Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Functional changes in temperature-sensitive mutants of the adenovirus single-stranded DNA-binding protein are accompanied by structural alterations.

Adenovirus requires the virus-encoded single-stranded DNA-binding protein (DBP) to replicate its DNA. We have previously shown (M. Tsuji, P. C. van der Vliet, and G. R. Kitchingman, J. Biol. Chem. 266:16178-16187, 1991) that the inability of three temperature-sensitive (ts) mutant DBPs (Ad2+ ND1ts23, Ad2ts111A, and Ad5ts125) to support DNA replication at the nonpermissive temperature was associated with impaired ability to bind to DNA. In this study, we examined these mutant proteins for structural alterations that might be linked to the functional changes. All three ts mutants, but not the wild-type protein, showed different proteolytic cleavage patterns before and after heating at 40 degrees C (the nonpermissive temperature), suggesting a possible conformational change during heating. The Ad2+ND1ts23 and Ad2ts111A DBPs have single amino acid changes located in a putative zinc finger subdomain (positions 282 and 280). In the presence of zinc ions, these ts mutants showed significantly increased resistance to inactivation at 40 degrees C. Surprisingly, however, the stabilizing effect of zinc was also observed with the Ad5ts125DBP, which contains a mutation located more than 100 amino acids from the zinc finger. Other related metal ions, such as cobalt, cadmium, and mercury, did not protect the ts DBPs from inactivation at 40 degrees C. These results indicate that functional changes of the ts DBPs in DNA replication and DNA binding are accompanied by structural alterations in the protein and that zinc and the metal-binding subdomain may play an important role in the structure and/or function of the DBP.     

Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells.

The genomes of the two nondefective adenovirus 2/simian virus 40 (Ad2/SV 40) hybrid viruses, nondefective Ad2/SV 40 hybrid virus 1 (Ad2+ND1) and nondefective hybrid virus 3 (Ad2+ND3), WERE FORMED BY A DELETION OF ABOUT 5% OF Ad2 DNA and insertion of part of the SV40 genome. We have compared the cytoplasmic RNA synthesized during both the early and late stages of lytic infection of human cells by these hybrid viruses to that expressed in Ad2-infected and SV40-infected cells. Separated strands of the six fragments of 32P-labeled Ad2 DNA produced by cleavage with the restriction endonuclease EcoRI (isolated from Escherichia coli) and the four fragments of 32P-labeled SV40 DNA produced by cleavage with both a restriction nuclease isolated from Haemophilus parainfluenzae, Hpa1, and EcoRI were prepared by electrophoresis of denatured DNA in agarose gels. The fraction of each fragment strand expressed as cytoplasmic RNA was determined by annealing fragmented 32P-labeled strands to an excess of cellular RNA extracted from infected cells. The segment of Ad2 DNA deleted from both hybrid virus genomes is transcribed into cytoplasmic mRNA during the early phase of Ad2 infection. Hence, we suggest that Ad2 codes for at least one "early" gene product which is nonessential for virus growth in cell culture. In both early Ad2+ND1 and Ad2+ND3-infected cells, 1,000 bases of Ad2 DNA adjacent to the integrated SV40 sequences are expressed as cytoplasmic RNA but are not similarly expressed in early Ad2-infected cells. The 3' termini of this early hybrid virus RNA maps in the vicinity of 0.18 on the conventional SV40 map and probably terminates at the same position as early lytic SV40 cytoplasmic RNA. Therefore, the base sequence in this region of SV40 DNA specifies the 3' termini of early messenger RNA present in both hybrid virus and SV40-infected cells.     

Temperature-sensitive mutants of adenovirus single-stranded DNA-binding protein. Inability to support DNA replication is associated with an altered DNA-binding activity of the protein.

The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+ND1ts23, Ad2ts111A, and Ad5ts125) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 degrees C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP.dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 degrees C. The functional change at 40 degrees C was irreversible, as the ts DBPs preincubated at 40 degrees C were no longer active when assayed at 30 degrees C. Upon heating to 40 degrees C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these three ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.     

Adenovirus E1A gene induction of susceptibility to lysis by natural killer cells and activated macrophages in infected rodent cells.

