Structural studies of human enteric adenovirus type 41

Enteric adenoviruses of serotypes 40 and 41 possess some specific structural features, one of which is the presence on the virion of two fibers of different lengths and primary sequences. These viruses are notoriously difficult to grow under laboratory conditions. In this paper the successful growth and purification of Ad41 are presented in detail. Structural Ad41 proteins were analyzed by biochemical methods, mass spectrometry, and electron microscopy (EM), in order to identify and localize them on polyacrylamide denaturing gels and to assess the proportion of short and long fibers in the virion. Surprisingly, the three proteins composing virus short and long pentons did not totally enter the denaturing polyacrylamide gels, which is probably due in part to their high pI. The pentons were separately purified and their dimensions were estimated from EM data. The EM images suggest that there are the same amounts of short and long fibers in each virion.     

Analysis of early region 1 of porcine adenovirus type 3

To identify the proteins encoded by the porcine adenovirus 3 (PAV-3) E1 region, rabbit antisera were prepared using a bacterial fusion protein encoding E1A, E1B(small), or E1B(large) protein. Sera against E1A, E1B(small), and E1B(large) immunoprecipitated a protein of 35, 23, and 53 kDa, respectively, in in vitro translated and transcribed mRNA and PAV-3 infected cells. To determine the role of E1 proteins in PAV-3 replication, we constructed vectors with a deletion(s) in the E1 region. Mutant PAV211, containing deletions in E1A and E3, grew to titers similar to wild-type in VIDO R1 cells (E1A complementing) but not in swine testicular (ST) cells. No early protein (E1B(small), DNA binding protein) expression could be detected in PAV211 infected ST cells by Western blots. Mutant PAV212, containing deletions in E1B(small) and E3, grew to wild-type titers in VIDO R1 or ST cells. These deletions were successfully rescued, resulting in recombinant PAV214, containing deletions in E1A, E1B(small), and E3. However, mutant PAV-3, containing a triple stop codon inserted in the E1B(large) coding sequence, could not be isolated. Next, we constructed a recombinant PAV216 by inserting the green fluorescent protein gene flanked by a promoter and a poly(A) in the E1A region of the PAV214 genome. Both PAV214 and PAV216 replicate as efficiently as wild-type in VIDO R1 cells. These results suggested that (a) E1A is essential for virus replication and is required for the activation of other PAV-3 early genes, (b) E1B(small) is not essential for replication of PAV-3, and (c) E1B(large) is essential for virus replication. Moreover, the PAV216 vector can be used for the expression of a transgene.     

Transcription mapping of mouse adenovirus type 1 early region 3

Early region 3 (E3) of mouse adenovirus type 1 was analyzed using S1 nuclease protection and primer extension assays, cDNA sequencing, and genomic sequencing. We present the genomic sequence from 79 to 83 map units of the viral genome, the precise ends and splice sites of the E3 mRNAs, and the predicted protein sequence encoded by the mRNAs. Three major classes of early mRNAs were identified; all were approximately 1 kb long, consisted of three exons, and shared 5' and 3' ends. The three classes had alternative splicing at the junction between the second and third exon. The three proteins predicted by the three mRNAs were slightly similar to the E3 19K glycoprotein of human adenovirus type 3; the longest of the three was the most similar. Open reading frames corresponding to late proteins were also identified in the translated mouse adenovirus type 1 DNA sequence. In mouse adenovirus, as in the human adenoviruses, L4 overlaps E3, and L5 starts just downstream of the E3 region.     

