Human 143B Cells Research Articles

Efficient Elimination of mtDNA from Mammalian Cells with 2',3'-Dideoxycytidine.

Mammalian cell lines devoid of mitochondrial DNA (mtDNA) are indispensable in studies aimed at elucidating the contribution of mtDNA to various cellular processes or interactions between nuclear and mitochondrial genomes. However, the repertoire of tools for generating such cells (also known as rho-0 or ρ0 cells) remains limited, and approaches remain time- and labor-intensive, ultimately limiting their availability. Ethidium bromide (EtBr), which is most commonly used to induce mtDNA loss in mammalian cells, is cytostatic and mutagenic as it affects both nuclear and mitochondrial genomes. Therefore, there is growing interest in new tools for generating ρ0 cell lines. Here, we examined the utility of 2',3'-dideoxycytidine (ddC, zalcitabine) alone or in combination with EtBr for generating ρ0 cell lines of mouse and human origin as well as inducing the ρ0 state in mouse/human somatic cell hybrids. We report that ddC is superior to EtBr in both immortalized mouse fibroblasts and human 143B cells. Also, unlike EtBr, ddC exhibits no cytostatic effects at the highest concentration tested (200 μM), making it more suitable for general use. We conclude that ddC is a promising new tool for generating mammalian ρ0 cell lines.

Characterization of human mitochondrial PDSS and COQ proteins and their roles in maintaining coenzyme Q10 levels and each other's stability

Mutations of many PDSS and COQ genes are associated with primary coenzyme Q10 (CoQ10) deficiency, whereas mitochondrial DNA (mtDNA) mutations might cause secondary CoQ10 deficiency. Previously, we found that COQ5 and COQ9 proteins are present in different protein complexes in the mitochondria in human 143B cells and demonstrated that COQ5 and COQ9 knockdown suppresses CoQ10 levels. In the present study, we characterized other PDSS and COQ proteins and examined possible crosstalk among various PDSS and COQ proteins. Specific antibodies and mitochondrial localization of mature proteins for these proteins, except PDSS1 and COQ2, were identified. Multiple isoforms of PDSS2 and COQ3 were observed. Moreover, PDSS1, PDSS2, and COQ3 played more important roles in maintaining the stability of the other proteins. Protein complexes containing PDSS2, COQ3, COQ4, COQ6, or COQ7 protein in the mitochondria were detected. Two distinct PDSS2-containing protein complexes could be identified. Transient knockdown of these genes, except COQ6 and COQ8, decreased CoQ10 levels, but only COQ7 knockdown hampered mitochondrial respiration and caused increased ubiquinol:ubiquinone ratios and accumulation of a putative biosynthetic intermediate with reversible redox property as CoQ10. Furthermore, suppressed levels of PDSS2 and various COQ proteins (except COQ3 and COQ8A) were found in cybrids containing the pathogenic mtDNA A8344G mutation or in FCCP-treated 143B cells, which was similar to our previous findings for COQ5. These novel findings may prompt the elucidation of the putative CoQ synthome in human cells and the understanding of these PDSS and COQ protein under physiological and pathological conditions.

Potential Molecular Mechanisms of AURKB in the Oncogenesis and Progression of Osteosarcoma Cells: A Label-Free Quantitative Proteomics Analysis.

Our previous study indicated that knockdown of Aurora-B inhibit the proliferation of osteosarcoma cells. But the function and molecular mechanisms of Aurora-B in osteosarcoma cells growth and metastasis remains unclear. The aim of this study was to investigate the molecular mechanisms of Aurora-B in the progression of osteosarcoma. Osteosarcoma cells (U2-OS and 143B) were treated with specific Lentivirus-Vectors (up or downregulation Aurora-B). The ability of cells proliferation, migration, and invasion was measured using Cell-Counting Kit-8, wound healing and transwell invasion assays. Furthermore, based on label-free quantitative proteomic analysis of potential molecular mechanisms of Aurora-B in human 143B cells. A total of 25 downregulated and 76 upregulated differentially expressed proteins were screened in terms of the change in their expression abundance. We performed functional annotation and functional enrichment analyses. Gene ontology enrichment, KEGG analysis, and protein–protein interaction networks were constructed and analyzed. We found that the PTK2 may play an important role in the progression of osteosarcoma cells. Finally, Western blot revealed that expression of PTK2, AKT, PI3K, and nuclear factor-kappaB increased after over expression of Aurora-B. Overall, these data highlight that Aurora-B may promote the malignant phenotype of osteosarcoma cells by activating the PTK2/PI3K/AKt/nuclear factor-KappaB pathway.