Rodent cells immortalized by the E1A gene of nononcogenic adenoviruses are susceptible to lysis by natural killer (NK) cells and activated macrophages. This cytolysis-susceptible phenotype may contribute to the rejection of adenovirus-transformed cells by immunocompetent animals. Such increased cytolytic susceptibility has also been observed with infected rodent cells. This infection model provided a means to study the role of E1A gene products in induction of cytolytic susceptibility without cell selection during transformation. Deletion mutations outside of the E1A gene had no effect on adenovirus type 2 (Ad2) or Ad5 induction of cytolytic susceptibility in infected hamster cells, while E1A-minus mutant viruses could not induce this phenotype. E1A mutant viruses that induced expression of either E1A 12S or 13S mRNA in infected cells were competent to induce cytolytic susceptibility. Furthermore, there was a correlation between the accumulation of E1A gene products in Ad5-infected cells and the level of susceptibility of such target cells to lysis by NK cells. The results of coinfection studies indicated that the E1A gene products of highly oncogenic Ad12 could not complement the lack of induction of cytolytic susceptibility by E1A-minus Ad5 virus in infected cells and also could not block induction of this infected-cell phenotype by Ad5. These data suggest that expression of the E1A gene of nononcogenic adenoviruses may cause the elimination of infected cells by the immunologically nonspecific host inflammatory cell response prior to cellular transformation. The lack of induction of this cytolysis-susceptible phenotype by Ad12 E1A may result in an increased persistence of Ad12-infected cells in vivo and may lead to an increased Ad12-transformed cell burden for the host.     

Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2(+)ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2(+)ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2(+)ND1 either by the cleavage patterns produced by Escherichia coli R.RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2(+)ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2(+)ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2(+)ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40.     

Cell surface location of simian virus 40-specific proteins on HeLa cells infected with adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2.

HeLa cells infected with the nondefective adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 or Ad2+ND2 were analyzed for cell surface location of the SV40-specific hybrid virus proteins by indirect immunofluorescence microscopy. Two different batches of sera from SV40 tumor-bearing hamsters, serum from SV40 tumor-bearing mice, or two different antisera prepared against purified sodium dodecyl sulfate-denatured SV40 T-antigen, respectively, were used. All sera were shown to exhibit comparable T- and U-antibody titers and to specifically immunoprecipitate the SV40-specific proteins from cell extracts of Ad2+ND2-infected cells. Whereas analysis of living, hybrid virus-infected HeLa cells did not yield conclusive results, analysis of Formalin-fixed cells resulted in positive cell surface fluorescence with both Ad2+ND1- and Ad2+ND2-infected HeLa cells when antisera prepared against sodium dodecyl sulfate-denatured SV40 T-antigen were used as first antibody. In contrast, sera from SV40 tumor-bearing animals were not or only very weakly able to stain the surfaces of these cells. The fact that the tumor sera had comparable or even higher T- and U-antibody titers than the antisera against sodium dodecyl sulfate-denatured T-antigen but were not able to recognize SV40-specific proteins on the cell surface suggests that SV40 tumor-specific transplantation antigen may be an antigenic entity different from T- or U-antigen.     

The relationship between region E1a and E1b of human adenoviruses in cell transformation

Baby rat kidney (BRK) cells were transfected either with intact region E1 DNA of adenovirus type 5 (Ad5) or with mixtures of DNA fragments containing the separated E1a and E1b regions. The results showed that mixtures of regions E1a and E1b transform with a similar efficiency as intact region E1. DNA fragments containing region E1b alone have no detectable transforming activity in primary BRK cells nor in established rat cell lines. When region E1a and Ad5 was combined with region E1b and Ad12 complete transformation was also obtained. Characterization of the cell lines transformed by separated E1a and E1b regions have led to the following conclusions: (1) Expression of region E1b is not dependent on specific linkage to region E1a as it occurs in the intact E1 region. (2) Region E1b is normally expressed into the corresponding major adenovirus T antigens (65,000 and 19,000 Mr with region E1b of Ad5; 60,000 and 19,000 Mr with E1b or AD12). (3) Region E1b of Ad12 can be activated by region E1a of Ad5 indicating that the Ela regions of both serotypes are functionally similar in transformation. (4) Cell lines containing region E1b of Ad5 are weakly oncogenic in nude mice whereas cells containing E1b of Ad12 are highly oncogenic in nude mice, indicating that the degree of oncogenicity is determined by region E1b.     

Simian virus 40 (SV40)-specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U-antigen determinants.

