Complementation of translational defect for growth of human adenovirus type 2 in Simian cells by a Simian virus 40-induced factor

The defective step which leads human adenovirus type 2 infection of African green monkey kidney cells (clone C14) to be abortive and its complementation in simian virus 40-transformed cells (clone T22) were studied by comparing the synthesis and function of macromolecules in these cell lines. Neither a quantitative nor a qualitative difference was detected in virus DNA replication and in virus mRNA synthesis in these cells, while a definite difference was observed in protein synthesis. The capsid proteins, such as hexon or penton, were synthesized in T22 cells but not in C14 cells. Inability of polyribosomes to synthesize the capsid proteins in C14 cells infected with adenovirus type 2 may not be due to a defect in elongation of nascent polypeptides or their release, since nascent polypeptides pulse-labelled with [³H]leucine were completely released from polyribosomes after the chase. The electrophoretic analysis of proteins synthesized in vitro with polyribosomes from either infected T22 or C14 cells using the pH 5 enzyme and S100 fraction from T22 cells revealed that hexon was synthesized with polyribosomes from T22 cells but not from C14 cells, thereby suggesting that the defect is not ascribed to a component in the pH 5 enzyme and S100 fraction, but resides in polyribosomes. The analysis of late adenovirus mRNA associated with polyribosomes in the infected T22 and C14 cells by hybridization competition or by sedimentation revealed that all the species of virus mRNA were present in the cytoplasm of these cells; however, certain species of virus mRNA larger than 20 S were absent in polyribosomes of the infected C14 cells. Sedimentation analysis of late adenovirus mRNA following separation on poly(U)-Sepharose or by membrane filtration gave the same results. These results suggest that the defect of C14 cells to support growth of adenoviruses is due to the inability of ribosomes to associate with certain species of late virus mRNA to form polyribosomes and suggest that a factor complementing this defect is induced by simian virus 40.     

Improved Production of Gutted Adenovirus in Cells Expressing Adenovirus Preterminal Protein and DNA Polymerase

Production of gutted, or helper-dependent, adenovirus vectors by current methods is inefficient. Typically, a plasmid form of the gutted genome is transfected with helper viral DNA into 293 cells; the resulting lysate is serially passaged to increase the titer of gutted virions. Inefficient production of gutted virus particles after cotransfection is likely due to suboptimal association of replication factors with the abnormal origins found in these plasmid substrates. To test this hypothesis, we explored whether gutted virus production would be facilitated by transfection into cells expressing various viral replication factors. We observed that C7 cells, coexpressing adenoviral DNA polymerase and preterminal protein, converted plasmid DNA into replicating virus approximately 50 times more efficiently than did 293 cells. This property of C7 cells can be used to greatly increase the efficiency of gutted virus production after cotransfection of gutted and helper viral DNA. These cells should also be useful for generation of recombinant adenovirus from any plasmid-based precursor.     

Adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro.

The adenovirus precursor to the terminal protein (pTP), expressed in a vaccinia virus expression system or in native adenovirus, was assayed for its ability to interact with the nuclear matrix. Biochemical function was measured by determining the relative amount of pTP protein or of adenovirus DNA that remained associated with the nuclear matrix after extensive washing. pTP was retained on the matrix whereas beta-galactosidase was not, as assayed by quantitative immunoblot analysis. Nuclear matrix isolated from adenovirus-infected HeLa cells retained bound adenovirus DNA even when washed with 1 M guanidine hydrochloride; this interaction could be inhibited by added purified pTP protein. Analogous experiments with matrix isolated from HeLa cells infected with a recombinant vaccinia virus that expressed pTP showed a similar retention of pTP protein; this association could also be inhibited by added pTP protein. Binding of pTP to nuclear matrix isolated from uninfected cells was saturable, with an apparent Kd of 250 nM and an estimated 2.8 x 10(6) sites for pTP binding per cell nucleus. The association of pTP with matrix is postulated to help direct adenovirus replication complexes to the appropriate locale within the nucleus.     