Study on the inhibitory effect of rhodioside on osteosarcoma 143 cells invasion, migration and potential mechanism

Objective To investigate the effect of rhodioside on the proliferation, invasion and migration of human osteosarcoma 143B cells and its possible mechanism. Methods Osteosarcoma 143B cells were cultured in dulbeeeo modified eagle medium (DMEM) medium (including 10% serum and 1% double resistance). Osteosarcoma 143B cells were divided into control group, 30 and 60 μmol/L rhodioside treatment group. Cell proliferation was tested by methyl thiazol tetrazolium (MTT) assay at 24, 36, 72 h, Transwell method was used to detect three groups of cell invasion; migration ability were detected by cell scratch assay. After 24 h treatment, calculation of cell migration rate. After 24 h treatment, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the relative expression level of matrix metalloproteinase (MMP)-2 and MMP-9 mRNA. The protein expression level of MMP-2 and MMP-9 in each group was detected by Western blotting. The autophagy after 24 h was detected by transmission electron microscopy. Results After the treatment of 24 h, the absorbance value of 60 mol/L group (0.038±0.002) and 30 mol/L group (0.054±0.002) 143B cells was lower than that of the control group. Compared with the control group, rhodioside with concentration of 30 and 60 mol/L at 48 h and 72 h inhibited the proliferation of 143B cells, respectively, with a dose-dependent and time dependent manner. The number of transmembrane of 143B cells in 60 μmol/L (48.7±8.2) and 30 μmol/L (76.8±12.8) rhodioside group was less than that of the control group (94.5±11.9), t1=8.056, P=0.000; t=2.472, P=0.038, respectively. The migration rates of 143B cells in 60 μmol/L [(52.9±6.8)%] and 30 μmol/L [(82.6±3.7)%] rhodioside group were lower than those of the control group [(98.7±0.3)%, t=12.576, P=0.000, t=9.794, P=0.000], respectively. The relative expression of protein and mRNA of MMP-2 and MMP-9 in 60 mol/L and 30 mol/L rhodioside treatment group was lower than that of the control group. The number of autophage in the 60 mol/L rhodioside group was (2.6±0.5) more than that of the control group (1.3±0.4, t=3.517, P=0.025). Conclusion Rhodioside inhibits the proliferation, migration and invasion of osteosarcoma cells, and its mechanism may be related to the induction of autophagy and the inhibition of the expression of MMP family protein. Key words: Osteosarcoma; Rhodioside; Migration; Invasion; Autophagy

Comparison on the Roles of COQ3 and COQ7 Proteins in Maintaining Coenzyme Q 10 Levels, the Stability of Other COQ Proteins, and Mitochondrial Functions in Human Cells

