Sequences from the beginning of the fiber messenger RNA of adenovirus-2.

Small restriction fragments, from around co-ordinate 86.6 on the adenovirus-2 genome, have been used as primers for direct DNA sequence analysis by Sanger's (Sanger et al., 1977) chain termination method with Ad2² DNA as template. The genomic sequences obtained have been compared with sequences deduced using fiber messenger RNA from Ad2 or Ad2⁺ ND5-infected cells as template. With one primer, Hha 54, the sequences complementary to mRNA match those of the genome for 10 nucleotides but then differ from those found on the genome because this primer hybridizes near the point at which the leader sequence becomes joined to the main body of fiber mRNA. Using Ad2⁺ ND5 fiber mRNA as template, the sequence beyond the point of divergence matches the known sequence of the third leader component of one of the late Ad2 mRNAs, that encoding the hexon polypeptide. With Ad2 fiber mRNA, a heterogeneous sequence continuation is found, in accordance with earlier findings that two major species of fiber mRNA are present, which differ in the nature of the leader component joined to the main body of fiber mRNA (Chow &; Broker, 1978). Nevertheless, the data suggest that both leader components appear to become joined to the same nucleotide at the start of the main body of fiber mRNA.The AUG codon, which probably encodes the N-terminus of the fiber polypeptide, occurs just two nucleotides beyond the point at which these leader segments become spliced to the main body of fiber mRNA.     

The genes of influenza virus

The 5′ terminal sequences of several adenovirus 2 (Ad2) mRNAs, isolated late in infection, are complementary to sequences within the Ad2 genome which are remote from the DNA from which the main coding sequence of each mRNA is transcribed. This has been observed by forming RNA displacement loops (R loops) between Ad2 DNA and unfractionated polysomal RNA from infected cells. The 5′ terminal sequences of mRNAs in R loops, variously located between positions 36 and 92, form complex secondary hybrids with single-stranded DNA from restriction endonuclease fragments containing sequences to the left of position 36 on the Ad2 genome. The structures visualized in the electron microscope show that short sequences coded at map positions 16.6, 19.6 and 26.6 on the R strand are joined to form a leader sequence of 150–200 nucleotides at the 5′ end of many late mRNAs. A late mRNA which maps to the left of position 16.6 shows a different pattern of second site hybridization. It contains sequences from 4.9?6.0 linked directly to those from 9.6?10.9. These findings imply a new mechanism for the biosynthesis of Ad2 mRNA in mammalian cells.     

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.     

Visualization and mapping of late nuclear adenovirus RNA

Nuclei of KB cells harvested at late stages of productive infection with adenovirus type 2 (Ad2) harbor RNA molecules which measure up to 13 μm in length, as determined by electron microscopy of denatured RNA. While some of the molecules display features of secondary structure that are characteristic for precursor rRNA, our interest was in those showing almost no intramolecular folding. When hybridized to double-stranded viral DNA under conditions which favor RNA:DNA duplex formation, nuclear AD2 RNA displaces the homologous DNA region and generates R loop structures whose size is proportional to the length of the hybridizing RNA. Slowly sedimenting RNA forms small R loops, whereas RNA of high sedimentation velocity generates loops that span a large proportion of the DNA length. Using SV40 sequences within Ad2⁺ND₄ hybrid DNA as a position marker, we oriented many of the R loops on the conventional Ad2 map. Our analysis was restricted to the most abundant sequences of late Ad2 nuclear RNA participating in R loop formation. A small but significant proportion of large RNA generates loops between map positions 0.3 and 0.9. The much more frequent RNA of intermediate size (although larger than mRNA) hybridizes with midpoints near map positions 0.55 and 0.88 — that is, near the gene locations for hexon and fiber. Our findings are compatible with the idea that the nuclear RNAs visualized in this study are intermediates in a processing pathway leading to mature forms of late Ad2 mRNA.     

Adenovirus type 2 fiber mRNA synthesis: no evidence for a cytoplasmic processing pathway.

Adenovirus type 2 fiber mRNA exists in several forms in the cytoplasm which differ in the presence or absence of extra 5'-leader segments (L. T. Chow and T. R. Broker, Cell 15:497-510, 1978). We have investigated the possibility that forms possessing extra leader segments serve as precursors to the mature form in the cytoplasm. Pulse-labeled fiber mRNA became considerably shorter (150 to 250 bases) during a chase; however, most of the pulse-labeled species failed to hybridize to DNA fragments known to encode extra leader segments. Moreover, the entire decrease in size appeared to be due to extensive shortening of the polyadenylic acid tail. Mature-sized fiber mRNA was synthesized normally in cells infected with the nondefective adenovirus type 2-simian virus 40 hybrid virus Ad2+ND5, in which the region encoding the extra leader segments is deleted. These results indicate that the additional 5'-leader segments present in wild-type adenovirus type 2 fiber mRNA are not required for the production of mature fiber mRNA and that species that possess them are not cytoplasmic precursors to the mature form.     

