mRNAs from human adenovirus 2 early region 4

The molecular structure of the mRNAs from early region 4 of human adenovirus 2 has been studied by Northern blot analysis, S1 nuclease analysis, and sequence analysis of cDNA clones. The results make it possible to identify four different splice donor sites and six different splice acceptor sites. The structure of 12 different mRNAs can be deduced from the analysis. The mRNAs have identical 5' and 3' ends and are thus likely to be processed from a common mRNA precursor by differential splicing. The different mRNA species are formed by the removal of one to three introns, and they all carry a short 5' leader segment. The introns appear to serve two functions; they either place a 5' leader segment in juxtaposition with an open reading frame or fuse two open translational reading frames. The early region 4 mRNAs can encode at least seven unique polypeptides.     

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.     

Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs

A primary site of infection by human adenoviruses is lymphoid cells. However, analysis of the viral control elements and the cellular factors that regulate adenoviral gene expression in lymphocytes has not been reported. The adenovirus early region 3 (ES) gene products are involved in the maintenance of viral persistence by complexing with the class I MHC antigens, thus preventing their cell surface expression with a resultant decrease in host immunologic destruction. To determine whether different cellular factors were involved in E3 regulation in lymphocytes as compared with HeLa cells, both DNA binding and transfection analysis with the E3 promoter in both cell types were performed. These studies detected two novel domains referred to as L1 and L2 with a variety of lymphoid but not HeLa extracts. Each of these domains possessed strong homology to motifs previously found to bind the cellular factor NF-kappa B. Transfections of E3 constructs linked to the chloramphenicol acetyltransferase gene revealed that mutagenesis of the distal NF-kappa B motif (L2) had minimal effects on promoter expression in HeLa cells, but resulted in dramatic decreases in expression by lymphoid cells. In contrast, mutagenesis of proximal NF-kappa B motif (L1) had minimal effects on gene expression in both HeLa cells and lymphoid cells but resulted in a small, but reproducible, increase in gene expression in lymphoid cells when coupled to the L2 mutation. Reversing the position and subsequent mutagenesis of the L1 and L2 domains indicated that the primary sequence of these motifs rather than their position in the E3 promoter was critical for regulating gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of CD4 gene expression on adenovirus replication.

The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.     

Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane-cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.     

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo.

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.     

Organization and expression of the E4 region of adenovirus 2.

The E4 region of Adenovirus 2 is a leftward transcribed part of the viral genome. Its nucleotide sequence has already been analysed. From this sequence several open reading frames were defined, which could be used in the coding of the E4 proteins. Using S1 digestion of mRNA-DNA hybrids a precise mapping of donor and acceptor sites was done. Their use in various combinations allows the synthesis of mRNAs, able to direct the synthesis of at least 7 polypeptides, ranging in size from 9K to 34K. Comparison of the sequences of the different acceptor sites indicates that they all conform to the consensus sequence. Analysis of the ATG surrounding sequence shows that initiator ATG may be positively selected according to Kozak's rule.     

Mutation affecting late gene expression in polyoma virus maps in the late region.

A mutation in polyoma virus strain 3049 which results in the overproduction of capsid proteins has been mapped to the late region of the genome between the HindIII site at 45.0 map units and the BamHI site at 58.6 map units. This region contains the coding sequence for VP3 and a portion of VP2, but does not include the late promoters or the coding sequence for the late leaders. The possible role of VP2 or VP3 in the regulation of genetic expression in polyoma virus is discussed.