The distribution of simian virus 40 (SV40)-specific proteins in nuclear subfractions of pulse-chase-labeled HeLa cells infected with nondefective adenovirus type 2 (Ad2)-SV40 hybrid viruses was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND5 specifically associate with the nuclear matrix and are virtually absent from the high-salt nuclear extract. In Ad2+ND4-infected HeLa cells, the SV40-specific proteins with molecular weights of 64,000 (64K) and lower also specifically associate with the nuclear matrix. The SV40-specific 72K, 74K, and 95K proteins were found both in the nuclear matrix and in the high-salt nuclear extract. Analyses of the nuclear matrices isolated from hybrid virus-infected cells by immunofluorescence microscopy showed that SV40 U-antigen-positive sera from SV40 tumor-bearing hamsters react with SV40-specific proteins integrated into nuclear matrices of HeLa cells infected by Ad2+ND1, Ad2+ND2, and Ad2+ND4, but not with nuclear matrices of HeLa cells infected by Ad2+ND5. This suggests that SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND4 integrated into the nuclear matrix carry SV40 U-antigen determinants. The apparent discrepancy in the subcellular localization of SV40-specific proteins in hybrid virus-infected cells when analyzed by biochemical cell fractionation procedures and when analyzed by immunofluorescence staining is discussed.     

Purification and functional characterization of adenovirus ts111A DNA-binding protein. Fluorescence studies of protein-nucleic acid binding

The adenovirus single-stranded DNA (ssDNA)-binding protein (DBP) is necessary for the elongation step in viral DNA replication. In an attempt to characterize the putative ssDNA-binding domain of the DBP, we purified and characterized the Ad2ts111A DBP, which contains a glycine-to-valine substitution at amino acid 280. This mutation is adjacent to that in the previously studied Ad2+ND1ts23. Ad2+ND1ts23 exhibits a temperature-sensitive defect in DNA replication, and its DBP has previously been shown to bind ssDNA with reduced affinity. Ad2ts111A DBP, like Ad2+ND1ts23, does not support adenovirus DNA replication in vitro at elevated temperatures. However, the Ad2ts111A DBP binds ssDNA more tightly than does Ad2+ND1ts23 and is not temperature sensitive in this function. To determine the nucleic acid-binding properties of DBP, we applied spectrofluorometric techniques, which had not been used previously to study adenovirus DBP. Using the homopolynucleotide poly(1,N6)-ethenoadenylic acid (poly(r epsilon A], we have determined that the binding site size is approximately 16 nucleotides. In 20 mM NaCl, the Ad2wt, Ad2ts111A, and Ad2+ND1ts23 DBP proteins all bound stoichiometrically to poly(r epsilon A) with overall apparent affinities above 108 M-1. Based on titrations carried out at higher salt concentrations, however, the stability of these complexes did appear to increase in the order Ad2+ND1ts23 less than Ad2ts111A less than Ad2wt. By these techniques, we have confirmed also that the DBP of another temperature-sensitive mutant, H5ts107, like the Ad2ts111A DBP, retains its ability to bind ssDNA even at a restrictive temperature utilizing the salt concentration compatible with adenovirus DNA replication in vitro. The H5ts107 DBP, which contains an amino acid substitution at position 413, is defective for in vitro replication at nonpermissive temperature but is not temperature sensitive for binding to ssDNA. In summary, our results indicate that the replication defects of the Ad2ts111A are similar to those of H5ts107 and cannot be attributed to defective, nonspecific ssDNA binding by the DBP. It appears that ssDNA binding by itself is not sufficient to account for the role of DBP in adenovirus DNA replication.     

Studies on Nondefective Adenovirus-Simian Virus 40 Hybrid Viruses I. A Newly Characterized Simian Virus 40 Antigen Induced by the Ad2+ND1 Virus

