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S Mak

Publications and source records attributed to S Mak.

At least 37 records · Page 2Linked to original sources
PubMed1992

Adenovirus type 12 early region 1B proteins and metabolism of early viral mRNAs.

Early region 1B (E1B) of human adenoviruses encodes two major proteins. The 19-kDa polypeptide appears to prevent E1A-induced cytolysis and DNA degradation. The larger E1B product of approximately 55 kDa, which is essential for viral replication, plays a role in the accumulation and stability of viral mRNAs and the late shutoff of host metabolism. For serotype 12 (Ad12), this 482-residue (482R) protein is essential for viral DNA replication. In the present report we have used a series of mutants to examine the roles of Ad12 482R and the 19-kDa, 163R protein in the metabolism of early viral mRNAs. No specific effects on the accumulation of early (or late) mRNAs were detected with any of the mutants affecting 163R. With mutant dl42, which encodes an altered 482R product that lacks residues 114-155, both viral DNA replication and late viral protein synthesis were defective. Accumulation of E1A transcripts in the nucleus and cytoplasm resembled wt. The levels of mRNAs from early regions E1B, E2A and E3 at later times during infection were somewhat lower than those of wt, but this decrease may have been due to the absence of progeny viral DNA in dl42-infected cells. However, the accumulation of both E2B and E4 mRNAs at all times was severely reduced. These data suggested that the requirement of 482R for Ad12 DNA replication may be related to its specific role in the metabolism of E2B and E4 mRNAs that encode products necessary for viral DNA synthesis.

Adenovirus E1B Proteins↗
PubMed1992

Overexpression of the E1B 55-kilodalton (482R) protein of human adenovirus type 12 appears to permit efficient transformation of primary baby rat kidney cells in the absence of the E1B 19-kilodalton protein.

To analyze the structure and function of the E1B 19,000-molecular-weight protein (19K protein) (163R) of human adenovirus type 12, mutants were produced at various positions across the 163R-coding sequence. Viruses bearing mutations within the first 100 or so amino acids yielded unstable 163R-related products, induced DNA degradation and enhanced cytopathic effect (cyt/deg phenotype) in KB cells, and transformed primary rodent cells at much lower efficiencies than wild-type (wt) virus. Deletion of the final 16 residues at the carboxy terminus had no phenotypic effect. Alteration of residue 105 reduced transforming efficiency significantly, suggesting that this region of 163R is functionally important. Disruption of the AUG initiation codon at nucleotide 1542 blocked production of 163R completely but resulted in higher levels of E1B 55K-482R protein synthesis and a transforming efficiency similar to that of wt virus. These data suggested that while 163R is of some importance, normal transforming efficiencies can be obtained in its absence if 482R is overexpressed.

Adenovirus Early Proteins↗
PubMed1990

Separate regions of an adenovirus E1B protein critical for different biological functions.

The E1B region of Ad12 encodes two major proteins, the 482R (55K) and 163R (19K). In this report we showed that the E1B 482R is multifunctional, in that its structure may somehow contribute to its own stability, in viral DNA and virus replication, in transformation of primary cells, and in tumorigenicity. Deletion of the first 24 amino acids and of aa residues 114-155 (dl42) results in an instability of the 55K protein. The N-terminal 24 aa residues (pm1852) or amino acids residues 80-96 (dl17) are not required for viral DNA or virus replication, whereas amino acid residues between 114 and 155 (dl42) are absolutely necessary for viral DNA synthesis. Deletion of amino acid residues 1-24, 80-96, and 114-155 (dl42) greatly reduces the transforming ability of both virus and plasmids containing any one of these deletions. One of the critical regions for tumorigenicity residues within amino acid residues 80-96, since cells transformed by this plasmid are nontumorigenic. On the other hand the region bounded by amino acid residues 114-155 (dl42) is not required for tumorigenicity in immunocompetent animals.

Adenovirus Early Proteins↗
PubMed1988

Expression of the cellular p53 protein in cells transformed by adenovirus 12 and viral DNA fragments.

