Genomic and bioinformatics analysis of human adenovirus type 37: New insights into corneal tropism
© Robinson et al; licensee BioMed Central Ltd. 2008
Received: 07 December 2007
Accepted: 09 May 2008
Published: 09 May 2008
Human adenovirus type 37 (HAdV-37) is a major etiologic agent of epidemic keratoconjunctivitis, a common and severe eye infection associated with long-term visual morbidity due to persistent corneal inflammation. While HAdV-37 has been known for over 20 years as an important cause, the complete genome sequence of this serotype has yet to be reported. A detailed bioinformatics analysis of the genome sequence of HAdV-37 is extremely important to understanding its unique pathogenicity in the eye.
We sequenced and annotated the complete genome of HAdV-37, and performed genomic and bioinformatics comparisons with other HAdVs to identify differences that might underlie the unique corneal tropism of HAdV-37. Global pairwise genome alignment with HAdV-9, a human species D adenovirus not associated with corneal infection, revealed areas of non-conserved sequence principally in genes for the virus fiber (site of host cell binding), penton (host cell internalization signal), hexon (principal viral capsid structural protein), and E3 (site of several genes that mediate evasion of the host immune system). Phylogenetic analysis revealed close similarities between predicted proteins from HAdV-37 of species D and HAdVs from species B and E. However, virtual 2D gel analyses of predicted viral proteins uncovered unexpected differences in pI and/or size of specific proteins thought to be highly similar by phylogenetics.
This genomic and bioinformatics analysis of the HAdV-37 genome provides a valuable tool for understanding the corneal tropism of this clinically important virus. Although disparities between HAdV-37 and other HAdV within species D in genes encoding structural and host receptor-binding proteins were to some extent expected, differences in the E3 region suggest as yet unknown roles for this area of the genome. The whole genome comparisons and virtual 2D gel analyses reported herein suggest potent areas for future studies.
Adenoviruses (AdV) in the Adenoviridae family have been divided into four genera: Mastadenovirus, Aviadenovirus, Atadenovirus, and Siadenovirus . The AdV was first isolated from human adenoids and characterized by two different research teams [2, 3]. Human AdV (HAdV) fall within the genus Mastadenovirus, and cause a wide array of diseases including acute respiratory disease, gastroenteritis, and ocular surface infection [4–6]. The AdV is non-enveloped with a double stranded linear genome that ranges from 26 to 45 kb in size. The icosahedral shaped capsid ranges from 70 to 100 nanometers in diameter . There are 51 known HAdV serotypes classified into 6 species (A-F), based on restriction enzyme analysis and hemaglutination assays, later confirmed by genome analyses and phylogenetic calculations. Recently, a proposed fifty-second HAdV serotype was identified and placed into a new species G .
HAdV-37 was originally isolated in 1976 from 62 eyes and 9 genitourinary sites, and subsequently characterized as a new serotype in 1981 . HAdV-37 is a major etiologic agent of epidemic keratoconjunctivitis, an explosive and highly contagious infection of the conjunctiva and cornea, and continues to cause outbreaks . HAdV-37 also was recently implicated in the pathogenesis of obesity .
Although HAdV species D contains the most serotypes, complete sequence is available for only 6 – HAdV-9, HAdV-17, HAdV-26, HAdV-46, HAdV-48, and HAdV-49 – and none of these have been associated with epidemic keratoconjunctivitis. In this study, we have sequenced the complete genome of HAdV-37 and describe its overall organization. The HAdV-37 genome appears in most respects typical of other HAdV. However, global pairwise genome alignments, phylogenetic analyses, and in silico comparisons of putative viral proteins revealed unique characteristics of the genome, including areas of non-conserved sequence in the penton, hexon, E3, and fiber regions, and differences in size and/or pI of select predicted HAdV-37 proteins. Understanding the disparities between the HAdV-37 genome and those of species D HAdV with dissimilar tissue tropisms may lead to improved understanding of the genomic determinants of infection.
The 5' and 3' termini of the HAdV genome are composed of inverted terminal repeat (ITR) sequences which for HAdV-37 were determined to be 159 bp in length. These sites serve as replication origins for the virus . The motif located at the extreme termini of the HAdV-37 genome consists of a CATCATCATAAT, which is unique among previously sequenced HAdV serotypes. Unique sequences for the extreme termini have been also observed in other HAdVs including HAdV-4 . The conserved ATAATATACC motif within the ITR, which interacts with the terminal protein precursor (pTP) and polymerase complex during DNA replication , was determined at base pairs 8–17. A NFIII/Oct-1 recognition site (TATGCAAAT) was identified within the ITR of HAdV-37 at nucleotides 40–48. A Sp1 binding site (GGGGCGGA) was identified at nucleotides 73–80. Also, a NFI/CTFI (TGGGGCGGAGCCA) site was located at overlapping nucleotides 72–84.