Coenzyme Q (CoQ) is a lipid-soluble antioxidant, with the oxidized (ubiquinone) and the reduced (ubiquinol) forms, and also an electron carrier for the electron transport chain. CoQ6 is synthesized by at least nine proteins (Coq1--9), and many of them form the Coq protein complex in the mitochondria in yeast. CoQ is present in the form of CoQ10 in humans. Yeast Coq1 corresponds to PDSS1 and PDSS2 proteins in humans. Mutations in PDSS or COQ genes are associated with primary CoQ10 deficiency. Secondary CoQ10 deficiency is associated with mitochondrial DNA mutation. We have demonstrated that the human COQ5, present as a mature protein without mitochondrial targeting sequence in the mitochondria, is required for maintaining CoQ10 levels. The maturation of COQ5 protein and the stability of the COQ5-containing protein complex in the mitochondria were suppressed by the conditions of mitochondrial energy deficiency. Here we further investigated the roles of COQ3 and COQ7 proteins in maintaining CoQ10 levels and in the mitochondria. Our results showed that both COQ3 and COQ7 were required for maintaining CoQ10 levels in human 143B cells, but the degree of the decrease in CoQ10 levels was greater after COQ7 knockdown than that after COQ3 knockdown. After COQ7 knockdown, but not after COQ3 knockdown, the ubiquinol-10:ubiquinone-10 ratio was increased and the peaks corresponding to the putative substrate of the COQ7 enzyme, 5-demethoxyubiquinone-10 (DMQ10/DMQ10H2), appeared on the HPLC chromatograms. The CoQ10 levels in the mitochondria were also decreased after COQ3 or COQ7 knockdown, and the putative DMQ10 was present in the mitochondria after COQ7 knockdown. Moreover, COQ3 or COQ7 knockdown caused decreased mitochondrial respiratory functions. Only COQ3 knockdown could decrease the stability of PDSS2 and several COQ proteins. Furthermore, mature forms of COQ3 and COQ7 were found in the mitochondria. Levels of the mature COQ7 protein, but not COQ3, were suppressed in 143B cells treated with the chemical uncoupler FCCP and in the cybrids harboring a pathogenic mitochondrial DNA mutation, which both had diminished CoQ10 levels. All these novel findings may further elucidate the functions of COQ proteins and their roles in human diseases.

142 - Alterations of Endogenous Coenzyme Q10 Levels in Human Cells after the Induction of Lipid Peroxidation by an Organic Peroxide

Coenzyme Q10 (CoQ10) is an essential electron carrier in the mitochondrial electron transport chain and also an endogenously synthesized lipid-soluble antioxidant. We have previously demonstrated that hydrogen peroxide increased CoQ10 levels, but disruption of mitochondrial membrane potential by the chemical uncoupler FCCP suppressed CoQ10 levels in the human 143B cells. Mor bioeover, we have also shown that the COQ5 protein, which is required for CoQ biosynthesis in yeast, is essential for maintaining CoQ10 levels in 143B cells. In this study, we investigated how the condition of enhanced lipid peroxidation affected levels of the reduced form (ubiquinol-10) and oxidized form (ubiquinone-10) of CoQ10. The organic peroxide tert-butyl hydroperoxide (TBHP) was used to induce lipid peroxidation in 143B cells. Levels of esterified F2-isoprostanes (F2-IsoPs) in cells, detected by the gas chromatography/mass spectrometry method, were confirmed to be elevated by a low, medium, or high dose of TBHP as early as after 4 h of treatment. The levels remained augmented after 18 h of TBHP treatment but the degree of the increase was less. Only the medium-dose and high-dose TBHP treatments enhanced total CoQ10 levels after 18 h or 24 h of treatment, but the high-dose TBHP treatment also enhanced ubiquinol-10 levels after 4 h of treatment. Transient knockdown of COQ5, which decreased CoQ10 levels, suppressed the increase of both ubiquinol-10 and ubiquinone-10 levels after 24-h TBHP treatment. These results demonstrate for the first time that specific augmentation of lipid peroxidation could up-regulate formation of endogenous CoQ10 levels and ubiquinol-10 could be the initially generated form.

Disruption of the human COQ5-containing protein complex is associated with diminished coenzyme Q10 levels under two different conditions of mitochondrial energy deficiency

BackgroundThe Coq protein complex assembled from several Coq proteins is critical for coenzyme Q6 (CoQ6) biosynthesis in yeast. Secondary CoQ10 deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ10 levels and COQ5 protein maturation in human 143B cells. MethodsThis study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined. ResultsA high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ10 levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids. ConclusionsA novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ10 levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation. General significanceThe findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ10 deficiency in human cells.

Human METTL20 Methylates Lysine Residues Adjacent to the Recognition Loop of the Electron Transfer Flavoprotein in Mitochondria

In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria.