Expression of the p53 cellular phosphoprotein was examined in rat cells transformed by adenovirus 12 (Ad12) virions and by fragments of Ad12 DNA. p53 was detected in all the cell lines examined. Steady-state levels of p53 were highest in cells transformed by the entire E1 region rather than by E1A alone. Physical association between p53 and the Ad12 E1B 55K protein was not detected. The Ad12 E1B-encoded 55K protein, but not the Ad12 E1B 17K and 19K proteins, appears to participate in regulating p53 protein levels.

Adenovirus Early Proteins↗
PubMed1986

Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential.

The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome by transfecting human cultured fibroblasts with the pSV3-gpt and pSV3-neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers. Transformed human fibroblasts were obtained at a frequency of about 10(-5) with both selection systems. These transformed cells showed a twofold increase in growth rate and three to tenfold increase in cell number at confluence. The improved growth characteristics were associated with the expression of the SV40 T-antigen detected with immunoprecipitation. These cell lines also changed from their usual spindle shapes to an epithelioid morphology characteristic of transformed cells. From 60 to 100% of the cells transfected with pSV3 plasmid DNA demonstrated numerical and structural abnormalities in their karyotypes. Cells transfected with DNA from a similar plasmid, pSV2-neo, which differed from the pSV3-neo plasmid only by missing the sequence encoding the complete early region of SV40, neither expressed T-antigen nor showed any change in morphology, improvement in growth characteristics or abnormalities in karyotype. However, they were still selectable with the aminoglycoside G-418. Therefore, by appropriate choice of vector plasmids, dominant selection markers and improved growth characteristics can be imparted separately or simultaneously to human fibroblasts. The morphological, biochemical and chromosomal changes resulting from such transformations must be recognized in using this approach for biochemical and genetic studies.

Antigens, Viral, Tumor↗
PubMed1986

Inhibition of protein synthesis enhances transformation of primary cells by viral DNA.

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK-) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.

Adenoviruses, Human↗
PubMed1984

19-kDa tumor antigen coded by early region E1b of adenovirus 2 is required for efficient synthesis and for protection of viral DNA.

The adenovirus E1b region (mp 4.5-11.2) codes for two major tumor antigens of 53 and 19 kDa. The lp+ locus maps within the 19-kDa tumor antigen-coding region and has been shown to play an essential role in cell transformation. We have investigated the role of the 19-kDa tumor antigen during productive virus growth using a specific Ad2 mutant (dl250) lacking most of the 19-kDa tumor antigen-coding region. Mutant dl250 grows more slowly and yields about 100-fold less progeny virus than Ad2 wt in human KB cells. In cells infected with mutant dl250, viral DNA is present at only about one-half the level of that of Ad2 wt. The defect in DNA accumulation appears to be both at the level of DNA synthesis and stability of newly synthesized DNA. In mutant-infected cells, newly replicated viral as well as cellular DNA are extensively degraded during late stages of viral infection. We have mapped a class of Ad12 (highly oncogenic group A) mutants (cyt) that are nononcogenic in newborn hamsters and induce DNA degradation to the 19-kDa tumor antigen-coding region by intertypic complementation analysis. These results strongly suggest that the 19-kDa tumor antigen plays an essential role in efficient viral DNA synthesis and protection of newly replicated viral DNA against cellular nucleases in addition to its role in cell transformation and tumorigenesis.

Adenoviridae↗
PubMed1984

Adenovirus cyt+ locus, which controls cell transformation and tumorigenicity, is an allele of lp+ locus, which codes for a 19-kilodalton tumor antigen.

