Email updates

Keep up to date with the latest news and content from BMC Genomics and BioMed Central.

Open Access Research article

Unique sequence features of the Human Adenovirus 31 complete genomic sequence are conserved in clinical isolates

Soeren Hofmayer1, Ijad Madisch12, Sebastian Darr1, Fabienne Rehren1 and Albert Heim1*

Author Affiliations

1 Institut für Virologie, Medizinische Hochschule Hannover, Hannover, Germany

2 Current address: Department of Radiology, Massachusetts General Hospital Harvard Medical School 100 Charles River Plaza, Suite 400 Boston, Massachusetts 02114, USA

For all author emails, please log on.

BMC Genomics 2009, 10:557  doi:10.1186/1471-2164-10-557

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2164/10/557


Received:26 June 2009
Accepted:25 November 2009
Published:25 November 2009

© 2009 Hofmayer et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Human adenoviruses (HAdV) are causing a broad spectrum of diseases. One of the most severe forms of adenovirus infection is a disseminated disease resulting in significant morbidity and mortality. Several reports in recent years have identified HAdV-31 from species A (HAdV-A31) as a cause of disseminated disease in children following haematopoetic stem cell transplantation (hSCT) and liver transplantation. We sequenced and analyzed the complete genome of the HAdV-A31 prototype strain to uncover unique sequence motifs associated with its high virulence. Moreover, we sequenced coding regions known to be essential for tropism and virulence (early transcription units E1A, E3, E4, the fiber knob and the penton base) of HAdV-A31 clinical isolates from patients with disseminated disease.

Results

The genome size of HAdV-A31 is 33763 base pairs (bp) in length with a GC content of 46.36%. Nucleotide alignment to the closely related HAdV-A12 revealed an overall homology of 84.2%. The genome organization into early, intermediate and late regions is similar to HAdV-A12. Sequence analysis of the prototype strain showed unique sequence features such as an immunoglobulin-like domain in the species A specific gene product E3 CR1 beta and a potentially integrin binding RGD motif in the C-terminal region of the protein IX. These features were conserved in all analyzed clinical isolates. Overall, amino acid sequences of clinical isolates were highly conserved compared to the prototype (99.2 to 100%), but a synonymous/non synonymous ratio (S/N) of 2.36 in E3 CR1 beta suggested positive selection.

Conclusion

Unique sequence features of HAdV-A31 may enhance its ability to escape the host's immune surveillance and may facilitate a promiscuous tropism for various tissues. Moderate evolution of clinical isolates did not indicate the emergence of new HAdV-A31 subtypes in the recent years.

Background

Adenoviridae are non-enveloped, double-stranded DNA viruses with an icosahedral capsid [1]. Human Adenoviruses (HAdV) belong to the genus Mastadenovirus and are classified into six species (HAdV-A to HAdV-F) that were defined historically as subgenera on the basis of hemagglutination properties [2,3]. Subsequently, oncogenic properties in rodents and DNA homology were also used to define the subgenus (species) [1]. Recently a new strain of HAdV was discovered and has been classified as HAdV-52, representing a putative new species G [4].

Human adenoviruses have long been recognized as pathogens causing a broad spectrum of different diseases depending on the type-related organotropism and virulence. For example, infections of the upper respiratory tract are caused by HAdV-C1, -C2, -C5, -B3, and -B7 [5,6], the more dangerous infections of the lower respiratory tract mainly by HAdV-B3, -B7, -B21, and -E4 [7-9]. The types HAdV-D8, -D19, and -D37 are closely associated with severe epidemic keratoconjunctivitis. Gastroenteritis and diarrhoea are caused by the enteric adenoviruses HAdV-F40,-F41 and HAdV-A31, which are frequently found in infants and children.

Moreover, immunocompromised patients can develop a sepsis-like, disseminated adenovirus syndrome that is associated with high levels of immunosuppression (for example, lymphocyte counts <300/μl) as a crucial risk factor [10]. A wide range of organs can be affected and an effective antiviral therapy is not yet available. Consequently, mortality rates of up to 60% were reported [10-12]. Disseminated disease is mainly caused by species C adenoviruses. However, in recent decades, HAdV-A31 has been increasingly reported as a etiologic agent for dissemination in immunosuppressed children following allogenic haematopoietic stem cell transplantation [10,13-15].

One of the essential sequence features for HAdV types causing dissemination may be the viral RGD (integrin binding) motif of the penton base protein, because HAdV-F types lacking the RGD motif have never caused a disseminated infection (with exception of a single case report) in spite of their high prevalence [16-18]. In addition, recent studies have shown that the binding of blood coagulation factor (F) X to the HAdV-C5 hexon protein facilitates infection of the liver and could also foster virus dissemination [19]. Similar to HAdV-C5, F IX binding may promote HAdV-A31 infection of epithelial cells [20].

The outstanding clinical relevance of HAdV-A31 is also documented by its significant association with immunosuppressed patients in comparison to immunocompetent patients [16]. This high incidence may be explained by reactivations of latent (or persisting) HAdV infections, which have been recently described for species C HAdV [21]. A similar mechanism may be suspected for HAdV-A31 although any conclusive data on its persistence is still lacking. So far, genetic analysis of HAdV-A31 strains isolated from hSCT patients showed significant differences even in the same clinical centre, suggesting reactivations rather than infection chains of de novo HAdV-A31 infections [14,22]. However, HAdV-A31 may also be transmitted easily in a nosocomial setting between immunosuppressed patients, as high amounts of HAdV-A31 are spread with faeces [23].

In spite of this increasing clinical relevance of HAdV-A31, the virus had not yet been completely sequenced. Therefore, we determined the complete nucleotide sequence of the HAdV-A31 prototype strain in order to search for unique sequence motifs which may be associated with its high virulence. In addition, we compared several virulence associated gene regions (E1A, E3, E4, fiber knob, penton base, protein IX, and pX) of seven clinical isolates and the HAdV-A31 prototype.

Results

General properties

The complete genomic sequence of HAdV-A31 is 33,763 base pairs in length and was submitted to GenBank as AM749299. The plus strand has a base composition of 22.89% G, 23.48% C, 27.49% A and 26.14% T. The GC content is 46.36%. As other Mastadenoviruses, HAdV-A31 is organized into four early, one intermediate and five late transcription units. We identified 34 coding regions that are homologue to previously described gene products of other human adenoviruses (Figure 1). The annotation of the predicted coding gene regions is listed in Table 1.

Table 1. HadV-A31 annotation of coding regions

thumbnailFigure 1. Map of the genome organization and transcription units of HAdV-A31. Early and late transcription units are represented in different colors, intermediate gene products in white. The block arrows represent the predicted protein, titled either by protein name or predicted molecular size. Orientation of the arrows indicates the direction of transcription.

Phylogeny

Phylogenetic analysis of the whole genomic sequence of HAdV-A31 was performed by using the neighbor-joining method. Representative members of all HAdV species were included in the analysis. HAdV-A31 clustered as expected to the species HAdV-A, close to HAdV-A12 (Figure 2).

thumbnailFigure 2. Phylogenetic analysis of all available complete genomic HAdV sequences representing all human adenovirus species (A to G), including the newly generated HAdV-A31 sequence. The tree was generated with MEGA 3.1 using neighbor-joining method, bootstrap values (%) were generated with 1,000 pseudoreplicates. For nucleotide accession numbers see Methods section.

