Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.     

Comparison of viral RNA sequences in adenovirus 2-transformed and lytically infected cells

The complementary strands of fragments of ³²P-labelled adenovirus 2 DNA generated by cleavage with restriction endonucleases EcoRI or Hpa1 were separated by electrophoresis. Saturation hybridization reactions were performed between these fragment strands and unlabelled RNA extracted from the cytoplasm of adenovirus 2-transformed rat embryo cells or from human cells early after adenovirus 2 infection. The fraction of each fragment strand complementary to RNA from these sources was measured by chromatography on hydroxylapatite. Maps of the viral DNA sequences complementary to messenger RNA in different lines of transformed cells and early during lytic infection of human cells were constructed.Five lines of adenovirus 2-transformed cells were examined. All contained the same RNA sequences, complementary to about 10% of the light strand of EcoRI fragment A. DNA sequences coding for this RNA were more precisely located using Hpa1 fragments E and C and mapped at the left-hand end of the genome. Thus any viral function expressed in all adenovirus 2-transformed cells, tumour antigen, for example, must be coded by this region of the viral genome. Two lines, F17 and F18, express only these sequences; two others, 8617 and REM, also contain mRNA complementary to about 7% of the heavy strand of the right-hand end of adenovirus 2 DNA; a fifth line, T2C4, contains these and many additional viral RNA sequences in its cytoplasm.The viral RNA sequences found in all lines of transformed cells are also present in the cytoplasm of human cells during the early phase of a lytic adenovirus infection. The additional cytoplasmic sequences in the 8617 and REM cell lines also correspond to “early” RNA sequences.     

Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2-infected and transformed cells.

The concentrations, in copies per cell, of viral RNA sequences complementary to different regions of the genome were determined at 8, 18 and 32 hours after infection of human cells with adenovirus type 2: separated strands of fragments of ³²P-labelled adenovirus 2 DNA, generated by cleavage with restriction endonucleases EcoR1, Hpa1 and BamH1, were added to reaction mixtures at sufficient concentrations to drive hybridizations with infected or transformed cell RNA. Under these conditions, the fraction of ³²P-labelled DNA entering hybrid is directly proportional to the absolute amount of complementary RNA in the reaction.At 8 hours after infection in the presence of cytosine arabinoside, “early” viral messenger RNA sequences are present at a frequency of 300 to 1000 copies per cell. The abundance of early mRNA sequences in different lines of adenovirus 2-transformed rat cells is markedly lower than their concentration in lytically infected cells. Moreover, the abundance of early mRNA in a given transformed rat cell line reflects the number of copies of its template DNA sequences per diploid quantity of cell DNA. After the onset of the late phase of the lytic cycle, the abundance of one early mRNA species, that coding for a single-stranded DNA binding protein required for viral DNA replication, is amplified. Viral RNA sequences complementary to regions of the genome coding for other early mRNA sequences remain at the level observed at 8 hours after infection.Exclusively “late” viral mRNA sequences are present over a range of concentrations, 500 to 10,000 copies per cell, depending on the region of the genome. By 18 hours after infection, the nucleus contains approximately three times as much total, viral RNA as the cytoplasm. The abundant nuclear, viral RNA sequences at 18 hours are transcribed from a contiguous region, 65% of the genome in length. In some cases, viral RNA sequences complementary to mRNA sequences are very abundant in the nucleus. When cytoplasmic and nuclear fractions are mixed and incubated under annealing conditions, some mRNA sequences will anneal with more abundant, anti-messenger nuclear RNA sequences to form double-stranded RNA. Such annealing of nuclear, viral RNA to early, cytoplasmic mRNA sequences probably accounts for the inability to detect, by filter hybridization, certain classes of early mRNA sequences during the late stage of infection.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.     

Transcription of the genome of adenovirus type 12. IV. Maps of stable late RNA from productively infected human cells.

From human KB cells productively infected with adenovirus type 12, mRNA and stable nuclear RNA were isolated late (42 h) after infection. Using restriction endonuclease fragments of adenovirus type 12 DNA, mRNA and stable nuclear RNA sequences were mapped on the viral genome. Late after infection, preferentially the r (= rightward) strand is transcribed into stable nuclear RNA, whereas the l (= leftward) strand is expressed only to a minor extent. Adenovirus type 12-specific mRNA originates from the following sections on the viral genome: 0 to 0.11, 0.18 to 0.20, 0.27 to 0.49, 0.56 to 0.63, 0.68 to 0.84, and 0.89 to 0.92 fractional length units on the r strand and 0.11 to 0.16, 0.22 to 0.27, 0.50 to 0.54, 0.62 to 0.66, 0.855 to 0.865, and 0.93 to 1.0 fractional length units on the l strand. Self-complementary viral RNA isolated at 42 h postinfection anneals to 70 to 80% of each strand of the viral genome.     

Isolation and Characterization of Simian Virus 40 Ribonucleic Acid

Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization in formamide was used to isolate simian virus 40-specific RNA. Early in the lytic cycle, a 19S viral RNA species was observed. Late in the lytic cycle, 16S and 19S viral species were found. The 16S and 19S species of viral RNA were localized in the cytoplasm. High-molecular-weight heterogeneous RNA, containing viral sequences, was isolated from the nuclear fraction of infected cells late in the lytic cycle. This RNA may contain non-viral sequences linked to viral sequences. The formamide hybridization technique can be used to isolate intact late lytic viral RNA which is at least 99% pure.     

Relationship of mRNA from Productively Infected Cells to the Complementary Strands of Adenovirus Type 2 DNA

The complementary strands of adenovirus type 2 (Ad2) DNA were separated by buoyant density gradient centrifugation with poly (U, G). The complementary strand DNA was shown to remain intact through the course of strand separation. The l-strand of Ad2 DNA, appearing in the less dense complex with poly (U, G) in neutral CsCl density gradients, was shown to have a buoyant density in alkaline (pH 12.5) CsCl density gradients which is 2 to 3 mg per ml greater than that of its complement (h-strand). Renaturation of purified complementary strand DNA was observed only in mixtures of h- and l-strand DNA, and then with the second-order reaction rate expected for Ad2 DNA. Hybridization of the complementary strands of Ad2 DNA with cytoplasmic mRNA isolated from infected HeLa cells was performed in liquid phase and analyzed by hydroxylapatite chromatography. Before viral DNA synthesis (6 h after infection), 13 to 18% of the h-strand and 30 to 35% of the l-strand were represented in viral mRNA. Late (18 h) after infection the mRNA represented 20 to 25% and 63 to 68% of the h- and l-strands, respectively. Most, if not all sequences present in viral mRNA before viral DNA synthesis were also present in the cytoplasm late in infection.