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), does not induce heat-labile SV40 T antigen but does induce a previously uncharacterized heat-stable SV40 antigen-the SV40 "U" antigen. This antigen is detectable by both immunofluorescence and complement fixation by using sera from hamsters with SV40 tumors. Sera from hamsters bearing SV40 tumors can be divided into two groups, those that react with both SV40 T and U antigens (T(+)U(+) sera) and those that react with SV40 T antigen only (T(+)U(-) sera). SV40 U-specific sera from monkeys immunized with Ad2(+)ND(1)-infected cells do not react with SV40 T antigen by immunofluorescence but do react with an antigen in the nucleus of SV40-transformed cells and with an early, cytosine arabinoside-resistant antigen present in the nucleus of SV40-infected cells. A heat-stable SV40 antigen detectable by complement fixation with T(+)U(+) hamster sera is present in extracts of SV40-induced hamster tumors and in cell packs of SV40-infected or -transformed cells. SV40 U-antigen synthesis by Ad2(+)ND(1) virus is partially sensitive to inhibitors of deoxyribonucleic acid synthesis, whereas U-antigen synthesis by SV40 virus is an early cytosine arabinoside-resistant event. As an early SV40 antigen differing from SV40 T antigen, U antigen may play a role in malignant transformation mediated by SV40.     

Depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza A (H5N1) virus isolated from humans

Previously, we observed that several virulent influenza A (H5N1) viruses which caused severe or fatal disease in humans were also lethal in BALB/c mice following dissemination of the virus to solid organs, including the brain. In contrast, one particular human H5N1 virus was nonlethal in mice and showed no evidence of systemic spread. To compare H5N1 viruses of varying pathogenicity for their ability to alter the mammalian immune system, mice were infected with either influenza A/Hong Kong/483/97 (HK/483) (lethal) or A/Hong Kong/486/97 (HK/486) (nonlethal) virus and monitored for lymphocyte depletion in the blood, lungs, and lymphoid tissue. Intranasal infection with HK/483 resulted in a significant decrease in the total number of circulating leukocytes evident as early as day 2 postinfection. Differential blood counts demonstrated up to an 80% drop in lymphocytes by day 4 postinfection. In contrast, nonlethal HK/486-infected mice displayed only a transient drop of lymphocytes during the infectious period. Analysis of lung and lymphoid tissue from HK/483-infected mice demonstrated a reduction in the number of CD4(+) and CD8(+) T cells and reduced synthesis of the cytokines interleukin-1beta and gamma interferon and the chemokine macrophage inflammatory protein compared with HK/486-infected mice. In contrast, the cytokine and chemokine levels were increased in the brains of mice infected with HK/483 but not HK/486. Evidence of apoptosis in the spleen and lung of HK/483-infected mice was detected in situ, suggesting a mechanism for lymphocyte destruction. These results suggest that destructive effects on the immune system may be one factor that contributes to the pathogenesis of H5N1 viruses in mammalian hosts.     

Evidence for Simian Virus 40 (SV40) Coding of SV40 T-Antigen and the SV40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus Type 2-SV40 Hybrid Viruses

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND1, Ad2(+)ND2, Ad2(+)ND4, and Ad2(+)ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2(+)ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2(+)ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.     

The adenovirus type 2-simian virus 40 hybrid virus Ad2+ND4 requires deletion variants to grow in monkey cells.

The Ad2+ND4 virus is an adenovirus type 2 (Ad2)-simian virus 40 (SV40) recombination. The Ad2 genome of this recombinant has a rearrangement within early region 3; Ad2 DNA sequences between map positions 81.3 and 85.5 have been deleted, and the SV40 DNA sequences between map positions 0.11 and 0.626 have been inserted into the deletion in an 81.3-0.626 orientation. Nonhybrid Ad2 is defective in monkey cells; however, the Ad2+ND4 virus can replicate in monkey cells due to the expression of the SV40-enhancing function encoded by the DNA insert. Stocks of the Ad2+ND4 hybrid were produced in primary monkey cells by using the progeny of a three-step plaque purification procedure and were considered to be homogeneous populations of Ad2+ND4 virions because they induced plaques in primary monkey cells by first-order kinetics. By studying the kinetics of plaque induction in continuous lines (BSC-1 and CV-1) of monkey cells, we have found that stocks (prepared with virions before and after plaque purification) of Ad2+ND4 are actually heterogeneous populations of Ad2+ND4 virions and Ad2+ND4 deletion variants that lack SV40 and frequently Ad2 DNA sequences at the left Ad2-SV40 junction. Due to the defectiveness of the Ad2+ND4 virus, the production of progeny in BSC-1 and CV-1 cells requires complementation between the Ad2+ND4 genome and the genome of an Ad2+ND4 deletion variant. Since the deletion variants that have been obtained from Ad2+ND4 stocks do not express the SV40-enhancing function in that they cannot produce progeny in monkey cells, we conclude that they are providing an Ad2 component that is essential for the production of Ad2+ND4 progeny. These data imply that the Ad2+ND4 virus is incapable of replicating in singly infected primary monkey cells without generating deletion variants that are missing various amounts of DNA around the left Ad2-SV40 junction in the hybrid genome. As the deletion variants that arise from the Ad2+ND4 virus are created by nonhomologous DNA recombination, the generation of deletion variants in monkey cells infected with Ad2+ND4 may be a useful model for studying this process.     