Global pairwise alignment
Genome Organization of HAdV-37
Protein Length (aa)
hypothetical 20.5 kDa
hypothetical 34.76 kDa
hypothetical 16.57 kDa
DNA binding protein
hypothetical 19.29 kDa
hypothetical 11.4 kDa
hypothetical 8.58 kDa
hypothetical 9.29 kDa
hypothetical 31.6 kDa
Percent identities/similarities of select HAdV-37 proteins and their homologs
DNA binding protein
E1B proteins potentiate viral replication by blocking apoptosis. E1B 19K blocks the mitochondrial apoptosis pathway by inactivating BAK and BAX . E1B 55K inhibits the ability of p53, the host tumor suppressor protein, to initiate cell cycle arrest [20, 21]. The putative TATA box for the E1B messages was predicted at nucleotide 1525. Two predicted proteins of molecular weights of 21.1 and 55.2 kDa were identified within E1B which correspond to 19- and 55-kDa proteins, respectively, as reported for HAdV-9. Amino acid sequence analysis revealed that the predicted 21.1 kDa protein was 99% identical and 100% similar to the 19 kDa homologue found in the HAdV-9 genome (Table 2). The polyadenylation signal for these transcripts was predicted at nucleotide 3863.
The E2 region of the genome consists of two transcription units, E2A and E2B, which encode three proteins that are required for viral replication . These three proteins are known as the DNA binding protein (DBP), terminal protein precursor (pTP), and DNA polymerase. The E2A 54.9 kDa DNA binding protein was identified on the complementary strand between nucleotides 21305 and 22777. Also on the complementary strand, but located within the E2B region, we identified the pTP and DNA polymerase. The polyadenylation signal for these transcripts was not identified.
The HAdV E3 region encodes proteins that modulate the host immune response to infection but are not required for viral growth in vitro [22, 23]. HAdVs within species D have previously been suggested to encode eight ORFs within the E3 region [24, 25]. Seven classical and one hypothetical E3 ORFs were identified in our annotation of HAdV-37. The predicted molecular weights for these are 12.2, 21.8, 18.6, 48.9, 31.6, 10.47, 14.7, and 14.8 kDa. The TATA box was predicted at nucleotide 25879 with a TATAAA motif. One polyadenylation signal for this transcription unit was identified at nucleotide 30837.
Open reading frames located in the E4 transcription unit produce proteins that have a wide variety of functions . For example, E4 ORF 3 and E4 ORF 6 enhance the stability of late viral mRNAs and increase their export from the nucleus thereby increasing viral mRNA accumulation in the cytoplasm . E4 ORF 6 also binds to p53 and can block apoptosis [27, 28]. We found 6 predicted ORFs in HAdV-37 located on the complementary strand. Surprisingly, the E4 ORF 1 from the HAdV-37 genome was predicted at 65 amino acids in length corresponding to a molecular weight of 7.4 kDa. In contrast, the HAdV-9 homologue of E4 ORF 1 is 125 amino acids in length, and contains three regions essential for tumor transformation (region I, residues 34 to 41; region II, residues 89 to 91; region III 122 to 125). The E4 ORF 1 of HAdV-9 has structural similarity to other viral dUTPase enzymes [29, 30]. ClustalW analysis of the HAdV-37 E4 ORF 1 compared to the HAdV-9 homologue revealed a 100% similarity from residues 61–125, including regions II and III and a truncated dUTPase domain. Further work will be needed to evaluate the significance of this truncation. The TATA box for this region was identified at nucleotide 34665 and the polyadenylation signal at nucleotide 32184.
The intermediate genes of HAdV are IVa2 and IX. The IVa2 protein interacts with L1 52/55K during viral DNA packaging, and assists in the activation of the major late promoter (MLP) [14, 31–33]. The HAdV-37 IVa2 gene, found on the complementary strand, was predicted using the splice site finder , with a 448 amino acid protein and 99% amino acid homology to HAdV-9 IVa2. The IX protein is a minor capsid protein and also assists in the activation of the major late promoter [35, 36]. A coding sequence for a 13.7 kDa protein corresponding to IX was found at nucleotides 3454–3858.