Conditional lethal mutants of adenovirus type 2-simian virus 40 hybrids. II. Ad2+ND1 host-range mutants that synthesize fragments of the Ad2+ND1 30K protein.

Adenovirus type 2 (Ad2) grows 1,000 times less well in monkey cells than in human cells. This defect can be overcome, not only upon co-infection of cells with simian virus 40 (SV40), but also when the relevant part of the SV40 genome is integrated into the adenovirus genome to form an adenovirus-SV40 hybrid virus. We have used the nondefective Ad2-SV40 hybrid virus Ad2+ND1, which contains an insertion of 17% of the SV40 genome, to isolate host-range mutants which are defective in growth on monkey cells although they grow normally on human cells. Like Ad2, these mutants are defective in the synthesis of late proteins in monkey cells. A 30,000-molecular-weight protein (30K), unique to Ad2+ND1-infected cells, can be synthesized in vitro, using Ad2+ND1 mRNA that contains SV40 sequences. 30K is not seen in cells infected with those host-range mutants that are most defective in growth on monkey cells, and translation in vitro of SV40-specific mRNA from these cells produces new unique polypeptides, instead of 30K. Genetic and biochemical analyses indicate that these mutants carry point mutations rather than deletions.     

Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA.

Fiber mRNA of adenovirus 2 has been used as a template for RNA-dependent DNA polymerase. The resulting cDNA/RNA hybrids have been inserted at the Pst I site of the plasmid vector pBR322 after A:T tailing. One recombinant plasmid, pJAW 43, has been characterized in detail and shown to contain sequences from the main body of fiber mRNA, the three leaders common to most late adenoviral mRNAs and a fourth leader found in some species of fiber mRNA. The complete DNA sequence of the leader region has been determined and does not contain the initiation codon AUG, although this codon does occur immediately downstream from the junction between the fourth leader and the main body of the fiber mRNA. The first leader (map coordinate 16.6) is 41 nucleotides long, the second (from 19.6) is 71 nucleotides, the third (from 26.6) is 88 nucleotides and the fourth (from 78.5) is 181 nucleotides. The location of junctions between viral leaders and intervening sequences has been determined by reference, where possible, to sequences of the adenovirus 2 genome. Although the presence of short repeated sequences at the boundaries of intervening sequences and leaders makes it impossible to locate the splice point unambiguously, all of the leader-intervening sequence junctions can be arranged to stress a common feature--the presence of the dinucleotides GT and AG at the 5' and 3' ends, respectively, of the intervening sequences. This prototype sequence, which has also been recognized at or near the splice points in other eucaryotic systems, is possibly part of a larger unit which serves as a recognition site for specific excision-ligation events that ultimately lead to the production of mature mRNAs.     

Methylation of late adenovirus 2 nuclear and messenger RNA.

Methylation of adenovirus 2 (Ad 2) late RNA was studied. RNA was double-labeled with [³H-methyl]-methionine and [¹⁴C]-uridine 15–20 h postinfection. Nuclear RNA (rRNA) and cytoplasmic RNA (mRNA) was extracted, and fractionated into polyA(+) and (?) molecules using poly(U)-Sepharose. Ad 2 specific RNA was purified by 2 cycles of hybridization to and elution from Ad 2 DNA immobilized on filters. The Ad 2 polyA(+) and (?) nRNA and mRNA fractions had the same ${}^3\text{H}{}^{14}\text{C}$ ratios, and were estimated to contain a minimum of 1.4 methylated nucleotides per 1000 bases. Viral RNA was digested with RNase T₂ and chromatographed on DEAE-Sephadex in 7 M urea at pH 7.6. All four Ad RNA fractions contained methylated constituents consistent with: (1) two classes of methylated “capped” 5′-termini with general structures m⁷ GpppN^mpNp and m⁷ GpppN^mpN^mpNp; (2) internal base methylations; (3) minor amounts of internal ribose 2′-0-methylations. Two classes of 5′-termini have previously been reported for animal cell mRNA, but not for mRNA from a variety of viruses. Internal methylations may be unique to RNA molecules transcribed in the nucleus, since they have not been found in RNA from cytoplasmic viruses. No gross differences were observed in the DEAE-Sephadex elution profiles of the methylated constituents of the four types of Ad 2 RNA. These results suggest that the majority of methylation events occur in the nucleus, and raise the possibility that Ad 2 methylated late nRNA may differ significantly from SV40 late nRNA (Lavi, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. USA $\text{72}$, 2012–2016).     

Simian virus 40-specific polypeptides in AD2+ ND1- and Ad2+ ND4-infected cells.

