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The Ad2 E3-10.4K protein is required together with the E3-14.5K protein to down-regulate the epidermal growth factor receptor in adenovirus-infected cells. Both proteins are also required to prevent tumor necrosis factor cytolysis under certain conditions. 10.4K is a 91 amino acid membrane-associated protein that migrates as two bands, upper and lower, on SDS-PAGE. We show here that the upper band is the primary translation product which initiates at AUG 2173 in the E3 transcription unit of Ad2. The upper band is processed slowly (>4 hr to complete) into the lower band by proteolytic cleavage between residues Ala 22 and Ala 23 by a microsome-associated protease. The upper and lower bands become equal in abundance, after which they are very stable. The N-terminus of the in vivo-derived upper band is not blocked to sequencing and it retains its initiating Met. 10.4K has a hydrophobic domain (H1) near its N-terminus that is probably a signal sequence for membrane insertion; cleavage of this signal is atypical because it was not contranslational in vivo and it was not complete. 10.4K has a second hydrophobic domain (H2) located within residues 35–60. H2 appears to be a transmembrane (stop transfer) domain because both the upper and the lower 10.4K bands remained associated with membranes after extraction at pH 11.5, because both bands were extracted into the detergent phase with Triton X-114, and because both bands were only partially reduced in size when 10.4K-containing microsomes were digested with proteinase K. These proteinase K-digested bands were immunoprecipitated with an antipeptide antiserum against residues 19–34 but not with an antiserum against residues 68–80 or 77–91, indicating that both 10.4K bands are orientated in the membrane with the C-terminus in the cytoplasm. We conclude that the lower band of 10.4K is a type I bitopic membrane protein and suggest that the upper band is a polytopic membrane protein with both the H1 and the H2 hydrophobic domains spanning the membrane.

The Ad2 proteinase, which is thought to be encoded by a 23-kDa open reading frame located at the end of the L3 family of late mRNAs, is expressed poorly even late after infection. To obtain sufficient proteinase for biochemical characterization, a DNA fragment containing the 23-kDa open reading frame was cloned into plasmids that permit efficient expression in Escherichia coli. Polyclonal antiserum specific for the Ad2 proteinase was produced by immunizing rabbits with a fusion protein that included the entire proteinase open reading frame, and this antiserum was used to show that the product of the 23-kDa reading frame is assembled into virions. Bacterial products corresponding to the complete 204 amino acid proteinase reading frame, to a 9 amino acid proteinase deletion, and to a proteinase fusion protein of 227 amino acids were used to determine the size of the proteinase polypeptide in Ad2 virions and in infected HeLa cell extracts. A single proteinase polypeptide that migrated during SDS-polyacrylamide gel electrophoresis with the 204 amino acid recombinant proteinase was detected in wild-type and H2ts1 virions, and in infected cell extracts. Immunoblot titrations showed that a wild-type Ad2 virus particle contains about 10 proteinase polypeptides; an H2ts1 virion has approximately fivefold less proteinase. In virions, the proteinase was associated primarily with the virus core. The 204 amino acid proteinase produced in E. coli permitted cleavage of the major core protein precursor, P-VII, to mature, authentic VII, but the proteinase deletion lacking 9 amino acids from near the amino-terminus was inactive. These results are inconsistent with autocatalytic processing of the Ad2 proteinase as was reported by Chatterjee and Flint (1987, Proc. Natl. Acad. Sci. USA 84, 714-718).

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

Human adenoviruses (e.g., Ad2, Ad5) establish chronic infections in human lymphoid-derived cell lines, including Raji and Jijoye ( R. E. Wallace, 1969 , Proc. Soc. Exp. Bich Med. 130, 702–710; N. Faucon, G. Ogier, and Y. Chardonnet, 1982 , J. Nad. Cancer Inst. 69, 1215–1220); however, the mechanisms by which chronic infections are established and maintained are not understood. When Raji or MOLT-3 cell cultures were infected with Ad2 at high multiplicity, these cell lines continued to grow exponentially and produced only small amounts of infectious virus. Virus-specific antigens, including the DNA-binding protein and hexon, were expressed in only 5% of the Ad2-inoculated cultures. All Raji and MOLT-3 cells were found to have adenovirus receptors, but the Ad2 virions that adsorbed to most Raji cells were sequestered in caps, suggesting that most cells fail to internalize adsorbed Ad2. Cell synchronization experiments showed a correlation between the proportion of cells that became productively infected and the proportion of cells in mitosis at the time of infection. In contrast, primary blood lymphocytes had few, if any, Ad2 receptors and were not productively infected by Ad2.