The late transcription units of HAdVs are transcribed from the MLP, which consists of an inverted CAAT box (5777–5780 bp) and TATA box (5827–5832 bp). The late mRNAs have been grouped into five families (L1 to L5), based on the location of the polyadenylation signal. Proteins expressed by these five families are involved in capsid production for mature virions . The L1 transcription unit encodes two proteins, 52/55K and IIIa. The 52/55K protein is involved in scaffolding of the capsid and therefore facilitates virus assembly . The 52/55K protein also interacts with the intermediate gene product IVa2 to facilitate DNA packaging [31, 33, 38]. Polypeptide IIIa is a structural protein that has been located on the inner capsid surface below the penton base . The 52/55K and polypeptide IIIa in HAdV-37 were predicted to have molecular weights of 42.2 kDa and 26.6 kDa, respectively. The predicted polyadenylation signal for the L1 region was found at nucleotide 13484.
The proteins encoded on the L2 transcription unit also are involved in capsid formation . The penton base (protein III) is found at each of the 12 vertices of the virion . The penton base contains an Arg-Gly-Asp (RGD) sequence which interacts with host integrins to induce internalization of the virus . The HAdV-37 penton base is located at nucleotides 13530–15089. The length of the protein was predicted to be 519 amino acids with an estimated molecular weight of 58.4 kDa. The RGD sequence was located at amino acid position 309–311. The predicted protein was 100% identical to the previously published penton base protein identified for HAdV-37 . The HAdV-37 penton base homologue is 90% identical and 95% similar to the predicted HAdV-9 penton base protein (Table 2). The V, VII, and X proteins constitute the HAdV core proteins and facilitate packaging of viral DNA within the capsid . The HAdV-37 pVII protein-coding sequence was identified at nucleotides 15093–15683, and the protein was predicted to have a molecular weight of 21.7 kDa. Proteins with an amino acid length of 334 and 74 were predicted in the HAdV-37 genome for proteins V and X at nucleotides 15716–16720 and 16750–16974, respectively. The L2 transcripts share a putative polyadenylation signal at nucleotide 16982.
Three open reading frames corresponding to pVI, hexon, and protease proteins, with respective molecular weights of 25.5, 106.8, and 23.4 kDa, were identified within the L3 transcription unit. The pVI protein contains two nuclear export signals and two nuclear localization signals, and plays a role in transporting the hexon protein to the nucleus for viral assembly . The C terminus of this protein has also been implicated in regulation of the viral protease . The HAdV-37 pVI protein was located at nucleotides 17030–17734. This predicted 234 amino acid protein was 100% identical to its HAdV-17 homologue. The hexon protein, the most abundant virion component, constitutes 240 of the 252 subunits of the protein shell of the virus . The HAdV-37 hexon protein is 949 amino acids in length and was nearly identical to that predicted by Ebner et al. . Our sequence data suggests an additional 10 amino acids on the N-terminus of the protein, similar to the predicted hexon gene for HAdV-46 and HAdV-9, both within species D. The final protein encoded in the L3 transcription unit is the 23 kDa viral protease protein. The HAdV-37 homologue was predicted to be 207 amino acids in length. This protein cleaves other viral proteins allowing for assembly and viral maturation , and the transcript shares a predicted polyadenylation signal at nucleotide 21256 with other L3 transcripts.
Three L4 proteins, 100 kDa, pVIII, and 22 kDa, were predicted from our annotation. The 100 kDa protein is a nonstructural protein that assists in the translation of late viral mRNAs, and inhibits translation of cellular mRNAs [47, 48]. More recently this protein has been implicated as a scaffold for trimerization of the hexon . The predicted 100 kDa protein for HAdV-37 genome was 732 amino acids in length and had a molecular weight of 82.3 kDa. This protein is 98% identical to the published HAdV-46 100 kDa protein. Protein VIII is a minor capsid protein that plays a role in the stability of the virion capsid . The pVIII protein of HAdV-37 is 24.6 kDa in molecular weight and has a 99% identity to the published HAdV-46 pVIII protein. The 22 kDa protein is involved in the packaging of HAdV DNA . A 22 kDa homologue was identified in HAdV-37 with a predicted protein of 137 amino acids and molecular weight of 15.8 kDa. Its highest percent identity was to the HAdV-9 22 kDa protein (99%). The predicted polyadenylation signal for the L4 transcription unit is at nucleotide 26507.
