Mutant Of Human Adenovirus Type Research Articles

Impact of the adenoviral E4 Orf3 protein on the activity and posttranslational modification of p53.

Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1-17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076-1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3 ubiquitin ligase that contains the viral E1B 55-kDa and E4 Orf6 proteins, while the E4 Orf3 protein has been reported to block its ability to stimulate expression of p53-dependent genes. The comparisons reported here of the posttranslational modifications and activities of p53 populations that accumulate in infected normal human cells in the absence of both mechanisms of inactivation or of only the E3 ligase revealed little impact of the E4 Orf3 protein. These observations indicate that E4 Orf3-dependent disruption of Pml bodies does not have a major effect on the pattern of p53 posttranslational modifications in adenovirus-infected cells. Furthermore, they suggest that one or more additional viral proteins contribute to blocking p53 activation and the consequences that are deleterious for viral reproduction, such as apoptosis or cell cycle arrest.

Replication of an adenovirus type 34 mutant dna containing tandem reiterations of the inverted terminal repeat

A mutant of human adenovirus type 34 (Ad34) has been isolated which contains DNA molecules with tandem reiterations of from two to eight copies of a 131-bp sequence within the right-sided inverted terminal repetition. Terminal heterogeneity was not eliminated by repeated plaque purifications indicating that the population of DNA molecules with various numbers of reiterations could rapidly evolve from the DNA of a single virus particle. These enlarged DNA molecules were capable of replication both in vivo and in vitro. The nucleotide sequence of the mutant Ad34 inverted terminal repetitions contained most of the essential features of the Ad origin of DNA replication. These features include the ATAATATACC sequence which is present between the highly conserved bases 9–18 in all human adenoviruses, as well as the consensus sequences for the binding of nuclear factor I and nuclear factor III. However, the reiterated sequences lacked a dG appropriately placed on the template strand to serve as a potential site for internal initiation. It appears that the rapid amplification of two to eight copies of the reiterated terminal sequences does not arise from internal initiation during replication but probably from homologous recombination.

Adenovirus early region 4 is required for efficient virus particle assembly.

H2dl807, a defective deletion mutant of human adenovirus type 2 lacking parts of early regions 3 and 4 and all of late region 5, was severely defective for virus particle assembly on HeLa cells, producing about 1% of the normal yield of particles. On Vero cells, H2dl807 produced only 5% as many particles as wild type, while on W162 cells, a Vero cell derivative which supports the growth of early region 4 mutants, H2dl807 produced nearly 40% of the wild-type level of particles. Two other defective deletion mutants, H2dl802 and H5dl1021, which lack parts of early region 3 and which are incapable of making fiber, the product of late region 5, were wild type for virus assembly. These data suggest that the cause of the assembly defect of H2dl807 is the lack of a diffusible early region 4 product. H2dl807-infected Vero cells accumulated nearly wild-type amounts of viral late proteins in the nucleus and cytoplasm. Thus, the defect of the mutant in assembly on Vero cells is not due to a general lack of late proteins. Finally, the fact that H2dl802 and H5dl1021 make wild-type amounts of virus particles suggests that fiber is not essential for adenovirus assembly.

Adenovirus E1A gene autorepression: revertants of an E1A promoter mutation encode altered E1A proteins.

Revertants have been isolated from Ad3hr15, a mutant of human adenovirus type 3 that carries a defective E1A promoter. Transcription of these revertant E1A genes is restored--from nil for Ad3hr15 mutant to levels exceeding that of the wild-type virus. The mutant Ad3hr15 virus and the revertants all have an aberrant E1A promoter that contains two short tandem duplications of viral DNA sequence. The E1A gene-coding region of the mutant is the same as that for wild-type adenovirus type 3, whereas the revertants are characterized by short in-frame deletions within the 5' exon region of their E1A genes. Location of these reverting, second-site deletions is discussed in relation to E1A gene autoregulation and the evolved diversity of E1A-related oncogenic potential among different human adenoviruses.

