Rapid intracellular turnover of adenovirus 5 early region 1A proteins

The half-life of the adenovirus 5 early region 1A (E1A) proteins was examined in productively infected and transformed cells. In HeLa cells infected with adenovirus 5, the half-life of the E1A proteins was approximately 30 min; in the transformed 293 cells, the constitutively expressed E1A proteins had a half-life of approximately 120 min. In HeLa cells, the E1A proteins produced by an adenovirus mutant that expresses only the 13S mRNA had a half-life of about 35 min; E1A proteins produced by a mutant that express only the 12S mRNA had a half-life of about 80 min. This difference in the stability of these two classes of E1A proteins helps explain why the steady-state level of the 12S class is usually equal to or greater than that of the 13S class, despite the fact that the concentration of the 13S mRNA is about four times greater than the concentration of the 12S mRNA.     

mRNAs from human adenovirus 2 early region 4

The molecular structure of the mRNAs from early region 4 of human adenovirus 2 has been studied by Northern blot analysis, S1 nuclease analysis, and sequence analysis of cDNA clones. The results make it possible to identify four different splice donor sites and six different splice acceptor sites. The structure of 12 different mRNAs can be deduced from the analysis. The mRNAs have identical 5' and 3' ends and are thus likely to be processed from a common mRNA precursor by differential splicing. The different mRNA species are formed by the removal of one to three introns, and they all carry a short 5' leader segment. The introns appear to serve two functions; they either place a 5' leader segment in juxtaposition with an open reading frame or fuse two open translational reading frames. The early region 4 mRNAs can encode at least seven unique polypeptides.     

Translation efficiency of adenovirus early region 1A mRNAs deleted in the 5'' untranslated region

Adenovirus deletion mutants were studied to examine the influence of the 5' untranslated sequence on the translation of early region 1A mRNAs. Alterations of the 5' untranslated sequence, including complete deletion of the wild-type 5' untranslated sequence, did not significantly affect the rate of translation.     

Isolation of a nondefective recombinant between adenovirus type 5 and early region 1A of adenovirus type 12.

A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.     

Production of a monospecific antiserum against the early region 1A proteins of adenovirus 12 and adenovirus 5 by an adenovirus 12 early region 1A-beta-galactosidase fusion protein antigen expressed in bacteria.

Antisera were prepared against the amino acid sequences encoded within the N-terminal half of the adenovirus 12 (Ad12) early region 1A (E1A) gene. This was accomplished by construction of a plasmid vector which encoded the N-terminal 131 amino acids of Ad12 E1A joined in frame to the coding sequence of beta-galactosidase. After induced synthesis in Escherichia coli, the Ad12 E1A-beta-galactosidase fusion protein (12-1A-FP) was extracted with urea and used to raise antibodies in rabbits. The 12-1A-FP antisera immunoprecipitated major phosphoproteins of 39,000 and 37,000 apparent molecular weights from Ad12-transformed and infected cells. The 12-1A-FP antisera also immunoprecipitated E1A phosphoproteins from Ad5-transformed and infected cells. Immunospecificity of the 12-1A-FP antisera was demonstrated by the ability of 12-1A-FP antigen to block immunoprecipitation of E1A proteins. Furthermore, E1A proteins immunoprecipitated from in vivo-labeled cells comigrated with those translated in vitro by RNA that had been hybridization selected to E1A DNA.     

Nucleotide sequences and mapping of novel heterogenous 5''-termini of adenovirus 2 early region 4 mRNA.

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.     

Activation of human c-raf-1 by replacing the N-terminal region with different sequences.

Two transformants of NIH 3T3 cells, obtained by the transfection of human colon cancer and normal colon DNAs, contained activated c-raf-1. In both the activated c-raf-1, the 5' half of the c-raf-1 sequence was replaced by sequences other than c-raf-1 as a result of recombinations which occurred at the intron between exons 7 and 8. It was suggested, however, that these recombinations, which conferred the transforming activity on the c-raf-1, occurred during the transfection. In one case analyzed, characteristic sequences were found near the breakpoint and these may be involved in the recombination. It was found, upon analysing the structure of the cDNA derived from one of the activated c-raf-1, that fused mRNA had been transcribed from the recombined gene comprising the non-raf gene and c-raf-1. The mRNA possibly encodes a fused protein. One cDNA clone was derived from alternatively spliced mRNA, although its physiological role is unclear. On comparing the structure of the two human activated c-raf-1 and the rat activated c-raf which we have reported previously, it was revealed that, in these three cases, the sequences joined to the truncated c-raf(-1)1 were different. It was suggested from data which we and others have previously reported that various sequences could be capable of activating c-raf(-1) by replacing its 5' half.     

Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products

Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins.     

Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.

Peptide-specific antisera were developed to analyze the products encoded by adenovirus type 5 early region 4 (E4) open reading frames 6 and 7. Reading frame 6 previously was shown to encode a 34-kilodalton polypeptide (34K polypeptide) that forms a complex with the early region 1B (E1B)-55K antigen and is required for efficient viral growth in lytic infection. Antisera that were generated recognized the E4-34K protein as well as a family of related polypeptides generated by the fusion of open reading frames 6 and 7. These polypeptides shared amino-terminal sequences with the 34K protein. Short-pulse analysis suggested that the heterogeneity observed with the 6/7 fusion products resulted from differential splicing patterns of related E4 mRNAs. An antiserum directed against the amino terminus of reading frame 6 recognized only the free form of the 34K antigen that was not associated with the E1B-55K protein. This observation allowed the determination of the stability of the free and complexed form of this polypeptide. Pulse-chase analyses demonstrated that both forms of the 34K protein had half-lives greater than 24 h, suggesting that complex formation did not result in stabilization of this gene product. The half-lives of the 6/7 fusion products were approximately 4 h. The 34K protein also was shown to have a nuclear localization within infected cells. Finally, analysis of a mutant carrying deletions in both the E4-34K and E1B-55K polypeptides indicated that the complex formed between these two proteins was a functional unit in lytic infection.     

Comparison of the nucleotide sequences of early region Elb DNA of human adenovirus types 12, 7 and 5 (subgroups A,B and C)

The nucleotide sequences of human adenovirus serotypes 12 (Ad 12, oncogenic subgroup A), 7 (Ad7, weakly oncogenic subgroup B) and 5 (Ad5, non-oncogenic subgroup C) DNAs have been compared. The region studied stretches from the termination codon of region Ela (m.p. 4.2) and comprises the entire region of the three viral DNAs. ending at the polyadenylation signal of the gene for viral polypeptide IVa2 (m.p. 11.2). The homology in the sequences encoding the Elb proteins of M_r 20000 and M_r 55000 is 55–60%, when two serotypes are compared, and about 45% in a three-strain comparison. However, a short internal segment encoding the C terminus of the M_r 20000 protein, and at the same time amino acids 23-120 of the M_r 55000 protein show a much higher degree of divergence in the three strains, as do the noncoding areas. The present study does not reveal how the three serotypes are phylogenetically related.     

Sequence of the immunoregulatory early region 3 and flanking sequences of adenovirus type 35

Adenovirus type 35 (Ad35) is an important pathogen in immunosuppressed individuals such as AIDS patients and bone marrow transplant recipients. Ad35, a member of Ad subgroup B, differs with respect to pathogenic properties from the more fully characterized subgroup C Ad, such as Ad2 and Ad5. One region of human Ad which varies between subgroups and which may influence Ad pathogenesis is early region 3 (E3), a region which appears to modulate the immune response to Ad infection. In order to begin to characterize the differences between the Ad35 E3 and the E3 of other Ad, the complete DNA sequence of the Ad35 E3 promoter and coding sequence along with two flanking structural proteins, pVIII and fiber, has been determined. Ad35 contains open reading frames which are unique to the subgroup B Ad in addition to the four characterized immunoregulatory proteins encoded by the subgroup C Ad. Further evaluation of the sequence of one of these proteins, 18.5K, which is the class-I major histocompatibility complex (MHC) binding protein of 18.5 kDa, demonstrates that the amino acid sequence of this Ad2 gp19K homologue fits a proposed model of gp19K-MHC interaction. Analysis of promoter sequences demonstrates that an NF-κB site found in the subgroup C E3 promoter is absent from the Ad35 E3 promoter. In addition, the fiber genes of Ad35 and other subgroup B Ad have been shown to diverge in an unexpected way, yielding three clusters of fiber homology.     

Hybridization maps of early and late messenger RNA sequences on the adenovirus type 2 genome.