A global pairwise alignment was constructed using the mVISTA Limited Area Global Alignment of Nucleotides (LAGAN) [24] in order to compare the predicted whole genomic sequence of HAdV-A31 to the representative types of each species. The graphical alignments showed the close relationship between HAdV-A31 and -A12 with the exception of the coding regions for immunogenic determinants (hexon, fiber). Interestingly, the early transcription unit E3, which is not under selective pressure by the immune system, also showed a significant divergence between HAdV-A31 and HAdV-A12 (Figure 3).

thumbnailFigure 3. Global pairwise sequence alignment of the HAdV-A31 genome with representative types of each HAdV species. The x axis shows the genome position, the y axis shows the sequence conservation in percent. Arrows on top display the transcription units and the direction of their transcription.

ITR

HAdV's inverted terminal repeats (ITR) and its flanking DNA regions exhibit several binding sites for viral proteins and a set of cellular factors for efficient adenoviral DNA replication [25]. HAdV-A31's ITRs are 148 bp in length. A nuclear factor III (NF-III) binding site described as conserved among most human adenoviruses as a 5'ATGNNAATGA 3' sequence motif (ATGCAAATAA in HAdV-E4) [26] was not identified in the HAdV-A31 ITR, but a 5'ATGAAGTGGG 3' sequence at position bp 46-55 may function as a binding site for NF-III. HAdV-A12 also lacks the classical NF-III binding motif but has the same 5'ATGAAGTGGG 3' motif as HAdV-A31 at bp 46-55. The conserved NF-I binding site (5'TGGACTTGAGCCAA 3') was predicted for HAdV-A31 at position 25-38.

HAdV-A31 ITRs also revealed binding sites for the transcription factors SP1 and ATFs. The binding of ATFs to a 5'TGACGT 3' motif has been shown to be important for efficient viral growth of HAdV-C5 [27]. Whereas all other human adenoviruses (including HAdV-A12) reveal exactly this ATF binding motif, HAdV-31's putative single ATF binding site between bp 113 - 117 showed a C instead of the commonly observed T at the end of the motif. An SP1 binding site was present as a GC-rich region between bp 52 - 61 (5' TGGGCGGAGT 3'). The extreme termini of the ITRs had the common 5'CTATCTATAT 3' motif that is required for viral replication and also protects the viral genome by ORP-A binding from DNAse-I digestion. The core origin of DNA replication, which binds the complex of preterminal protein (pTP) and DNA polymerase, was present in the highly conserved motif 5' ATAATATACC 3' between bp 9 and 18 in HAdV-A31.

Human elongation factor 1-alpha (EF-1A) is known to be an efficient factor for enhancement of E1A gene region transcription. HAdV-A31 revealed only two putative EF-1A binding motifs (5'GCCGGATGT 3'), which are located within the ITR region at bp 105-113 and 136-144, whereas the five EF-1A binding sites are located upstream from the E1A promoter region of HAdV-C5 [28].

E1A coding region

E1A is the first gene expressed after adenoviral infection [3]. The predicted gene product of the HAdV-A31 E1A ORF was 266 amino acids in length with a molecular weight of 29.4 K. Most of the functional sites of E1A proteins are located in conserved regions CR1 - CR4 [29]; homologue amino acid sequences at corresponding positions were found in HAdV-A31. The predicted E1A protein of HAdV-A31 revealed a sequence (EQDENGMAHVSAAAAAAAANRER) at position 122 - 144 that is homologue to the E1A protein of HAdV-A12. The latter was previously identified as a repressor for the presentation of MHC class I molecules on the surface of infected cells. Essential for this function of HAdV-A12 is a stretch of 23 amino acids, which could not be found in the E1A proteins of other HAdV species [30].

HAdV-A31 revealed the essential Rb-protein binding motif as a LLCYE sequence at residues 107-111. A C-terminal binding protein (CtBP) interacting motif (PVDLS), which is probably equivalent to PLDLS in other HAdV types, was found near the C-terminus of the E1A protein. Two zinc finger motifs CSLC and CKSC, both essential for transactivation of transcription [31] are located in the CR3 region.

E1B coding region

Two ORF in the E1B gene region coded for predicted proteins of 18.3 K and 53.1 K molecular weight. The predicted 18.3 K protein was 156 amino acids in length and homologue to the small t-antigen, which is known to have anti-apoptotic features [32]. While the stretch of the first 145 amino acids of the HAdV-A31 E1B 18.3 K protein shared a homology of 93.1% to the corresponding gene product of HAdV-A12, the C-terminus is highly divergent (only 27.7% identity).

The second E1B gene product was a predicted 53.1 K protein, 472 amino acids in length and homologue to the large t-antigen. It defends the virus against the p53 mediated antiviral host cell response by binding to the DNA linked p53 protein directly and repressing its function as a transcriptional activator. In addition to this direct interaction, a complex comprising the large t-antigen, the E4 ORF 6 gene product and a set of cellular cofactors build an E3-ligase-complex that also degrades the p53 protein [33,34]. Essential for the stability of this complex is a BC-Box binding motif ((A,P,S,T)LxxxCxxx(A,I,L,V)), present in the predicted E1B 53 K gene product of HAdV-A31 as an ALRPDCTYKI motif at amino acid position 156-164.

Amino acid comparison with the predicted large t-antigen of HAdV-A12 revealed a high sequence divergence (only 55.5% identity) between residues 38 - 100. However, the C-terminus of large t-antigen, which was described as essential for repressing the function of p53 in HAdV-A12 infected cells, showed a high sequence homology to HAdV-A12.

E2 coding region

E2 is divided into two transcriptional units, E2A and E2B. Transcription of the E2 region is controlled by a well characterized RNA polymerase II promoter (major promoter) on the complementary strand, which is transactivated by E1A and E4 ORF6/7 gene products. The E2 promoter region of HAdV-A31 was located between bp c25299-c25431. It lacked at least one of the typical E2F (bp c25422-25429, TTTCCCGC) binding motifs as described for HAdV-C2 and -C5. However, a putative binding motif for E4F1 (base pair c25430-c25436, ACGTCAC) was predicted. E4F1 is another cellular transcription factor that stimulates the transcription of E4 genes mediated by E1A gene products [35]. Moreover, the E2 promoter of HAdV-A31 had a binding motif for ATF (bp c25431 - c25439, TGACGTCAC) and a TATA-like sequence for binding TBP (bp c25381-c25386, TTAAGC). Within the E2A region an ORF for a predicted protein of 55.3 K was identified. The product was homologue to the DNA binding protein (DBP) and revealed a zinc-binding domain at amino acid position 229-242 (HxC8CxH).

The E2B region of HAdV-A31 encoded a predicted 134.6 K protein homologue to the HAdV DNA polymerase protein, and a 73.1 K gene product homologue to the precursor of the terminal protein (pTP), both on the complementary strand. The common nuclear localization signal of pTP was present as RLPVRRRRRRLP motif at amino acid position 351-362.

E3 coding region

The E3 region is known to code for a set of proteins that are not essential for virus replication in vitro but are important factors for interfering with the host immune response [36,37]. Six ORFs were identified in the E3 transcription unit of HAdV-A31, encoding putative gene products of the following sizes: 12.1 K, 28.6 K, 29.4 K, 10.5 K, 12.5 K, and 14.6 K. The organization is similar to the E3 region of the closely related HAdV-A12 [38].