Isolation and partial characterization of a human adenovirus type 4 temperature-sensitive mutant (Mastadenovirus h 4 tsl).

A random selection procedure was used to isolate a temperature-sensitive (ts) mutant of human adenovirus type 4 (Mastadenovirus h 4 tsl) from nitrous acid mutagenized virus stock. The mutant displayed restricted growth at the nonpermissive temperature of 39 degrees C. Analysis of the mutant grown at 39 degrees C, by two-dimensional immunoelectrophoretic analysis, showed the mutant to be defective in the expression of the penton base and fibre structural components. The mutant was, however, capable of synthesizing immunologically reactive hexon components. Temperature-shift experiments revealed detectable fibre and penton to be present following shift-down from 39 to 32 degrees C. Time-sequence analysis of shift-down experiments suggested a possible defect in processing of the components, as indicated by an increase of immunologically detectable penton base. The ability of the mutant to assemble viruslike particles at 39 degrees C was confirmed by electron microscopy. Though the particles assembled appeared as mature virions, crystalline arrays of packed particles were less in number and somewhat smaller in size than those observed at 32 degrees C.

A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2.

Cell lines that produce viral gene products and that can support the growth of viral mutants lacking those products have been valuable in the genetic analysis of the transforming regions of several animal viruses. To extend the advantages of such complementing cell systems to regions of the adenoviral genome not directly involved in transformation, we have constructed a cell line that will support the growth of a defective adenoviral deletion mutant, H2dl808, that lacks most of early region 4 (E4). The right-hand terminal adenovirus 5 EcoRI restriction fragment, which contains all of E4, was first inserted into the vector pSV2gpt, and the recombinant plasmid was introduced into Vero cells by calcium phosphate precipitation. Clones containing the hybrid plasmid were selected by their resistance to mycophenolic acid. Five mycophenolic acid-resistant clones were then tested for the ability to support the growth of H2dl808. One of the five lines, W162, permits plaque formation by H2dl808 at an efficiency that is greater than 10(6)-fold higher than that of the parental Vero cells and allows the production of high-titer, helper-free H2dl808 stocks. Thus, W162 cells are permissive for at least one defective E4 mutant. The line carries, as expected, an intact E4, detected by hybridization. Using an H2dl808 lysate produced on W162 cells, we have accurately mapped the 808 deletion. It extends from between BclI and SmaI sites at positions 91.4 and 92.0, respectively, to just beyond a HindIII site at position 97.2 and, therefore, falls entirely within E4. H2dl808 and W162 should be of value in determining the physiological role of E4 in adenoviral infection.

Interactions between initiating chemical carcinogens, tumor promoters, and adenovirus in cell transformation.

Cell culture systems that respond to the combined action of initiating chemical carcinogens, tumor promoters, and transforming viruses represent useful model systems for studying the complex multifactor nature of the carcinogenic process. We have utilized both secondary rat embryo (2 degrees RE) and a clonal population of established Fischer rat embryo (CREF) cells to study the effect of multiple agents on the process of adenovirus transformation. In the present review we summarize our investigations on the effects of polycyclic aromatic hydrocarbons, tumor promoters, a bee venom polypeptide-melittin (MEL), and the polypeptide hormone epidermal growth factor (EGF) on transformation of rat embryo cells by a temperature-sensitive mutant of human adenovirus type 5 (H5ts125). We also describe the effect of tumor promoters on the progression of the transformed phenotype, ie the temporal acquisition of anchorage-independent growth in adenovirus-transformed clones and the ability of MEL and EGF to elicit effects similar to those of tumor promoters in adenovirus-transformed cells.