Suppression of coenzyme Q10 levels and the induction of multiple PDSS and COQ genes in human cells following oligomycin treatment

Endogenous coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant and essential for the electron transport chain. We previously demonstrated that hydrogen peroxide enhanced CoQ10 levels, whereas disruption of mitochondrial membrane potential by a chemical uncoupler suppressed CoQ10 levels, in human 143B cells. In this study, we investigated how CoQ10 levels and expression of two PDSS and eight COQ genes were affected by oligomycin, which inhibited ATP synthesis at Complex V without uncoupling the mitochondria. We confirmed that oligomycin increased the production of reactive oxygen species (ROS) and decreased mitochondria-dependent ATP production in 143B cells. We also demonstrated that CoQ10 levels were decreased by oligomycin after 42 or 48 h of treatment, but not at earlier time points. Expression of PDSS2 and COQ2–COQ9 were up-regulated after 18-hour oligomycin treatment, and the expression of PPARGC1A (PGC1-1α) elevated concurrently. Knockdown of PPARGC1A down-regulated the basal mRNA levels of PDSS2 and five COQ genes and suppressed the induction of COQ8 and COQ9 genes by oligomycin, but did not affect CoQ10 levels under these conditions. N-acetylcysteine suppressed the augmentation of ROS levels and the enhanced expression of COQ2, COQ4, COQ7, and COQ9 induced by oligomycin, but did not modulate the changes in CoQ10 levels. These results suggested that the condition of mitochondrial dysfunction induced by oligomycin decreased CoQ10 levels independent of oxidative stress. Up-regulation of PDSS2 and several COQ genes by oligomycin might be regulated by multiple mechanisms, including the signaling pathways mediated by PGC-1α and ROS, but it would not restore CoQ10 levels.

Interference of Co-Amplified Nuclear Mitochondrial DNA Sequences on the Determination of Human mtDNA Heteroplasmy by Using the SURVEYOR Nuclease and the WAVE HS System

High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detecting human mtDNA variants and found that its performance was slightly better than that of denaturing high-performance liquid chromatography (DHPLC). The potential interference from co-amplified NUMTs on screening mtDNA heteroplasmy when using these 2 highly sensitive techniques was further examined by using 2 published primer sets containing a total of 65 primer pairs, which were originally designed to be used with one of the 2 techniques. We confirmed that 24 primer pairs could amplify NUMTs by conducting bioinformatic analysis and PCR with the DNA from 143B-ρ0 cells. Using mtDNA extracted from the mitochondria of human 143B cells and a cybrid line with the nuclear background of 143B-ρ0 cells, we demonstrated that NUMTs could affect the patterns of chromatograms for cell DNA during SN-WAVE/HS analysis of mtDNA, leading to incorrect judgment of mtDNA homoplasmy or heteroplasmy status. However, we observed such interference only in 2 of 24 primer pairs selected, and did not observe such effects during DHPLC analysis. These results indicate that NUMTs can affect the screening of low-level mtDNA variants, but it might not be predicted by bioinformatic analysis or the amplification of DNA from 143B-ρ0 cells. Therefore, using purified mtDNA from cultured cells with proven purity to evaluate the effects of NUMTs from a primer pair on mtDNA detection by using PCR-based high-sensitivity methods prior to the use of a primer pair in real studies would be a more practical strategy.

The Human Mitochondrial Transcriptome

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).

Effect of mitochondrial dysfunction and oxidative stress on endogenous levels of coenzyme Q10 in human cells

Little is known about the regulation of endogenous CoQ(10) levels in response to mitochondrial dysfunction or oxidative stress although exogenous CoQ(10) has been extensively used in humans. In this study, we first demonstrated that acute treatment of antimycin A, an inhibitor of mitochondrial complex III, and the absence of mitochondrial DNA suppressed CoQ(10) levels in human 143B cells. Because these two conditions also enhanced formation of reactive oxygen species (ROS), we further investigated whether oxidative stress or mitochondrial dysfunction primarily contributed to the decrease of CoQ(10) levels. Results showed that H(2)O(2) augmented CoQ(10) levels, but carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a chemical uncoupler, decreased CoQ(10) levels in 143B cells. However, H(2)O(2) and FCCP both increased mRNA levels of multiple COQ genes for biosynthesis of CoQ(10) . Our findings suggest that ROS induced CoQ(10) biosynthesis, whereas mitochondrial energy deficiency caused secondary suppression of CoQ(10) levels possibly due to impaired import of COQ proteins into mitochondria.

Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

A possible involvement of plasma membrane NAD(P)H oxidase in the switch mechanism of the cell death mode from apoptosis to necrosis in menadione-induced cell injury.

The effects of inhibitors of plasma membrane NADPH oxidase on menadione-induced cell injury processes were studied using human osteosarcoma 143B cells. The intracellular level of superoxide in the cells treated with menadione for 6 h reached a maximum followed by an abrupt decrease. The population of apoptotic cells detected by Annexin V and propidium iodide double staining also reached its maximum at 6 h of menadione-treatment while that of necrotic cells increased continuously reaching 90% of the total population at 9 h of the treatment. Pretreatment of the cells with inhibitors of NADPH oxidase, including diphenyliodonium chloride, apocynin, N-vanillylnonanamide and staurosporine was effective in lowering the menadione-induced elevations of superoxide, and also in the suppression of the switch of the cell death mode from apoptosis to necrosis in menadione-treated cells except for the case of staurosporine. These results strongly suggest that superoxide generated by NADPH oxidase, besides that generated by the mitochondria, may contribute to the remarkable increase in the intracellular level of superoxide in the cells treated with menadione for 6 h resulting in the switch from apoptosis to necrosis, although a direct evidence of the presence of active and inactive forms of NADPH oxidase in control and menadione-treated 143B cells is lacking at present.

Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse

We constructed a series of cosmid vectors that carry two tandemly arranged λ cos and mammalian selective markers. We achieved cloning efficiencies of 1–3 × 10 7 and greater than 10 6 colony-forming units per μg of insert, using a cloned 42-kb BamHI fragment and Sau3AI fragments of 40–50 kb from mouse genomic DNA, respectively. The modified Ca-phosphate coprecipitation method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607–616] considerably improved the efficiency of gene transfer of cosmids into cultured mammalian cells: when genes encoding thymidine kinase from herpes simplex virus type 1 and aminoglycoside 3′-phosphoribosyltransferase from Tn5 were selected, the efficiencies of gene transfer into mouse L cells were about 10 −6. The mouse genome contains one copy of the functional gene for elongation factor 2 ( EF2) per haploid genome and multiple copies of the EF2-related gene. We isolated a cosmid that carried functional full-length mouse EF2 from a cosmid library of L-cell genomic DNA, by colony hybridization and subsequent gene transfer of candidate cosmids into human 143B cells.

Induction of cell surface expression of HLA antigens by human IFN-gamma encoded by recombinant vaccinia virus.

A recombinant vaccinia virus (VV) encoding human IFN-gamma (VV-huIFN-gamma) was constructed and its effects on MHC Ag expression in human and murine cells in vitro analyzed by flow cytometry. At high multiplicities of infection (5 pfu/cell) the IFN-gamma expressed by vaccinia was not able to overcome the profound decrease of MHC concentration, normally associated with VV infection, in any of the cells tested. However, at successively decreasing multiplicities of infection, a gradual increase in MHC class I concentration above control levels was observed in human 143B cells but not in murine L929 cells, thus indicating that the species specificity of IFN-gamma is preserved in VV-huIFN-gamma-infected cells. We infer from these data that the IFN-gamma secreted by infected 143B cells is able to exert an MHC upregulating effect on uninfected cells in the vicinity. Antiviral activity of the IFN-gamma expressed by the virus was also assessed. Pretreatment for 24 to 48 h of human 143B cells with IFN-gamma containing supernatants had a significant antiviral effect comparable to rhuIFN-gamma. However, when added 1 h after virus infection, antiviral activity was much less evident. Also, the IFN-gamma secreted by infected 143B cells in monolayers infected at low multiplicity did not efficiently inhibit spread of infection to other cells in the vicinity.