E1B Region Research Articles

Polypeptides encoded by transforming region E1b of human adenovirus 2: Immunoprecipitation from transformed and infected cells and cell-free translation of Elb-specific mRNA

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [ 35S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.

Different mRNAs from the transforming region of highly oncogenic and non-oncogenic human adenoviruses.

Only a fraction of the genome of adenovirus, a DNA tumour virus, is required for transformation in vitro1–4. The leftmost 11% of the genome, including the two transcription units E1A and E1B, appear to be sufficient for transformation of rat embryo cells in tissue culture for highly, weakly and non-oncogenic adenovirus serotypes5,6. Various spliced mRNAs, orginating from the transforming region, have been identified7-11 and in some cases the corresponding protein products found12–14 but it is unknown what renders some serotypes highly oncogenic for baby hamsters compared with others that are non-oncogenic although able to transform cells in vitro. We have studied the mRNAs from the transforming region of the highly oncogenic serotype 12 (Ad12) in the hope of finding novel mRNAs which could account for the oncogenic properties of the virus. We have previously failed to find any differences between the spliced mRNAs in the transcription unit of the E1A region of Ad12 and the corresponding mRNAs from the non-oncogenic type 2 (Ad2)15. Here we have extended our analysis to the E1B region and show that the highly oncogenic Ad12 encodes mRNAs which have different structures from those encoded by Ad2.

Viral messenger RNAs in six lines of adenovirus-transformed cells

We have compared the proteins translated in vitro using virus-specific mRNA from six lines of adenovirus-transformed cells (F4, F17, F19, REM, T2C4, and 293) with those made using RNA from productively infected cells. The mRNAs for proteins E1A-58K, 54K, 48K, and 42K, and for E1B-15K, were detected in all cell lines and for E1B-57K in at least five of the lines. Neither of the minor E1A mRNAs (for proteins 25K and 28K) found under specific conditions in infected cells were found in any transformed cell line. Two cell lines, F4 and T2C4, contained small amounts of other mRNAs encoded within region E1B including in the latter case one not detected in infected cells. The lines F4, REM, and T2C4 contain at least some E4 mRNAs, again including in the latter case one not detected in infected cells. T2C4 cells also contain E3 mRNAs, E2A-72K protein mRNA, and two E2B mRNAs, although the E2B proteins are not identical to any found in infected cells. The F19 and T2C4 lines express mRNAs encoded between 11 and 17 map units, the “IVa 2 region,” but do not include the mRNA for polypeptide IVa 2 itself. Some early mRNAs, including the “immediate early” mRNAs, have not been detected in any of these lines of transformed cells: nor did the cell lines contain any mRNAs found exclusively at late times during productive infection. Comparison of these results with the properties of the cells tentatively suggest a correlation (although not necessarily causal) between the expression of additional viral genes and increased tumorigenicity.

The 2.2 kb E1b mRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets

By nucleotide sequence analysis and S1 nuclease mapping we have determined the structural organization of early region E1b of Ad12. We have also revised the nucleotide sequence of the E1b region of Ad5. Both regions have an identical structural organization and show considerable homology at the nucleotide level. The major tumor antigens (Ad12, 19 and 54 kilodaltons [kd]); Ad5, 21 and 55 kd) are encoded in two overlapping reading frames. A single mRNA of 2.2 kilobases codes for both these proteins, depending on which AUG triplet serves as the start codon: the 19–21 kd protein initiates at the 5′-proximal AUG; the 54–55 kd protein initiates at the second AUG in another reading frame. Peptide mapping shows that the small and large tumor antigens do not share common tryptic peptides, in accordance with the nucleic acid sequence data. In addition, the 19–21 kd protein can also by synthesized from a one kilobase mRNA. Finally, the gene for the Ad12 analog of protein IX is characterized.

