Transcription of the genome of adenovirus type 12. III. Maps of stable RNA from productively infected human cells and abortively infected and transformed hamster cells.

The adenovirus type 12-specific mRNA and the stable nuclear RNA from productively infected KB cells, early postinfection, from abortively infected BHK-21 cells, and from the adenovirus type 12-transformed hamster lines T637 and HA12/7 have been mapped on the genome of adenovirus type 12. The intact separated heavy (H) and light (L) strands of adenovirus type 12 DNA have been used to determine the extent of complementarity of the mRNA or nuclear RNA from different cell lines to each of the strands. More precise map positions have been obtained by the use of the H and L complements of the fragments of adenovirus type 12 DNA which were produced with the EcoRI and BamHI restriction endonucleases. The results of the mapping experiments demonstrate that the mRNA's isolated early from productively and abortively infected and from two lines of transformed cells are derived from the same or similar regions of the adenovirus type 12 genome. The map positions on the adenovirus type 12 genome for the mRNA from the cell lines as indicated correspond to regions located approximately between 0 and 0.1 and 0.74 and 0.88 fractional length units on the L strand and to regions between 0.63 and 0.74 and 0.89 and 1.0 fractional length units on the H strand. The HA12/7 line lacks mRNA complementary to the region between 0.74 and 0.88 fractional length units on the L strand. Similar data are found for the nuclear RNA, except that the regions transcribed are more extensive than those observed in mRNA. The polarity of the H strand has its 3'-end on the right terminus in the EcoRI A fragment, and the L strand has its 3'-end on the left terminus in the EcoRI C fragment. Thus, the H strand is transcribed from right to left (1 = leftward strand); and the L strand is transcribed from left to right (r = rightward strand). The designations H and L refer to the relative heavy and light densities of the two strands in polyuridylic-polyguanylic acid-CsCl density gradients. The EcoRI C-H and D-H complements have been shown to be part of the intact L strand; thus, there is a "reversal in heaviness" on the left terminus of the viral DNA.     

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.     

Mappings of adenovirus type 7 cytoplasmic RNA species synthesized early in lytically infected cells and synthesized in transformed cells.

Early virus-specific RNA synthesized in KB cells infected with adenovirus type 7 and virus-specific RNA synthesized in rat embryo cells (71JY1-2) transformed by the adenovirus type 7 HindIII-I.J fragment (left-hand 8.1% of the viral genome) have been mapped on the viral genome. About 25% of the viral genome, four discrete regions, two on each strand of the viral genome, are expressed as "early" mRNA. Almost similar regions in the left-hand 8.1% of the viral genome are transcribed both in KB cells at early times after infection and in 71JY1-2 cells.     

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.     

In vitro translation of adenovirus type 12-specific mRNA isolated from infected and transformed cells.

The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.     

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.     

Location of sequences on the adenovirus genome coding for the 5.5S RNA.

The origin of a low molecular weight virus-associated RNA (VA-RNA) was mapped by hybridization of VA-RNA to specific fragments of adenovirus type 2 DNA, obtained after cleavage with three different restriction endonucleases. VA-RNA was found to hybridize exculsively to the l-strand [strand with low buoyant density in CsCl when complexed with poly(U,G)] of a segment of the viral DNA which is located between positions 0.27 and 0.32 on the unit map of the adenovirus type 2 genome.     

The gene and messenger RNA for adenovirus polypeptide IX

A small RNA species, distinct from the VA RNAs, has been identified in HeLa cells infected with adenovirus type 2. The RNA, which has been purified using a novel screening procedure, is polyadenylated, sediments at 9S and has an estimated length of 550 nucleotides. In a cell-free translation system, the 9S RNA directs the synthesis of virion polypeptide IX, molecular weight 12,000 daltons. The location of its gene has been established by hybridization of the RNA to fragments of viral DNA produced by cleavage with restriction endonucleases: it spans position 10.0 on the r strand of the viral genome. These results unexpectedly place the gene for a “late” protein within a region of the genome which is transcribed early during infection.     

The low molecular weight of RNAs of adenovirus 2-infected cells

The cytoplasm of HeLa cells infected with adenovirus type 2 contains many species of low molecular weight RNA, including several of viral origin. In addition to a 9 S messenger RNA, the viral genome gives rise to two species of virus-associated RNA: the major species is 5.5 S RNA or virus-associated RNA_I, and the minor species is 5.2 S RNA or virus-associated RNA_II. Virus-associated RNA_I occurs in the cytoplasm in several electrophoretically separable forms, and its sequences are also present in high molecular weight nuclear RNA but not in cytoplasmic mRNA. The structure of virus-associated RNA_II is shown to be distinct from that of the major species, and the position of its gene is mapped on the viral genome. The two virus-associated RNA genes are located on the r strand near position 30 of the adenovirus type 2 physical map, and are separated by a spacer of about 75 base-pairs.     

Transcription of the genome of adenovirus type 12. Viral mRNA in productively infected KB cells.

In human KB cells productively infected with adenovirus type 12, viral DNA replication starts between 12 and 14h postinfection. Virus-specific, polysome-associated mRNA was investigated early (6-8h) and late (26-28h) after infection. Most of the viral mRNA was polyadenylated and accounted for 0.46% and 24.1% of the mRNA synthesized early and late postinfection, respectively. The viral-specific mRNA isolated both early and late after infection falls into several distinct size-classes, ranging in molecular weights between 0.3X10(6) and 1.5X10(6) for the early RNA and between 0.6X10(6) and 2.3X10(6) for the RNA synthesized late in the infection.     

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.