The early region E1b of adenovirus type 2 (Ad2) codes for two major tumor antigens of 53 and 19 kilodaltons (kd). The adenovirus lp+ locus maps within the 19-kd tumor antigen-coding region (G. Chinnadurai, Cell 33:759-766, 1983). We have now constructed a large-plaque deletion mutant (dl250) of Ad2 that has a specific lesion in the 19-kd tumor antigen-coding region. In contrast to most other Ad2 lp mutants (G. Chinnadurai, Cell 33:759-766, 1983), mutant dl250 is cytocidal (cyt) on infected KB cells, causing extensive cellular destruction. Cells infected with Ad2 wt or most of these other Ad2 lp mutants are rounded and aggregated without cell lysis (cyt+). The cyt phenotype of dl250 resembles the cyt mutants of highly oncogenic Ad12, isolated by Takemori et al. (Virology 36:575-586, 1968). By intertypic complementation analysis, we showed that the Ad12 cyt mutants indeed map within the 19-kd tumor antigen-coding region. The transforming potential of dl250 was assayed on an established rat embryo fibroblast cell line, CREF, and on primary rat embryo fibroblasts and baby rat kidney cells. On all these cells, dl250 induced transformation at greatly reduced frequency compared with wt. The cells transformed by this mutant are defective in anchorage-independent growth on soft agar. Our results suggest that the 19-kd tumor antigen (in conjunction with E1a tumor antigens) may play an important role in the maintenance of cell transformation. Since we have mapped the low-oncogenic or nononcogenic Ad12 cyt mutants within the 19-kd tumor antigen-coding region, our results further indicate that the 19-kd tumor antigen also directly or indirectly plays an important role in tumorigenesis of Ad12. Our results show that the cyt+ locus is an allele of the lp+ locus and that the cyt phenotype may be the result of mutations in specific domains of the 19-kd tumor antigen.

Adenoviruses, Human↗
PubMed1984

Adenovirus 12 nononcogenic mutants: oncogenicity of transformed cells and viral proteins synthesized in vivo and in vitro.

Several nononcogenic cyt mutants of adenovirus type 12 induced the same E1A (55,000 [55K] and 25K) and E1B polypeptides (55K, 19K, and 17K) as did the wild-type virus, except that cyt 68 did not induce the E1B 19K protein. Tumorigenicity tests showed cells transformed by cyt 68 to be highly oncogenic in vivo. Therefore, it was concluded that the E1B 19K polypeptide is not necessary for tumor induction but may be involved in the efficiency of transformation.

Adenoviruses, Human↗
PubMed1983

Transformation of rat cells by cyt mutants of adenovirus type 12 and mutants of adenovirus type 5.

Several mutants with much reduced oncogenicity (spontaneous mutants H12 cyt 52 and H12 cyt 70 and UV-induced mutants H12 cyt 61, H12 cyt 62, and H12 cyt 68) of the highly oncogenic adenovirus type 12 (Ad12) were studied for their ability to transform primary baby rat kidney cells. Four of the mutants showed much reduced capacity to transform cells in vitro, while H12 cyt 61 transformed cells as efficiently as the wild-type virus. Viral gene expression in several cell lines established from cultures infected by cyt mutants was studied, and it was found that viral sequences belonging to the left 16% of Ad12 were always transcribed. These results suggest that the function of the transformed state is not defective in the cyt mutants studied. Heterotypic complementation studies showed that the defect(s) in a cyt mutant can be corrected by an Ad7 function. Ad5 dl 313, with a deletion between 3.5 and 10.5 map units, transformed rat cells only at high multiplicity. These results suggest that the region E1B of adenoviruses may be required for efficient transformation of rat cells.

Adenoviruses, Human↗
PubMed1982

Adenovirus-12 genes undetectable in human retinoblastoma.

Retinoblastoma is a rare eye tumor found predominantly in young children. Intra-ocular injection of adenovirus-12 (Ad-12) into newborn recipients induces in rats and in baboons tumors morphologically similar to human retinoblastoma tumors. In addition, Ad-12 DNA can transform human embryonic retinal cells and the transformed cells form retinoblastoma-like tumors in nude mice. These observations have led to the suggestion that Ad-12 may also be involved in the induction of human retinoblastoma tumors. To test this possibility, DNA and RNA were isolated from six human tumors. Analysis of RNA and DNA by blotting with labelled Ad-12 failed to reveal viral sequences in human retinoblastoma.