Construction of adenoviral vectors

Recombinant adenovirus vectors have proven to be useful tools in facilitating gene transfer. Construction of such vectors requires a knowledge of the adenovirus genome structure and its life cycle. A commonly used recombinant adenovirus involves deletion of the E1 region; such a recombinant is traditionally produced by overlap recombination after contransfection of 293 cells with a plasmid shuttle vector and a large right-end restriction fragment of viral DNA. The shuttle vector contains a cassette for a transgene placed in region E1 and flanking sequences from adenovirus for recombination. Normally, a high background of parental virus results because of the difficulty in separating right-end restriction fragment length DNA from uncut DNA. This paper describes a negative selection based on the traditional cotransfection method using viral DNA from an E1-deleted adenoviral recombinant that expresses green fluorescent protein (GFP). In situ fluorescent microscopy is used to distinguish the recombinant plaques (white or nonfluorescent) from the parental virus plaques (green or fluorescent). In addition, this system allows for the detection of contaminating parental virus at later stages when production lots of the recombinant vector are being made.     

Adenovirus DNA replication in vitro: Origin and direction of daughter strand synthesis

We have characterized a soluble enzyme system from adenovirus-infected cells that is capable of replicating exogenously added adenovirus DNA in vitro. Maximal DNA synthesis is observed when DNA-protein complex, isolated from purified adenovirus virions, is added as template. Under these conditions DNA replication starts at or near either end of the template. Daughter strand synthesis then proceeds in the 5′ to 3′ direction displacing the parental strand of the same polarity. Thus, the r daughter strand is synthesized from right to left on the conventional map of the adenovirus genome, and the l daughter strand is synthesized from left to right. This course of events is the same as that which occurs during adenovirus DNA replication in vivo. In contrast, when deproteinized adenovirus DNA is added to the in vitro system, the limited DNA synthesis that is observed appears to be due to a repair-like reaction. In particular, synthesis can begin at many sites within the template, and the synthetic product consists largely of short DNA chains that are covalently linked to template DNA strands.     

Differential cell membrane labilizing effect of adenovirus type 5 and 12 observed during productive and abortive infection.

Culture fluid of human epitheloid (HEp-2) cells was examined for extracellular lactate dehydrogenase activity as an indicator of cell damage during a 48 h period in which virus replication and changes in cell morphology occurred. Uninfected and adenovirus type 5-infected cells had the same levels of extracellular enzyme activity both before and after the appearance of morphological changes in cells due to virus infection, whereas adenovirus type 12-infected cells showed increased extracellular enzyme activity. Cells infected with either adenovirus type 5 or type 12 had the same total cellular and extracellular lactate dehydrogenase activity. Hydrocortisone, a membrane stabilizing agent, prevented abnormal leakage of lactate dehydrogenase from adenovirus type 12-infected cells, but had no effect on virus replication or total enzyme activity of infected cells. After inoculation of monkey kidney (Vero) cells the yield of progeny adenovirus type 5 virions was greatly reduced and there was no production of adenovirus type 12 virions. The pattern of extracellular lactate dehydrogenase activity of uninfected and adenovirus type 5- and type 12-infected Vero cells was like that with HEp-2 cells. Therefore, production of adenovirus type 12 virions is not necessary for the virus-cell interaction causing cell membrane labilization.     

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.     

Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors

The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.     

Role for the adenovirus IVa2 protein in packaging of viral DNA

Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant, pm8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.     

A second specific endonuclease from Haemophilus aegyptius.

A second restriction-like endonuclease has been partially purified from Haemophilus aegyptius. This enzyme cleaves bacteriophage λ DNA and adenovirus 2 DNA at many sites, but cleaves simian virus 40 DNA at only one site.     

Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.     

Latent infection of KB cells with adeno-associated virus type 2.

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.     