Adenovirus early function required for protection of viral and cellular DNA

Studies were done to characterize a DNA-negative temperature-sensitive (ts) mutant of human adenovirus type 2, H2 ts111. The temperature-sensitive defect, which was reversible on shift-down in the absence of protein synthesis, was expressed as early as 2 h postinfection, and the results of density-labeling experiments are in agreement with at least a DNA replication initiation block. On shift-up, after allowing viral DNA synthesis at permissive temperatures, the newly synthesized viral DNA and the mature viral DNA were cleaved into fragments which sedimented as a broad peak with a mean coefficient of 10-12S. This cleavage was more marked in the presence of hydroxyurea as the DNA synthesis inhibitor. Parental DNA in infected cells was degraded to a much lesser extent regardless of the incubation temperature. In contrast, the parental DNA was strongly degraded when early gene expression was permitted at 33 degrees C before shift-up to 39.5 degrees C. Furthermore, cellular DNA was also degraded at 39.5 degrees C in ts111-infected cells, the rate of cleavage being related to the multiplicity of infection. This cleavage effect, which did not seem to be related to penton base-associated endonuclease activity, was also enhanced when early gene expression was allowed at 33 degrees C before shift-up. The ts111 defect, which was related to an initiation block and endonucleolytic cleavage of viral and cellular DNA, seemed to correspond to a single mutation. The implication of the ts111 gene product in protection of viral and cellular DNA by way of a DNase-inhibitory function is discussed.

Isolation and characterization of a specific deletion mutant of human adenovirus type 2

A specific deletion mutant of human adenovirus type 2 (Ad2) was detected when pools of wild-type Ad2 were grown at high multiplicity of infection in HeLa cells. This deletion mutant, designated dl-Ad2, was enriched when the defective Ad2-SV40 hybrids of the Ad2 ++HEY population were cloned in monkey cells in the presence of an added excess of wild-type Ad2. Electron microscope heteroduplex analysis and restriction endonuclease examination established the Ad-specific nature of the dl-Ad2 DNA and revealed a single, homogeneous deletion of about 0.08 to 0.09 fractional genome length situated between 0.78 and 0.87 Ad2 map unit. This genome structure is very similar to that of several other incomplete adenoviruses already described. The deleted segment encompasses one of the four early genome regions of Ad2. dl-Ad2 particles are not infectious in both human and monkey cells, although the mutant DNA is replicated in these cell types. dl-Ad2 is able to interfere efficiently with SV40 DNA replication in coinfected monkey cells. Furthermore, virus populations could be cloned consisting exclusively of Ad2 ++HEY hybrid viruses and dl-Ad2 “helper” viruses, indicating that dl-Ad2 can complement sufficiently for the large Ad2 DNA deletion in the Ad2 ++HEY hybrid genomes.

A host range mutant of human adenovirus type 5 defective for growth in hamster cells.

A host-range mutant of adenovirus type 5, which grows in human cells but not in hamster cells, has been isolated. This mutant is complemented in mixed infection in hamster cells at 38.5 degrees C by temperature-sensitive mutants of type 5 belonging to seventeen complementation groups, and may constitute a new group. In mixed infection of human cells at 32 degrees C with the host-range and temperature-sensitive mutants, recombination takes place and by a series of two factor crosses the host-range mutation has been approximately located on the adenovirus genetic map.

Temperature-sensitive mutant of adenovirus type 2 blocked in virion assembly: accumulation of light intermediate particles.

A temperature-sensitive mutant of human adenovirus type 2, ts112, was isolated and characterized, ts112 was blocked in a late function required for virus maturation. At restrictive temperature, it accumulated light precursor particles that were able to mature into infectious virions upon temperature shift-down. Use of a mild extraction procedure and a reversible fixation by a cleavable diimido ester permitted the isolation and analysis of these labile intermediates in the adenovirus assembly. These accumulated particles had a sedimentation coefficient of about 600S and a buoyant density of 1.315 g/cm3 in CsCl. They contained a DNA fragment of 7--11S and two nonvirion proteins having molecular weights of 50,000 (50K) and 39.000 (39K), respectively. They resembled in composition and morphology the light intermediate particles found in wild-type adenovirus 2, which were identified as precursors of heavy intermediates, preceding the young virions. The ts112 lesion was apparently located at the exit of either the 50K and/or 39K proteins and at the entry of viral DNA.

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.

