Nuclei of adenovirus 2-infected cells contain an RNA species that corresponds to an intron excised intact from mRNA precursors.

We used Northern and S1 nuclease analyses to identify a nuclear RNA species of the structure predicted for an intron excised from the precursor of adenovirus 2 E2A early mRNA. The structure of this excised intron demonstrated that the splicing of E2A mRNA can involve the removal of the intron sequences as single intact molecules. The concentration of the excised intron is 30 copies per nuclei, comparable to the levels of E2A polyadenylated splicing precursors, but 30-fold less than nuclear E2A mRNA. Late in infection, when the production of E2A early mRNA is dramatically elevated (Goldenberg et al., J. Virol. 38:932-939, 1981), the excess intron was not detected, indicating that either its stability or the mechanism of splicing is altered. We also identified a spliced nonpolyadenylated E2A nuclear RNA species with a structure similar to the mRNA, indicating that splicing may normally occur in the absence of polyadenylation.

Splice junctions in adenovirus 2 early region 4 mRNAs: multiple splice sites produce 18 to 24 RNAs.

We localized the splice junctions in adenovirus 2 early region 4 (E4) mRNAs. Processing of the E4 precursor RNA positioned the donor splice site of the 5' leader sequence adjacent to acceptor sites near the 5' ends of five of the six open reading regions in the E4 transcription unit. Of particular interest among the E4 mRNAs is an extensively spliced class which includes multiple species with sizes ranging from 1.1 to 0.75 kilobases (kb). Purified 1.1- to 0.75-kb mRNAs specified at least 10 polypeptides in vitro. We detected eight acceptor and two donor splice sites utilized in the deletion of the intron from the 3' portion of these mRNAs. E4 RNAs were isolated from the cytoplasm of infected cells at 5, 9, 12, and 18 h after infection. The E4 mRNAs were present throughout infection, but different members of the 1.1- to 0.7-kb class were predominant at each time assayed. Alternate splicing of the 3.0-kb E4 precursor RNA can generate as many as 25 mRNAs that encode at least 16 polypeptides.

Identification of adenovirus genes that require template replication for expression.

The relationship between adenovirus type 2 DNA replication and expression of intermediate stage viral genes was investigated. The 1.03-kilobase mRNA from early region 1b (E1b) and the mRNAs coding for proteins IX and IVa2 were first detected between 6 and 8 h postinfection. Inhibition of viral DNA replication with hydroxyurea prevented expression of the IX and IVa2 mRNAs, but not of the E1b mRNA. Pulse-labeling experiments demonstrated that the block of IX and IVa2 expression in hydroxyurea-treated cells was at the level of transcription. By a series of superinfection experiments, it was determined that the viral and cellular factors present during the late stage of adenovirus infection are insufficient to activate IX gene expression. The viral DNA template must first replicate before IX transcription can begin.

Expression of adenovirus-2 early region 4: assignment of the early region 4 polypeptides to their respective mRNAs, using in vitro translation.

Adenovirus-2 early region 4 (E4; map positions 91.3 to 99.1) encodes six 5' and 3' coterminal, differently spliced mRNAs, which are 2.5, 2.1, 1.8, 1.5, 1.2, and 0.8 kilobases (kb) long. Hybridization selection with five cloned viral DNA fragments that hybridize with subsets of E4 mRNAs was used to purify these six mRNAs and a previously unreported 3.0-kb mRNA from virus-infected cells. E4 mRNAs which were purified by hybridization selection with cloned EcoRI fragment C (map positions 89.7 to 100) were also fractionated by size. The purified mRNAs were then translated in rabbit reticulocyte or wheat germ lysate systems. The full complement of E4 mRNAs specified as many as 16 different polypeptides, with molecular weights ranging from 24,000 (24K) to 10K. The most abundant E4 mRNA, which was 2.1 kb long, specified an 11K polypeptide. The 1.5-kb mRNA, which differed from the 2.1-kb mRNA only by deletion of a second intron from the 3' untranslated region, also specified an 11K polypeptide. The second most abundant mRNA, which was 1.8 kb long, and the 1.2-kb mRNA, which had an intron deleted from the 3' untranslated region, specified a 15K polypeptide. This polypeptide was labeled more intensely with [5,6-(3)H]leucine than with [35S]methionine. The 3.0- and 2.5-kb mRNAs specified four polypeptides (24K, 22K, 19K, and 17K). Translation of E4 mRNAs with a mean size of 0.8 kb, which accumulated preferentially in the presence of cycloheximide, yielded at least 10 polypeptides that migrated in polyacrylamide gels with apparent molecular weights ranging from 21,800 to 10,000. On the basis of translation in wheat germ lysates and the distribution of polypeptides encoded by size-fractionated mRNAs, we concluded that the 0.8-kb mRNA size class includes a heterogeneous mixture of mRNAs which are probably formed as the result of utilization of alternate splice acceptor and donor sites during removal of the second intron. Our polypeptide assignments for the 2.1-, 1.8-, 1.5-, and 1.2-kb mRNAs are compatible with locations of two open coding regions in the DNA sequence (Herisse et al., Nucleic Acids Res. 9:4023-4042, 1981). The relationship between the four polypeptides encoded by the 3.0- and 2.5-kb mRNAs and the two open coding regions is discussed. The production of multiple polypeptides from a heterogenous mixture of mRNAs in the 0.8-kb size class is compatible with two large open coding regions in that part of the sequence. Thus, nearly all of the potential coding information in the leftward strand of E4 is expressed in translatable form during infection. Moreover, alternate splicing of the 0.8-kb mRNA size class can produce multiple polypeptides with common amino-terminal and different carboxy-terminal amino acid sequences, which may have the same function but different specificities.

In vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.

Early region 2 of the adenovirus 2 genome (map position 61-75) specifies two poly(A)+ nuclear RNAs (28S and 23S) that appear to be precursors of the 20S cytoplasmic mRNA [Goldenberg, C. J. & Raskas, H. J. (1979) Cell 16, 131-138]. Splicing of these nuclear RNAs in vitro has been obtained with a whole cell extract prepared from MOPC-315 mouse myeloma cells. The in vitro reaction excises sequences from two introns and attaches 5' sequences to the mRNA body. The splicing reaction was demonstrated by two procedures: (i) hybridization of pulse-labeled RNA fractionated by size and (ii) annealing of RNAs with radioactive DNA probes followed by nuclease digestion. The first procedure provided evidence that sequences from the large 2300-nucleotide intron (74.6-68.8) were excised and 5' transcripts were spliced to the mRNA body. Utilizing both S1 and Exo VII nucleases, the second procedure demonstrated excision of sequences from the smaller 720-nucleotide intron (68.5-66.3), the splicing of sequences from the second leader (68.8) to the mRNA body, and the formation of an mRNA body of 1700 nucleotides, the size found in vivo. These findings provide evidence that an in vitro system that splices viral RNAs to yield products comparable to those found in vivo is now available.

Transfection of KB cells by liposomes containing adenovirus type 2 DNA.

Adenovirus type 2 DNA was entrapped in liposomes which were then used to transfect KB cells with an efficiency of approximately 4,000 plaques per mug of encapsulated DNA.

Regulation of integrated adenovirus sequences during adenovirus infection of transformed cells.

A human cell line (293) transformed by adenovirus type 5 encodes mRNA's and proteins from the early region 1 (E1) of the viral genome. These products correspond to those synthesized early after adenovirus infection of normal cells. This pattern of expression is different from that observed at later times in the lytic cycle. We have determined whether integrated sequences can undergo the early-late transition during infection of transformed cells. Cultures of 293 cells were infected with mutants of adenovirus type 5 that have deletions in EI genes. In such infections, the integrated sequence complements the deletion mutants so that viral DNA replication, late mRNA and protein synthesis, and viral assembly occur. Because the infecting genomes lack EI sequences, the products synthesized from the integrated DNA could be analyzed. In contrast to the early-late transition that occurs with EI DNA in free viral genomes, the pattern of mRNAs and proteins made from the integrated sequences was restricted to the early pattern. Assuming that the viral sequences in 293 cells have not become altered during the history of the cells, our results suggest that regulation of integrated adenovirus genes may not be determined exclusively by nucleotide sequence recognition. Apparently, during infection certain factors prevent the integrated viral genes from responding to the regulatory signals which control late expression from free EI DNA. The distinction between integrated and free viral sequences might reflect the different fates of viral and host transcripts during the lytic cycle of adenovirus.

Transfection of KB cells by polyethylene glycol-induced fusion with erythrocyte ghosts containing adenovirus type 2 DNA.

Cell ghosts were formed by hypotonic haemolysis and subsequent isotonic re-sealing of human erythrocytes in the presence of adenovirus type 2 DNA. The ghosts entrapped virus DNA with an efficiency which depended on the salt concentration employed during haemolysis and on the concentration of the DNA itself. The entrapped DNA was largely protected from digestion by deoxyribonuclease I and could be recovered intact by phenol extraction. Erythrocyte ghosts containing 32P-adenovirus type 2 DNA were fused with KB cells during brief treatment with polyethylene glycol (PEG). Following fusion, counts equivalent to an average of 25 molecules of labelled DNA were micro-injected and transported to each KB cell nucleus. Less than 1/4 as many DNA counts were recovered from nuclei when PEG treatment was omitted. A direct immunofluorescent assay demonstrated virus replication in some cells following their fusion with DNA-containing ghosts. The efficiency of transfection was considerably lower than that expected from the large number of successfully micro-injected DNA molecules. This suggests that most of the micro-injected DNA molecules were degraded before a successful infection could be completed.

pMB9 plasmids bearing the Salmonella typhimurium his operon and gnd gene.

