Induction of gene expression by exon 2 of the major E1A proteins of adenovirus type 5.

We have constructed an adenovirus type 5 (Ad5) E1A mutant, dl1119/520, that produces essentially only exon 2 of the major E1A proteins. In infected primary baby rat kidney cells, this mutant induced expression of the E1B 55-kDa protein, and in infected human KB cells, it induced expression of this protein, the E2A 72-kDa protein, and hexon. In KB cells, this mutant grew substantially better than Ad5 dl312, which lacks E1A, and as well as Ad5 dl520, an E1A mutant producing only the 243-residue protein. These results suggest that exon 2 of E1A proteins on its own was able to activate gene expression. We also constructed mutants of dl1119/520, containing small deletions in regions of exon 2 that others found to be associated with effects on the properties of E1A transformants. None of these deletions destroyed gene activation completely, indicating that there may be some redundancy among sequences in exon 2 for inducing gene expression. The two deletions that decreased induction the most, residues 224 to 238 and 255 to 270, were in regions reported to be associated with the expression of a metalloprotease and with enhanced transformation, suggesting that exon 2 may regulate expression of genes governing cell growth. It is remarkable that all sections of E1A proteins, exon 1, the unique region, and exon 2, have now been found to affect gene expression.     

The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells.

Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein.     

Induction of susceptibility to tumor necrosis factor by E1A is dependent on binding to either p300 or p105-Rb and induction of DNA synthesis.

The introduction of the adenovirus early region 1A (E1A) gene products into normal cells sensitizes these cells to the cytotoxic effects of tumor necrosis factor (TNF). Previous studies have shown that the region of E1A responsible for susceptibility is CR1, a conserved region within E1A which binds the cellular proteins p300 and p105-Rb at nonoverlapping sites. Binding of these and other cellular proteins by E1A results in the induction of E1A-associated activities such as transformation, immortalization, DNA synthesis, and apoptosis. To investigate the mechanism by which E1A induces susceptibility to TNF, the NIH 3T3 mouse fibroblast cell line was infected with viruses containing mutations within E1A which abrogate binding of some or all of the cellular proteins to E1A. The results show that TNF susceptibility is induced by E1A binding to either p300 or p105-Rb. E1A mutants that bind neither p300 nor p105-Rb do not induce susceptibility to TNF. Experiments with stable cell lines created by transfection with either wild-type or mutant E1A lead to these same conclusions. In addition, a correlation between induction of DNA synthesis and induction of TNF sensitivity is seen. Only viruses which induce DNA synthesis can induce TNF sensitivity. Those viruses which do not induce DNA synthesis also do not induce TNF sensitivity. These data suggest that the mechanisms underlying induction of susceptibility to TNF by E1A are intimately connected to E1A's capacity to override cell cycle controls.     

Complementary functions of E1a conserved region 1 cooperate with conserved region 3 to activate adenovirus serotype 5 early promoters.

The amino-terminal region of the adenovirus type 5 E1a protein including conserved regions (CRs) 1 and 2 binds the 105-kDa retinoblastoma protein and a second, 300-kDa, cellular protein. We show that mutant viruses with deletions of CR1 which release the binding of either p105 or p300 still activate early promoters and infect cells productively. However, mutations which disrupt binding of both proteins disrupt early promoter activity and block the viral life cycle. Ela CR3, which has an established role in early promoter activation, can act in trans to the amino-terminal functions. This suggests that the amino terminus provides distinct, redundant functions related to p300 and Rb binding that synergize with CR3 to transactivate early genes.     

p53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G₁. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39°C. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.     

Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts

Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+, Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 and pmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

Induction of the cell cycle in baby rat kidney cells by adenovirus type 5 E1A in the absence of E1B and a possible influence of p53.

From previous studies on the induction of DNA synthesis in quiescent primary baby rat kidney cells by adenovirus type 5 (Ad5) E1A deletion mutants, we concluded that induction is prevented only when cellular proteins p300 and pRb are both uncomplexed with E1A (J.A. Howe, J.S. Mymryk, C. Egan, P.E. Branton, and S.T. Bayley, Proc. Natl. Acad. Sci. USA 87:5883-5887, 1990). We have now examined induction by these same mutants in virus lacking the E1B region, so that cellular p53 was no longer complexed to the E1B 55-kDa protein. E1A mutants that fail to bind pRb induced DNA synthesis at a significantly lower level in Ad5 lacking E1B than in Ad5 containing E1B. Apparently, therefore, uncomplexed p53 can partially replace p300 in cooperating with pRb to suppress DNA synthesis in baby rat kidney cells.     

The E1B 19-kilodalton protein is not essential for transformation of rodent cells in vitro by adenovirus type 5.