Purification and characterization of an early protein (E14K) from adenovirus type 2-infected cells.

One adenovirus type 2 (Ad2) early protein, with an apparent molecular weight of 14,000 in sodium dodecyl sulfate-polyacrylamide gels (E14K), was purified to homogeneity. Purification involved fractionation of cytoplasmic extracts, precipitation at low pH, and DEAE-cellulose, phosphocellulose, and hydroxylapatite chromatography. The yield was around 12 microgram of purified protein per 10(9) HeLa cells. The two Ad2 DNA binding proteins with molecular weights of 75,000 and 45,000 (E75K and E45K) were purified by the same procedure. Tryptic peptide analyses indicated that the E14K protein is unrelated to the DNA binding proteins. The purified E14K protein has a high content of basic amino acids and a sedimentation coefficient of 5.5S in the native state, corresponding to a molecular weight of around 95,000. Pulse-chase experiments suggest that the E14K polypeptide is a primary translation product. Immunoprecipitation with a monospecific antiserum against the E14K protein revealed that it is exclusively localized in the cytoplasm of infected cells. E14K started to be synthesized at 2 hpostinfection, with a maximal rate of synthesis at 4 to 6 h postinfection. Immunoprecipitation of cell extracts from four different Ad2-transformed hamster embryo cell lines revealed that only one (Ad2HE4) of them expresses this protein. The adenovirus-simian virus 40 hybrid virus (Ad2ND1) does not express this protein, suggesting that the gene for the E14K protein is located in the part of the Ad2 genome which is deleted in this hybrid virus.     

Conditional lethal mutants of adenovirus type 2-simian virus 40 hybrids. II. Ad2+ND1 host-range mutants that synthesize fragments of the Ad2+ND1 30K protein.

Adenovirus type 2 (Ad2) grows 1,000 times less well in monkey cells than in human cells. This defect can be overcome, not only upon co-infection of cells with simian virus 40 (SV40), but also when the relevant part of the SV40 genome is integrated into the adenovirus genome to form an adenovirus-SV40 hybrid virus. We have used the nondefective Ad2-SV40 hybrid virus Ad2+ND1, which contains an insertion of 17% of the SV40 genome, to isolate host-range mutants which are defective in growth on monkey cells although they grow normally on human cells. Like Ad2, these mutants are defective in the synthesis of late proteins in monkey cells. A 30,000-molecular-weight protein (30K), unique to Ad2+ND1-infected cells, can be synthesized in vitro, using Ad2+ND1 mRNA that contains SV40 sequences. 30K is not seen in cells infected with those host-range mutants that are most defective in growth on monkey cells, and translation in vitro of SV40-specific mRNA from these cells produces new unique polypeptides, instead of 30K. Genetic and biochemical analyses indicate that these mutants carry point mutations rather than deletions.     

Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus

Background

Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed.

Methodology/Principal Findings

In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ² =  44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive.

Conclusions/Significance

The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.     

Differences in DNA-binding proteins isolated from normal and transformed human cells

When the DNA-binding proteins (DBPs) of WI38 normal human fibroblasts and their SV40-transformed counterpart were compared, two DBPs were present in greater amounts in the transformed cells. These two DBPs, P5a and P6b, were also present in greater amounts in HeLa cells versus WI38 cells and in chemically transformed human liver cells versus normal liver cells. Therefore, these DBP differences do not appear to be specific for transformation by SV40. Increased amounts of P5a were present in 7 of 9 transformed cell lines examined. The two tumor cell lines lacking the P5a change were sensitive to density-dependent inhibition of replication, whereas the other seven cell lines were not. This correlation suggests that the increase in P5a may play a role in the release from density-dependent inhibition of replication observed in most transformed cells.