The 12.1 K predicted protein was homologue to a 12.5 K protein which is present in all HAdV types beside the enteric HAdV-F40 and -F41. The sequence identity between HAdV-A31 and -A12 was comparatively high (96.1%).

The second and third reading frames, coding for a 28.6 K (CR1 alpha) and a 29.4 K (CR1 beta) protein, revealed comparatively low identities of 76.1% and 72.1% to homologue E3 gene products of HAdV-A12. CR1 alpha and beta were described as species HAdV-A specific gene products [39]. Prediction of transmembrane domains suggested that both gene products were type Ia transmembrane proteins. Protein Blast search of CR1 beta showed homologies between the putative gene product of HAdV-A31 and proteins of the SLAM (signalling lymphocyte activation molecule) family. Patterns of predicted functional domains of HAdV-A31 in comparison to HAdV-A12 and -F40 are shown in Figure 4A.

thumbnailFigure 4. Schematic view of the predicted E3 CR1 beta (A) and the E3 RID beta (B) proteins of HAdV-A31, -A12 and -F40 or -C5, respectively. (A): a V-Set domain (red box) was only predicted within the N-terminal region of the E3 CR1 beta protein of HAdV-A31. (B): N-terminal phosphorylation sites (small red boxes) were predicted both for the HAdV-A31 and -C5 RID beta proteins, but not for HAdV-A12. Domain predictions were carried out using web based Pfam, ProSite and BLASTp.

The 10.5 K and 12.5 K ORF of HAdV-A31 were identified to be homologue to the known RID (receptor internalization and degradation) alpha and beta proteins, which are present in the E3 transcription units of all HAdV. Both proteins are non-covalently associated integral membrane proteins [40]. The N-terminal phosphorylation sites pattern of HAdV-A31 RID-beta was more similar to HAdV-C5, whereas HAdV-A12 lacked these phosphorylation sites completely (Figure 4B). YxxO motifs and proline rich sequence stretches near the C-terminus, which are conserved among all HAdV and may be part of a protein interacting domain [39], were identified in the predicted RID beta protein of HAdV-A31 (Figure 4B). YxxO motifs function as signals for transport and internalization into lysosomes/endosomes.

RID alpha is a hydrophobic protein and appears in two isoforms [39]. Depending on cleavage of the signal peptide, it either functions as a type I or a type II transmembrane protein. An analogue cleavage pattern was predicted for the RID alpha protein of HAdV-A31 by using the web based TMHMM v. 2.0 software. As described for other human adenoviruses dileucine, dileucine-like and YxxO motifs are also present in the cytoplasmatic portion of the HAdV-A31 RID alpha gene product.

The last ORF of the E3 region encodes a 14.6 K protein that is homologue with a sequence identity of 88.2% to the 14.7 K protein of HAdV-A12. A corresponding protein is present in all species of HAdV species [41]. It has been shown to be located in the cytosol and nucleolus, functioning as an inhibitor of TNF mediated cell lysis. Structure and function analysis of the 14.7 K of HAdV-C5 indicate that its biological function does not depend on single conserved subdomains but that critical amino acids are distributed throughout the entire protein. It has been shown that a set of three cysteine residues between amino acid 40 and amino acid 120 are essential for the function of the protein [36,37]. Corresponding cysteine residues were identified in the predicted 14.6 K protein of HAdV-A31.

E4 coding region

Transcription of the E4 region on the complementary strand is stimulated by the E1A gene product and the cellular transcription factor E4F1. An E4F1 binding motif ACGTCAC (of the consensus sequence ACGTMAC) was identified in HAdV-A31 at bp c33513-c33519 located upstream of the putative TATA box at bp c33479-c33502.

Corresponding to the E4 transcription units of other human adenoviruses (including the closely related HAdV-A12), six ORFs were predicted within the genome of HAdV-A31. These ORFs encoded for respective proteins of 13.9 K, 14.9 K, 13.4 K, 13.7 K, 33.9 K and 13.9 K (spliced gene product ORF6/7). The predicted gene product of the HAdV-A31 ORF6/7 exhibited only a single BC-Box like motif (SAWPECNSLT) slightly different to the consensus motif sequence (A,P,S,T)LxxxCxxx(A,I,L,V). This is in contrast to HAdV-C5, which shows two BC-Box motifs that are both required for degradation of p53 [34].

Virus associated RNA

Sequence coding for the VA RNA was predicted by comparison with HAdV-A12 and is located at bp10146 - 10286 in the genome of HAdV-A31.

Intermediate genes

Two proteins are encoded in the adenoviral intermediate gene region, IX and IVa2. The protein IX of HAdV-A31 was predicted as a 14.9 K protein of 144 amino acids in length. It is a structural component of the virus and influences hexon-hexon interaction. A stretch of 32 amino acids is conserved among all HAdVs and is supposed to be crucial for the incorporation of the protein IX into the capsid. Corresponding amino acids were identified in the protein IX of HAdV-A31 at position 14-45. Surprisingly, we identified an RGD motif in the amino acid sequence of HAdV-A31 protein IX at amino acid position 102-104 (Figure 5). This RGD motif is only 40 amino acids distant from the C-terminus that is assumed to be exposed on the surface of the virus [42]. Moreover, the RGD motif is located at the N- terminal end of a coiled coil region predicted by the COILS web based software [43]. This prediction suggested that the RGD motif is possibly solvent exposed and may be functional in binding to alpha integrins.

thumbnailFigure 5. Multiple alignment of the protein IX amino acid sequences of HAdV-A31, -A12, -C5, -F40 and -G52. The RGD motif found in the protein IX of HAdV-A31 is highlighted.

The IVa2 ORF is transcribed from the complementary strand and codes for a 50.9 K protein of 449 amino acids in length. The protein IVa2 of human adenoviruses binds the A-repeat sequences at the left end of the genome and is involved in the process of viral DNA packaging and virus assembly. Furthermore, it is assumed to have a function as a transcriptional activator of the late adenoviral genes [44].

Late genes

The major late transcription unit (MLTU) encodes the majority of the virus structural proteins and is organized into five subregions L1-L5. The initiation of late gene transcription is controlled by a promoter region that is present in all human adenoviruses and termed as major late promoter (MLP) [3]. Based on sequence comparison, the putative inverted CAAT box (TGATTGGTT) was identified at bp 5633-5641 and the TATA box at position 5684-5690 in the genome of HAdV-A31. The L1 transcription unit encodes the 52/55 K and the pIIIa protein. An ORF coding for a 41.8 K protein as a homologue of 52/55 K that is known to be functionally relevant in the process of virion assembly was predicted. A second ORF encodes the putative pIIIa protein of 64.4 K that is associated with the hexon protein and present on the outer surface of the virion.

Four ORFs within the L2 region were identified: coding for the penton base protein (III), proteins V, pVII and the pX. The predicted penton base protein of HAdV-A31 is 505 amino acids in length and exhibits an integrin (αvβ3 and αvβ5) binding RGD motif at amino acid position 301-303. In addition to the RGD sequence, an LDV motif was present at amino acid position 285-287 of HadV-A31. LDV motifs were described as interacting with another group of integrins (α4β1 and α4β7) [45-47].

