Region E3 Of Adenovirus Research Articles

Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters.

Recombinant adenoviruses carrying the hepatitis B virus surface antigen coding sequence in the adenovirus E3 region were constructed using DNA from either adenovirus type 5 or an adenovirus type 5 E3-region deletion mutant. Both of these recombinant adenoviruses replicated as efficiently as wild-type adenovirus in all human cells tested, including the human diploid cell strain WI-38. This indicates that insertion of the hepatitis B virus surface antigen gene into the E3 region does not significantly affect viral replication. Human cells infected with these recombinant adenoviruses secreted immunoreactive hepatitis B virus surface antigen. Since a practical small animal model for human adenoviruses was lacking, a hamster model was developed to evaluate the immunogenic potential of these recombinant adenoviruses. Upon intranasal inoculation, both wild-type adenovirus and the adenovirus E3-region deletion mutant replicated in the lungs of these animals and induced an antibody response against adenovirus. Hamsters similarly immunized with the live recombinant adenoviruses produced antibody against both adenovirus and hepatitis B virus surface antigen.

Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.

Evidence that AG [formula omitted]AU [formula omitted]A and CC [formula omitted]AG [formula omitted]A initiate translation in the same mRNA in region E3 of adenovirus

We describe a simple method to introduce site-specific mutations into region E3 of adenovirus (Ad). Mutations are made in cloned Ad2 EcoRI-D (map position 76–83), then ligated between Ad5 EcoRI-A (map position 0–76) and EcoRI-B (map position 83–100) to complete the viral genome. We have used this method to isolate a viable virus mutant ( dl702) that is relevant to the problems of translation initiation and gene organization in the E3 complex transcription unit. mRNA a in region E3 encodes an abundant glycoprotein termed gp19K. There are two AUGs in mRNA a that are 5′ to AUG 1204 which initiates gp19K. One of these, AUG 1022, could initiate a 6.7K protein, although this protein has not been identified in infected cells. Mutant dl702 has a deletion such that the 6.7K gene is fused in-frame to the gp19K gene. We report that the 6.7K-gp19K fusion protein is synthesized both in dl702-infected cells and after cell free translation of infected cell RNA. The quantity of fusion protein made is much less than that of wild type gp19K. The sequence context of AUG 1022 for 6.7K is AG U AU AUG A, and that of AUG 1204 for gp19K is CC A AG AUG A. The consensus sequence of eukaryotic initiation codons is CC Pu CC AUG G, with the Pu at −3 being important (M. Kozak, Nucleic Acids Res. 12, 857–872, 1984). Our results suggest that (i) AUG 1022 can initiate translation in vivo and therefore the 6.7K protein probably is made in infected cells, (ii) that mRNA a is a dicistronic mRNA encoding the 6.7K and gp19K proteins, and (iii) that the initiation codon for 6.7K may be much less efficient than that for gp19K. Thus, the E3 genes may be organized such that the relative abundance of the 6.7K and gp19K proteins is controlled by the efficiency of their initiation codons in the same mRNA.

Novel deletion mutants that enhance a distant upstream 5′ splice in the E3 transcription unit of adenovirus 2

Region E3 of adenovirus is a "complex" transcription unit: i.e. different mRNAs and proteins arise by differential RNA 3' end selection and differential splicing of the primary transcript. We are using viable virus mutants to understand the controls that dictate the specificity and efficiency of the RNA processing signals. We describe a novel class of deletion mutations that enhance a natural 5' splice site located approximately 740 nucleotides (nt) upstream. In particular, deletions within nt 1691-2044 in the E3 transcription unit result in a 5-fold enhancement of the 5' splice site at nt 951 (as reflected in steady-state mRNA). The effect is specific, because the deletions do not affect the 5' splice site at nt 372, and because deletions within nt 2044-2214 do not affect either the 951 or the 372 5' splice sites. As a consequence of the enhanced splicing at the 951 5' site, synthesis of the major E3 mRNA and the major E3 protein (gp19K) are dramatically reduced. At least one of the natural 3' splice sites, located at nt 2157, is the recipient of the enhanced splicing at the 951 5' splice site. We conclude that sequences located within nt 1691-2044 affect (probably in cis) splicing at the 951 5' splice site. We speculate that nt 1691-2044 includes a splicing control region which functions to suppress splicing at the 951 5' splice site.