Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins

Nuclear membrane fractions were prepared by two procedures from KB cells pulse labeled with [(3)H]thymidine for 5 min late after infection with adenovirus 2: (i) the M-band technique, which yields a sharp peak containing most of the newly synthesized viral DNA, and (ii) the discontinuous sucrose gradient method, which yields three membrane fractions, one which bands at the interface between sucrose layers at density 1.18 and 1.20 g/ml and contains most of the newly synthesized viral DNA. Studies using cycloheximide to inhibit protein synthesis showed that proteins whose synthesis begins early after infection and occurs in the absence of viral DNA replication are required for viral DNA synthesis late after infection. To study the nature of these proteins, nuclear membrane fractions were isolated from cells labeled with [(3)H]leucine from 6 to 24 h postinfection in the presence of arabinosyl cytosine to block viral DNA replication, and were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Two proteins of molecular weights 75,000 and 45,000 were the major labeled polypeptides in the nuclear membrane fractions prepared from infected cells both by the M-band and the discontinuous sucrose gradient methods. These two proteins were not found in nuclear membrane fractions from uninfected cells. It is suggested that the 75,000 and 45,000 proteins may be early viral gene products that may play a role in the viral DNA replication.     

Inhibition of adenovirus DNA replication by vesicular stomatitis virus leader RNA.

Vesicular stomatitis virus (VSV) leader RNA and a synthetic oligodeoxynucleotide of the same sequence were found to inhibit the replication of adenovirus DNA in vitro. In contrast, the small RNA transcribed by the VSV defective interfering particle DI-011 did not prevent adenovirus DNA replication. The inhibition produced by leader RNA was at the level of preterminal protein (pTP)-dCMP complex formation, the initiation step of adenovirus DNA replication. Initiation requires the adenovirus pTP-adenovirus DNA polymerase complex (pTP-Adpol), the adenovirus DNA-binding protein, and nuclear factor I. Specific replication in the presence of leader RNA was restored when the concentration of adenovirus-infected or uninfected nuclear extract was increased or by the addition of purified pTP-Adpol or HeLa cell DNA polymerase alpha-primase to inhibited replication reactions. Furthermore, the activities of both purified DNA polymerases could be inhibited by the leader sequence. These results suggest that VSV leader RNA is the viral agent responsible for inhibition of adenovirus and possibly cellular DNA replication during VSV infection.     

Uncoating of adenovirus type 2.

The uncoating of adenovirus type 2 and a temperature-sensitive mutant, tsl, was studied. HEp-2 cells were infected with 32P- OR 125I-labeled purified virions for various lengths of time, and the nuclear and cytoplasmic fractions were analyzed by sucrose gradient velocity sedimentation and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Within 1 h of infection, virions were converted into three subviral structures: (1) subviral structures in the cytoplasm with a density greater than virions but which qualitatively still contained all virus polypeptides; (ii) corelike structures associated with both the nuclear and cytoplasmic fractions and composed of viral DNA and polypeptides VIa2, V and PVII; and (iii) putative DNA-terminal protein complexes in the nuclei. The kinetic and compartmentalization studies suggested that the DNA-terminal protein complex is the end product of uncoating. The virions which were synthesized by tsl at the nonpermissive temperature and contained the precursor polypeptides PVI and PVII were found to be blocked in uncoating at the corelike stage. This block in uncoating provides the explanation for the lack of infectivity of these virions. A model for the uncoating of adenovirus is proposed.     

Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells.

During the S phase of the eukaryotic cell cycle and in virus-infected cells, DNA replication takes place at discrete sites in the nucleus, although it is not clear how the proteins involved in the replicative process are directed to these sites. Nuclear factor I is a cellular, sequence-specific DNA-binding protein utilized by adenovirus type 2 to facilitate the assembly of a nucleoprotein complex at the viral origin of DNA replication. Immunofluorescence experiments reveal that in uninfected cells, nuclear factor I is distributed evenly throughout the nucleus. However, after a cell is infected with adenovirus type 2, the distribution of nuclear factor I is dramatically altered, being colocalized with the viral DNA-binding protein in a limited number of subnuclear sites which bromodeoxyuridine pulse-labeling experiments have identified as sites of viral DNA replication. Experiments with adenovirus type 4, which does not require nuclear factor I for viral DNA replication, indicate that although the adenovirus type 4 DNA-binding protein is localized to discrete nuclear sites, this does not result in the redistribution of nuclear factor I. Localization of nuclear factor I to discrete subnuclear sites is therefore likely to represent a specific targeting event that reflects the requirement for nuclear factor I in adenovirus type 2 DNA replication.     

Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

Parental adenovirus type 2 genomes recovered early or late in infection possess terminal proteins.