Specific fragments of adenovirus type 2 DNA, generated by cleavage with restriction endonucleases endoR.EcoRI, endoR.HpaI and endoR.HindIII were used in hybridization-mapping experiments. The complementary strands of individual cleavage fragments were separated by the method of Tibbetts &; Pettersson (1974). Liquid hybridizations were performed with ³²P-labeled separated strands of cleavage fragments and messenger RNA extracted from cells early and late after adenovirus infection. The fraction of each fragment strand which was represented in “early” and “late” messenger RNA was determined by chromatography on hydroxylapatite. Early messenger RNA was found to be derived from four widely separated regions, two on the 1- and two on the h-strand (h- and l- refer to the strand with heavy and light buoyant density in CsCl when complexed with poly(U, G)). Messenger RNA, present exclusively late after infection, is derived from several locations, predominantly from the l-strand with a major block of continuous sequences extending between positions 0.25 and 0.65 on the unit map of the adenovirus type 2 genome.     

Stabilities and interrelations of multiple species of human adenovirus type 5 early region 1 proteins in infected and transformed cells.

Early region 1A (E1A) of human adenovirus type 5 (Ad5) produces two mRNAs coding for phosphoproteins of 289 and 243 residues (289R and 243R). Each of these products has been shown to migrate on sodium dodecyl sulfate gels as two major and two minor species. In the present study, the stabilities of E1A polypeptides, as well as those of some other early Ad5 proteins, were studied in infected KB cells that were pulse-labeled with [35S]methionine and then chased in the presence or absence of cycloheximide. The E1B 58,000- and 19,000-molecular-weight proteins (58K and 19K proteins; 496R and 176R) as well as the E2A 72K DNA-binding protein were relatively stable over the 4-h chase period; turnover was less than 30%. The E1A species were considerably more unstable, with an overall half-life of about 60 min. Interestingly, it was found that when cycloheximide was present during the chase, E1A proteins were much more stable, and the half-life increased to about 240 min. Analysis of the stabilities of individual E1A species indicated that the products of the 1.1-kilobase mRNA (289R) had half lives (about 55 min) somewhat shorter than those (about 90 min) of the 0.9-kilobase mRNA products (243R). In addition, the faster-migrating species produced from each mRNA (molecular weights, 48,500 and 45,000) had significantly shorter half-lives than did the slower-migrating species (52,000 and 50,000). In the presence of cycloheximide, the faster-migrating species were still quite short-lived, but the half-lives of the 52K and 50K species were considerably increased. An examination of the kinetics of turnover of the various E1A species suggested that the faster-migrating forms may be precursors to the slower-migrating ones. Somewhat similar stabilities were also found for the various E1A species in Ad5-transformed 293 cells.     

The nucleotide sequence of the transforming early region E1 of adenovirus type 5 DNA

The sequence of the leftmost 11.3% of the non-oncogenic human adenovirus type 5 (Ad5) DNA has been determined. This segment contains the entire early region E1 of the Ad5 genome which has been shown to be involved in in vitro transformation of non-permissive rodent cells (Van der Eb et al., 1980). From the DNA sequence, and from the mRNA sequence data obtained by Perricaudet et al, (1979, 1980) for the E1 mRNAs from the closely related adenovirus type 2 (Ad2), it is possible to predict the primary structure of the polypeptides encoded by this region. The function of these proteins in cell transformation is discussed. From the positions of mapped restriction endonuclease sites and termini of RNA segments in the nucleotide sequence the length of the Ad5 DNA is estimated to be 36.6 kb.     

Acylation of the 176R (19-kilodalton) early region 1B protein of human adenovirus type 5.

Antipeptide sera were prepared in rabbits against synthetic peptides corresponding to the predicted amino and carboxy termini of the early region 1B 176R (19-kilodalton [kDa]) protein of human adenovirus type 5. Both antisera specifically immunoprecipitated the 19- and 18.5-kDa forms of the 176R protein observed previously with antitumor sera. These data suggested that both species are full-length molecules of 176 residues. To identify posttranslational modifications that could explain the formation of these multiple species and possibly their known association with membranes, studies were carried out to determine whether they are glycosylated or acylated. Neither the 19- nor the 18.5-kDa species appeared to be a glycoprotein, however, they were labeled with [3H]palmitate and [3H]myristate, indicating that both species are acylated. Thus, whereas acylation does not appear to be the cause of the multiple species, it could play a role in the membrane association of these viral proteins. The acylation of 176R was found to be unusual. The fatty acid linkage was resistant to treatment with hydroxylamine or methanol-KOH, suggesting that acylation was through an amide bond. In addition, both palmitate and myristate were present in 176R, suggesting either a lack of specificity in the acylation reaction or the existence of more than one acylation site.