Species dependence of the major late promoter in adenovirus type 12 DNA.

In hamster cells human adenovirus type 12 (Ad12) is deficient in DNA replication and late gene expression whereas adenovirus type 2 (Ad2) can replicate. Functions located in the E1 region of the Ad2 or adenovirus type 5 (Ad5) genome can complement the deficiencies of the Ad12 genome in hamster cells, but, infectious viral particles are not produced. We have now investigated the activity of the major late promoter of Ad2 and of Ad12 DNA in human and hamster cells. This promoter governs the expression of most of the late viral functions. We have inserted the major late promoter (MLP) of Ad2 or of Ad12 DNA in front of the chloramphenicol acetyl transferase gene in the pSVO-CAT construct. Upon transfection into uninfected human and hamster cells, the pAd12MLP-CAT construct shows no significant activity; the pAd2MLP-CAT construct exhibits low activity. In Ad12-infected human cells, both constructs are active. These findings support the notion that other viral factors are required for MLP activity of Ad2 or Ad12 DNA in permissive human cells. In Ad2-infected hamster cells, both the pAd2MLP-CAT and the pAd12MLP-CAT constructs are active. Apparently, the Ad12 MLP can be activated by Ad2 functions, as already demonstrated for the entire Ad12 genome in double-infected cells or in Ad2- or Ad5-transformed cells superinfected with Ad12. In Ad12-infected hamster cells, however, the MLP of Ad12 DNA is inactive but that of Ad2 DNA shows activity. Thus the MLP of Ad12 DNA somehow differentiates between cellular auxiliary functions of different species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a nondefective recombinant between adenovirus type 5 and early region 1A of adenovirus type 12.

A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.     

Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences.

A cell-free system of nuclear extracts from BHK21 cells has been developed to catalyse recombination in vitro between the DNA of adenovirus type 12 (Ad12) and two different hamster preinsertion sequences. The pBR322 cloned 1768 bp fragment p7 and the 3.1 kbp fragment p16 from BHK21 hamster DNA had previously been identified as the preinsertion sites corresponding to the junctions between Ad12 DNA and hamster DNA in cell line CLAC1 and in the Ad12-induced tumour T1111(2), respectively. Preinsertion sequences, which had recombined previously with foreign (Ad12) DNA, might again be recognized by the recombination system even in a cell-free system. PstI cleaved Ad12 DNA and the circular or the EcoRI linearized p7 or p16 preinsertion sequences were incubated with nuclear extracts. Recombinants were isolated by transfecting the DNA into recA- Escherichia coli strains and by screening for Ad12 DNA-positive colonies. Without a selectable eukaryotic marker, all Ad12 DNA positive recombinants were registered. Out of a total of greater than 90 p7-Ad12 DNA recombinants, 21 were studied by restriction-hybridization, and four by partial nucleotide sequence analyses. Among the p16-Ad12 DNA recombinants, four were analysed. The sites of linkage between Ad12 DNA and p7 or p16 hamster DNA were all different and distinct from the original CLAC1 or T1111(2) junction site between Ad12 and hamster DNA. The in vitro recombinants were not generated by simple end-to-end joining of the DNA fragments used in the reaction but by genetic exchange. Thirteen of the 25 recombinants were derived from the 61-71 map unit fragment of Ad12 DNA. Recombination experiments between Ad12 DNA and four randomly selected unique or repetitive hamster DNA sequences of 1.5-6.2 kbp in length did not yield recombinants. Apparently, the p7 and p16 hamster preinsertion sequences recombined with Ad12 DNA with a certain preference.     

Dependence of tumor-forming capacities of cells transformed by recombinants between adenovirus types 5 and 12 on expression of early region 1.