Requirement of the adenovirus IVa2 protein for virus assembly

The adenovirus L1 52/55-kDa protein is required for viral DNA packaging and interacts with the viral IVa2 protein, which binds to the viral packaging sequence. Previous reports suggest that the IVa2 protein plays a role in viral DNA packaging and that this function of the IVa2 protein is serotype specific. To further examine the function of the IVa2 protein in viral DNA packaging, a mutant virus that does not express the IVa2 protein was constructed by introducing two stop codons at the beginning of the IVa2 open reading frame in a full-length bacterial clone of adenovirus type 5. The mutant virus, pm8002, was defective for growth in 293 cells, although it replicated its DNA and produced early and late viral proteins. Electron microscopic and gradient analyses revealed that the mutant virus did not assemble any viral particles in 293 cells. In 293-IVa2 cells, which express the IVa2 protein, infectious viruses were produced, although the titer of the mutant virus was lower than that of the wild-type virus, indicating that these cells may not fully complement the mutation. The mutant viral particles produced in 293-IVa2 cells were heterogeneous in size and shape, less stable, and did not traffic efficiently to the nucleus. Marker rescue experiments with a wild-type IVa2 DNA fragment confirmed that the only mutations present in pm8002 were in the IVa2 gene. The results indicate that the IVa2 protein is required for adenovirus assembly and suggest that virus particles may be assembled around the DNA rather than DNA being packaged into preformed capsids.     

Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.     

表达超抗原SEA基因的溶瘤腺病毒载体的构建与鉴定

目的:构建表达超抗原SEA基因的溶瘤腺病毒载体并鉴定.方法:采用PCR技术,从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中获得SEA全长基因序列,酶切后克隆入pCA13质粒,构建重组病毒质粒pCA13-SEA.将鉴定正确的pCA13-SEA与含有腺病毒右臂的质粒pBHGE3通过Lipofectamine2000共转染HEK293细胞,经同源重组产生重组腺病毒Ad-SEA.Ad-SEA在293细胞中大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度.结果:经PCR扩增、酶切鉴定、序列测定证实,SEA基因成功克隆到溶瘤腺病毒载体中,可实现SEA基因的表达.结论:成功构建了表达超抗原SEA基因的溶瘤腺病毒载体,为进一步研究该病毒对膀胱肿瘤靶向治疗的作用奠定了基础.     

Letter: Some unusual properties of replicating adenovirus type 2 DNA

Replicating adenovirus type 2 DNA was isolated from KB cells 13 hours after infection. The buoyant density in caesium chloride of the replicating DNA was found to be 5 to 10 mg/cm² greater than that of mature adenovirus type 2 DNA. The single-strand specific nuclease from Neurospora crassa released 25 to 30% of the radioactivity from replicating DNA and the density difference between replicating and mature adenovirus DNA was eliminated after digestion with this enzyme, but not after digestion with RNase or pronase. The results suggest that the complementary strands of adenovirus type 2 DNA are replicated asynchronously.     

The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner

During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system.     

Constitutive episomal expression of polypeptide IX (pIX) in a 293-based cell line complements the deficiency of pIX mutant adenovirus type 5.

The human adenovirus type 5 capsid is composed of a number of distinct polypeptides. It has been shown previously that one of these, polypeptide IX (pIX), is not absolutely required for the production of viable virus. However, viruses lacking this polypeptide have a significantly reduced packaging limit and, in the one case studied, also show a thermolabile virion phenotype. This report describes the use of eukaryotic episomal vectors based on the Epstein-Barr virus replicon to generate cells which stably express pIX. These cells provide pIX that is efficiently incorporated into virions that are genetically pIX-; such enhanced thermostability. These cells have also been used to isolate a genetically pIX- virus having a genome of length some 2.3 kbp in excess of the previously defined packaging limit for pIX- virus; the resulting virions have wild-type thermostability. These cells expand the theoretical capacity of adenovirus vectors for foreign DNA to around 9.2 kbp and may therefore be useful in gene therapy applications in which vector capacity is limiting.     

Biological effects accompanying the microinjection of adenovirions, adenoviral and plasmid DNA into mammalian cells

Microinjection of either type 1 human adenovirus, type SA7 monkey adenovirus virions or circular adenovirus DNA, obtained by the treatment of DNA-terminal protein complexes with glutaraldehyde, into nuclei of permissive cells results in the complete cycle of virus reproduction. Microinjection of neither linear native, condensed adenovirus DNA nor the DNA-terminal protein complexes under the same conditions initiates the adenovirus reproduction thought the synthesis of early and some late viral antigens is observed in the injected cells. Integration of injected adenovirus DNA into the cellular DNA occurs as far as 30 min after injection. Microinjection of either adenovirus DNA or its oncogene containing fragments into nuclei of semipermissive cells induces the transformation of these cells. In this case the time of the first appearance of transformation foci is decreased.