Adenovirus 2 virions contain a small, highly basic protein known as μ (mu). Partial sequence analysis of mu labeled with radioactive amino acids showed that it is derived from an 11-kDa virion precursor protein, L2–79R. Amino acid analysis, direct microsequence analysis, time-of-flight mass spectrometer analysis, and chemical synthesis demonstrated that mu is the unmodified, 19 amino acid peptide obtained from the 79-residue precursor by adenovirus-encoded proteinase-mediated cleavage after glycine31 and glycine50. Mu bound tightly to DNA and was located in the virion core. In vitro, mu could precipitate DNA fragments, suggesting that it may have a role in viral chromosome condensation.

Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a single (virus-encoded?) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381–3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.

The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor of the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. We have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glycine amino acid pair not previously reported to be a viral cleavage site.

Interserotypic recombinants are formed in crosses between temperature-sensitive ( ts ) mutants of adenovirus type 5 (Ad5) and the nondefective adenovirus 2-SV40 hybrid virus Ad2 + ND1. The positions of Ad2 + ND1 and Ad5 DNA sequences in recombinant genomes can be determined by analysis of their DNA with restriction endonucleases and the sites of the mutation in many ts mutants have been mapped [Grodzicker, T., Williams, J. F., Sharp, P. A., and Sambrook, J. (1974), Cold Spring Harbor Zymp. Quant. Biol. 39 , 439–446; Sambrook, J., Williams, J., Sharp, P. A., and Grodzicker, T. (1975), J. Mol. Biol. 97 , 369–390]. We show here that several Ad5 polypeptides can be distinguished from their Ad2 + ND1 counterparts by SDS-polyacrylamide gel electrophoresis. Analysis of the polypeptides encoded by recombinants of known genetic constitution has allowed us to locate several structural genes on the adenovirus genetic and physical maps and to assign ts mutants to specific genes. Furthermore, we have physically mapped the early Ad5 mutant ts 125 which has a defect in the gene for the adenovirus-specific single-stranded binding protein. In addition, we show that the virus-associated RNAs of Ad2 and Ad5 can be separated electrophoretically, permitting rapid and simple determination of the origin of the DNA surrounding position 0.30 in interserotypic recombinants.

An attempt was made to isolate spontaneous mutants of adenovirus type 2 and of the adenovirus-SV40 hybrids, Ad2 + ND3 and Ad2 + ND5, that would grow efficiently on monkey cells. Virus stocks were serially passaged through the semipermissive established monkey libe CV-1. After five serial passages in the absence of intentional mutagenesis, only stocks of Ad2 + ND3 yielded significant numbers of variants that plaqued with similar efficiency on human and on monkey cell monolayers. Four independent Ad2 + ND3 variants, designated hr600, hr601, hr602, and hr603, have been isolated and partially characterized. Each grows and plaques on CV-1 monolayers with at least 100-fold greater efficiency than does the parent Ad2 + ND3. No differences was found between the genomes of these variants and the genome of parental Ad2 + ND3 by restriction enzyme analysis or by the analysis of heteroduplexes between Ad2 + ND3 (or variant) DNA and DNA of the hybrid Ad2 + ND1. Monkey cells infected with each of the variants synthesize as much fiber protein and component IIIa protein as do monkey cells productively infected with Ad2 + ND1. Phenotypically, these variants resemble adenovirus 2 host-range variants selected by Klessig ( J. Virol. 21 , 1243–1246, 1977) after nitrous acid treatment of adenovirus 2 stocks.