Recombinants between an adenovirus type 5 (Ad5) deletion mutant and the Ad12 DNA fragment containing early region 1 (E1) were isolated from cells cotransfected with the EcoRI-C fragment of Ad12 DNA and Ad5 dl312 (deletion in E1A) DNA (rcA) and from cells cotransfected with the SalI-C fragment of Ad12 DNA and Ad5 dl312 DNA (rcB). No recombinant was isolated from cells cotransfected with Ad5 dl313 (deletion in E1B) DNA and restriction fragments of Ad12 DNA. Both rcA and rcB are defective and able to replicate in human embryo kidney (HEK) and KB cells with complementation by dl312. Both rcA and rcB formed Ad12 T antigen g, but not T antigen f, in infected HEK and KB cells. In rcA- and rcB-infected cells, Ad5 E1B and Ad12 E1A genes are transcribed. Heteroduplex and size analyses of rcA-1 or rcB-1 DNA fragments hybridized with Ad12 DNA revealed that rcA-1 DNA has a deletion between 5 and 15 map units with an insertion of a portion of Ad12 DNA (10%) and that rcB-1 DNA has a deletion between 70 and 80 map units with an insertion of a portion of Ad12 DNA (10%). The transformed cell lines, RCAY and RCBY, were established after infection of rat 3Y1 cells with rcA and rcB, respectively. Both Ad5 and Ad12 DNA sequences are contained in these cells. In RCAY cells, Ad12 T antigen g is detected, but Ad12 T antigen f is not. In RCBY cells, both Ad12 T antigen g and f are detected. Only the Ad12 E1A gene is transcribed in RCAY cells, whereas Ad5 E1B, Ad12 E1A, and Ad12 E1B genes are transcribed in RCBY cells. In soft-agar cultures, RCBY cells form large colonies, whereas RCAY cells form only tiny colonies. RCBY cells form tumors as efficiently as 12WY cells in transplanted rats. RCAY cells formed tumors inefficiently. Ad5-transformed 5WY cells do not form tumors. These observations indicate that the efficient tumor formation by RCBY cells is dependent on the expression of the Ad12 E1A and E1B genes, whereas the inefficient tumor formation by RCAY cells is due to the expression of only the Ad12 E1A gene.     

Overproduction of adenovirus DNA polymerase and preterminal protein in HeLa cells

Adenovirus (Ad) DNA polymerase (AdPol) and the preterminal protein (pTP) form a complex that is involved in the in vitro initiation of Ad DNA replication. Recombinant vaccinia viruses (vv) were constructed in which the genes encoding AdPol and pTP were cloned into a vaccinia/T7 hybrid expression-based vector downstream from the T7 promoter (pT7)/encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR). HeLa cells infected with the recombinant vv-AdPol or vv-pTP or a mixture of both, together with the vv expressing T7 RNA polymerase produced significant levels of pTP and AdPol which were biologically active in the in vitro initiation of Ad DNA replication. These amounts of pTP and AdPol were only about two-fold less than the levels produced in insect cells infected with the recombinant baculovirus constructs expressing AdPol and pTP.     

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.     

Chromosomal distribution of the hamster intracisternal A-particle (IAP) retrotransposons

The retrotransposon-like elements of the intracisternal A-particle (IAP) sequences occur in about 900 copies per haploid hamster cell genome. By applying the fluorescent in situ hybridization (FISH) technique and four different, cloned segments of the IAP element as hybridization probes, these elements were found to be distributed in specific patterns over many of the 44 hamster chromosomes. The hybridization patterns were very similar regardless of whether all four probes or only the IAPI probe carrying the long terminal repeat (LTR) region were used. The IAP elements were found most abundantly, though not exclusively, on the short arms of at least 12 of the autosomes. Of the sex chromosomes, the shorter Y chromosome was stained on both arms, and the X chromosome on one arm by the IAP probes. Primary Syrian hamster cells, the established Syrian hamster cell line BHK21, and the adenovirus type 12 (Ad12)-transformed BHK21 cell line T637 yielded very similar results. In Chinese hamster ovary (CHO) or 3T3 mouse cells, signals could not be elicited by FISH using the Syrian hamster IAP probes. On Southern blots, the DNAs from these cell lines hybridized very weakly, if at all, to the IAP sequences. Thus, IAP sequences were retroposed after Syrian hamster and mouse or Syrian and Chinese hamsters had diverged in evolution.     

Episodic evolution mediates interspecies transfer of a murine coronavirus.