At both early (3 h) and late (18 h) times after infection of KB cells with adenovirus 2, more than 90% of parental nuclear viral genomes exist as complexes which contain terminally linked proteins. Density shift experiments employing 5-bromo-2'-deoxyuridine indicate that these parental DNA molecules remain complexed with terminal proteins after DNA replication. The persistent linkage of proteins to the termini of intranuclear viral DNA suggests that these proteins have an essential role in adenovirus replication.     

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.     

Induction of pre-early nuclear antigen(s) in HEL cells infected with human cytomegalovirus.

Human cytomegalovirus (HCMV)-specific nuclear antigen could be detected within 1 hr after infection in human embryo lung cells by the anticomplement immunofluorescence (ACIF) test. This antigen has been named the pre-early nuclear antigen (PENA) in this paper. Serum absorption tests suggested that PENA is immunologically different from the early antigen and the major nuclear inclusion antigens detected by the indirect immunofluorescence test before and after viral DNA replication, respectively. PENA-forming ability of the virus corresponded to its plaque forming ability. PENA formation was not affected by phosphonoacetate but was inhibited by the addition of inhibitors of RNA and protein syntheses or by UV-irradiation of infecting virus, suggesting that the formation of PENA depends on the expression of infecting virus gene functions. Virus-specific proteins were isolated by indirect immunoprecipitation from HCMV-infected cells exposed to 35S-methionine. SDS-polyacrylamide gel electrophoresis of the immunoprecipitate showed that at least two species of virus-specific polypeptides with molecular weights o.f 70,000 and 30,000 were synthesized de novo within 3 hr after infection.     

Electron Microscopy of Adenovirus 12 Replication 1. Fine Structural Changes in the Nucleus of Infected KB Cells

The ultrastructure of KB cells infected with oncogenic adenovirus 12 was studied at various intervals from 4 to 72 hr after viral inoculation. At 12 hr after infection, the nucleus and the nucleolus became hypertrophic. At 16 hr, bundles of fibers digestable by proteolytic enzymes were seen in the nucleus; they are considered as the early viral antigens identified immunologically by others. Between 24 and 26 hr, four types of nuclear inclusions appeared. Their sequence of appearance and fine structure are described. On the basis of their sensitivity to proteolytic digestion in thin sections, and the results of immunoferritin studies made by others, some of these inclusions are believed to represent viral structural antigens. Throughout the cycle of viral replication, the nucleolus displayed prominent and constant changes in the form of focal condensations and loosening of the nucleolonema, followed by atrophy and fragmentation. It is suggested that the early nucleolar changes reflect an active participation of the nucleolus in the synthesis of adenovirus 12. A hitherto unknown striated structure with definite periodicity, which is easily digested by proteolytic enzymes, was found in the nuclei during the late stages of adenovirus 12 replication.     

Adenovirus ubiquitin-protein ligase stimulates viral late mRNA nuclear export

Theadenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular proteins to form an E3 ubiquitin-protein ligase that polyubiquitinates p53 and probably one or more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their proteasomal degradation. The MRN complex is required for cellular DNA double-strand break repair and induction of the DNA damage response by adenovirus infection. To determine if the ability of E1B-55K and E4orf6 to stimulate viral late mRNA nuclear export requires the ubiquitin-protein ligase activity of this viral ubiquitin-protein ligase complex, we designed and expressed a dominant-negative mutant form of cullin 5 in HeLa cells before infection with wild-type Ad5 or the E1B-55K null mutant dl1520. The dominant-negative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitin-protein ligase but had no effect on viral early mRNA synthesis, early protein synthesis, or viral DNA replication. However, expression of the dominant-negative cullin 5 protein caused a decrease in viral late protein synthesis and viral nuclear mRNA export similar to the phenotype produced by mutations in E1B-55K. We conclude that the stimulation of adenovirus late mRNA nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein ligase activity of the adenovirus ubiquitin-protein ligase complex.     

Human cytomegalovirus-induced immediate early antigens: analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation.

Immediate early antigen (IEA) induced in human lung fibroblasts by human cytomegalovirus was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation with IEA-positive human sera. Two polypeptides of 76,000 daltons (76K) and 82K were detectable within 90 min after infection. Polypeptides of similar molecular weight were also found in immunoprecipitates of human cytomegalovirus-infected cells nonpermissive for virus replication. IEA is located within the nucleus, although some of the 76K material appears to be located on the outer nuclear membrane. Raising salt concentrations in the extraction buffer increased antigen extraction. The contribution of these IEA polypeptides to IEA nuclear fluorescent staining is discussed.     