A comparison of the proteins synthesized in human cells at late times after infection with adenovirus (Ad2) and with the adeno-simian virus 40 (SV40) hybrid viruses revealed polypeptides of 30,000 and 92,000 molecular weight specific for the hybrid viruses Ad2+ND1 and Ad2+ND4, respectively. Cell-free translation of SV40-specific mRNA, prepared from these cells by hybridization of total cytoplasmic RNA to SV40 DNA, showed that the mRNA's specifying these two polypeptides were at least partially encoded by the SV40 portion of the hybrid viruses. Cell-free translation of SV40-specific mRNA prepared from monkey cells infected with SV40 produced polypeptides of 40,000, 43,000, 48,500, and 92,000 molecular weight. The SV40 and Ad2+ND4 92,000-molecular-weight polypeptides made in vitro were very similar in electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to the polypeptide precipitated by Tegtmeyer (1974) with SV40 anti-T serum.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses II. Relationship of Adenovirus 2 Deoxyribonucleic Acid and Simian Virus 40 Deoxyribonucleic Acid in the Ad2+ND1 Genome

A nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), has been plaque-isolated from an Ad2-SV40 hybrid population. This virus, unlike the defective Ad-SV40 hybrid populations previously described, replicates without the aid of nonhybrid adenovirus helper. Consequently, the hybrid virus deoxyribonucleic acid (DNA) can be obtained free of nonhybrid adenovirus DNA. The DNA of the Ad2(+)ND(1) virus was shown by ribonucleic acid (RNA)-DNA hybridization to consist of nucleotide sequences complementary to Ad2- and SV40-specific RNA. Techniques of equilibrium density and rate zonal centrifugation were employed to demonstrate that these Ad2 and SV40 nucleotide sequences were linked together in the same DNA molecules by alkali-resistant bonds. Calibration curves were established relating the amount of tritium-labeled SV40-specific RNA (prepared in vitro or in vivo) bound to given amounts of SV40 DNA in a hybridization reaction, and these curves were employed to determine the equivalent amount of SV40 DNA in the Ad2(+)ND(1) molecule. From the results obtained, it was estimated that 1% of the Ad2(+)ND(1) DNA consists of SV40 nucleotide sequences.     

Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells.

The genomes of the two nondefective adenovirus 2/simian virus 40 (Ad2/SV 40) hybrid viruses, nondefective Ad2/SV 40 hybrid virus 1 (Ad2+ND1) and nondefective hybrid virus 3 (Ad2+ND3), WERE FORMED BY A DELETION OF ABOUT 5% OF Ad2 DNA and insertion of part of the SV40 genome. We have compared the cytoplasmic RNA synthesized during both the early and late stages of lytic infection of human cells by these hybrid viruses to that expressed in Ad2-infected and SV40-infected cells. Separated strands of the six fragments of 32P-labeled Ad2 DNA produced by cleavage with the restriction endonuclease EcoRI (isolated from Escherichia coli) and the four fragments of 32P-labeled SV40 DNA produced by cleavage with both a restriction nuclease isolated from Haemophilus parainfluenzae, Hpa1, and EcoRI were prepared by electrophoresis of denatured DNA in agarose gels. The fraction of each fragment strand expressed as cytoplasmic RNA was determined by annealing fragmented 32P-labeled strands to an excess of cellular RNA extracted from infected cells. The segment of Ad2 DNA deleted from both hybrid virus genomes is transcribed into cytoplasmic mRNA during the early phase of Ad2 infection. Hence, we suggest that Ad2 codes for at least one "early" gene product which is nonessential for virus growth in cell culture. In both early Ad2+ND1 and Ad2+ND3-infected cells, 1,000 bases of Ad2 DNA adjacent to the integrated SV40 sequences are expressed as cytoplasmic RNA but are not similarly expressed in early Ad2-infected cells. The 3' termini of this early hybrid virus RNA maps in the vicinity of 0.18 on the conventional SV40 map and probably terminates at the same position as early lytic SV40 cytoplasmic RNA. Therefore, the base sequence in this region of SV40 DNA specifies the 3' termini of early messenger RNA present in both hybrid virus and SV40-infected cells.     

Characterization of the simian virus 40-specific messenger RNAs isolated from HeLa cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4

Previous work has shown that cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses, Ad2⁺ND2 and Ad2⁺ND4 synthesize more than one SV40⁴ large T antigen-related protein. These proteins overlap in amino acid sequence and have their carboxy-terminal sequences in common (Mann et al., 1977). We have characterized the messenger RNAs coding for these SV40-specific proteins. By translating in vitro SV40-specific mRNA isolated from cells infected with these viruses we have shown that each SV40-specific protein can incorporate ³⁵S-labeled formyl methionine at its N-terminus donated by [³⁵S]-fmet-tRNA_f^met, demonstrating that each protein results from a de novo initiation event. Furthermore, analysis of the N-terminal tryptic peptides of these proteins indicates that each protein has a unique N-terminal peptide and therefore a unique initiation site for protein synthesis, with the possible exception of the 74,000 and 95,000 molecular weight proteins, which may have the same N-terminal sequence. Therefore, these proteins cannot be derived by proteolytic cleavage of a large precursor protein.The messenger activities for many of the hybrid virus proteins can be resolved by gel electrophoresis, demonstrating the presence of multiple SV40-specific mRNA species. This result is consistent with the possibility that each SV40-specific protein is coded by a distinct species of RNA.