Nuclear RNA is spliced in the absence of poly(A) addition

Splicing of newly formed nuclear RNA transcripts has been demonstrated during adenovirus type 2 (Ad2) mRNA formation in HeLa cells in the presence of “cordycepin,” 3′ deoxyadenosine, a drug that stops poly(A) addition to nuclear RNA. Nuclear RNA prepared from Ad2-infected cells after a 30 min label time in the presence or absence of 3′ deoxyadenosine was hybridized to and eluted from Ad2 DNA sequences in the transcription units of region E1b and region E2. The nuclear RNA from the 3′ dA-treated cells did not contain poly(A) but did contain Ad2-specific molecules ∼200 to 250 bases shorter than the spliced mRNAs of the control infected cells. In addition, the ∼2 kb RNA from Ad2 region E2 was shown to have sequences that lie more than 3.5 kb apart on the DNA, suggesting that correct cutting and splicing of the primary transcript in the absence of poly(A) synthesis had occurred. Therefore, although poly(A) addition usually precedes splicing during mRNA formation, poly(A) is not required for splicing.

Structure of two adenovirus type 12 transforming polypeptides and their evolutionary implications.

The human adenoviruses are classified according to their nucleotide sequence homology and their oncogenic potential in rodents. The left-hand end of the genome of the adenovirus types 5 (ad5), 7 (ad7) and 12 (ad12), which are respectively, non-oncogenic (subgroup C), weakly oncogenic (subgroup B) and highly oncogenic (subgroup A), contains all the genetic information needed to induce and maintain the transformed phenotype. This part of the genome contains the early transcription unit designated E1 which is subdivided in two transcription units E1A and E1B. Two spliced mRNAs are transcribed from the E1A region which codes for several phosphorylated polypeptides. These polypeptides play a key role by controlling the expression of the other early transcription units. The major role of region E1A in adenovirus cell transformation might not be activate the true transforming genes of the region E1B. An additional role probably consists of the activation of some cellular genes as a restriction fragment containing this region can immortalize rodent cells in vitro. An important question is why some adenoviruses are oncogenic and others are not. We report here differences in the structures of the E1A polypeptides from ad7 and ad12, compared to ad5, which may partially account for their differing oncogenicity.

Predicted structure of two adenovirus tumor antigens.

Early adenovirus type 2(Ad2) mRNA sequences have been cloned by using the pBR322 plasmid as a vector. Two clones that include sequences from region E1B were identified and their DNAs were characterized by hybridization, restriction enzyme cleavage, and DNA sequence analysis. The results showed that the clones were derived from two different spliced mRNAs. By combining our results with the established DNA sequence for region E1B of the closely related adenovirus type 5[Maat, J., van Beveren, C.P. & van Ormondt, H. (1980) Gene, in press] it was possible to deduce the structure of a 13S and a 22S mRNA. The two mRNAs differ from each other by the size of their intervening sequences. If translation starts at the first AUG following the cap, the 22S mRNA encodes a Mr 67,000 polypeptide that is terminated by a UGA stop codon located immediately before the splice, whereas the 13S mRNA encodes a Mr 20,000 polypeptide that is translated in different reading frames before and after the splice. The Mr 20,000 and 67,000 polypeptides correspond in molecular weight to two proteins that invariably are precipitated from infected cell extracts by antisera from animals carrying adenovirus-induced tumors.

Early viral proteins in HeLa cells infected with adenovirus type 5 host range mutants

Several classes of adenovirus type 5 host range mutants have been isolated, These mutants replicate in Ads-transformed human embryonic kidney cells (293 cells) but at low multiplicities of infection fail to replicate in HeLa cells. Experiments are presented that demonstrate that these host range mutants form two complementation group;: group I maps in region E1a of the adenovirus genome and contains mutants hr1, hr2, dl311, and dl312; group II maps in region Elb and includes mutants hr6, hr7, dl313, and sub315. Mutants dl314 and sub316 both fail to efficiently complement any of the group I and II host range mutants. At low multiplicities of infection most group I mutants failed to produce detectable levels of virus proteins derived from regions E1, E2, or E3 of the viral genome. This group I phenotype was markedly depen(lent upon the multiplicity of infection; at a multiplicity of 100 PFU/cell, hr1-infected HeLa cells contained region E1b, E2, and E3 proteins, Group II mutants failed to produce only region E1b proteins; some of these mutants overproduced (two- to fivefold) region E2 and E3 proteins,