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 and P3 regions of the polyprotein. Sequence analysis of stepwise Edman degradation show that these proteins are 3b/9 (Mr 77,000) and X/9 (Mr 50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.

Each of types 2, 5 and 12 human adenovirus (Ad) induces the synthesis of single-strand-specific DNA-binding proteins at early times after infection of human or monkey cells. The molecular weights of these proteins are: Ad2, 72,000; Ad5, 72,000; and Ad12, 58,000. In addition, each of these viruses produces a collection of two or three lower molecular weight (41,000–47,000), single-strand-specific DNA-binding proteins that appear in variable amounts after infection. Tryptic fingerprints of the peptides derived from [ 35 S]methionine-labeled 72,000-MW (Ad2 and 5), 58,000-MW (Ad12) and 41,000–47,000-MW (Ad2, 5 and 12) DNA-binding proteins have been obtained. The results of this analysis permit the following conclusions: (i) The 72,000-MW (Ad2 and 5) and 58,000-MW (Ad12) proteins synthesized in adenovirus-infected cells are distinct from a 72,000-MW cellular DNA-binding protein found in uninfected or mock-infected cells. (ii) The [ 35 S]methionine-labeled peptides derived from the 41,000–47,000-MW proteins (Ad2, 5 and 12) are similar if not identical to the [ 35 S]methionine-labeled tryptic peptides derived from the larger (Ad2 and 5, 72,000-MW; Ad12, 58,000-MW) DNA-binding proteins. (iii) The [ 35 S]methionine-labeled peptides derived from the adenovirus types 2 and 5, 72,000-MW proteins are quite similar but distinct from the adenovirus type 12, 58,000-MW protein. (iv) The peptide fingerprints of the adenovirus type 2, 72,000-MW protein synthesized in either human or monkey cells are very similar or identical.

Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the “i”-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the Header segment.

We have investigated the relationships among the major proteins encoded by early region 1A of Ad2 by comparative analysis of their [ 35 S]methionine-labeled tryptic peptides using reverse phase high-performance liquid chromatography. In some instances the purified peptides were subjected to automatic sequential Edmund degradation in order to determine the position at which the methionine residues occur within the peptides. These peptides have then been located in the amino acid sequence predicted from the DNA sequence of the leftmost 4.5% of the viral genome. The data show that the 50,000, 46,000, and 42,000 molecular weight E1A proteins (all of which focus at ca. pH 6.0) obtained from Ad2-infected HeLa cells are derived from the 1.1 kb region 1A mRNA. Similarly, it is shown that two polypeptides, 58,000 (58K) and 48,000 (48K), result from cell-free translation of the 1.1 kb mRNA. The data are also consistent with the hypothesis that the in vivo 46,000, 42,000, and 38,000 molecular weight E1A proteins (all of which focus at ca pH 5.9) are derived from the 0.9 kb region 1A mRNA. Cell-free translation of this mRNA yields two polypeptides, 54,000 (54K) and 42,000 (42K).

The partial amino-terminal amino acid sequence has been determined for four adenovirus 2 proteins: hexon, fiber, component IX, and early protein E1B-15K. A comparison of these sequences with the nucleotide sequence of the region of the genome encoding each of these proteins has identified the initiation sites for protein synthesis. Each protein is initiated at the AUG codon nearest the 5′ end of its mRNA. The initiating methionine is retained by fiber and component IX while it is removed from hexon and protein E1B-15K.

A c-DNA clone containing the complete sequence information for the murine p53 protein, from embryonal carcinoma cells, has been isolated. The nucleotide sequence of this clone reveals an open reading frame encoding a protein of 390 amino acids with a molecular weight of 43,364 Da. The NH2-terminal domain of this protein is acidic whereas the carboxyl terminus is rich in basic amino acid residues. These terminal domains are separated by a proline-rich, hydrophobic run of amino acids. Proline comprises approximately 10% of the total amino acid residues. Two tryptic peptides, derived from p53 protein radiolabeled with either methionine or proline, were purified and the position of these labeled residues in the peptide was determined. The positions of three methionine and five proline residues in these two peptides matched the amino acid sequence of the predicted open reading frame determined from the c-DNA clone.