Unique species of mRNA from adenovirus type 7 early region 1 in cells transformed by adenovirus type 7 DNA fragment.

Adenovirus type 7 (Ad7) early region 1 mRNA species transcribed in rat cell lines transformed by the HindIII-I . J fragment (the left 7.8% of the viral genome) and in human KB cells infected with Ad7 were mapped on the viral genome, using S1 nuclease gel and diazobenzyloxymethyl paper hybridization techniques. At the early stage of productive infection, two mRNA's (950 and 840 nucleotides long) with the common 5' and 3' ends but different internal splicings were mapped from region 1A (map units 1.4 to 4.3), and one mRNA (2,310 nucleotides long, with the internal splicing between map units 9.9 to 10.1) was mapped from region 1B (map units 4.6 to 11.4). At the late stage, these early spliced mRNA's were also found and at least three additional Ad7 mRNA's were identified: 700-nucleotide-long mRNA in region 1A; and 1,100- and nucleotide-long mRNA's in region 1B. In transformed rat cell lines, two early region 1A mRNA's (950 and 840 nucleotides long) were also transcribed. Surprisingly, in addition, several unique Ad7 mRNA's, not found in productivity infected cells, were identified in all of the transformed cell lines. Their molecular sizes and coding sequences varied in individual cell lines. However, these mRNA's had the 5' end-proximal portion in region 1B and the 3' end-proximal portion in region 1A, these portions being transcribed by extending from region 1B to 1A on viral DNA fragments joined in a tandem array in transformed cells.     

Identification of early adenovirus type 2 RNA species transcribed from the left-hand end of the genome.

Unique fragments of adenovirus type 2 DNA generated by cleavage with endonuclease R-Eco RI or endonuclease R-Hsu I (Hin dIII) were used to map cytoplasmic viral RNAs transcribed early in productive infection. Radioactive early viral RNA was first fractionated by polyacrylamide gel electrophoresis. Eluted viral RNAs were then tested for hybrid formation with DNA fragments. The Eco RI DNA fragment (Eco RI-A) which contains the left-hand 58% of the genome hybridized 13S and 11S RNAs. More detailed mapping of these RNAs was achieved by hybridization to the seven Hsu I fragments of Eco RI-A. The early RNA annealed only to Hsu I-G and C, two fragments which comprise the extreme left-hand 17% of the genome. Viral RNA migrating as 13S and 11S annealed to Hsu I-G, and 13S RNA annealed to Hsu I-C. A 13S RNA is transcribed from Eco RI-A late in infection (18 h). Hybridization-inhibition studies with Eco RI-A DNA, early cytoplasmic RNA, and 3H-labeled 13S late RNA demonstrated that this RNA synthesized at late times is an early RNA species which continues to be synthesized in large amounts at 18 h. This 13S RNA synthesized at 18 h hybridized to Hsu I-C but not to Hsu I-G DNA. These results establish that the 13S RNAs transcribed from Hsu I-G and C at early times must be different species.     

Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.     

mRNA from the transforming segment of the adenovirus 2 genome in productively infected and transformed cells.

We have identified two mRNA species transcribed from the adenovirus 2 genome section (HindIII-G fragment) believed to harbor genes for initiation and maintenance of cell transformation. The HindIII-G fragment occupies the left 7.5% of the genome and is transcribed from left to right [poly(U:G) r strand]. Poly(A)-terminated labeled mRNA was isolated from polyribosomes of adenovirus 2 early infected KB cells and from the transformed cell line 8617, hybridization purified using the HindIII-G fragment, and electrophoresed on formamide-polyacrylamide gels. Viral mRNA's of 24S (1.2 X 10(6) daltons) and 14S (4.5 X 10(5) daltons) were isolated from early infected cells and of 22S (1.0 X 10(6) daltons) and 14S from 8617 cells. Hybridization competition indicated that HindIII-G-specific mRNA was present in the polysomes at one-sixth the concentration late after infection as compared with early, indicating that the proteins coded by the transforming segment may be synthesized at reduced amounts during late stages. Only 1/10 the amount of RNA labeled late annealed to the G fragment as compared with that labeled early (per weight of RNA). Thus, synthesis of transforming gene mRNA is probably "turned off" late after infection. Both 24S (22S) and 14S mRNA's from infected and 8617 cells were complementary to the Hpa I-E fragment (left 4.1% of genome). The Hpa I-E fragment is too small to encode 24S and 14S species, which implies that the 5'-terminal regions of both species are coded by the same DNA 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.     

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.     

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.     

Coordinate regulation of two cytoplasmic RNA species transcribed from early region 2 of the adenovirus 2 genome

Early region 2 (E2) of the adenovirus 2 genome specifies a 72,000-dalton DNA-binding protein that is required for viral DNA replication. Electron microscopy studies have detected two major forms of 20S E2 mRNA, one species with a 5' leader from map position 75 and a second form having a leader from position 72 (Chow et al., J. Mol. Biol. 134:265-303, 1979). Only the species with a leader from position 75 was detected at early times; however, both forms were found at late times. We have analyzed the temporal regulation of E2 expression by documenting mRNA accumulation in the cytoplasm. Kinetic studies of pulse-labeled RNAs demonstrated a peak of E2 cytoplasmic RNa synthesis at 10 to 12 h, coinciding with the time of maximal synthesis of the 72,000-dalton DNA binding protein and viral DNA. To estimate the relative abundances of the two major E2 RNA species at various times during infection, total E2 cytoplasmic and polysomal 20S RNAs were isolated by hybridization-selection with specific DNA probes. The leader sequences in the selected RNAs were then quantitated by further RNA-DNA hybridization. We found that the elevated accumulation rate for E2 cytoplasmic RNA at late times reflected an increase in formation of both major species. Moreover, for all time points examined 66% of the mRNA species had a 5' end from map position 75, and 33% had a 5' terminus from position 72. Continuous labeling experiments provided evidence that both RNA forms have comparable half-lives. The results suggest that the two major species encoded by E2 are regulated in a coordinate fashion late in infection.