The L5 region of the HAdV genome consists entirely of the fiber protein gene. Fiber protein trimerizes to produce the functional unit which projects from the 12 penton vertices of the virus capsid. The fiber protein's carboxyl (C)-terminal globular domain, known as the fiber knob, acts as the primary ligand for host cell receptor binding. The HAdV-37 fiber genome sequence was previously reported, and our predicted protein of 365 amino acids was identical . The HAdV-37 fiber was only 76% identical and 89% similar to its homologue in the HAdV-9 genome (Table 2). The polyadenylation signal for this transcript was predicted at nucleotide 32143. Nucelotide sequence encoding a potential heparan binding site, previously reported in the fiber shaft of HAdV-5, was not present in the HAdV-37 fiber gene [53–55].
Most HAdVs contain two virus-associated (VA) RNA genes, VA RNAI and VA RNAII. VA RNAI acts against cellular antiviral defense by blocking the activation of the protein kinase PKR, which when activated turns off protein synthesis in infected cells . VA RNAII binds to RNA helicase A and NF90, the latter a component of the nuclear factor of activated T cells (NFAT) . These VA RNAs also have been recently shown to suppress RNA interference . The VA RNA genes for HAdV-37 were previously identified . Our sequence for VA RNAI is located at nucleotides 10253–10410 and is 99% identical to the previously reported sequence, differing by only one base pair. VA RNAII is located at nucleotides 10471–10620 and was 100% identical to that previously reported.
Protein and phylogenetic analysis
Molecular weight/pI of select HAdV-37 proteins and their homologs in other HAdV species
DNA binding protein
Conserved hypothetical HAdV-37 Proteins
HAdV-5 Hypothetical 12 kDa
HAdV-7 Hypothetical 10.4 kDa
HAdV-7 13.6 Agnoprotein
HAdV-16 Hypothetical 12.6 kDa
HAdV-3 Hypothetical 9.7 kDa
HAdV-7 Hypothetical 11.3 kDa
HAdV-7 Hypothetical protein
HAdV-9 E3 orf3 33.1 kDa
We have determined the complete 35,213 base pair genome of HAdV-37 and identified 35 putative adenoviral genes along with 8 hypothetical ORFs conserved with at least one other HAdV for each ORF. Comparison of the HAdV-37 genome to that of HAdV-9, another species D virus, identified areas of substantial divergence in the penton, hexon, E3, and fiber regions. Disparities between these two HAdV species D viruses in genes encoding structural and host receptor-binding proteins were somewhat expected and also consistent with known differences in host tissue tropism, for example the propensity of HAdV-37 to cause corneal infection, as compared to the association of HAdV-9 with urethritis and follicular conjunctivitis [7, 65]. Differences between HAdV-9 and 37 in the E3 region, known to be important to immune evasion and regulation by the virus, but not essential to viral replication in vitro, suggest as yet undiscovered functions for this region [22, 23]. Divergence in the E3 region, possibly relevant to cellular and tissue specificity during infection, might be due to positive selection. Sequencing of other HAdVs within species D would provide further insight into this area of the HAdV genome.
By phylogenetic analyses and paired comparisons of predicted proteins, HAdV-37 and HAdV-9 of species D appeared most closely related to HAdV-7 of species B and HAdV-4 of species E. Subsequent virtual 2D gel analyses suggested that for a few proteins, a relatively few amino acid substitutions between otherwise similar proteins conferred significant effects on protein charge. If our analyses prove correct, such differences suggest that the function of such proteins in HAdV species D could be quite different than previously described for serotypes of other HAdV species. We acknowledge that our predictions represent a first approximation of protein characteristics, and could be subject to over-interpretation for at least two reasons. First, our comparisons to other viruses are only as reliable as the quality of GenBank viral sequence and annotation. Secondly, post-translational modifications may alter both charge and molecular weight of any given protein. Actual 2D gel analysis will be necessary to confirm such predicted differences.
There is growing concern over the accuracy of in silico ORF prediction in AdVs due to splice variants, as well as inconsistencies in banked annotations . To address such concerns, we compared HAdV-37 annotation using three different methods: NCBI ORF finder, JCVI's annotation engine, and GeneMark Heuristic model. We narrowed our annotation to 35 ORFs by comparison with previously determined adenoviral annotations, but we consider our annotation provisional. We identified 8 hypothetical ORFs similar to those previously identified in other HAdV species. The very suggestion of hypothetical proteins implies that our understanding of the HAdV is far from complete. Transcriptome analysis using viral microarrays may help to clarify the best annotation . We suggest that the true transcriptome and proteome of HAdV-37 remain to be determined.