The predicted homologue of protein V has a molecular weight of 39.8 K and is 347 amino acids in length. A 20.4 K gene product is homologue to the pVII, and a predicted product of 7.9 K represents the pX protein of HAdV-A31. All three proteins are described as being core proteins and are associated with the virus DNA [48]. The sequence generated for pX protein of HAdV-A31 was highly divergent from the HAdV-A31 pX sequence previously available in GenBank [GenBank: U14653] (nucleic acid sequence homology 70.4%, amino acid 53.4%). Therefore, this region was re-sequenced a second time and additionally sequenced for all seven clinical isolates; our previous result was confirmed. Most likely, the databank sequence relates to a subtype of HAdV-A31 or is mislabelled.

The L3 transcription unit encodes three proteins; we could identify ORF for pVI, the hexon protein (II) and the virus encoded protease. The predicted pVI protein of HAdV-A31 is 260 amino acids in length and has a molecular weight of 28.5 K. It revealed two nuclear localization signals (KRPRP at amino acid 136-140 and KRRR at amino acid 255-258) close to its C-terminus. Corresponding motifs of HAdV-C2 play an important role in directing cytoplasmatic proteins to the nucleolus and thus might be functionally active as nuclear localization signals [49]. The mature pVI protein is a minor capsid component.

The predicted hexon protein of HAdV-A31 has a molecular weight of 103.7 K and a length of 922 amino acids. Due to its serotype defining main neutralization determinant ε, it was highly divergent from the closely related HAdV-A12.

The predicted ORFs of the L4 region of HAdV-A31 encode putative proteins of 86.8 K (as equivalent to100 K), 20.2 K (as equivalent to 22 K), 23 K (as equivalent to 33 K), 25.2 K (as equivalent to pVIII), respectively.

Only one ORF is present in the L5 region of HAdV-A31, encoding a 58.9 K protein of 556 amino acids in length, representing the fiber protein (IV), a major structural protein with the highly variable hemagglutination determinant in its terminal knob structure. The fiber shaft was found to be 31 amino acids (two pseudorepeats) shorter than the fiber shaft of HAdV-A12. We detected a deletion in the 3rd non-consensus β-repeat of HAdV-A31 fiber shaft and a KLGXGHXFS motif in the penultimate repeat instead of the classical KLGXGLXFD/N flexibility consensus motif of other adenovirus serotypes. This could affect the function of the flexibility regions and influence the cell attachment.

Comparison of clinical isolates to the prototype sequence

The coding regions of the E1A, E3, E4, penton base, fiber knob, protein IX and pX genes of seven clinical isolates were sequenced and compared. All strains have been isolated from paediatric patients with disseminated infections following hSCT. Overall, the pX, IX, E3 RID alpha, E4 ORF 3 and ORF 4 nucleic acid sequences of all isolates were 100% identical to the prototype. The lowest amino acid identity was observed in the E3 CR1 beta gene with 99.2%. In Table 2, gene product and amino acid substitutions of clinical strains are listed. Lowest S/N ratios were calculated for the E3 CR1 beta (2.36), whereas only synonymous mutations were observed in the penton base protein of several clinical isolates. Two isolates revealed an amino acid substitution in the penton protein at position 305 (tyrosine residue instead of phenylalanine), which is close to the functional RGD motif and may influence integrin binding. Interestingly, all wild type strains revealed the additional RGD motif in protein IX of the HAdV-A31 prototype strain. All motifs described for the HAdV-A31 prototype strain were conserved in the clinical isolates.

Table 2. Sequence comparison of clinical isolates to the HAdV-A31 prototype sequence

Discussion

We determined the complete 33,763 base pair genome of the HAdV-A31 prototype strain and identified 34 putative genes. HAdV-A31 is a highly significant pathogen, which has been frequently isolated from severely affected hSCT recipients, and frequently presents itself as a disseminated disease, which is usually caused by species HAdV-C [10]. Experimental data strongly suggested that species HAdV-C types have the ability to establish latent infections in mucosal lymphocytes and that stimulation of those cells can cause viral reactivation in cases of immunosuppression [21]. Especially the early gene products (e.g. E3), which counteract host anti-viral defence mechanisms, might play a key role in the process of persistence and reactivation [39,50,51]. A similar mechanism of persistence and reactivation can be suspected in case of HAdV-A31, which would explain its high incidence in immunosuppressed patients. Therefore, the early coding regions E1A, E1B and E3 of the newly generated HAdV-A31 prototype sequence were analyzed in detail for functional motifs. Moreover, these genome regions of seven HAdV-A31 wild type strains isolated from immunosuppressed patients were also sequenced in order to clarify whether a highly pathogenic subtype of HAdV-A31 was circulating in recent years. However, analysis of nucleic acid and predicted amino acid sequences of seven HAdV-A31 clinical isolates revealed high identity to the prototype strain (Table 2). Non-synonymous mutations in clinical isolates clustered in the E3 region, but did not affect previously described and predicted functional sites and motifs (Figure 4) [37,39]. An S/N ratio of 2.36 within the species HAdV-A specific CR1 beta protein suggested selection of a potentially highly functional E3 protein, which is assumed to interact with the immunosurveillance of adenovirus infected cells [39]. Unfortunately, experimental data about the functions of the E3 gene products CR1 alpha and beta were not available. Therefore, in silico protein analysis of the predicted E3 CR1 alpha and beta proteins of HAdV-A31 were performed. Interestingly, the E3 CR1 beta protein of the HAdV-A31 prototype and all analyzed clinical isolates were predicted to exhibit an immunoglobulin-like (V-set) domain, which was predicted neither for the closely related HAdV-A12 nor for the corresponding E3 gene products of the related enteric species F adenoviruses. Immunoglobulin-like domains are described to be involved in cell-cell interaction of the immune system [52]. A similar domain was previously described as involved in a novel feature of the soluble 49 K E3 gene product of HAdV-D19a adenoviruses [37]. Functional studies of the 49 K protein of HAdV-D19a demonstrated proteolytic processing and secretion of the type Ia transmembrane protein [37]. Furthermore, a NK cell binding activity was detected and the immunoglobulin-like domain of the HAdV-D19a 49 K protein was assumed to interact directly with NK cells, protecting infected cells against lyses [37]. For comparison, in silico protein analysis of HAdV-A31 CR1 beta also predicted a type Ia transmembrane domain, a (signalpeptide-)cleavage probability of ~83%, a C-terminal sorting motif and various glycosylation sites, all of which are analogous to the confirmed predictions for HAdV-D19a E3 49 K protein.

In contrast to these predictions for CR1 beta, potential functionality of the predicted CR1 alpha protein of HAdV-A31 have remained obscure, since all performed analyses and predictions did not reveal similarities with functional sites or motifs of any E3 counterparts of other HAdV species. Overall, CR1 alpha and beta amino acid sequence comparison between HAdV-A31 and -A12 showed a particularly low identity of only 76.1% and 72.1%, respectively. This is in considerable contrast to corresponding E3 gene products of HAdV-F40 and -41, which had a high intraspecies homology of 98.8% and 99.2%, respectively. Significant differences between both species A adenoviruses were also identified in the theoretical molecular weight and isoelectric point (pI) for CR1 alpha and beta gene products computed and visualized in virtual 2D gel analysis (data not shown). These considerable differences between HAdV-A31 and -A12 in the primary structure, protein size, pI and predicted functional domains consequently indicated differences in protein function and might be an important feature in explaining the described higher virulence of HAdV-A31.

