Search

Mutational analysis within the amino-terminal (N-t) domain of the adenovirus DNA-binding protein (DBP) defined a region (aa 2-38) important for DBP function. Several viruses carrying lesions in this region of DBP showed reduced accumulation of viral DNA and infectious virions. Characterization of one of these mutants, H5in800, indicated that the N-t domain affects viral DNA synthesis in vivo. The reduction in DNA synthesis was not due to a change in the amount or nuclear location of the H5in800 DBP. Expression of other early genes in H5in800-infected cells was similar to that seen in wild-type Ad5-infected cells, suggesting that the depression of DNA synthesis was not due to disruption of DBP's role in early gene expression. The H5in800 and wild-type DBP also had comparable affinities for single-stranded DNA and functioned with similar efficiencies in two DNA elongation assays. Prior studies have shown that the carboxyl-terminal (C-t) domain of DBP was responsible for these two activities. Together these results suggest that DBP has at least two separable functions in viral DNA replication in vivo and that both domains of the protein are necessary for full activity. The intragenic complementation between the N-t mutant H5in800 and the C-t mutant H5in804 supports this model.

Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.

Adenovirus (Ad) type 2 DNA synthesis can be initiated in the presence of a soluble extract of uninfected HeLa cell nuclei, a 25-60% saturated ammonium sulfate fraction of infected cytoplasm and viral DNA covalently linked to a 5'-terminal protein (Ad DNA-prot). As the purification, from either the nuclei or cytoplasm, of factors active in DNA replication proceeded, various nonreplicative reactions which incorporate labeled deoxynucleotides were uncovered. In order to distinguish replicative from repair reactions, an assay was developed in which the Ad DNA-prot was digested with Xba I, all of the fragments so produced were used (without separation) in a replication reaction, and the products were assayed by electrophoresis on neutral agarose gels. In replicative reactions, most of the radioactivity was incorporated into the terminal fragments, with the internal fragments remaining unlabeled. Infected cytoplasm contains a "discrimination" function in addition to specific factors for Ad DNA replication. The discrimination factors inhibit the nonspecific nucleotide incorporation by uninfected HeLa nuclear extracts on Ad DNA-prot. The specific replicative incorporation into the terminal Ad DNA-prot fragments has also allowed an independent assay for reinitiation of progeny molecules synthesized in vitro. After the first round of replication, the 5' strand of the progeny duplex from each end is labeled. These same labeled strands will be displaced during the second round of replication and appear in new bands which have been shown to be the single-strand equivalents of the terminal fragments. Thus, at least two rounds of Ad DNA synthesis can initiate at each terminus in vitro. The appearance of displaced single strands requires DNA replication because the addition of dideoxycytidine triphosphate after the first round of synthesis prevents the displacement reaction. Both the progeny single- and double-stranded DNA appear to be linked to protein.

A soluble replication system that synthesizes full-sized adenovirus DNA molecules has been developed. The extraction of infected HeLa nuclei solubilizes approximately 25% of the viral replicating DNA and leaves the HeLa chromatin associated with the insoluble nuclear material. Both the extracted viral DNA and the DNA product synthesized in vitro are intact and identical to the adenovirus DNA or its replicating intermediates produced in whole infected cells. The direction of elongation and the termini of replication of progeny DNA in vitro are essentially identical to those observed in vivo. Nuclear extract replication is dependent on Mg2+ and the four deoxyribonucleoside triphosphates and partially stimulated by ATP.

Genetic and biochemical studies of adenovirus (Ad) DNA synthesis in vitro demonstrate that the Ad DNA binding protein (Ad DBP) is not necessary for the initiation of Ad DNA synthesis but is required for chain elongation. The DBP, which enhances early elongation to the 26th deoxynucleotide by approximately two- to fourfold, is absolutely required as chain elongation proceeds further. Ad DNA synthesis was assayed in a system requiring Ad DNA covalently linked at each 5' terminus to a protein (Ad DNA-pro), various fractions of Ad-infected cytoplasm, and an extract of uninfected Hela nuclei. Initiation of Ad DNA replication was measured by the formation of a covalent complex between the 80,000 dalton preterminal protein (pTP) and 5' dCMP. DNA binding proteins from two ts mutants, H5ts125 and H5ts107, have been purified and shown to be functional at 30 degrees but inactive at 38 degrees in an in vitro elongation system dependent on purified proteins. Chymotryptic cleavage of the 72K wild-type Ad2 DBP produces a 34K carboxyl terminal fragment which retains full activity in the in vitro elongation of Ad DNA.

A soluble adenovirus DNA replication system has been prepared in extracts of HeLa cells infected with the temperature-sensitive mutant H5ts125. These nuclear extracts synthesize full-sized viral DNA at 30 degrees but are temperature sensitive in this function at 38 degrees. A 72,000-dalton DNA-binding protein purified from cells infected with wild-type virus complements the temperature-sensitive defect and restores viral DNA replication to 74% of normal values.