Future sequencing of HAdVs may permit new insights into viral origin, evolution, and pathogenesis. Recently, HAdV-22 was isolated for the first time from an outbreak of epidemic keratoconjunctivitis. The HAdV-22 isolate was shown to contain both HAdV-8 fiber gene and HAdV-37 penton base gene . These recombination events apparently conferred corneal tropism to HAdV-22, a virus not normally known to infect the cornea. As more HAdV species D viruses are sequenced, new insights into tropism and pathogenesis are likely to emerge.
In summary, the complete genome sequence of HAdV-37 was determined and annotated. The organization of the HAdV-37 genome is similar to other human species D adenoviruses except in the penton, hexon, E3, and fiber regions. Phylogenetic analysis of HAdV-37 proteins revealed close relation to species B and E human adenoviruses, while virtual 2D gel analysis identified differences in proteins thought to function similarly. The availability of the HAdV-37 complete genome sequence will facilitate future studies into the pathogenicity of this important human pathogen.
Cells, virus stock, DNA purification
HAdV-37 strain GW was obtained from the American Type Culture Collection (ATCC). Virus stocks were grown in A-549 cells (CCL-185), a human alveolar epithelial cell line that was previously shown to support HAdV-1 virion production . Virus was purified by CsCl gradient and subsequent dialysis, and stored at -80°C. DNA extraction was accomplished by the addition of proteinase K, phenol:chloroform extraction, and finally ethanol precipitation.
Standard PCR methodology was used to amplify regions of the genome to be sequenced. HAdV type 17 was used as a reference strain for the design of initial PCR primers. To close gaps in the sequence and improve overall sequence quality, Primer 3  and CONSED  software were used to design primers from newly acquired sequence. Shrimp alkaline phosphatase and exonuclease I treatment were used to dephosphorylate and degrade residual PCR primers present together with the PCR products. Sequencing was performed using the ABI BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). The sequencing reaction mixture was purified using Sephadex G-50 (Sigma Aldrich, St. Louis, MO), and the reaction products analyzed on ABI 3700 or ABI 3730 XL capillary electrophoresis DNA sequencers (Applied Biosystems). To sequence the viral inverted terminal repeat (ITR) ends, primers were designed from newly determined adjacent sequence, and direct sequencing was performed using whole genome DNA as the template .
Sequence analysis and genome annotation
Sequence data was filtered using LUCY (JCVI, Rockville, MD), and data assembly performed with Phred/Phrap, using default assembly parameters [71–73]. Genome assembly contained 664 high quality reads with an average length of 834 bps. The fold coverage for both strands of the genome was 15. The Phrap average quality score was 89.0. Genome annotation was performed using JCVI's automated annotation system , and the data was stored in a MySQL database. Manatee  was used to manually review the data from the annotation engine. Additionally, we used GeneMark Heuristic Models gene prediction , and NCBI's ORF Finder  to examine the sequence. Open reading frames were searched against available databases in GenBank, PIR, SWISS-PROT, and JCVI's CMR database. Splice sites were predicted using a splice site finder program . An online sequence alignment program, mVISTA LAGAN  was used for global pair-wise sequence alignment . CpG analysis was performed with FUZZNUC .
Nucleotide sequence accession numbers
The nucleotide sequence for the following HAdVs can be found in GenBank: HAdV-2 [AC_000007], HAdV-4 [AY487947], HAdV-7 [AC_000018], HAdV-9 [AJ854486], HAdV-12 [AC_000005], HAdV-17 [AC_000006], HAdV-40 [L19443]. Previously sequenced HAdV-37 penton base protein, hexon protein, fiber protein, and VA RNA gene accession numbers are AAG00906, ABA00016, AAB71734, and U10679, respectively. The GenBank accession number for HAdV-37 is DQ900900.
In silico protein analysis
Percent identities and similarities between proteins of HAdV-37 and other HAdVs were determined using Fasta3 [60, 79] and Blastp software . Proteins from the GenBank database were analyzed by an in silico 2D gel program JVirGel 2.2.3b . Phylogenetic analysis was performed with Molecular Evolutionary Genetics Analysis (MEGA) 3.1 . Bootstrap confirmed neighbor joining phylogenetic trees were designed with MEGA 3.1 with 500 replicates.
We thank Nicole Benton for her technical assistance in sequencing as well as Jeremy Zaitshik for his bioinformatics assistance. Research support was provided through NIH grants EY013124, EY015222, EY012190, P20 RR017703, P20 RR015564, T32 A1007633 and a Research to Prevent Blindness Physician-Scientist Merit Award (to JC).
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