Block in entry of enteric adenovirus type 41 in HEK293 cells

Human species F adenoviruses, HAdV-40 and HAdV-41, display characteristic gut tropism in vivo as well as poor infectivity in cell culture. To address the hypothesis that poor infectivity of HAdV-40/41 reflects a partial block prior to genome delivery, the internalization and trafficking of HAdV-41, HAdV-5 (species C) and HAdV-35 (species B) were compared in 293 (human embryonic kidney) cells, which complement E1B function in HAdV-40/41, and in A549 (lung epithelial) cells. Unlike fluorescently labeled HAdV-5 virions which were transported towards the nucleus and HAdV-35 virions which colocalized with LAMP-1, HAdV-41 virions appeared to be scattered throughout the cytoplasm but did not colocalize with markers of late endosomes/lysosomes (cathepsin B, LAMP-1) or with caveolin 1. Fluorescent dextran was released from vesicles in only 10% of HAd41-infected cells that took up dextran, compared to 70% of HAdV-5-infected cells, suggesting inefficient disruption of endosomes by HAdV-41 or uptake of HAdV-41 virions into a different compartment than HAdV-5 virions. Quantitative transmission electron microscopy, which showed greater binding of HAdV-41 virions to 293 cells than to A549 cells, identified a major block in uptake of HAdV-41 virions from the surface of both cell lines. More than 80% of virions remained on the surface 60 min p.i. and as late as 4h p.i. In contrast to HAdV-5 and HAdV-35 virions, which associated mostly with clathrin-coated pits, HAdV-41 virions associated mostly with caveolar-like invaginations and, to a lesser extent, with larger non-clathrin-coated pits, suggesting internalization by pathways other than clathrin-mediated endocytosis.     

Human enteric adenovirus type 41 binding to HEp-2 cells is specific

Properties of human enteric adenovirus type 41 (Ad41) binding to its receptor on the surface of HEp-2 cells were investigated. The binding was found to be temperature-dependent, saturable, and specific. Analysis of the binding data showed a single class of 4.3 x 10(4) receptor sites per cell, an equilibrium dissociation constant of 21.0 nM, and no cooperativity among receptor sites. Trypsin-treated HEp-2 cells subsequently grown in the presence of tunicamycin or 2-deoxyglucose recovered full Ad41 binding activity, but could not if subsequently grown in the presence of cycloheximide. These data indicate that a single type of virus receptor, likely protein in nature, is present on the surface of HEp-2 cells to specifically bind Ad41.     

Restriction analysis of the prototype strain of enteric adenovirus type 41 using exonuclease III.

Enteric adenoviruses 40 and 41 (Ad40 and Ad41) are a prominent cause of gastroenteritis in young children. Diagnosis of these enteric types by conventional means is complicated by their fastidious growth characteristics. Enteric adenovirus growth was enhanced by cocultivation. Typing of enteric isolates currently entails analysis of the extracted viral DNA with restriction enzymes. Restriction endonuclease fragments of the Ad41 strain Tak genome were ordered by (i) double digestion, (ii) release of restriction fragments from plasmids containing 84% of the Ad41 genome in EcoRI fragments A, B and C, (iii) hybridization of Southern blotted Ad41 fragments with EcoRI fragment containing plasmids and various segments of the Ad2 genome, (iv) sequential reduction of the genome beginning with terminal restriction fragments with exonuclease III and S1 nuclease. The termini of adenovirus genomes are difficult to clone and the use of exonuclease III is a useful alternative to conventional restriction mapping. DNA restriction patterns, fragment sizes and restriction maps of the Ad4 1 strain Tak with enzymes BamHI, BglII, ClaI, EcoRI, HindIII, PstI, SalI, SmaI and XhoI are presented. Prototype strain restriction maps should enable better understanding of adenovirus type 41 and its epidemiology.     