Viral DNA synthesis in vitro with nuclei isolated from adeno-associated virus type 1-infected cells

Electron microscope autoradiograms revealed that grains (viral DNA) were found in the nuclei of cells coinfected with adeno-associated virus type 1 (AAV1) and a temperature-sensitive mutant of human adenovirus type 31 (H31 tsA13) at the nonpermissive temperature (40°). Nuclei isolated from cells coinfected with AAV1 and H31 tsA13 at 40° were active in AAV1-DNA synthesis in vitro. Alkaline sucrose gradient analysis of the in vitro products showed that DNA synthesized with nuclei isolated from coinfected cells sedimented faster than marker AAV1-DNA, although DNA synthesized with nuclei isolated from cells coinfected in the presence of cycloheximide cosedimented with the marker DNA. This fast-sedimenting DNA shifted to the position of the marker DNA after treatment with papain, trypsin, and sodium dodecyl sulfate followed by extraction with phenol. This observation suggests the involvement of a protein in the formation of fast-sedimenting viral DNA.

Multiplication of adeno-associated virus type 1 in cells coinfected with A temperature-sensitive mutant of human adenovirus type 31

Abstract A temperature-sensitive mutant (tsA13) of human adenovirus type 31 (H31), defective in viral DNA replication, was able to support growth of adeno-associated virus type 1 (AAV1) at the nonpermissive temperature (40°). With the use of this system, the multiplication of AAV1 and AAV1-specific changes were investigated. The latent period of AAV1 growth was shortened by preinfection of cells with H31tsA13 10 hr before superinfection with AAV1. The rate of DNA synthesis began to rise at about 6 hr postinfection (p.i.) with AAV1 and reached its maximum at 16 hr p.i. In cells coinfected with H31tsA13 and AAV1, only AAV1 DNA was detected without the presence of adenovirus DNA at 40°. Replicative intermediates of AAV1 DNA were larger than AAV1 DNA in neutral and alkaline sucrose gradients. Specific inclusions induced by AAV1 were observed in the nucleus of coinfected and stained cells. Microscopic autoradiogram of coinfected cells revealed that grains (viral DNA) were found before the appearance of the inclusions of AAV1 in the interior of the nucleus. The AAV1 virion antigen first appeared in the nucleus at about 6 hr p.i. with AAV1 and spread into the cytoplasm within 12 hr p.i. The electron microscopic examination of infected cells revealed that the inclusions were aggregates or crystalline arrays of AAV1 particles in the nucleus. However, neither adenovirus inclusions nor particles were observed. AAV1 DNA replication proceeded in the presence of cycloheximide. The time interval between AAV1 infection and the peak of DNA synthesis became shorter, when AAV1 was superinfected at 16 hr or later after infection with H31tsA13. These observations suggest that AAV1 lacks its own early protein, and that the lack of the early protein is complemented by a factor(s) induced in adenovirus-infected cells.

Decreased transforming ability of a temperature-sensitive mutant of human adenovirus type 12

A mutant of human adenovirus type 12, temperature-sensitive for virus replication an for the enhancement of thymidine incorporation in resting human embryonic kidney cells, is described. The mutant was also temperature-sensitive for transformation of rat cells. The results indicate that a viral gene required for the virus growth cycle is also required for transformation.

Conditionally defective helper adenoviruses and satellite virus replication.

ADENO-ASSOCIATED satellite viruses (ASV) are extremely defective in that they need a helper adenovirus to complete their replication cycle in susceptible cells1–3. Although the helper virus is usually not defective there have been reports of systems which are at least conditionally defective. Smith and Gehle4 found that a canine adenovirus, ICH, which did not seem to replicate in human amnion cells (essentially a non-permissive system) could be used to pass the satellite serially in these cells if the passage was reinfected each time with helper virus. Ito et al.5 reported that a temperature-sensitive mutant of human adenovirus type 31, ts 13, defective in viral DNA synthesis, could complement a cycle of satellite virus replication at the non-permissive temperature.