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.

Role of interferon in natural kill of HSV-1-infected fibroblasts.

The production of interferon during natural killer (NK) assays against HSV-1-infected fibroblasts (NK(HSV-1)) was studied to determine whether this interferon was responsible for inducing the preferential lysis of herpes-virus-infected target cells over uninfected target cells. The interferon produced during NK(HSV-1) assays was analyzed and found to have the properties of HU-IFN-alpha. Little or no IFN was produced during NK assays against uninfected fibroblasts (NK(FS)) or K562 (NK(K562)) cells. Although the appearance of interferon in the culture supernatants seemed to parallel the development of cytotoxicity during NK(HSV-1) assays, the levels of cytotoxicity and IFN generated did not correlate, arguing against a strict quantitative dependence of cytotoxicity upon IFN production. NK(K562) and NK(FS) cytotoxicity developed with little or no production of IFN. When IFN-pretreated effector cells were used, there was still a preferential lysis of infected over uninfected target cells. This preferential lysis by IFN-treated effector cells of infected over uninfected targets was seen as early as 2 hr into the assay. Anti-IFN antibodies added to the NK assays, although neutralizing all the IFN produced during the assays, had no effect on NK(FS) or NK(K562) cytotoxic activity and caused a slightly reduction of NK(HSV-1) activity only in one of three experiments. We conclude that although IFN is generated during NK(HSV-1) assays, this IFN cannot solely account for the increased lysis of infected over uninfected cells and that NK(HSV-1) activity is in some other way dependent on the virus infection.

Identification of Phosphotyrosine-Containing Proteins in Untransformed and Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts

Phosphorylation on tyrosine residues mediated by pp60src appears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a 32P-labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid around M(r)'s of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defective src deletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded to M(r)'s of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60src or the combined action of pp60src and pp60src-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.