Caspase-mediated cleavage of adenovirus early region 1A proteins

Adenovirus 2 and 12 early region 1A (Ad2 and Ad12 E1A) proteins were cleaved during cisplatin-induced apoptosis of Ad-transformed rat and human cells. Cleavage was inhibited in the presence of caspase inhibitors such as Z-VAD-FMK. In Ad12 transformants both 13S and 12S E1A proteins were cleaved at a similar rate. In Ad2 transformants the E1A 13S component was appreciably less stable than the 12S component. In in vitro studies Ad2 and Ad12 E1A 13S and Ad2 12S proteins were rapidly cleaved by caspase 3 whereas Ad12 12S E1A and Ad12 13S E1A were rapidly degraded by caspase 7. Cleavage sites in Ad12 13S proteins for caspase 3 have been determined. Initial cleavage occurred at D24 and D150; this was followed by cleavage at D204 and D242. Caspase-3-mediated cleavage of Ad12 13S E1A destroyed its ability to bind to CBP and TBP but interaction between C terminal E1A polypeptides and CtBP was observed. During viral infection Ad5 and Ad12 E1A 12S proteins were markedly more stable than 13S proteins but no difference was observed in Ad E1A levels in the absence or presence of the caspase inhibitors Z-VAD-FMK or Z-D(OMe)-E(OMe)-V-D(OMe)-CH(2)F. Limited caspase 3 and 10 activation occurred during infection with the E1B 19K(-) virus Ad2 pm1722 but little or no activation of caspase 3 was observed during wt virus infection. Examination of protein cleavage during viral infection of A549 cells showed proteolysis of lamin B and PARP in response to Ad5 wt and Ad2 pm1722. Protein degradation in response to both viruses was partially inhibited by Z-VAD-FMK. Following infection of human skin fibroblasts lamin B was degraded, although only limited changes in PARP levels were observed. We have concluded that Ad E1A is cleaved by caspases during apoptosis but not during viral infection. However, some of the processes commonly associated with apoptosis occur during viral infection, particularly with E1B 19K(-) mutants, although apoptosis per se is not evident.     

Detection of cellular proteins associated with human adenovirus type 5 early region 1A polypeptides

Antisera prepared against synthetic peptides corresponding to the amino and carboxy termini of human adenovirus type 5 (Ad5) early region 1A (E1A) proteins were used to identify polypeptides that are associated with these viral species in lytically infected KB cells. Proteins were sought which coprecipitated with E1A polypeptides using both sera and which were not recognized in extracts from mock-infected cells by either serum. Four such species were identified with apparent molecular weights of 68K, 65K, and a doublet at about 105K. A fifth species migrating with a molecular weight in excess of 250K was also identified consistently with E1A-C1 but not E1A-N1 serum. Addition of an excess of the appropriate synthetic peptide to the immunoprecipitation mixtures prevented the precipitation of all of these species. Mixing experiments demonstrated that all species were cellular proteins expressed in normal uninfected KB cells and in addition showed that an association with E1A proteins could take place in vitro. Studies carried out with the mutants pm975 and hr1 indicated that while the 105K doublet and the greater than 250K species were found with the products of both the 1.1- and 0.9-kb E1A mRNAs, 65K and 68K appeared to be primarily associated with those of the 1.1-kb mRNA. Finally, the 105K doublet and greater than 250K were shown to be phosphoproteins. These data indicated that Ad5 E1A proteins may function in a complex with cellular polypeptides which includes species of 105K, 68K, 65K, and possibly a large protein of greater than 250K.     

Enzyme-linked immunosorbent assay for detection of enteric adenovirus 41

An enzyme-linked immunosorbent assay (ELISA) for direct detection of enteric adenovirus 41 (Ad41) in stool specimens was developed and compared with an Ad40-specific ELISA described previously [Johansson et al, 1980]. Rabbit antiserum to Ad41 was obtained by immunization with purified virions. To eliminate genus-specific reactivity the serum was passed through an immunosorbent column containing soluble adenovirus components of members of subgenera A to E. The anti-Ad41 serum still displayed high reactivity against Ad40 and had to be immunoabsorbed with soluble virus components of Ad40 to be rendered type-specific. The absorbed antiserum was used in an indirect ELISA and proved to be specific for Ad41. No heterotypic reactivity against Ad40 or Ad1 through Ad35 was found. The Ad41-specific ELISA proved to be of equal sensitivity to electron microscopy. The type-specific ELISAs for Ad40 and Ad41 were evaluated by testing 76 stool specimens containing enteric adenoviruses originating from England and Scandinavia. All specimens could be typed--41 (54%) as Ad40 and 35 (46%) as Ad41. These results were confirmed by DNA restriction site analysis. The type-specific ELISA proved to be a specific, sensitive, and a rapid technique for detection of Ad41 and allowed clear-cut discrimination from Ad40 in clinical specimens.     

Differences in the interactions of oncogenic adenovirus 12 early region 1A and nononcogenic adenovirus 2 early region 1A with the cellular coactivators p300 and CBP

Association with the cellular coactivators p300 and CBP is required for the growth-regulatory function of adenoviral (Ad) early region 1A (E1A) proteins. E1A regions necessary for these interactions overlap with domains involved in the induction of tumours in immunocompetent rodents through highly oncogenic Ad12. Differences in the association of cellular factors with the respective E1A domains of Ad12 and nononcogenic Ad2 might therefore be involved in serotype-specific oncogenicity. We analyzed the interaction of the Ad12 E1A 235R protein with p300 and CBP. Here we demonstrate that in the case of Ad12, but not Ad2/5, amino acids (aa) 1-29 of E1A proteins are sufficient to bind the p300-C/H3 domain in vivo and wild-type p300 in vitro. The conserved arginine-2, which is essential for the interaction between Ad2 E1A and p300, was dispensable for the Ad12 E1A 235R-p300 interaction in vitro. In addition to the p300-C/H3 region, we identified a second domain within p300 (aa 1999-2200) binding to the 235R protein. Contrary to p300, the amino-terminus and CR1 are necessary to associate with CBP. The aa 1-29 of the 235R protein but not CR1 are essential for the repression of colTRE-driven gene expression. This repression function is strictly dependent on p300 but not on CBP.     

Analysis of early region 4 of porcine adenovirus type 3

The early region 4 (E4) of porcine adenovirus (PAdV)-3, located at the right-hand end of the genome is transcribed in a leftward direction and has the potential to encode seven (p1-p7) open reading frames (ORFs). To determine the role of each protein in viral replication, we constructed full-length PAdV-3 genomic clones containing deletions of individual E4 ORF or combined deletions of the neighboring ORFs. Transfection of swine testicular (ST) cells with individual E4 mutant plasmid DNAs generated PAdV-3 E4 mutant viruses except with plasmids containing a deletion of ORF p3, ORF p2+ p3 or ORF p3+ p4. Each of the mutants was further analyzed for growth kinetics, and early/late protein synthesis. Mutant viruses carrying deletions in ORF p1, ORF p2 or ORF p4 showed growth characteristics similar to that of wild-type PAdV-3. Early/late protein synthesis was also indistinguishable from that of wild-type PAdV-3. However, mutant viruses carrying deletions in ORF p5, ORF p6 or ORF p7 showed a modest effect in their ability to grow in porcine cells and express early proteins. These results suggest that the E4 ORF p3 (showing low homology with non-essential human adenovirus (HAdV)-9-E4 ORF1 encoded proteins) is essential for the replication of PAdV-3 in vitro. In contrast, the E4 ORF p7 (showing homology to essential HAdV-2 34 kDa protein) is not essential for replication of PAdV-3 in vitro. Moreover, successful deletion of 1.957 kb fragment in E4 region increased the available capacity of replication-competent PAdV-3 (E3 + E4 deleted) to approximately 4.3 kb and that of replication-defective PAdV-3 (E1 + E3 + E4 deleted) to approximately 7 kb. This is extremely useful for the construction of PAdV-3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.     

Characterization of adenovirus type 40 E1 region

The left-most 3.9 kb of adenovirus type 40 (Ad40) DNA has been sequenced using cloned viral DNA fragments. The Ad40 E1 region is deduced to code for at least four polypeptides, 221 and 249 amino acids as E1A products in addition to 166 and 475 amino acids as E1B products. E1B polypeptides share about 50% homology with well-defined adenovirus types, 2/5, 7, and 12, throughout the E1B sequences. E1A homology of Ad40 to these types is relatively lower than that of E1B, while highly conserved regions of E1A are retained to a certain level in Ad40 as well. Activity for morphological transformation of Ad40 E1A on 3Y1 cells is considerably lower when compared to that of Ad5 and Ad12 E1A genes. Transient chloramphenicol acetyltransferase (CAT) expression assay shows that Ad40 E1A has a trans-acting function, though lower than that of other E1A genes, on adenovirus early promoter. The Ad40 E1A promoter also holds only a little cis-acting activity in 3Y1 cells. Lower activities of both Ad40 E1A promoter and certain E1A functions may explain in part the difficulty in propagation of Ad40.     