Comparison of the other E3 gene products, 12.5 K, RID alpha, RID beta and 14.7 K, revealed sequence identities between 80 and 96% with HAdV-A12. With the exception of predicted phosphorylation sites of RID beta (Figure 4B), previously described functional sites and motifs are conserved in HAdV-A31 and HAdV-A12, suggesting a comparable functionality. As determined for HAdV-A12 and species F adenoviruses, the E3 transcription unit of HAdV-A31 also lacked the extensively studied GP 19 K protein, which down regulates the expression of MHC I molecules and NK activation receptors [53-55]. As a substitute for this important immune escape mechanism, the E1A gene product of HAdV-A12 was identified to down regulate the expression of MHC class I molecules by interfering with the transcription of MHC I gene products [56]. This unique feature can be assumed for the E1A protein of HAdV-A31 as well, because amino acid stretches of HAdV-A12, which have been identified as essential for this mechanism, were identified in the predicted E1A gene product of HAdV-A31 at corresponding positions. While the E1A protein is known to have pro-apoptotic features, the E1B 19 K gene product of HAdV-C5 shares homology with the cellular anti-apoptotic Bcl-2 protein and interferes with a set of different cellular pro-apoptotic proteins (Bak, Bax and Nbk/Bik), thus protecting infected cells against apoptosis [32]. Comparison of the E1B 19 K small t- antigen homologue of HAdV-A31 with HAdV-A12 revealed high divergences of the C-terminus. Since the E1B 19 K C-terminus of HAdV-C5 has been identified as exhibiting a functional domain that influences the lateral viral spread of HAdV-C5 by interfering with cellular apoptotic pathways [57], the observed divergence between HAdV-A31 and -A12 might have functional relevance.

In addition to the capability of persistence and of reactivation by interfering with the host immune response, the capacity for dissemination seems to be essential for a highly pathogenic HAdV subtype. For example, an outbreak of the strictly enterotropic HAdV-F41 did not cause any fatalities in paediatric hSCT recipients [17], which might be due to the missing integrin binding RGD motif within the penton protein of species F adenoviruses. Since fatal dissemination of adenoviruses affects various organic systems, a more promiscuous behaviour in cell attachment and entry can be assumed to be favourable [58]. Interestingly, the protein IX of HAdV-A31 revealed an RGD motif at amino acid position 102 - 106, which is conserved within all seven analyzed clinical isolates. This RGD motif is only 40 residues distant from the C - terminus, and it is unique among all sequenced human adenoviruses. As described previously, the C-terminus of HAdV protein IX is exposed on the outer surface of the virion [42,59]. It was shown for protein IX of BAdV-3, which is 125 amino acids in length that the N-terminus (13 - 32) and the central region (61 - 80) have immunogenic sites but are not exposed on the outer surface of the virion [60]. So far, no structural data about the exposed region of the C-terminus of protein IX of HAdV-A31 is available, but the observations for BAdV-3 indicate that the amino acid stretch of the protein IX of HAdV-A31 that contains the RGD motif might be present on the outer surface of the virion. As the HAdV-A31 fiber shaft is shorter and probably less flexible than the fiber shaft of HAdV-A12, a functional binding of the secondary cellular receptor to the RGD motif of protein IX instead of the RGD motif in the penton base may be possible or even preferred. Moreover, the construction of adenoviral vectors with an incorporated RGD motif within the C - terminus of protein IX has demonstrated that the additional RGD motif resulted in a significant augmentation of fiber independent infection of CAR-deficient cell types [60]. Therefore, the additional RGD motif within the protein IX HAdV-A31 might lead to a more effective targeting and internalization, and could be a factor in increased transmission and infectivity of the virus.

In addition to protein IX, sequence comparison of the cellular receptor binding sites of the penton protein of the clinical isolates with the HAdV-A31 prototype revealed an amino acid substitution (F305Y) in two clinical isolates. This substitution is close to the functional RGD motif and may influence integrin binding. Moreover, a low S/N ratio of 2.78 (with exception of clinical isolate number: 95/8866) for the fiber knob indicated selective pressure on a major structural protein of the clinical isolates.

Overall, the sequence divergence of the isolated clinical strains in comparison to the prototype sequence was determined to range between 99.2 and 100%. These results suggested that all isolated strains were closely related to the prototype; a single outbreak subtype associated with severe disease in stem cell transplant recipients was not identified (Table 2). This is in congruence with previous results of RFLP analysis of 79 HAdV-A31 wild type isolates from immunocompromised and immunocompetent hosts, where a wide variety of slightly genetically different subtypes of HAdV-A31 was described [22].

Conclusion

Overall, studying the HAdV-A31 prototype seemed to be sufficient to elucidate the high incidence and disease burden in immunosuppressed patients because HAdV-A31 strains recently circulating in immunocompromised patients were closely related to the prototype. Unique motifs of the HAdV-A31 E1 and E3 regions may provide immune modulation and perhaps virus persistence. The additional RGD motif of protein IX may promote promiscuous tropism for various tissues and enhance dissemination. Further studies of its biological relevance in vivo/in vitro are necessary to clarify its potentially unique characteristics.

Methods

Sequencing strategy

Previously published partial nucleotide sequences of HAdV-A31 and the genomic HAdV-A12 sequence were used to design the PCR primer for the production of genome fragments of the HAdV-A31 up to 5000 base pairs in length. Depending on the size of the amplicon, these fragments were either cloned and sequenced subsequently or sequenced directly from the amplicon. Both strands were sequenced by primer walking with overlapping sequencing reactions. Furthermore, we resequenced the HAdV-A31 sequences already available in GenBank (with exception of the hexon gene [61]). On the basis of the newly generated complete nucleotide sequence of HAdV-A31, we designed the PCR and sequencing primers for sequencing the E1A, E3, E4, fiber knob and penton base coding regions of seven clinical HAdV-A31 isolates.

HAdV prototype strain, wild type strains and cells

The HAdV-A31 (ATTC) prototype strain was obtained from the American Type Culture Collection (ATCC). Wild type strains of HAdV-A31 had been isolated between 1995 - 2004 from cases of severe HAdV disease in France and Germany (Table 2) [14].

All viruses were propagated on the human lung cancer cell line A549 (ATCC, CCL-185) on 75-cm2 or 25 cm2 culture flasks using DMEM medium with 5% of FBS (Biowest, Nuaillé, France) and 1% Penicillin/Streptomycin (Cytogen, Sinn, Germany) added. When the cytopathogenic effect was above 50%, cells were washed with PBS- (Cytogen) and lysed using Trypsin/EDTA (Cytogen). The DNA was extracted with the Qiagen blood kit (Qiagen, Hilden, Germany).