The newly constructed adenovirus type 5 mutant in1 carries a single AT base pair insertion immediately after nucleotide position 1715 in the E1B gene sequence which destroys the proximal AUG normally present in E1B messages and prevents production of intact E1B 19-kDa protein in infected cells. We have used in1, variants of in1 containing mutant alleles of viral genes known to enhance transformation frequency, and adenovirus type 5 mutant dl337 (S. Pilder, J. Logan, and T. Shenk, J. Virol. 52:664-671, 1984), in which the sequence between nucleotides 1770 and 1916 within the 19-kDa reading frame is deleted, to test the generally accepted hypothesis that this E1B protein is essential for the transformation of rodent cells and maintenance of the transformed phenotype. We find that these mutants transform rat embryo cells, rat kidney and mouse kidney primary cells, and cells of the 3Y1 rat line with decreased frequencies only when virus is added to these various cells at high input multiplicities of infection. In contrast, when lower doses of virus are used, the mutants transform with wild-type frequencies. Cells infected with higher doses of mutant virus show increased levels of DNA degradation and cell killing compared with those of cells infected with the same levels of wild-type virus, and these effects most likely contribute to the decreased transformation frequencies observed. On the basis of these results and the results of phenotypic analyses of numerous transformants, we propose that the E1B 19-kDa protein is not required for induction and/or maintenance of transformed-cell characteristics in rodent cells infected with adenovirus type 5.     

Disruption of the coordinate expression of muscle genes in a transfected BC3H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein.

Mouse BC3H1 myoblasts were stably transfected with the adenovirus 5 E1A gene. One clonal line, BC3E7, was found to differ in some important respects from those previously reported for E1A-transformed myoblasts. In contrast to BC3H1 cells which differentiate when confluent in medium containing 0.5% fetal calf serum (FCS), BC3E7 cells failed to elongate and align, to express acetylcholine receptor and creatine kinase, and to down-regulate expression of beta- and gamma-actins and tropomyosin isoform (TM) 1. However, increased synthesis of TMs 2, 3, and 4, and myosin light chain 1 associated with differentiation in BC3H1 still occurred in BC3E7 cells, and most surprisingly, alpha-actin was produced at a significant level in both proliferating and confluent BC3E7 cells. Interestingly, myogenin was expressed in confluent BC3E7 cells in 0.5% FCS, but not in 20%. The level of E1A expression in BC3E7 cells was found to be very low by analysis of mRNA, by immunoprecipitation of E1A protein, and by the ability of BC3E7 cells to complement the E1A-deficient adenovirus mutant dl312. These results suggest that different levels of E1A may be needed to repress different promoters and that E1A does not block myogenic differentiation by repressing myogenin expression, but represses each muscle gene independently.     

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).     

Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.     

trans-dominant interference of type 5 adenovirus E1a mutants in cell transformation.

Two type 5 adenovirus (Ad5) early region 1a (E1a) mutants, H5in104 and H5dl105, were impaired in viral replication and cell transformation. In addition, these mutants trans dominantly inhibited the frequency with which H5sub309, a phenotypically wild-type mutant, and H5dl520, a high-frequency transformation mutant, transformed CREF cells. Inhibition of transformation varied in proportion to the input ratio of mutant to coinfecting virus. It was found that H5in104, but not H5dl105, could not complement Ad5 E1b mutants that failed to synthesize 19- or 55-kDa E1b product. H5dl105 yielded 10-fold less virus than the wild-type did in 293 cells, which constitutively express E1a and E1b products; similar low yields were also observed with H5in104 and H5dl105 in another E1a- and E1b-expressing transformed cell line, KB16. Marker rescue and DNA sequence analyses, however, indicated that the phenotypes of H5in104 and H5dl105 were the result of their respective E1a mutations. The data presented are the first to demonstrate that mutants of animal viruses can effect dominant interference with the viral function(s) that produce cell transformation.     

Assembly of ROMK1 (Kir 1.1a) inward rectifier K+ channel subunits involves multiple interaction sites.

The ROMK1 (Kir 1.1a) channel is formed by a tetrameric complex of subunits, each characterized by cytoplasmic N- and C-termini and a core region of two transmembrane helices flanking a pore-forming segment. To delineate the general regions mediating the assembly of ROMK1 subunits we constructed epitope-tagged N-terminal, C-terminal, and transmembrane segment deletion mutants. Nonfunctional subunits with N-terminal, core region, and C-terminal deletions had dominant negative effects when coexpressed with wild-type ROMK1 subunits in Xenopus oocytes. In contrast, coexpression of these nonfunctional subunits with Kv 2.1 (DRK1) did not suppress Kv 2.1 currents in control oocytes. Interactions between epitope-tagged mutant and wild-type ROMK1 subunits were studied in parallel by immunoprecipitating [35S]-labeled oocyte membrane proteins. Complexes containing both wild-type and mutant subunits that retained H5, M2, and C-terminal regions were coimmunoprecipitated to a greater extent than complexes consisting of wild-type and mutant subunits with core region and/or C-terminal deletions. The present findings are consistent with the hypothesis that multiple interaction sites located in the core region and cytoplasmic termini of ROMK1 subunits mediate homomultimeric assembly.