Adenoviruses, Human↗
PubMed1982

Mapping of an adenovirus function involved in the inhibition of DNA degradation.

A function involved in the inhibition of DNA degradation has been assigned through complementation tests to a product of region E1b of the adenovirus genome (between 4.5 and 10.5 map units). DNA degradation induced by the adenovirus type 12 (Ad12) cyt mutant H12cyt70 and the Ad5 early deletion mutant dl313 (with the deletion between 3.5 and 10.7 map units) was inhibited by coinfection with Ad5 region E1a (between 0 and 4.5 map units) mutants dl312 and hr1 and region E1b mutant hr6. The defect of inhibition of DNA degradation in Ad5 dl313 was also complemented in 293 cells. This DNase-inhibitory function does not appear to involve polypeptide IX or the 58,000-dalton polypeptide. Wild-type Ad12 induced DNA degradation in hamster embryo cells, suggesting that the DNase-inhibitory function is not expressed in these nonpermissive cells. Additional evidence suggests the involvement of a second viral product which positively influences the DNase activity and which appears to be an early function.

Adenoviruses, Human↗
PubMed1981

Protein kinase activity immunoprecipitated from adenovirus infected cells by sera from tumor-bearing hamsters.

Earlier, we reported (N. J. Lassam, S. T. Bayley, F. L. Graham, and P. E. Branton, Nature (London) 277:241-243, 1979) detecting protein kinase activity when cytoplasmic extracts of human adenovirus type 5 (Ad5)-infected KB cells immunoprecipitated with 14b antitumor serum directed against the transforming proteins of Ad5, were incubated with [gamma-32P]ATP. Here we show that in the in vitro assay this kinase phosphorylated both the heavy chain of immunoglobulin G and polypeptide than comigrated on sodium dodecyl sulfate gels with the 58,000-dalton Ad5 antigen. It also phosphorylated added histone H3. Evidence is presented that the protein kinase activity found with extracts from Ad5-infected cells is not due to nonspecific trapping of cellular enzymes in immune complexes, but to an enzyme which is distinct from kinases detected at background levels in controls. Serine and threonine were the major phosphorylated amino acids, and essentially no phosphotyrosine was detected. Protein kinase activity detected in Ad12-infected cells immunoprecipitated by an antiserum derived from hamsters bearing Ad12-induced tumors appeared to be immunologically distinct from that immunoprecipitated from Ad5-infected cells by 14b serum.

Adenoviruses, Human↗
PubMed1981

Degradation of intracellular DNA in KB cells infected with cyt mutants of human adenovirus type 12.

A group of mutants (cyt mutants) with much reduced oncogenicity was isolated from the highly oncogenic human adenovirus type 12 (Takemori et al., Virology 36: 575-586, 1968). These mutants induce extensive cellular destruction during lytic infection of human cells and produce low yields of virions. We report here that human KB cells infected with cyt mutants synthesized a reduced amount of viral DNA as compared with cells infected with the parental virus. Furthermore, the newly synthesized viral and cellular DNAs were extensively degraded in mutant-infected cells. Viral DNA was first synthesized as complete genome size, and most of it was degraded to subgenomic size within 6 h after synthesis. This virus-induced DNA degradation function, as well as the low yield of virions, was prevented by co-infection with the parental virus.

Adenoviruses, Human↗
PubMed1979

Structure and function of adenovirus type 12 defective virions.

Purified human adenovirus type 12 preparations contain defective virions with a lighter density. These defective virions were isolated, and their biological functions and DNA were characterized. They can induce early and late antigens in infected cells and tumors in newborn hamsters with similar efficiency as complete virions. The majority of the DNA molecules from light virions contain deletions mapping near 16% from the left-hand end of the genome. Mechanisms for the generation of these molecules are discussed.

Adenoviruses, Human↗
PubMed1978

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.

Adenoviruses, Human↗