PCR amplification and cloning of DNA fragments

For the amplification of fragments up to 500 bp we used the HotStar Mix (Qiagen, Hilden, Germany) in a total volume of 50 μl with 1 μM of each primer and 5 μl of the purified genomic HAdV-A31 DNA. The PCR program began with the activation of the "hot start" DNA polymerase for 15 min at 95°C, followed by 40 cycles consisting of denaturation at 94°C for 20 s, a primer annealing temperature between 52 - 56°C depending on the composition of the used pair of primers for 20 s and elongation at 72°C for 40 s, followed by a final extension step of 72°C for 5 min. All fragments of larger size were amplified using the Expand High Fidelity PCR System from Roche Applied Science (Mannheim, Germany) which achieves a higher transcriptional fidelity by a proofreading activity. PCR reactions were performed in a total volume of 50 μl with 1 μM of each primer and 5 μl of the purified genomic DNA. The program starts with an initial denaturation step at a temperature of 94°C for 2 min, followed by 10 cycles consisting of denaturation at 94°C for 15 s, annealing, depending on the used primer pair, at temperatures between 52 - 56°C for 30 s and an elongation at 68°C for 1 - 4 min depending on the fragment length. These cycles were followed by 20 cycles with the same conditions but with 5 s extension of the elongation phase for each successive cycle. All PCR reactions were performed in a T-personal 48 thermocycler (Biometra, Goettingen, Germany).

Gel electrophoresis

PCR products were separated in a 1% agarose gel for 60 min at 120 V. DNA extraction from the agarose gels was performed with the Qiagen gel extraction kit according to the manufacturer's recommendations.

Cloning of PCR products

Depending on their length, extracted DNA fragments were either cloned before sequencing or they were sequenced directly. We performed AT-cloning reactions using the pGEM - T Easy Vector System I (Promega Corporation, Madison, WI, USA) and transfected replication competent E.coli bacteria. Plasmid DNA was extracted using the Qiagen mini prep kit according to the manufacturer's recommendations.

Sequencing

Cycle sequencing of both DNA strands was performed with rhodamine-labeled dideoxynucleotide chain terminator (DNA sequencing kit; ABI, Warrington, England) and analyzed on an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA, USA). PCR primers were used for the sequencing reactions.

DNA and protein analysis

Sequence assembly was carried out with the program SeqMan 5.00 from the DNASTAR software package. DNA and protein homology searches were performed using the NCBI BLAST program. DNA and protein sequence alignment were carried out using the ClustalW algorithm implemented in the BioEdit package (version 7.0.4.1). Genome annotation, analysis of non coding DNA motifs and functional protein motifs were performed by using the web based gene prediction software GENEMARK http://exon.biology.gatech.edu webcite, the DNASIS MAX 2.00.002.002 software and by sequence comparison.

Phylogenetic analysis was performed with the MEGA software package (version 3.1). The phylogenetic trees were constructed with the neighbor-joining method. Bootstrap analysis was performed with 1,000 pseudoreplicates. The graphical alignments were performed with LAGAN 2.0 software http://lagan.stanford.edu/lagan_web/index.shtml webcite[24].

In silico protein analysis and motif prediction were performed with the web based Pfam http://pfam.sanger.ac.uk webcite[62] and proSITE http://www.expasy.ch/prosite webcite software [63]. Transmembrane domains were predicted with the TMHMM software http://www.cbs.dtu.dk/services/TMHMM webcite[64]. Virtual 2D Gel analysis was carried out using the JVirGel Version 2.0 software http://www.jvirgel.de webcite[65,66]. Prediction of coiled coil regions was performed using the COILS software http://www.ch.embnet.org/software/COILS_form.html webcite[43].

Nucleotide sequence accession numbers

HAdV-1 [GenBank: AF534906], HAdV-2 [GenBank: NC_001405], HAdV-3 [GenBank: DQ086466], HAdV-4 [GenBank: AY599837], HAdV-5 [GenBank: AC_000008], HAdV-7 [GenBank: AC_000018], HAdV-8 [GenBank: AB448768], HAdV-9 [GenBank: AJ854486], HAdV-11 [GenBank: AC_000015], HAdV-12 [GenBank: AC_000005], HAdV-16 [GenBank: AY601636], HAdV-17 [GenBank: AC_000006], HAdV-19 [GenBank: AB448771], HAdV-21 [GenBank: AY601633], HAdV-22 [GenBank: FJ404771], HAdV-26 [GenBank: EF153474], HAdV-34 [GenBank: AY737797], HAdV-35 [GenBank: AY128640], HAdV-37 [GenBank: DQ900900], HAdV-40 [GenBank: L19443], HAdV-41 [GenBank: DQ315364], HAdV-46 [GenBank: AY875648], HAdV-48 [GenBank: EF153473], HAdV-49 [GenBank: DQ393829], HAdV-50 [GenBank: AY737798], HAdV-52 [GenBank: DQ923122].

The previously sequenced HAdV-31 hexon protein's accession number is GenBank: DQ149611, pX is GenBank: U14653.

Authors' contributions

SH carried out the laboratory work, molecular genetic studies, genome annotation, bioinformatic analysis and drafted the manuscript. IM participated in the design of the study and performed the phylogenetic analysis. SD participated in sequencing and analysis of the fiber gene regions. FR participated in sequencing and bioinformatic analysis. AH conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We would like to thank Anneliese Plentz (Institut für Medizinische Mikrobiologie, Universität Regensburg) and Veronique Venard (Laboratoire de Bactériologie-Virologie, Faculté de Médecine) for providing us with clinical isolates of HAdV-A31, Heidi Deppe and Gabi Harste for excellent technical assistance, and Hannah Elmer for critical reading of the manuscript.

References

  1. Swenson PD, Wadell G, Allard A, Hierholzer JC: Adenoviruses. In Manual of Clinical Microbiology. Volume 2. Edited by Murray PR, Baron EJ, Pfaller MA, Jorgensen JH, Yolken RA. Washington, D.C.: ASM Press; 2003::1404-1417. OpenURL

  2. Benkö M, Harrach B, Russell WC: Adenoviridea. In Virus taxonomy Seventh report of the international Commitee on Taxonomy of Viruses. New York, N.Y.: Academic Press; 2000. OpenURL

  3. Shenk T: Adenoviridae: The viruses and their replication. New York: Lippincott-Raven publishers; 2001.

  4. Jones MS, Harrach B, Ganac RD, Gozum MM, Dela Cruz WP, Riedel B, Pan C, Delwart EL, Schnurr DP: New Adenovirus Species Found in Patient Presenting with Gastroenteritis.

    J Virol 2007, 81(11):5978-5984. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  5. Crawford-Miksza LK, Nang RN, Schnurr DP: Strain variation in adenovirus serotypes 4 and 7a causing acute respiratory disease.

    J Clin Microbiol 1999, 37(4):1107-1112. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  6. Erdman DD, Xu W, Gerber SI, Gray GC, Schnurr D, Kajon AE, Anderson LJ: Molecular epidemiology of adenovirus type 7 in the United States, 1966-2000.

    Emerg Infect Dis 2002, 8(3):269-277. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  7. Bhat AM, Meny RG, Aranas EA, Yehia F: Fatal adenoviral (type 7) respiratory disease in neonates.

    Clin Pediatr (Phila) 1984, 23(7):409-411. PubMed Abstract | Publisher Full Text OpenURL

  8. Chuang YY, Chiu CH, Wong KS, Huang JG, Huang YC, Chang LY, Lin TY: Severe adenovirus infection in children.

    J Microbiol Immunol Infect 2003, 36(1):37-40. PubMed Abstract | Publisher Full Text OpenURL

  9. Dakhama A, Hegele RG, Laflamme G, Israel-Assayag E, Cormier Y: Common respiratory viruses in lower airways of patients with acute hypersensitivity pneumonitis.

