The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression.

While the products of the type 5 adenovirus E1A and E1B genes can initiate pathways leading to a transformed rodent cell, little is known about how the rate of viral early gene expression influences the efficiency of this process. An adenovirus mutant [E1a(r) virus] that expresses its viral E1A and E1B genes at as much as a 100-fold-reduced rate relative to wild-type virus in infected CREF or HeLa cells transforms CREF cells at an 8-fold-higher efficiency than wild-type virus. Additional studies show that the reduction in viral E1A gene expression is solely responsible for this transformation phenotype, and at this low rate of viral E1A gene expression both E1A gene products must be expressed. Unlike previously characterized viruses which transform CREF cells at frequencies greater than wild-type virus, the foci obtained following E1a(r) virus infection were indistinguishable from those arising from wild-type virus by several criteria (morphological characteristics and anchorage-independent growth). Surprisingly, an analysis of viral early gene expression from a panel of wild-type- and E1a(r) virus-transformed CREF cell lines showed similar average rates of both viral E1A and E1B gene expression. By using an adenovirus-transformed cell line that is cold-sensitive for maintenance of the transformed cell phenotype, we show that both wild-type and the E1a(r) viruses can transform these cells at equally high efficiencies at the nonpermissive temperature of 32 degrees C. Our findings suggest that the process leading to a fully transformed cell involves multiple stages, with an early stage being facilitated by a reduced rate of viral E1A gene expression.     

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.     

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.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

Alternative splicing of the human cytomegalovirus major immediate-early genes affects infectious-virus replication and control of cellular cyclin-dependent kinase

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.     

Change of DNA near the origin of replication enhances the transforming capacity of human papovavirus BK.

A turbid-plaque-forming mutant (pm522) of human papovavirus BK, which has a small deletion at about 0.7 map unit and grows somewhat more slowly in human cells than does wild-type BK virus, transformed hamster and rat cells in culture much more efficiently than did wild-type virus. Another plaque morphology mutant, pm525, forming turbid plaques larger than those of pm522 also had a high transforming capacity. The similar difference in transforming capability between wild-type and plaque morphology viruses was observed with DNAs extracted from virions. Recombinant viruses were constructed from the wild-type DNA fragment lacking HindIII-C (0.62 to 0.73 map unit) and pm522 HindIII-C (including the origin of replication) by the molecular cloning method. Characterization of the recombinants showed that the change near the origin of DNA replication was responsible both for the altered plaque morphology and for the enhanced transforming capacity of the BK virus mutant.     

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.     

Baculovirus lef-12 is not required for viral replication

The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.     

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.     

Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff.

To delineate the function of adenovirus early region 4 (E4) gene products, we constructed a set of mutant viruses which carry defined lesions within this coding region. Deletion and insertion mutations within six of seven known E4 coding regions had no measurable effect on virus growth in cultured cells. A variant carrying a deletion within the last coding region (encoding a 34,000-molecular-weight polypeptide) was modestly defective, and a mutant lacking the majority of the E4 region was severely defective for growth. The phenotypes of the two defective mutants are similar and complex. Both display perturbations in DNA replication, translation of the E2A mRNA, accumulation of late viral mRNAs, and host cell shutoff.     

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.     

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.