A plasmid containing the entire Salmonella typhimurium his operon was constructed from plasmid pM89 and an EcoRI fragment of phi 80 his imm lambda DNA. The recombinant pST41 also includes the glucose 6-phosphate dehydrogenase (gnd) gene and has one EcoRI endonuclease cleavage site in the integrated fragment. This plasmid served as a source for the construction of two additional plasmids, one carrying the OGDC-region of the his operon and the other a CBHAFIE segment of the his gene along with the gnd gene. The presence of the his operon in the constructed plasmids was confirmed by hybridization to S. typhimurium his RNA. The location of the gnd gene in the CBHAFIE fragment of the his gene was confirmed genetically: after transfection with the plasmid bearing the gnd gene, a gnd recipient gained the capacity to utilize gluconate as a sole carbon source. The DNAs of the three hybrid plasmids were analyzed by gel electrophoresis. By comparing the EcoRI endonuclease cleavage pattern of these three hybrid plasmids with the DNA cleavage pattern of phi 80 his imm lambda, phi 80 imm lambda and lambda phages, the EcoRI cleavage map of phi 80 his imm lambda was obtained.

In vitro translation products specified by the transforming region of adenovirus type 2.

Region 1 DNA sequences (map positions 0 to 11% on the linear adenovirus 2 genome) are expressed both early and late in lytic infection and are required for transformation by the virus. During productive infection six distinct cytoplasmic RNAs are synthesized from this region. These RNAs comprise two families, each consisting of three size classes that share 3' sequences. Region 1 RNA's were purified by hybridization selection, using restriction fragments bound to nitrocellulose membranes, and by size fractionation. The isolated RNAs were then translated in cell-free systems derived from wheat germ and rabbit reticulocytes. The family of RNAs specified by 0 to 4.4 sequences includes two RNAs, which are 12S and 13S in size. These RNAs were partially separated by molecular weight and translated. The 13S RNA produced 53,000-dalton (53K) and 41K peptides, and the 12S RNA synthesized 47K and 35K products. The family of RNAs mapping from 4.4 to 11.0 encodes three separate polypeptides, each of which can be assigned to a specific RNA. A 12K product that comigrates with structural polypeptide IX is synthesized from the 9S RNA as previously reported (U. Pettersson and M. B. Mathews, Cell 12:741-750, 1977). The 13S RNA encodes a 15K polypeptide that corresponds to a 15K polypeptide in infected cell extracts. The 22s RNA encodes a 52K protein distinct from the 0 to 4.4 polypeptides.

Nucleosome-like structural subunits of intranuclear parental adenovirus type 2 DNA.

The intranuclear structure of parental adenovirus 2 DNA was studied using digestion with micrococcal nuclease as a probe. When cultures were infected with 32P-labeled virions, at a multiplicity of 3,000 particles per cell, 14 to 21% of parental DNA penetrated the cell and reached the nucleus. Of this parental DNA, 60% could be solubilized by extensive digestion with micrococcal nuclease. The nuclease-resistant fraction contained viral deoxyribonucleoprotein monomers and oligomers. These nucleosome-like structures contained DNA fragments which are integral multiples of a unit-length DNA of approximately 185 base pairs. The monomeric DNA is similar in length to the unit-length DNA contained in cellular nucleosomes. However, the viral oligomers are slightly smaller than their cellular counterparts. DNA-DNA hybridization demonstrated that all segments of the viral genome, including those expressed as mRNA only at late times, are represented in the nucleosomal viral DNA. The amount of early intranuclear viral chromatin was proportional to multiplicity of infection up to multiplicities of 4,000 particles per cell. However, viral transcriptional activity did not increase in direct proportion to the amount of viral chromatin. Maximum accumulation of intranuclear viral chromatin was achieved by 3 h after infection. The intranuclear parental viral chromatin remained resistant to nuclease digestion even at late times in infection, after viral DNA replication had begun.

Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution.

A method is described for the preparation isolation of highly purified adenovirus RNA species. Cytoplasmic RNAs from cells infected with adenovirus 2 were selected by hybridization to viral DNA fragments bound to nitrocellulose membranes. A series of washes at elevated temperatures (50-70 degrees) determined conditions at which the true hybrids were stable but non-specific RNA was removed. This temperature has been found to correlate with the base composition of the DNA fragment. After washing at this predetermined temperature, the specific RNA was eluted at 85 degrees. The purity of the eluted RNA was greater than 95% as determined by size, sequence specificity, and template activity in an in vitro protein synthesizing system. The method described should be generally useful for purification of specific RNAs.

Parental adenovirus type 2 genomes recovered early or late in infection possess terminal proteins.

At both early (3 h) and late (18 h) times after infection of KB cells with adenovirus 2, more than 90% of parental nuclear viral genomes exist as complexes which contain terminally linked proteins. Density shift experiments employing 5-bromo-2'-deoxyuridine indicate that these parental DNA molecules remain complexed with terminal proteins after DNA replication. The persistent linkage of proteins to the termini of intranuclear viral DNA suggests that these proteins have an essential role in adenovirus replication.