Adenovirus sequesters phosphorylated STAT1 at viral replication centers and inhibits STAT dephosphorylation

Tyrosine phosphorylation and nuclear translocation of STAT1 indicate activation of interferon (IFN) signal transduction pathways. Here, we demonstrate that tyrosine-phosphorylated STAT1 is targeted by a unique mechanism in adenovirus (Ad)-infected cells. Ad is known to suppress IFN-inducible gene expression; however, we observed that Ad infection prolongs the tyrosine phosphorylation of STAT1 induced by alpha IFN in infected cells. To understand this paradoxical effect, we examined the subcellular localization of STAT1 following Ad infection and found that nuclear, tyrosine-phosphorylated STAT1 accumulates at viral replication centers. This form of STAT1 colocalized with newly synthesized viral DNA. Viral DNA replication, but not viral late gene expression, is required for the regulation of STAT1 phosphorylation. Our results indicate that Ad infection regulates STAT1 dephosphorylation rather than STAT1 phosphorylation. Consistent with this idea, we show that Ad infection disrupts the interaction between STAT1 and its cognate protein tyrosine phosphatase, TC45. Our findings indicate that Ad sequesters phosphorylated STAT1 at viral replication centers and inhibits STAT dephosphorylation. This report suggests a strategy employed by Ad to counteract an active form of STAT1 in the nucleus of infected cells.     

Protein Synthesis in a Lymantria dispar Cell Line Infected by Cytoplasmic Polyhedrosis Virus

The efficiency of replication of a cytoplasmic polyhedrosis virus isolated from a member of the order Lepidoptera, Euxoa scandens, was studied in eight different lepidopterean cell lines. Lymantria dispar cells, which were found to support viral replication, more efficiently, were used to follow the kinetics of appearance of viral-specific polypeptides by a 2-h pulse with [³⁵S]methionine. Five polypeptides (ca. 120,000 molecular weight [120K], 105K, 66K, 46K, and 28K) were identified as components of the polyhedral inclusion bodies, and two polypeptides (112K and 39K) were assigned as viral-particle polypeptides. All these polypeptides were present after 24 h and were still being produced 96 h after infection. The rate of synthesis of the major polyhedral polypeptide (28K) increased in the time course of infection, whereas the background of cellular polypeptides seemed to be unaffected. An indirect immunoperoxidase technique, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was blotted to a nitrocellulose membrane, showed that traces of the major polyhedral polypeptide were found from 8 h postinfection.     

寇氏隐甲藻（Crypthecodinium cohnii）细胞核骨架与中间纤维...

Nuclear matrix (NM) and intermediate filament (IF) scaffold in primitive eukaryote Crypthecodinium cohnii were shown using selective extraction together with embedment-free electron microscopy, whole mount cell preparation and immunoblot techniques. There exists a delicate NM-IF network spreading over cytoplasm and nucleus in dinoflagellate cells, however, nuclear lamina is undeveloped. The diameter of NM fiber is about 3-5 nm and IF is 10 nm. Chromosomes are connected with NM filament network. Immunoblot analysis showed that dinoflagellate contained keratin-like polypeptides (63 kD and 67 kD) while mammalian lamin antibodies did not crossreact with dinoflagellate total protein. Our experiment results demonstrated that a framework similar to NM-IF scaffold in mammalian cell appeared in primitive eukaryote. We propose that: (1) NM-IF scaffold is not restrict to vertebrate cell, and it may be originated from early stages of eukaryote evolution; (2) Keratin is probably very conservative; (3) Compared with IF, lamina might appear late in evolution, and some of primitive characteristics of dinoflagellate nucleus may be related to the lack of lamina.     

Transcription and Transport of Virus-Specific Ribonucleic Acids in African Green Monkey Kidney Cells Abortively Infected with Type 2 Adenovirus

The techniques of deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization and immunological precipitation were used to compare the synthesis of adenovirus-specific macromolecules in African green monkey kidney (AGMK) cells infected with adenovirus, an abortive infection, and coinfected with both adenovirus and simian virus 40 (SV40), which renders the cells permissive for adenovirus replication. When viral protein synthesis was proceeding at its maximum rate, the incorporation of (14)C-amino acids into adenovirus structural proteins was about 90 times greater in the doubly infected cells than in cells infected only with adenovirus. However, the rates of synthesis of virus-specific ribonucleic acid appeared to be comparable in the two infections at all times measured. A time-dependent increase in the rate of RNA synthesis observed late in the abortive infection was dependent upon the prior replication of viral DNA. Moreover, all virus-specific RNA species that are normally made late in a productive adenovirus infection (i.e., the true late and class II early RNA species) were also detected in the abortive infection. Adenovirus-specific RNA was detected by molecular hybridization in both the cytoplasm and nuclei of abortively infected cells. Comparable amounts of viral RNA were found in the cytoplasmic fractions of AGMK cells infected either with adenovirus or with both adenovirus and SV40. The results of hybridization-inhibition experiments clearly showed that there was a class of virus-specific RNA molecules, representing about 30% of the total, in the nucleus that was not transported to the cytoplasm. This class of RNA was also identified in similar amounts in productively infected human KB cells. The difference in the abilities of cytoplasmic and nuclear RNA to inhibit the hybridization of virus-specific RNA from whole cells was shown not to be due to a difference in the molecular size of the RNA species from the two cell fractions or to the specific loss of a cytoplasmic species during RNA extraction procedures.     

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.