Utilizing the isolated left and right halves of both adenovirus type 2 and the nondefective adenovirus simian virus 40 hybrid (Ad2(+)ND(1)), studies were undertaken to find the site on the DNA molecules at which replication begins. The data are consistent with several models which include an initiation event at both ends and bidirectional growth.

Aphidicolin inhibits adenovirus DNA replication in HeLa cells and in a cell-free, infected, nuclear extract in which viral DNA is elongated. The compound inhibits alpha DNA polymerase, extensively purified from HeLa cells, but has little or no effect on the beta or gamma DNA polymerases similarly purified. Aphidicolin does not affect thymidine uptake by cells nor does synthesis as it also inhibits DNA replication in uninfected cells. The inhibition by aphidicolin is reversible if the drug is removed within 18 hrs after addition to HeLa or Chinese Hamster Ovary cells but the cells are irreversibly affected if the drug remains for 48 hours.

Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP.

After short periods of labeling with [3H]thymidine, recently completed adenovirus DNA molecules were isolated and cleaved with restriction endonucleases. The strands (heavy and light) of most of the restriction endonuclease fragments were separated. The pattern of labeling clearly shows an asymmetry of radioactivity on the isolated strands of each restriction endonuclease piece. The data is consistent with replication proceeding in the 5' to 3' direction on each strand. Thus, there is an initiation point placed at or near each end of the molecule.

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60 degrees C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases alpha, beta, gamma and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.

An adenovirus (Ad) DNA replication complex extracted from infected HeLa nuclei could be purified free of the bulk of intracellular DNA polymerase activity by sedimetation in neutral sucrose gradients. However, the replication complex still retained some alpha and gamma DNA-polymerase activity. Since this complex is inhibited by 2', 3' dideoxythymidine-5'-triphosphate (ddTTP), an inhibitor of DNA polymerase gamma, a functional role for this enzyme in Ad DNA replication is suggested. Similar inhibition by ddTTP in intact Ad infected nuclei and comparable inhibition of Ad DNA synthesis in whole cells by dideoxythymidine (ddThy) are consistent with a role for DNA polymerase gamma. Uninfected HeLa nuclei or whole cells are not similarly inhibited by ddTTP or DDThy respectively. Such data does not rule out an additional functional role for other DNA polymerases, and recent experiments from this laboratory (1) suggest that DNA polymerase alpha is also involved in Ad DNA synthesis.

The early transcription region 3 (E3) of group B adenovirus type 35 (Ad35), a serotype isolated primarily from patients with acquired immunodeficiency syndrome and other immunodeficiency disorders, has been partially sequenced. We had previously identified an Ad35 29-kilodalton (kDa) early glycoprotein which, analogous to group C Ad2 E3-19K, associated with major histocompatibility complex class I antigens in the endoplasmic reticulum of infected cells. The open reading frame (ORF) of the Ad35 29-kDa protein has now been identified within a 2-kilobase-pair cloned Ad35 E3 fragment. The predicted amino acid sequence was very similar to that of group B Ad3 E3-19K. In contrast, homology between the Ad35 and Ad2 glycoproteins was limited to five cysteines in identical positions and a 20-amino-acid region proximal to the transmembrane domain. In addition, 20.3- and 20.6-kDa ORFs have been identified downstream from the ORF for the Ad35 glycoprotein. Analogous 20-kDa ORFs are present in the Ad3 E3 region but are not present in Ad2 and Ad5. In contrast, the region analogous to an Ad2 11.6-kDa ORF, which is 9 kDa in size in Ad3, was absent from the expected position within the Ad35 E3 region. Because the E3 region is likely to play an important role in the interaction between virus and host, analysis of the function of the Ad35 E3 proteins should further our understanding of adenovirus pathogenesis.

Adenovirus type 2 DNA synthesis, either in permissive human cells or nonpermissive monkey cells, becomes independent of protein synthesis after the appearance of progeny viral DNA. In the presence of cycloheximide, semiconservative replication and initiation of progeny molecules can occur.

Intermediates in the synthesis of adenovirus type 2 deoxyribonucleic acid (DNA) were studied in HeLa cells. Pieces of DNA smaller than the viral genome were demonstrated after labeling with (3)H-thymidine for 10 to 240 sec. Intermediates as small as the Okazaki fragments (8 to 10S) do not predominate at any of the above times. No detectable addition of nucleotides to parental genome could be shown, nor was there any breakdown of recently synthesized viral DNA. The DNA intermediates were of viral origin for they hybridized to viral DNA and were made at a stage of the cell cycle (G(2)) when host DNA is not synthesized.