Molecular mechanisms permitting the establishment and dissemination of a virus within a newly adopted host species are poorly understood. Mouse hepatitis virus (MHV) strains (MHV-A59, MHV-JHM, and MHV-A59/MHV-JHM) were passaged in mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine DBT cells. From MHV-A59/MHV-JHM mixed infection, variant viruses (MHV-H1 and MHV-H2) which replicated efficiently in BHK cells were isolated. Under identical treatment conditions, the parental MHV-A59 or MHV-JHM strains failed to produce infectious virus or transcribe detectable levels of viral RNA or protein. The MHV-H isolates were polytrophic, replicating efficiently in normally nonpermissive Syrian hamster smooth muscle (DDT-1), Chinese hamster ovary (CHO), human adenocarcinoma (HRT), primate kidney (Vero), and murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK) and porcine testicular (ST) cell lines. The variant virus, MHV-H2, transcribed seven mRNAs equivalent in relative abundance and size to those synthesized by the parental virus strains. MHV-H2 was an RNA recombinant virus containing a crossover site in the S glycoprotein gene. At the molecular level, episodic evolution and positive Darwinian natural selection were apparent within the MHV-H2 S and HE glycoprotein genes. These findings differ from the hypothesis that neutral changes are the predominant feature of molecular evolution and argue that changing ecologies actuate episodic evolution in the MHV spike glycoprotein genes that govern interspecies transfer and spread into alternative hosts.     

The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.     

Fastidious human adenovirus type 40 can propagate efficiently and produce plaques on a human cell line, A549, derived from lung carcinoma.

Human adenovirus type 40 (Ad40) cannot propagate in conventional established human cell lines such as KB or HeLa cells. However, it has been shown that Ad40 DNA replicates in KB18 cells which express Ad2 E1B genes, suggesting that Ad40 is defective in the E1B gene function in KB or HeLa cells. We show here that Ad40 can propagate and produce plaques on A549 cells which do not contain Ad E1B genes. Our experiments show that the levels of replication of Ad40 DNA and production of infectious Ad40 virus in A549 cells are the same as or higher than those in 293 or KB18 cells. Dot blot analysis shows that the levels of Ad40 E1A and E1B mRNAs expressed in A549 cells at early to intermediate times postinfection are at least 10-fold higher than those in KB or KB18 cells. Northern (RNA) blot analysis shows that large E1B mRNA species (approximately 24S to 26S) are synthesized prior to the onset of DNA replication in A549 cells. No E1B mRNA species are synthesized in KB or KB18 cells at early times postinfection, and no differences in the expression of E1B mRNAs are seen between KB and KB18 cells. The experiment suggests that A549 cells have a cellular factor(s) which activates Ad40 E1B mRNA synthesis and that the E1B mRNA synthesis helps Ad40 propagation. In contrast, Ad40 can propagate in KB18 cells by using Ad2 E1B gene products that are constitutively expressed in this cell line. Furthermore, this result shows that Ad40 cannot propagate in KB cells because of the failure in the expression of E1B genes at early times postinfection.     

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.     

Integration of the Deoxyribonucleic Acid of Adenovirus Type 12 into the Deoxyribonucleic Acid of Baby Hamster Kidney Cells

In a previous report, evidence was presented that the deoxyribonucleic acid (DNA) of adenovirus type 12 (Ad12) is integrated by covalent linkage into the DNA of baby hamster kidney cells (BHK-21 cells). These studies have been extended. The DNA of Ad12 and that of BHK-21 cells grown in medium containing 5-bromodeoxyuridine could be separated by equilibrium centrifugation in alkaline CsCl density gradients. BHK-21 cells were infected with (3)H-labeled Ad12, and the total intracellular DNA was analyzed at various times after infection in alkaline CsCl density gradients. The (3)H label in the position of cellular DNA hybridized predominantly with viral DNA and to a lesser extent also with cellular DNA. Replication of viral DNA could not be detected in BHK-21 cells. The appearance of viral (3)H label in the density stratum of cellular DNA was not significantly affected when DNA synthesis in Ad12-infected BHK-21 cells was inhibited >96% by cytosine arabinoside. These findings provided additional evidence for integration of Ad12 DNA into the DNA of BHK-21 cells. It could be calculated that 5 to 55 Ad12 DNA equivalents per cell are integrated. Replication of viral or cellular DNA was not required for integration. Inhibition of protein or ribonucleic acid synthesis interfered with integration only slightly.     

Functional Implications in Apoptosis by Interferon Inducible Gene Product 1-8D, the Binding Protein to Adenovirus Preterminal Protein

Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.