    Am J Respir Crit Care Med 1999, 159(4 Pt 1):1316-1322. PubMed Abstract | Publisher Full Text OpenURL

  10. Kampmann B, Cubitt D, Walls T, Naik P, Depala M, Samarasinghe S, Robson D, Hassan A, Rao K, Gaspar H, et al.: Improved outcome for children with disseminated adenoviral infection following allogeneic stem cell transplantation.

    Br J Haematol 2005, 130(4):595-603. PubMed Abstract | Publisher Full Text OpenURL

  11. Chakrabarti S, Mautner V, Osman H, Collingham KE, Fegan CD, Klapper PE, Moss PA, Milligan DW: Adenovirus infections following allogeneic stem cell transplantation: incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery.

    Blood 2002, 100(5):1619-1627. PubMed Abstract | Publisher Full Text OpenURL

  12. Hale GA, Heslop HE, Krance RA, Brenner MA, Jayawardene D, Srivastava DK, Patrick CC: Adenovirus infection after pediatric bone marrow transplantation.

    Bone Marrow Transplant 1999, 23(3):277-282. PubMed Abstract | Publisher Full Text OpenURL

  13. Walls T, Shankar AG, Shingadia D: Adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients.

    Lancet Infect Dis 2003, 3(2):79-86. PubMed Abstract | Publisher Full Text OpenURL

  14. Venard V, Carret A, Corsaro D, Bordigoni P, Le Faou A: Genotyping of adenoviruses isolated in an outbreak in a bone marrow transplant unit shows that diverse strains are involved.

    J Hosp Infect 2000, 44(1):71-74. PubMed Abstract | Publisher Full Text OpenURL

  15. Seidemann K, Heim A, Pfister ED, Köditz H, Beilken A, Sander A, Melter M, Sykora KW, Sasse M, Wessel A: Monitoring of adenovirus infection in pediatric transplant recipients by quantitative PCR: report of six cases and review of the literature.

    Am J Transplant 2004, 4(12):2102-2108. PubMed Abstract | Publisher Full Text OpenURL

  16. Madisch I, Wolfel R, Harste G, Pommer H, Heim A: Molecular identification of adenovirus sequences: a rapid scheme for early typing of human adenoviruses in diagnostic samples of immunocompetent and immunodeficient patients.

    J Med Virol 2006, 78(9):1210-1217. PubMed Abstract | Publisher Full Text OpenURL

  17. Mattner F, Sykora KW, Meissner B, Heim A: An Adenovirus Type F41 Outbreak in a Pediatric Bone Marrow Transplant Unit: Analysis of Clinical Impact and Preventive Strategies.

    Pediatr Infect Dis J 2008, 27(5):419-424. PubMed Abstract | Publisher Full Text OpenURL

  18. Slatter MA, Read S, Taylor CE, Crooks BN, Abinun M, Flood TJ, Cant AJ, Wright C, Gennery AR: Adenovirus type F subtype 41 causing disseminated disease following bone marrow transplantation for immunodeficiency.

    J Clin Microbiol 2005, 43(3):1462-1464. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  19. Waddington SN, McVey JH, Bhella D, Parker AL, Barker K, Atoda H, Pink R, Buckley SM, Greig JA, Denby L, et al.: Adenovirus serotype 5 hexon mediates liver gene transfer.

    Cell 2008, 132(3):397-409. PubMed Abstract | Publisher Full Text OpenURL

  20. Jonsson MI, Lenman AE, Frangsmyr L, Nyberg C, Abdullahi M, Arnberg N: Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells.

    J Virol 2009, 83(8):3816-3825. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  21. Garnett CT, Talekar G, Mahr JA, Huang W, Zhang Y, Ornelles DA, Gooding LR: Latent species C adenoviruses in human tonsil tissues.

    J Virol 2009, 83(6):2417-2428. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  22. Johansson ME, Brown M, Hierholzer JC, Thorner A, Ushijima H, Wadell G: Genome analysis of adenovirus type 31 strains from immunocompromised and immunocompetent patients.

    J Infect Dis 1991, 163(2):293-299. PubMed Abstract OpenURL

  23. Leruez-Ville M, Chardin-Ouachee M, Neven B, Picard C, Le Guinche I, Fischer A, Rouzioux C, Blanche S: Description of an adenovirus A31 outbreak in a paediatric haematology unit.

    Bone Marrow Transplant 2006, 38(1):23-28. PubMed Abstract | Publisher Full Text OpenURL

  24. Brudno M, Do CB, Cooper GM, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S: LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA.

    Genome Res 2003, 13(4):721-731. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  25. De Jong JC, Wermenbol AG, Verweij-Uijterwaal MW, Slaterus KW, Wertheim-Van Dillen P, Van Doornum GJ, Khoo SH, Hierholzer JC: Adenoviruses from human immunodeficiency virus-infected individuals, including two strains that represent new candidate serotypes Ad50 and Ad51 of species B1 and D, respectively.

    J Clin Microbiol 1999, 37(12):3940-3945. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  26. Purkayastha A, Ditty SE, Su J, McGraw J, Hadfield TL, Tibbetts C, Seto D: Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development.

    J Virol 2005, 79(4):2559-2572. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  27. Hatfield L, Hearing P: Redundant elements in the adenovirus type 5 inverted terminal repeat promote bidirectional transcription in vitro and are important for virus growth in vivo.

    Virology 1991, 184:265-276. PubMed Abstract | Publisher Full Text OpenURL

  28. Bruder JT, Hearing P: Cooperative binding of EF-1A to the E1A enhancer region mediates synergistic effects on E1A transcription during adenovirus infection.

    J Virol 1991, 65(9):5084-5087. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  29. Avvakumov N, Wheeler R, D'Halluin JC, Mymryk JS: Comparative sequence analysis of the largest E1A proteins of human and simian adenoviruses.

    J Virol 2002, 76(16):7968-7975. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  30. Huvent I, Cousin C, Kiss A, Bernard C, D'Halluin JC: Susceptibility to natural killer cells and down regulation of MHC class I expression in adenovirus 12 transformed cells are regulated by different E1A domains.

    Virus research 1996, 45:123-134. PubMed Abstract | Publisher Full Text OpenURL

  31. Geisberg JV, Lee WS, Berk AJ, Ricciardi RP: The zinc finger region of the adenovirus E1A transactivating domain complexes with the TATA box binding protein.

    Proc Natl Acad Sci USA 1994, 91(7):2488-2492. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  32. Berk AJ: Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus.

    Oncogene 2005, 24(52):7673-7685. PubMed Abstract | Publisher Full Text OpenURL

  33. Querido E, Blanchette P, Yan Q, Kamura T, Morrison M, Boivin D, Kaelin WG, Conaway RC, Conaway JW, Branton PE: Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex.

    Genes Dev 2001, 15(23):3104-3117. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  34. Blanchette P, Cheng CY, Yan Q, Ketner G, Ornelles DA, Dobner T, Conaway RC, Conaway JW, Branton PE: Both BC-box motifs of adenovirus protein E4orf6 are required to efficiently assemble an E3 ligase complex that degrades p53.