Mutational analysis of early region 4 of bovine adenovirus type 3

The primary objective of characterizing bovine adenovirus type 3 (BAV3) in greater detail is to develop it as a vector for gene therapy and vaccination of humans and animals. A series of BAV3 early region 4 (E4) deletion-mutant viruses, containing deletions in individual E4 open reading frames (Orf) or combinations of Orfs, were generated by transfecting primary fetal bovine retinal cells with E4-modified genomic DNA. Each of these mutants was further analyzed for growth kinetics, viral DNA accumulation, and early-late protein synthesis. Mutant viruses carrying deletions in Orf1, Orf2, Orf3, or Orf4 showed growth characteristics similar to those of the E3-deleted BAV3 (BAV302). DNA accumulation and early/late protein synthesis were also indistinguishable from those of BAV302. However, mutant viruses carrying a deletion in Orf5, Orfs 1-3 (BAV429), or Orfs 3-5 (BAV430) were modestly compromised in their ability to grow in bovine cells and express early/late proteins. E4 mutants containing larger deletions, Orfs 1-3 (BAV429) and Orfs 3-5 (BAV430), were further tested in a cotton rat model. Both mutants replicated as efficiently as BAV3 or BAV302 in the lungs of cotton rats. BAV3-specific IgA and IgG responses were detected in serum and at the mucosal surfaces in cotton rats inoculated with mutant viruses. In vitro and in vivo characterization of these E4 mutants suggests that none of the individual E4 Orfs are essential for viral replication. Moreover, successful deletion of a 1.5-kb fragment in the BAV3 E4 region increased the available insertion capacity of replication-competent BAV3 vector (E3-E4 deleted) to approximately 4.5 kb and that of replication-defective BAV3 vector (E1a-E3-E4 deleted) to approximately 5.0 kb. This is extremely useful for the construction of BAV3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.     

The role of adenovirus early region 1A in the regulation of early regions 2A and 1B expression

The role of the adenovirus early region 1A (E1A) in the regulation of expression of early regions 2A (E2A) and 1B (E1B) has been investigated by microinjection of cloned segments of viral DNA into cultured cells. Plasmids carrying adenoviral sequences that code for the DNA binding protein (DBP) associated with either the early or the late E2A promoter (at map coordinates 75 and 72, respectively), or both promoters, were injected into cell nuclei in the presence or absence of a plasmid DNA containing early region 1A. The injected cells were scored for the expression of the DNA binding protein by indirect immunofluorescence using specific antisera. These studies suggest that the product(s) encoded by early region 1A differentially control the expression of the DNA binding protein. DBP produced from the early promoter is stimulated by addition of E1A from the late promoter. It has been shown previously by a variety of studies that E1A regulates E1B expression. However, experiments in which E1B DNA was microinjected together with E1A and E2A suggest that the DNA binding protein may have a stimulatory effect on the synthesis of E1B products.     

A nonessential glycoprotein is coded by early region E3 of adenovirus type 7

Ad7 was shown to induce polypeptides of 66,000 daltons (66K), 28K, and 16K during early stages of infection. The 28K could be labeled with [2-³H]mannose and it bound to concanavalin A-agarose, indicating that it is a glycoprotein. Neither the glycoprotein nor the 16K polypeptide were induced by a viable early region E3 deletion mutant or an E3 insertion mutant, indicating that the polypeptides are coded by early region E3, and that they are nonessential for growth of Ad7 on cultured KB cells. The Ad7 glycoprotein was compared to the Ad2 E3-coded glycoprotein in terms of size and oligosaccharide linkage. The Ad7 glycoprotein has an apparent molecular weight of 28K versus 25K for the Ad2 glycoprotein, as estimated by SDS-PAGE. The Ad7 glycoprotein and the purified 25K Ad2 glycoprotein were digested with endo-β-acetylglucosaminidase H (endo H) to determine the type of oligosaccharide linkages as well as the apparent molecular weights of the polypeptide chains. Endo H reduced the apparent molecular weights of the Ad7 and Ad2 glycopolypeptides from 28K and 25K, to 17.5K and 19K, respectively. This establishes that both proteins have Asn-linked oligosaccharides of the high-mannose type, and that the Ad7 polypeptide chain is slightly smaller than that of Ad2, 17.5K versus 19K. Thus, the E3s of both Ad7 (group B) and Ad2 (group C) encode glycoproteins that are similar in apparent molecular weight, that have high-mannose oligosaccharides, and that are nonessential for virus growth on cultured KB cells.     

The effect of CtBP1 binding on the structure of the C-terminal region of adenovirus 12 early region 1A

Adenovirus early region 1A (AdE1A) binds to the C-terminal binding protein 1 (CtBP1) primarily through a highly conserved PXDLS motif located close to its C-terminus. Purified synthetic peptides equivalent to this region of AdE1A have been shown to form a series of beta-turns. In this present study the effect of CtBP1 binding on the conformation of C-terminal region of Ad12E1A has been investigated. Using one- and two-dimensional (1)H NMR spectroscopy, the conformation of 20-residue peptides equivalent to amino acids I(241)-V(260) and E(247)-N(266) of Ad12E1A were examined in the absence of CtBP1. Whilst the latter peptide forms a series of beta-turns in its C-terminal half as reported previously, the former peptide is alpha-helical over the region D(243)-Q(253). Upon interaction with CtBP1 the conformation of the backbone in the region (255)PVDLCVK(261) of the Ad12E1A E(247)-N(266) peptide reorganises from a predominately beta-turn to an alpha-helical conformation. This structural isomerisation is characterised by a shift upfield of 0.318 ppm for the delta-CH(3) proton resonance of V(256). 2-D NOESY experiments showed new signals in the amide-alpha region which correlate to transferred NOEs from the protein to the peptide residues E(251), V(256) and K(261). In further analyses the contribution of individual amino acids within the sequence (254)VPVDLS(259) was assessed for their importance in determining structure and consequently affinity of the peptide for CtBP. It has been concluded that Ad12E1A residues (255)P-V(260) serve initially as a recognition site for CtBP and then as an anchor through a beta-turns-->alpha-helix conformational rearrangement. In addition it has been predicted that regions N-terminal to the PXDLS motif in AdE1As from different virus serotypes and from mammalian proteins form alpha-helices.     

A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5

Early region 1 (E1) of the human adenoviruses has many intriguing properties which have prompted numerous mutational studies to help delineate and characterize the domains responsible for these functions. In mutational analyses being done currently, the E1 region is usually cloned into a bacterial plasmid where it is mutated and then the altered E1 sequences are "rescued" back into infectious virus. The most frequently used rescue procedures are somewhat tedious, requiring the purification and fractionation of linear viral DNA or DNA fragments, and often involve the screening of numerous plaque isolates. Several observations we have made recently on the properties of adenovirus DNA in infected cells and on infectious plasmids in transfected cells led us to design a new approach for rescuing E1 mutations into infectious viral genomes. We constructed a plasmid, pJM17, containing the entire Ad5 DNA molecule, with an insert in the E1 region that exceeds the packaging constraints of the adenovirus capsid. Following transfection of pJM17 into 293 cells the plasmid DNA is able to replicate but cannot be packaged into infectious virions. In contrast cotransfection of 293 cells with pJM17 plus an E1-containing plasmid carrying mutated sequences produces recombinant virions at high efficiencies. Neither plasmid needs to be linearized prior to contransfection. The technique eliminates the need to purify and manipulate infectious virion DNA and since no unique restriction sites are needed, both E1A and E1B mutants' as well as foreign gene inserts in the E1 region can be easily rescued into virus.