Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA.

The adenovirus 5 mutant H5dl337 lacks 146 base pairs within early region 1B. The deletion removes a portion of the region encoding the E1B 21,000-molecular-weight (21K) polypeptide, but does not disturb the E1B-55K/17K coding region. The virus is slightly defective for growth in cultured HeLa cells, in which its final yield is reduced ca. 10-fold compared with wild-type virus. The mutant displays a striking phenotype in HeLa cells. The onset of cytopathic effect is dramatically accelerated, and both host cell and viral DNAs are extensively degraded late after infection. This defect has been described previously for a variety of adenovirus mutants and has been termed a cytocidal (cyt) phenotype. H5dl337 serves to map this defect to the loss of E1B-21K polypeptide function. In addition to its defect in the productive growth cycle, H5dl337 is unable to transform rat cells at normal efficiency.     

The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.     

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.     

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.     

Diverse roles for E4orf3 at late times of infection revealed in an E1B 55-kilodalton protein mutant background

Species C human adenovirus mutants that fail to express open reading frame 3 of early region 4 (E4orf3) are phenotypically indistinguishable from the wild-type virus when evaluated in cells cultured in vitro. However, E4orf3 gene function has been productively studied in the context of additional viral mutations. This study identifies diverse roles for the E4orf3 protein that are evident in the absence of early region 1B 55-kDa protein (E1B-55K) function. In an E1B-55K-deficient background, the E4orf3 protein promotes viral replication by increasing both the burst size and the probability that an infected cell will produce virus. Early viral gene expression is not impaired in E1B-55K/E4orf3 double mutant virus-infected cells. Cells infected with the double mutant virus accumulated concatemers of viral DNA. However, the E1B-55K/E4orf3 double mutant virus did not replicate any better in MO59J cells, in which viral DNA concatemers did not accumulate, than in MO59K cells, in which viral DNA concatemers were produced, suggesting that viral DNA concatenation is not the primary growth defect of the E1B-55K/E4orf3 double mutant virus. Accumulation of viral mRNA in the nucleus and cytoplasm of E1B-55K/E4orf3 double mutant virus-infected cells was severely reduced compared to that on wild-type virus-infected cells. Thus, in an E1B-55K mutant background, the E4orf3 protein promotes the accumulation of late viral RNA and enhances late gene expression. Finally, within the context of an E1B-55K mutant virus, the E4orf3 protein acts to suppress host cell translation and preserve the viability of cells at moderately late times of infection.     

Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses

We have studied the proteins encoded by the transforming region of the closely related human adenovirus serotypes 2 and 5. Messenger RNAs complementary to the two parts of this region, E1A and E1B, were prepared separately by hybridization to cloned DNA fragments encompassing 0.8 to 4.5 map units (for E1A) and 9.8 to 11.1 map units (for E1B). These RNAs were further fractionated by electrophoresis through agarose gels containing methylmercuric hydroxide, and then translated in vitro to identify the proteins encoded by each RNA species. E1A and E1B RNAs isolated at early and at late times after infection were compared. Three size classes of E1A mRNA direct the synthesis of at least five proteins: a28K³ protein encoded by a 0.6 kb mRNA, 42K and 54K proteins encoded by a 0.9 kb mRNA(s), and 48K and 58K proteins encoded by a 1.1 kb mRNA(s). The mRNA for the 28K protein accumulates preferentially at late times. Three size classes of early E1B mRNA direct the synthesis of three proteins: a 15K protein encoded by a 0.9 kb mRNA, an 18K protein encoded by a 1.2 kb mRNA, and a 57K protein encoded by a 2.6 kb mRNA. The mRNA for the 15K protein continues to accumulate at late times, and an additional 22K protein is made, while the 18K and 57K proteins are synthesized poorly, if at all, with late RNA.Substantially different E1A and E1B proteins are encoded by RNA from cells infected with the adenovirus type 5 mutants dl311, dl312, dl313, dl314 and hr1, which are all defective for replication on human cells and, except for dl311, for transformation. dl312, dl314 and hr1 are also defective for early viral gene expression. No viral mRNA could be detected in either dl312 or dl314-infected cells. hr1-infected cells contain a 0.9 kb mRNA encoding E1A 54K and 42K, but instead of 58K and 48K, the 1.1 kb hr1-E1A mRNA is translated into a 26K protein. The E1B mRNAs are present in substantially decreased amounts in hr1-infected cells. dl311-infected cells contain E1A mRNAs of 1.1 and 0.9 kb, encoding 38K and 34K proteins, respectively, and normal E1B mRNAs. The dl313 mRNAs of 1.1 and 0.9 kb contained fused E1A and E1B sequences and were translated into 40K and 36K proteins, respectively. These results are related to the mRNA structures and the biological activity of regions of the individual proteins.     

Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery

During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-alpha 1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.     

p53-Independent and -Dependent Requirements for E1B-55K in Adenovirus Type 5 Replication

The adenovirus type 5 mutant dl1520 was engineered previously to be completely defective for E1B-55K functions. Recently, this mutant (also known as ONYX-015) has been suggested to replicate preferentially in p53(-) and some p53(+) tumor cell lines but to be attenuated in primary cultured cells (C. Heise, A. Sampson-Johannes, A. Williams, F. McCormick, D. D. F. Hoff, and D. H. Kirn, Nat. Med. 3:639-645, 1997). It has been suggested that dl1520 might be used as a "magic bullet" that could selectively lyse tumor cells without harm to normal tissues. However, we report here that dl1520 replication is independent of p53 genotype and occurs efficiently in some primary cultured human cells, indicating that the mutant virus does not possess a tumor selectivity. Although it was not the sole host range determinant, p53 function did reduce dl1520 replication when analyzed in a cell line expressing temperature-sensitive p53 (H1299-tsp53) (K. L. Fries, W. E. Miller, and N. Raab-Traub, J. Virol. 70:8653-8659, 1996). As found earlier for other E1B-55K mutants in HeLa cells (Y. Ho, R. Galos, and J. Williams, Virology 122:109-124, 1982), dl1520 replication was temperature dependent in H1299 cells. When p53 function was restored at low temperature in H1299-tsp53 cells, it imposed a modest defect in viral DNA replication and accumulation of late viral cytoplasmic mRNA. However, in both H1299 and H1299-tsp53 cells, the defect in late viral protein synthesis appeared to be much greater than could be accounted for by the modest defects in late viral mRNA levels. We therefore propose that in addition to countering p53 function and modulating viral and cellular mRNA nuclear transport, E1B-55K also stimulates late viral mRNA translation.     

Defective synthesis of early region 4 mRNAs during abortive adenovirus infections in monkey cells.

Human adenovirus 2 grows poorly in monkey cells, partly because of defects in late gene expression. Since deletions in early region 4 (E4) cause similar defects in late gene expression, we examined E4 mRNA expression in abortive infections. Processing of E4 mRNAs was defective during abortive infections, most likely at the level of splicing. At early times in productive infections in HeLa cells, the major E4 species produced is a 2-kb mRNA; at late times, a shift occurs so that smaller spliced E4 mRNAs are also produced. In CV-1 cells, a nonpermissive monkey cell line, this shift did not take place and only the 2-kb species was produced at late times, suggesting a defect in E4 mRNA splicing during abortive infections. The adenovirus DNA-binding protein (DBP) was required for normal processing of E4 mRNAs, since a host range mutant (hr602) containing an altered DBP gene showed a normal late E4 mRNA pattern in CV-1 cells; in addition, DBP was required during infections in HeLa cells for late E4 mRNA expression. DBP was not required for production of the late E4 pattern in transient expression assays in HeLa or 293 cells, suggesting that a second factor in addition to the DBP, present during infection but not transfection, modulates E4 mRNA processing.     

Analyses of single-amino-acid substitution mutants of adenovirus type 5 E1B-55K protein

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.     

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 E1B 55-kilodalton protein is required for both regulation of mRNA export and efficient entry into the late phase of infection in normal human fibroblasts

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).