    Mol Cell Biol 2004, 24(21):9619-9629. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  35. Lee KA, Green MR: A cellular transcription factor E4F1 interacts with an E1a-inducible enhancer and mediates constitutive enhancer function in vitro.

    Embo J 1987, 6(5):1345-1353. PubMed Abstract | PubMed Central Full Text OpenURL

  36. Horwitz MS: Function of adenovirus E3 proteins and their interactions with immunoregulatory cell proteins.

    The journal of gene medicine 2004, 6(Suppl 1):S172-183. PubMed Abstract | Publisher Full Text OpenURL

  37. Windheim M, Hilgendorf A, Burgert HG: Immune evasion by adenovirus E3 proteins: exploitation of intracellular trafficking pathways.

    Current topics in microbiology and immunology 2004, 273:29-85. PubMed Abstract OpenURL

  38. Sprengel J, Schmitz B, Heuss-Neitzel D, Zock C, Doerfler W: Nucleotide sequence of human adenovirus type 12 DNA: comparative functional analysis.

    J Virol 1994, 68(1):379-389. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  39. Burgert HG, Blusch JH: Immunomodulatory functions encoded by the E3 transcription unit of adenoviruses.

    Virus Genes 2000, 21(1-2):13-25. PubMed Abstract | Publisher Full Text OpenURL

  40. Krajcsi P, Tollefson AE, Anderson CW, Wold WS: The adenovirus E3 14.5-kilodalton protein, which is required for down-regulation of the epidermal growth factor receptor and prevention of tumor necrosis factor cytolysis, is an integral membrane protein oriented with its C terminus in the cytoplasm.

    J Virol 1992, 66(3):1665-1673. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  41. Davison AJ, Benko M, Harrach B: Genetic content and evolution of adenoviruses.

    J Gen Virol 2003, 84(Pt 11):2895-2908. PubMed Abstract | Publisher Full Text OpenURL

  42. Akalu A, Liebermann H, Bauer U, Granzow H, Seidel W: The subgenus-specific C-terminal region of protein IX is located on the surface of the adenovirus capsid.

    Journal of virology 1999, 73:6182-6187. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  43. Lupas A, Van Dyke M, Stock J: Predicting coiled coils from protein sequences.

    Science (New York, NY) 1991, 252(5009):1162-1164. OpenURL

  44. Lutz P, Kedinger C: Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter.

    Journal of virology 1996, 70:1396-1405. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  45. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR: Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment.

    Cell 1993, 73(2):309-319. PubMed Abstract | Publisher Full Text OpenURL

  46. Mathias P, Wickham T, Moore M, Nemerow G: Multiple adenovirus serotypes use alpha v integrins for infection.

    J Virol 1994, 68(10):6811-6814. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  47. Karayan L, Hong SS, Gay B, Tournier J, d'Angeac AD, Boulanger P: Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

    J Virol 1997, 71(11):8678-8689. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  48. Chatterjee NK, Samsonoff WA, Balasubramaniam N, Rush-Wilson K, Spargo W, Church TM: Isolation and characterization of adenovirus 5 from the brain of an infant with fatal cerebral edema.

    Clin Infect Dis 2000, 31(3):830-833. PubMed Abstract | Publisher Full Text OpenURL

  49. Honkavuori KS, Pollard BD, Rodriguez MS, Hay RT, Kemp GD: Dual role of the adenovirus pVI C terminus as a nuclear localization signal and activator of the viral protease.

    J Gen Virol 2004, 85(Pt 11):3367-3376. PubMed Abstract | Publisher Full Text OpenURL

  50. Mahr JA, Gooding LR: Immune evasion by adenoviruses.

    Immunol Rev 1999, 168:121-130. PubMed Abstract | Publisher Full Text OpenURL

  51. Burgert HG, Ruzsics Z, Obermeier S, Hilgendorf A, Windheim M, Elsing A: Subversion of host defense mechanisms by adenoviruses.

    Curr Top Microbiol Immunol 2002, 269:273-318. PubMed Abstract OpenURL

  52. Barclay AN BM, Law SKA, McNight AJ, Tomlinson MG, Merwe PA: The Leukocyte Antigen Factsbook. San Diego, CA, USA: Academic Press; 1997.

  53. McSharry BP, Burgert HG, Owen DP, Stanton RJ, Prod'homme V, Sester M, Koebernick K, Groh V, Spies T, Cox S, et al.: Adenovirus E3/19K promotes evasion of NK cell recognition by intracellular sequestration of the NKG2D ligands major histocompatibility complex class I chain-related proteins A and B.

    J Virol 2008, 82(9):4585-4594. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  54. Andersson M, Paabo S, Nilsson T, Peterson PA: Impaired intracellular transport of class I MHC antigens as a possible means for adenoviruses to evade immune surveillance.

    Cell 1985, 43(1):215-222. PubMed Abstract | Publisher Full Text OpenURL

  55. Burgert HG, Kvist S: An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens.

    Cell 1985, 41(3):987-997. PubMed Abstract | Publisher Full Text OpenURL

  56. Hall KT, Blair GE: The mechanism of down-regulation of major histocompatibility complex (MHC) class I antigens in highly oncogenic adenovirus 12-transformed cells.

    Biochem Soc Trans 1997, 25(2):353S. PubMed Abstract OpenURL

  57. Subramanian T, Vijayalingam S, Chinnadurai G: Genetic identification of adenovirus type 5 genes that influence viral spread.

    J Virol 2006, 80(4):2000-2012. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  58. Forstmeyer D, Henke-Gendo C, Bröcker V, Wildner O, Heim A: Quantitative temporal and spatial distribution of adenovirus type 2 correlates with disease manifestations and organ failure during disseminated infection.

    Journal of medical virology 2008, 80:294-297. PubMed Abstract | Publisher Full Text OpenURL

  59. Dmitriev IP, Kashentseva EA, Curiel DT: Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX.

    J Virol 2002, 76(14):6893-6899. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  60. Zakhartchouk A, Connors W, van Kessel A, Tikoo SK: Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX.

    Virology 2004, 320(2):291-300. PubMed Abstract | Publisher Full Text OpenURL

  61. Ebner K, Pinsker W, Lion T: Comparative sequence analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications.

    J Virol 2005, 79(20):12635-12642. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  62. Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer EL, et al.: The Pfam protein families database.

    Nucleic Acids Res 2008, (36 Database):D281-288. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  63. Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, de Castro E, Lachaize C, Langendijk-Genevaux PS, Sigrist CJ: The 20 years of PROSITE.

    Nucleic Acids Res 2008, (36 Database):D245-249. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  64. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.

    J Mol Biol 2001, 305(3):567-580. PubMed Abstract | Publisher Full Text OpenURL

  65. Hiller K, Grote A, Maneck M, Munch R, Jahn D: JVirGel 2.0: computational prediction of proteomes separated via two-dimensional gel electrophoresis under consideration of membrane and secreted proteins.

    Bioinformatics 2006, 22(19):2441-2443. PubMed Abstract | Publisher Full Text OpenURL

  66. Hiller K, Schobert M, Hundertmark C, Jahn D, Munch R: JVirGel: Calculation of virtual two-dimensional protein gels.

    Nucleic Acids Res 2003, 31(13):3862-3865. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL