Analysis of Adenovirus - host cellinteractions and comparative genomicsAdeel ur Rehman

Analysis of Adenovirus - host cell

interactions and comparative genomics

Adeel ur Rehman

Degree project in biology, Master of science (2 years), 2013 Examensarbete i biologi 30 hp till masterexamen, 2013

Biology Education Centre and Department of Medical Biochemistry and Microbiology, Uppsala University

Supervisor: Professor Catharina Svensson

External opponent: Roberta Biasiotto

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ABSTRACT

Human adenovirus (HAdV) is currently being developed as a promising therapeutic candidate for the

treatment of cancer. High gene transfer efficiency, relatively simple and efficient purification in large

scale and inherent ability to kill the cell following a completed infection cycle are the characteristics that

make adenovirus (Ad) a molecular tool of choice for many scientists. Consequently, Ad is among the

most frequently used viral vector in clinical trials. Tissue and tumor cell specificity, toxicity and host

immune responses are still main obstacles for the development of novel biological viral cancer

therapies. A good animal model is necessary to investigate these issues, but human Ad infection and

replication is restricted to human cells. Our lab recently identified a mouse cell line (NMuMG), which

shows full susceptibility to infection by human adenovirus serotype 2 (HAdV-2) but not to infection by

human adenovirus serotype 12 (HAdV-12). It is not known why some adenovirus serotypes are

susceptible while others are not. Nor is it known at what stage the virus infection cycle is blocked during

infection with the non-growing Ad serotypes. In this project, we showed that both human adenovirus

serotype 3 (HAdV-3) and human adenovirus serotype 11 (HAdV-11) efficiently deliver their genomes into

the nuclei of NMuMG cells. However no replication of DNA was found. In order to find differences

among Ad serotypes, a comparative bioinformatics analysis of HAdV-2, -3 and -12 was performed to

identify species-specific genes. We showed that there is a substantial genome variation in term of gene

content between the different Ad serotypes and a small number of novel species-specific genes were

identified. These results may suggest that specific viral proteins have a functionally important role on

host cell specificity.

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Table of Contents

ABSTRACT ... 1

ABBREVIATIONS ... 3

INTRODUCTION ... 4

Key features of Human adenovirus ... 4

Genotypes and Genomics of Human adenovirus ... 4

Oncolytic Virus and Host Immune Response ... 4

Life cycle of Adenovirus ... 5

Aims of this study ... 6

MATERIAL AND METHODS ... 7

Bioinformatics ... 7

Cell lines and viruses ... 7

Virus infection and transfection ... 7

Oligonucleotides... 7

Cytoplasmic and Nuclear DNA extraction ... 7

Virus DNA extraction by Hirt method and PCR ... 8

RNA isolation and RT-PCR ... 8

Protein extraction, SDS-PAGE and Western Blot... 9

RESULTS ... 10

Differential distribution of genes ... 10

Virus replication in infected NMuMG cells ... 12

DISCUSSION ... 15

ACKNOWLEDGMENTS ... 17

REFERENCES ... 18

APPENDIX I. List of PCR primers, master mix, reagents, stock volume and concentration ... 20

APPENDIX II. The final reciprocal BLAST search results list compiled by manual curation ... 22

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ABBREVIATIONS

HAdV: Human adenovirus Ad: adenovirus

HAdV-2: Human adenovirus serotype 2 HAdV-3: Human adenovirus serotype 3 HAdV-11: Human adenovirus serotype 11 HAdV-12: Human adenovirus serotype 12 CAR: coxackie-adenovirus receptor DSG-2: desmoglein-2

kb: kilo bases

GC-content: guanine-cytosine content Ab: antibodies

ADP: adenoviral death protein (E3) CAR: coxsackie and adenovirus receptor CTL: cytotoxic T lymphocytes

NMuMG cells: Normal mouse mammary epithelial cells A549 cells: Human lung adenocarcinoma epithelial cells DMEM: Dulbecco’s modified Eagle’s medium

FBS: fetal bovine serum; ORF: open reading frame RT: room temperature

FFU: florescence forming units

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INTRODUCTION

Key features of Human adenovirus

Oncolytic viruses have the potential to infect cancer cells, multiply selectively within cancer cells and cause death, with the release of new viruses that can infects neighboring cancer cells. Human adenovirus (HAdV) is the major promising candidate available against in cancer gene therapy.

So far, 1970 clinical gene therapy trials are being or have been performed worldwide in which, Adenoviruses (Ad) are the most frequently used viral vector with 23% of all the trials.

Several characteristics make HAdV an attractive cancer gene therapy candidate, e.g., broad cell tropism, high gene transfer efficiency in both dividing and non-dividing cells, relatively simple and efficient purification in large scale, and induction of oncolysis by a completed infection cycle.

Although first, second and third generation Ad vectors have been developed, tissue specificity, host immune response against virus infection and lack of animal model are the major problems.

Genotypes and Genomics of Human adenovirus

Human Adenoviruses (HAdV) belong to family Adenoviridae and are non-enveloped viruses with a comparatively small double standard linear genome of 34-37 kb (Table 1).

HAdV are classified into seven different species (A-G) on the basis of particular serology or more recently, of genome sequence (http://hadvwg.gmu.edu/). Up to now, 65 different types have been identified, of these types, HAdV-5 of specie C is the most studied type.

The Ad genome has two major groups of genes termed as early and late transcription units. Early transcription units encode for E1A, E1B, E2, E3 and E4 protein that are mostly involved in transcription activation, inhibition of apoptosis, DNA replication, immune response and viral reprogramming of host cell. Late proteins L1 to L5 are expressed from a major late promoter and are generated by alternative splicing of a single transcript.

Comparative genomics is a powerful tool to identify type specific markers for studies on viral replication, and can provide how the tissue specificity and host immune response are regulated. The genome wide variation of HAdV types is complex and remains largely unknown. So far only three types (HAdV-12, 5, 2) from two species (A, C) have been subject to whole proteome sequencing and well annotation.

Oncolytic Virus and Host Immune Response

Several viral species such as Ad, retrovirus, poxvirus, herpes simplex virus, measles, reovirus and vesicular stomatitis virus have been used as oncolytic virus or genetically reengineered vectors to achieve cancer specific replication.

The selective replication of different Ads has been studied by comparing different cell lines (human or mouse) in vitro.

Different cell lines have shown variable levels of susceptibility to Ad infection and replication.

HAdVs are relatively easy to grow and modify, but manipulations that will retain replication competence

are difficult because, these viruses interact with both innate and adaptive immune system as well as

many growth controlling functions of the host cell.

Many genetically engineered Ad can induce cancer

specific immune response

therefore, in order to evaluate tumor killing activities and host cell immune

response in vivo, immunocompetent animal models are required. Immunocompetent hosts trigger

robust immune responses against HAdV, characterized mostly by the production of neutralizing

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antibodies. In addition T-cell responses are often generated resulting in the production of interferons.

So far, a few studies have been done to evaluate the efficacy of immunocompetent animal model against HAdVs. Syrian hamster and cotton rats are two recently established semi permissive animal models,

but in general, HAdV infection and replication is restricted to human cells. Nude mice (athymic and lack in functional T-cells) are usually used as animal models involving human xenograft studies, but these models do not allow investigation of host immune responses. Therefore, a replication competent animal model could be a milestone in the development of cancer gene therapy. Our lab recently identified that human adenovirus serotype 2 (HAdV-2) can infect and replicate in mouse cell line (NMuMG; normal mouse mammary epithelial cells), while human adenovirus serotype 12 (HAdV-12) cannot cause infection in the same cell line. It is unknown at which stage the virus infection cycle is blocked during infection with the non-growing HAdV serotypes. A few characteristics of HAdV types 2, 3 and 12 are described in the table 1. These HAdV types were used in this study for comparative analysis.

Table 1. The HAdVs used for comparative analysis. The three different species of HAdV using different host cell receptors. e.g. HAdV-12 and 2 use coxackie-adenovirus receptor (CAR), whereas HAdV-3 use desmoglein-2 (DSG)

as primary receptor. Integrin αvβ serve as secondary receptor.

Kb, kilo bases; GC%, guanine-cytosine contents.

Species Serotype Receptor Size (Kb) GC% Genes Protein Site of infection

A 12 CAR, integrin αvβ 34 46.50 16 36 Intestine

B 3 DSG-2, integrin αvβ 35 51.10 17 39 Respiratory tract

C 2 CAR, integrin αvβ 36 55.20 - 36 Respiratory tract, Liver

Life cycle of Adenovirus

To develop an immunocompetent animal model, the life cycle of adenovirus needs to be considered and

compared to immune responses. During the first stage, the virus binds to surface receptor on the target

cell and enters into the cell via endocytosis. Lysosomal degradation is prevented by a viral escape from

early endosome and delivery of genome into the nucleus. Once in the nucleus, the viral gene expression

is sequential, starting with the early genes E1-E4 and followed by the late genesL1-L5. Viral DNA

replicates and virions progeny assembles in the infected cell nucleus.

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Figure 1. Life cycle of replicative adenovirus. The cycle consists three groups of events: host-cell interactions, host-organism interactions and viral replication. Ab: antibodies; ADP: adenoviral death protein (E3); CAR: coxsackie and adenovirus receptor; CTL: cytotoxic T lymphocytes. Picture taken from Jesus et al., 2000

.

Aims of this study

The aim of this study was to investigate the viral replication in mouse epithelial cells. First we analyzed

the viral genome replication and then Ad protein expression by infecting cells with HAdV-3, HAdV-11

and HAdV-12. Secondly, in order to investigate the difference between HAdV serotypes, a comparative

bioinformatics analysis was performed to identify species specific genes by comparing HAdV-2, HAdV-3

and HAdV-12. That could increase our understanding of the tropism and the viral strategies to evade

host defense systems.

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MATERIAL AND METHODS

Bioinformatics

HAdV-2, HAdV-3 and HAdV-12 were selected for whole genome comparative bioinformatics analysis.

Complete set of proteins thought to be expressed by HAdV-2 and HAdV-12 were taken from non- redundant database (http://www.uniprot.org/), all the sequences were reviewed i.e. all the sequences are high quality and manually curated. For, HAdV-3, all ORFs were taken from GenBank. Type specific genes were identified by reciprocal BLAST (The Basic Local Alignment Search Tool) searches, using BLASTP (Protein BLAST) and TBLASTN (translated nucleotide BLAST). The final gene lists were compiled by manual curation of BLAST search result (appendix II).

Cell lines and viruses

All cells were maintained at 37 °C in an atmosphere containing 5% CO2. Normal mouse mammary epithelial cells (NMuMG) and human lung adenocarcinoma epithelial cells (A549) were maintained in Dulbecco’s modified Eagle’s medium supplemented (DMEM) with 10 % fetal bovine serum (FBS). HAdV- 3, HAdV-11 and HAdV-12 crude lysate were stored in -80 °C freezer and thawed immediately before use.

Virus titre was determined using fluorescence forming units (FFU) assay.

Plasmid encoding E1A and VA RNA were provided by our lab. NMuMG cells were plated and transfected using TurboFect (Fermentas) according to manufacturer’s instructions.

Virus infection and transfection

Ten 10 fluorescence forming units (FFU) per cells were infected in serum free medium (DMEM). After 1 hour, cells were re-fed with medium containing 2 % FBS and incubated at 37 °C in humidified air with 5%

CO

. Cells were harvested at different time points by trypsinization followed by centrifugation (4000 RPM; 4-8

C; 10 min) and cell pellet was frozen -20 °C for further DNA, RNA and protein extraction.

Oligonucleotides

Primers for PCR experiments were designed and all primers were purchase from Invitrogen. The oligonucleotides were dissolved in ddH

O to a concentration of 20 µM. In order to get 10 µM mix of each primer pair, forward and reverse primer of each ORF were mixed in a ratio of 1:1. The specificity of each primer was tested by using genomic DNA of respective adenovirus serotype (data not shown).

Cytoplasmic and Nuclear DNA extraction

The cell pellets were resuspended in 500 µl Nonidet P-40 (NP40) solution (appendix I), incubate on ice

for 10 min and centrifuged (13000 RPM; 4 °C; 10min). For cytoplasmic DNA extraction, supernatant were

collected into new tube and the same volume of phenol/chloroform was added, mixed and incubate at

room temperature (RT) for 5 min. For nuclear DNA extraction, pellet was lysed in RIPA lysis buffer was

added, mixed and incubated for 1 h at 37 °C, after which phenol/chloroform was added. After the

centrifugation at 12000 RPM; RT; 10 min, a phase separation occurred. The upper aqueous phase

(containing cytoplasmic or nuclear DNA) was collected carefully into new tube and 2x amount of 99 %

ethanol and 1/10 amount of NaCl were added. Following incubation for 1h at RT, the precipitated DNA

was recovered by centrifugation (13000 RPM; RT; 10 min).

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The pellet was air dried at RT and resuspended in 20 µl H

O. The DNA concentration was measured using a Nanodrop spectrophotometer. The DNA was stored at -80 °C.

Virus DNA extraction by Hirt method and PCR

Harvested cells were lysed by adding cell lysis buffer (500 µl), proteinase K (250 µl) and 10 % SDS (50 µl) and incubated at 37 °C for 3h. 150 µl of NaCl (5 M) was added to the lysed cells and incubated at 4 °C for 2h. Following centrifugation (13000 RPM; 4 °C; 30 min) the supernatant was collected into a new tube, to which an equal volume of phenol was added. After vortexing and centrifugation (13000 RPM; 4 °C; 10 min) the phenol extraction step was repeated once, with a phenol/chloroform mixture 50:50. Finally, any residual phenol was extracted by an equal volume of chloroform. The aqueous phase was collected into a new tube and 2x 88 % ethanol was added and incubated at -20 °C for overnight. After a final centrifugation step (13000 RPM; 4 °C; 10 min), the supernatant was removed and the pellet air-dried at RT.

For PCR: 20 ng/µl of DNA and 0.4 µl of a 10 µM primer pair were added to PCR mix (appendix I) to a reaction volume of 20 µl. The PCR program was set to 5 min denaturation period at 95 °C followed by 30 cycles at 95 °C for 20 sec, 60 °C for 20 sec, and 72 °C for 20 sec. Extension was run for 3 min at 72 °C. PCR products were analyzed on 1.2 % agarose gel (appendix I). 100 kb ladder was used for determination of product sizes.

RNA isolation and RT-PCR

NMuMG and/or A549 cells infected with Ad were grown on 20 cm diameter plate, collected at indicated time points (Figure 6). All steps were performed on ice. The cells were washed with PBS and lysed in IsoB/NP40 (0.65 %). The cells were collected by the help of scraper and transfer into new eppendorf tubes followed by 20 sec vortexing and incubation on ice for 5 min. After centrifugation (11500 RPM;

4 °C; 5 min) the supernatant was transferred into a new tube. 130 µl 5xRPS (appendix I) and 600 µl

phenol was added followed by vortexing and centrifugation (13000 RPM; RT; 5 min). The upper phase

was carefully collected and 600 µl phenol was added and the tube was centrifuged (13000 RPM; RT; 5

min). The upper phase was collected again into new tube and 600 µl chloroform-isoamyl alcohol 24:1

was added and centrifuged at 13000 RPM, RT, 5 min. RNA was precipitated from aqueous phase by

adding 600 µl isopropanol and 15 µl NaCl, vortex and incubated for 30 min at -20 °C. RNA was collected

by centrifugation (12000 RPM; 4 °C; 10 min). 2 µg of cytoplasmic RNA was used to synthesize cDNA using

Superscript II Reverse transcriptase (Invitrogen) according to the manufacturer instructions.

9 Protein extraction, SDS-PAGE and Western Blot

Cells pellets were lysed in 100 µl of cell lysis buffer (appendix I) and incubated for 30 min on ice. After

centrifugation (12000 RPM; 4 °C; 10 min) supernatant was collected into a new tube. After addition of

25 µl 5x loading dye (appendix I) and boiling for 5 min, the protein was stored at -20 °C. The denatured

protein samples were separated in 10 % SDS-PAGE gel (appendix). The proteins were transferred to a

PVDF membrane (Millipore). After the transfer, membrane was incubated in Odyssey blocking buffer (LI-

COR Biosciences) for 1 h, washed three times for 5 min with PBS-1 % Tween 20 (1-3 %) and probed with

monoclonal antibody directed against E1A (diluted in 1:2000 in PBS 1 % BSA) for 2 hours After the

membrane was washed 3 times for 5 min with PBS 1 % Tween 20, followed by incubation with

secondary antibody (Odyssey). The membrane was washed again with PBS 1 % Tween20 and was

scanned on the Odyssey infrared imaging system by Odyssey 2.1 software (LI-COR Biosciences).

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RESULTS

Differential distribution of genes

Most HAdVs genomes are fairly similar and most genes/proteins are conserved. To identify most promising gene that could be involved in replication specificity or specific immune response in HAdV, a bioinformatics analysis was performed of the type 2 (HAdV specie C), 3 (HAdV specie B) and 12 (HAdV specie A).

Comparison of HAdV-2, 12, and 3 using BLASTP and TBLASTN searches showed a significance sequence divergence in E3 glycoprotein. In addition, all the 3 Ads contain type specific glycoprotein or death protein specific for each serotype (Table 2 and appendix II). Comparisons among the three HAdVs types revealed the presence of type specific gene. The HAdV-2 has 6 specific genes, while HAdV-3 and HAdV- 12 have 4 and 2, respectively (Table 2). The majority of identified specific genes have no sequence similarity to known sequences and their function is therefore unknown. In total 12 HAdVs open reading frames displayed weak hits (E-value higher than 1e-20) for domains or signatures and/or existence in other organisms in the BLAST searches were performed at NCBI non redundant database (Figure 2, Table 2, Appendix II). HAdV-2 and HAdV-12 both contain a specie specific isoform of early E1A gene (appendix II). Unique genes shared between two types were compiled in Table 3.

Figure 2. Shared and non-shared gene content comparison. Venn diagram display shared and non-

shared genes content of HAdV serotype 2, 3 and 12. The intersection shows the shared gene content

among 2 or three genomes. BLASTP and TBLASTN searches were used to identify specific genes. 45

represent core gene content of HAdV. HAdV-2 contains 6 serotype specific genes, 4 genes specific for

HAdV-3 and 2 genes for HAdV-12. HAdV-2 and HAdV-3 shared 4 genes that were non-existent in HAdV-

12. One gene is shared between HAdV-12 and HAdV-2.

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Table 2. A List of identified unique genes. Type gene identity (with link), protein names, gene name and length (amino acid) are indicated number.

Type Gene ID Protein Names Gene names Length

HAdV-2 P24935 Early E3A 11.6 kDa glycoprotein 101

HAdV-2 P03289 Uncharacterized protein F-112 112

HAdV-2 P03294 Uncharacterized protein F-121 121

HAdV-2 P03292 Uncharacterized protein C-168 168

HAdV-2 P03285 Uncharacterized protein D-172 172

HAdV-2 P03286 Uncharacterized protein E-95 95

HAdV-12 YP_002640220.1 Membrane glycoprotein E3 CR1-alpha (unknown) E3 264 HAdV-12 YP_002640221.1 Membrane glycoprotein E3 CR1-beta (unknown) E3 252 HAdV-3 YP_002213787.1 Membrane glycoprotein E3 CR1-alpha E3 146 HAdV-3 YP_002213789.1 Membrane glycoprotein E3 CR1-beta E3 179 HAdV-3 YP_002213790.1 Membrane glycoprotein E3 CR1-gamma E3 189 HAdV-3 YP_002213791.1 Membrane protein E3 CR1-delta E3 77

Table 3. Unique shared gene content between 2 serotypes. In this comparison, HAdV-2 and HAdV-3 share 4 genes while HAdV-2 and 12 share only one gene. No significant sharing was found between HAdV-12 and 3.

Shared GeneID Protein Name Length Gene ID Protein Name Length Ad2-Ad3 P68978 Early E3 18.5 kDa glycoprotein 159 P11323 Early E3 18.5 kDa glycoprotein 172 Ad2-Ad3 P0DJX2 U exon protein (UXP) 217 Q2KSJ3 U protein 53 Ad2-Ad3 P03290 Uncharacterized protein E-115 115 DQ086466.1

Ad2-Ad3 P03291 Uncharacterized protein F-215 215 Q2KSK5 Hypothetical 12.6 kDa protein 111 Ad2-Ad12 P03293 Uncharacterized protein B-137 137 X73487.1

12 Virus replication in infected NMuMG cells

To screen for mouse animal model that might be permissive to HAdV-3, 11 and 12 infection, 2 different primary cell cultures from human and mouse was established and infected with HAdV-3, 11, 12. Earlier work from our group showed that, in contrast to HAdV-12, HAdV-2 is able to infect mouse mammary epithelial cells.

In order to understand why some HAdVs lack activity in murine cells, we analyzed the amount viral DNA and to investigate at which step the HAdVs infectious cycle was blocked.

The amount of adenovirus viral DNA (divided in cytoplasmic and nuclear DNA extract) extracted from human (A549) and mouse (NMuMG) cell cultures 1, 2, 4, 12, 24 and 48 h postinfection were measured by PCR using E1A region primers (Figure 3). As positive control, HAdV-3 and 11 replication were analyzed in human A549 cells, which showed no substantial increase in the virus DNA. In contrast, Mouse cells (NMuMG) infected from HAdV3 shows no increased in viral DNA at 4, 12 and 24 h postinfection while NMuMG cells infected with HAdV-11 shows enter into the cytoplasm followed by nuclear entry but at 4, 12 and 24 h postinfection it revealed low amount of viral DNA.

Figure 3. HAdV-3 and 11 activity in NMuMG and A549 cells. L and M represent 100bp ladder and uninfected control respectively. PCR products from cytoplasmic and nuclear fractions are shown at different time after infection (Hours).

We redesigned new primers for HAdV-12, HAdV-3 and HAdV-11 E1A primers to confirm the results we

got above (Figure 3). PCR analysis was done using new E1A type specific primers (Appendix I). The PCR

gels (Figure 4) showed the results for each HAdV type. It’s not known why HAdV-3 and HAdV-11

cytoplasmic fractions showed strong bands at 48 and 72 hours after infection on PCR gel, probably

because the contamination between cytoplasmic and nuclear fractions. However, we also found two

strong bands in HAdV-12 nuclear fraction at 48 and 72 hours after infection. This is despite that previous

results showed that, HAdV-12 neither infects nor replicate in NMuMG cells

.

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Figure 4. The course of HAdV-3, HAdV-11, HAdV-12 infection in NMuMG cells by detecting E1A gene in cytoplasmic and nuclear extracts A) NMuMG cells infected with HAdV-3, B) NMuMG cells infected with HAdV-11, C) NMuMG cells infected with HAdV-12. E1A specie specific primers were used in each experiment. M represents uninfected mock.

In order to check expression of E1A gene in NMuMG cells, whether it increased permissivity of the HAdV infection in NMuMG cells or whether E1A helps HAdV-11 infection or not transfection complementation analysis was done. NMuMG cells were transfected with plasmids encoding (type 2) E1A or (type 2) VA RNA and followed by infection with HAdV-11 at indicated time points. PCR using (type 2) E1A primer revealed strong bands on PCR gels, when HadV-11 transfected with E1A or VA RNA in NMuMG cells (Figure 5), there was a marked increase in the amount of viral DNA.

We then examined HAdV early protein expression using E1A monoclonal antibody. Western blot showed that there was almost absence of early protein expression in all three HAdV (data not shown).

Quantitative PCR (Figure 6) produced very low CT value in all cases. These results suggest that E1A gene was transcribed but we could not confirm E1A translation in NMuMG cells due to high concentration of cDNA in our RT PCR experiment.

Figure 5. PCR analysis of the E1A gene in NMuMG cells transfected with plasmid expressing the E1A or

VA RNA; cells were infected with HAdV-11 at indicated time points.

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Figure 6. RT-PCR analysis of the expression of E1A in NMuMG cells transfected with plasmid expressing E1A or VA RNA; cells were infected with HAdV-11. 10FFU cells were transfected with E1A or VA RNA followed by infection with HAdV-11. Total cellular RNA was extracted from infected cells at indicated time point. Two microgram was reverse transcribed and 50 ng of cDNA was then subject to quantitative PCR using E1A (type 2) region primers. Uninfected mocked was used as a control.

13,00 13,50 14,00 14,50 15,00 15,50

36 hours 48 hours 60 hours

C(t) Mean

E1A expression of HAdV-11

E1A+VA RNA HAdV-11 E1A VA RNA

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DISCUSSION

The genome of different HAdVs are relatively similar in term of core gene content, but tend to differ with respect to genes responsible for antigenicity. In this study, comparative genomic analysis of HAdV- 2, HAdV-3 and HAdV-12 revealed that the membrane glycoprotein or Ad death proteins and hypothetical proteins were quite divergent. These results seem promising and candidate virus species- specific genes can be characterized in future and their impact on virus proliferation and host cell responses might improve the understanding of Ad host cell interactions.

The Ad genome contains a small fraction of hypothetical genes that code for protein of unknown function. The lack of significant hits to other virus genomes likely shows strong adaptation of virus to its host environment. Since a large number of adenovirus sequences are available and database continuously growing, several adenovirus proteins will likely to assign orthologs and annotate, but experimental efforts will be required to determine the function of these proteins.

Comparative analysis of HAdVs shows that E3 glycoproteins were quite divergent from each other. Low amino acid sequence identity were found in the E3 glycoprotein of human adenovirus that may suggest distinctive roles for these proteins.

Moreover structural homology searches of E3 glycoprotein did not identify any structure that align well with HAdV-2 E3 glycoprotein. This suggest that E3 glycoprotein may have a unique structure.

It is true that genomic data cannot alone resolve the question of host cell specificity and virus proliferation, but genomic sequence data provide an evolutionary insight into how virus genomes have been changed over the course of evolution. In addition, the identification of specie specific novel genes and gene variants will provide candidate for future functional studies.

Immunocompetent animal model are very important for the development and assessment of biological therapies. Specially, the efficacy of oncolytic viruses is directly affected by the host immune response. So far, there is no model organism available to study replication competent oncolytic adenovirus and immune response of the host. Our lab recently identified NMuMG, a non-transformed epithelial mammary mouse cell line as permissive for HAdV-2 replication but not to HAdV-12.

In addition, NMuMG cells support replication of HAdV-2 nearly as efficiently as A549 cells. The replication of HAdV- 11 and HAdV-3 was compared in A549 cells (Figure 3). No viral replication was detected in A549 cells. It could be due to short post infection time. In this report, we tested HAdV-3, HAdV-11 and HAdV-12 replication and infection specificity in NMuMG cells. We found that, HAdV-3 and HAdV-11 were able to enter NMuMG cells, but that no replication of DNA was found. However, when we use serotype specific E1A primer (appendix I), HAdV-3 and HAdV-11 cytoplasmic fraction show strong band on PCR gel after 48 h and 72 h of infection (Figure 4). It might due to contamination between cytoplasmic and nuclear fraction, though, we measured the amount of cells and virus used for each experiment, before every infection. But still it is difficult to quantify the exact amount of infected and non-infected cells.

E1A complementation efficiency was tested in NMuMG cells transfected with either the E1A encoding

plasmid or the VA RNA encoding plasmid, or both plasmids together. Figure 5 (left gel) showed strong

bands for E1A and VA RNA suggesting that complementation assay was efficient.

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We ran western blots to detect E1A protein, but we found uniform high background across the membrane, it could be due to high concentration of antibody. We also ran a RT-PCR but CT value was very low, this could be overcome by dilution cDNA (Figure 6).

In conclusion, there is genetic variation between adenovirus species and small number of type specific

genes have been identified. Future experiment could include characterization and structural homology

modeling of unique genes. Moreover, HAdV-3 and HAdV-11 can enter into the nucleus of NMuMG cells.

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ACKNOWLEDGMENTS

I would like to thanks to my supervisor Prof. Catharina Svensson for guideline, support, encouragement,

and precious time. Many thanks to Troy, Daniel and everyone in adenovirus group member for help,

support and friendliness.

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Proc Natl Acad Sci U S A 1999, 96(22):12816-12821.

11. Giberson AN, Davidson AR, Parks RJ: Chromatin structure of adenovirus DNA throughout infection. Nucleic Acids Res 2012, 40(6):2369-2376.

12. Gurlevik E, Schache P, Goez A, Kloos A, Woller N, Armbrecht N, Manns MP, Kubicka S, Kuhnel F: Meganuclease-mediated Virus Self-cleavage Facilitates Tumor-specific Virus Replication. Mol Ther 2013.

13. Alemany R, Balague C, Curiel DT: Replicative adenoviruses for cancer therapy. Nat

Biotechnol 2000, 18(7):723-727.

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14. Miura Y, Yamasaki S, Davydova J, Brown E, Aoki K, Vickers S, Yamamoto M: Infectivity- selective oncolytic adenovirus developed by high-throughput screening of adenovirus- formatted library. Mol Ther 2013, 21(1):139-148.

15. Thomas MA, Spencer JF, La Regina MC, Dhar D, Tollefson AE, Toth K, Wold WSM: Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors. Cancer Research 2006, 66(3):1270-1276.

16. Ganly I, Mautner V, Balmain A: Productive replication of human adenoviruses in mouse epidermal cells. J Virol 2000, 74(6):2895-2899.

17. Wang H, Li ZY, Liu Y, Persson J, Beyer I, Moller T, Koyuncu D, Drescher MR, Strauss R, Zhang XB et al: Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14. Nat Med 2011, 17(1):96-104.

18. 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.

19. Sirena D, Ruzsics Z, Schaffner W, Greber UF, Hemmi S: The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3. Virology 2005, 343(2):283-298.

20. Roberts RJ, Akusjarvi G, Alestrom P, Gelinas RE, Gingeras TR, Sciaky D, Pettersson U: A Consensus Sequence for the Adenovirus-2 Genome. In: Adenovirus DNA. Edited by Doerfler W, vol. 8: Springer US; 1986: 1-51.

21. Alestrom P, Akusjarvi G, Pettersson M, Pettersson U: DNA sequence analysis of the region encoding the terminal protein and the hypothetical N-gene product of adenovirus type 2. J Biol Chem 1982, 257(22):13492-13498.

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20

APPENDIX I. List of PCR primers, master mix, reagents, stock volume and concentration

Primers used for PCR experiments. Direction 5’ to 3’.

HAdV-2: E1A-F atgagacatattatctgccac HAdV-2: E1A-R ttacagactcgggaaaaatct HAdV-12: E1A-F atgagaactgaaatgactccc HAdV-12: E1A-R cattcaccgcctgttcattat HAdV-3: E1A-F atgagacacctgcgcttcctg HAdV-3: E1A-R ttcacagcttcctcattggga HAdV-11: E1A-F atgagagatttgcgatttctg HAdV-11: E1A-R agtatcaaaagtgtccaaagg

Cell lysis buffer

Reagents Final concentration

NaCl 100 mM

Tris-HCl (pH 8.0) 10 mM

EDTA (pH 8.0) 25 mM

SDS 0.5 %

Proteinase K 250 µg/ml

RIPA buffer

Reagents Final concentration

NaCl 150 mM

Tris-HCl (pH 7.4) 50 mM

deoxycholate 1 %

Tween-20 1 %

Protease inhibitor (Roche) 1 (Tablet)

PCR Mix preparation (20 µl)

Reagents Stocks Volume

H2O 14 µl

5xHF 4 µl

10mMdNTP 0.4 µl

Primer A 0.2 µl

Primer B 0.2 µl

Template 1 µl or 20ng/ µl

DMSO 0.6 µl

Polymerase 0.2 µl

2X loading dye preparation (10 ml)

21

Reagents Stocks Volume

Tris-HCl pH6.8 1ml

10 % SDS 4 ml

Glycerol 3.75 ml

BPB 250 µl

DDT 1M 1 ml

10% and 15% separating gel Preparation (10 ml)

Reagents Stocks volume (10%) Stocks volume (15%)

H2O 4 ml 3.3 ml

30% Acrylamide solution (Bio-Rad) 3.3 ml 4ml

1.5 M Tris (pH 8.8) 2.5 ml 2.5 ml

10% SDS 100 µl 100 µl

10% Ammonium persulphate 100 µl 100 µl

TEMED 4 µl 4 µl

5% Stacking gel Preparation (5 ml)

Reagents Stocks volume

H2O 3.4 ml

30% Acrylamide solution (Bio-Rad) 830 µl

1.5 M Tris (PH 6.8) 630 µl

10% SDS 50 µl

10% Ammonium persulphate 50 µl

TEMED 5 µl

1.2% agarose gel Preparation (100 ml)

Reagents Stocks volume

Agarose 1.2 g

0.5X TBE buffer 100 ml

Gel red 5 µl

5X TBE buffer Preparation (1000 ml)

Reagents Stocks volume

H2O 900 ml (raise volume to 1 lL)

Tris base 54 g

Boric acid 27.5 g

EDTA (pH 8.8) 20 ml

22

APPENDIX II. The final reciprocal BLAST search results list compiled by manual curation

Table 4. HAdV-2 searched against HAdV-12 and HAdV-3.

Ad2

Gene ID Protein Names Gene names Leng th Ad12

Gene ID Protein names Gene

names Lengt

h Identi

ty Score E-value Ad3 Gene ID Protein Names Gene names Length Iden

tity Score E-value

P03252 Protease L3 204 P09569 Adenain 206 75% 857 1E-115 P10381 Adenain 209 80% 872 1E-118

P03264 Early E2A DNA-binding protein DBP 529 P04498 Early E2A DNA-binding

protein DBP 484 50% 1,594 1E-156 Q2Y0H2 DNA binding protein (E2A DNA

binding protein DBP) E2A 517 54% 1,777 1E-175 P03263 I-leader protein 145 P36704 Probable DNA-binding

protein 205 43% 290 2E-30 Q2Y0I5 Probable DNA binding

agnoprotein 198 48% 331 9E-37

P03261 DNA polymerase POL 1056 P06538 DNA polymerase POL 1,061 77% 5,425 0 Q2Y0I9 DNA polymerase E2B 1,122 78% 5,540 0 P03254 Early E1A 32 kDa protein 289 P03259 Early E1A 29.5 kDa protein 266 40% 470 1E-54 Q8JSK4 29.1 kDa protein E1A 261 36% 458 5E-53 P03254-

2

Isoform early E1A 26 kDa protein of Early

E1A 32 kDa protein E1A 243 P03259-2

Isoform Early E1A 26 kDa protein of Early E1A 29.5 kDa protein

235 37% 299 3E-30 Q8JSK3 25 kDa protein E1A 230 36% 323 8E-34

P03254- 3

Isoform early E1A 6 kDa protein of Early

E1A 32 kDa protein E1A 55 No hits found, Specie specific gene No hits found

P03244 E1B protein, large T-antigen E1B 495 P04491 E1B protein, large T-antigen 482 48% 1,518 1E-148 Q2Y0J2 E1B 55-kDa E1B 492 53% 1,817 1E-180 P03244-

2

Isoform E1B-155R of E1B protein, large T- antigen (Isoform E1B-18K of E1B protein, large T-antigen) E1B

155 P04491

E1B protein, large T- antigen (E1B 55 kDa protein) (E1B-55K)

482 55% 247 3E-22 Q2Y0J2 E1B 55-kDa (Large T antigen)

E1B 492 65% 307 1E-30

P03244- 3

Isoform E1B-92R of E1B protein, large T- antigen (Isoform E1B-16K of E1B protein, large T-antigen) E1B

92 No hits found No hits found

P03244- 4

Isoform E1B-82R of E1B protein, large T- antigen (Isoform E1B-15K of E1B protein, large T-antigen) E1B

82 No hits found No hits found

P03247 E1B protein, small T-antigen (E1B 19 kDa

protein) (E1B-19K) (E1B-175R) E1B 175 P04492

E1B protein, small T- antigen (E1B 19 kDa protein) (E1B-19K)

163 47% 376 1E-43 Q2Y0J3 19 kDa small T antigen E1B 178 50% 408 3E-48

P15133 Early E3B 10.4 kDa protein 91 P36705 Early E3B 10.4 kDa protein 91 44% 210 1E-20 P11318 Early E3B 10.4 kDa protein 91 51% 257 9E-28

P24935 Early E3A 11.6 kDa glycoprotein 101 No hits found No hits found

P27311 Early E3A 12.5 kDa protein 107 P36706 Early E3A 12.1 kDa protein 105 61% 360 5E-43 P11319 Probable early E3 12.1 kDa

glycoprotein 106 55% 318 1E-36

P68976 Early E3B 14 kDa protein 128 Q65288 E3B 14.7 KD protein 128 48% 293 4E-32 P11315 Early E3 15.3 kDa protein 136 49% 324 9E-37 P03250 Early E3B 14.5 kDa protein 130 P36707 Early E3B 12.7 kDa protein 110 33% 158 6E-12 P11316 Early E3B 14.5 kDa protein 134 37% 232 8E-23 P68978 Early E3 18.5 kDa glycoprotein (E3-19K)

(E3gp 19 kDa) (E19) (GP19K) 159 No hits found P11323 Early E3 18.5 kDa glycoprotein 172 32% 201 2.E-17

P03241 Probable early E4 11 kDa protein 116 P36708 Probable early E4 11 kDa

protein 116 45% 275 9E-30 Q2Y0F3 11 kDa protein E4 117 49% 253 2.E-26

P03240 Probable early E4 13 kDa protein 114 P36709 Probable early E4 13 kDa

protein 120 40% 247 2E-25 Q2Y0F5 13.6 kDa protein E4 122 45% 275 1E-29

P03238

Probable early E4 17 kDa

protein [Cleaved into: Early E4 10 kDa protein]

150 P36710 Early E4 34 kDa protein 291 46% 98 3E-02 Q2Y0F7 E4 ORF6/7 E4 141 48% 336 4E-38

P03242 Early 31 kDa protein 283 S10861 hypothetical protein (96%) 127 52% 115 5E-36 Q2Y0F1 13.9 kDa protein E4 125 46% 258 6E-25 P0DJX0 Early 4 ORF2 protein 130 S10863 hypothetical protein 131 48% 135 1E-43 Q2Y0F2 E4 ORF2 E4 144 32% 177 2E-14 P03239 Early E4 34 kDa protein 294 P36710 Early E4 34 kDa protein 291 55% 873 1E-115 Q2Y0F6 33.2 kDa protein E4 299 59% 958 1E-128 P03275 Fiber protein L5 582 P36711 Fiber protein (pIV) PIV 587 34% 1,004 1E-89 EF176023.

1 fiber portein 286 30% 295 5E-22

P03279 Pre-capsid vertex protein L1 585 P36712 Peripentonal hexon- 582 73% 2,533 0 Q2Y0I0 L1 protein pIIIa L1 588 75% 2,712 0

23

associated protein (Protein IIIa) PIIIA

P03280 Pre-hexon-linking protein L4 227 P36713 Hexon-associated protein

(Protein VIII) (pVIII) PVIII 233 75% 931 1E-126 P11324 Hexon-associated protein PVIII 135 78% 549 2E-69 P03282 Hexon-interlacing protein IX 140 P03284 Hexon-associated protein

(Protein IX) (pIX) PIX 144 56% 365 1E-42 P68970 Hexon-associated protein PIX 138 51% 328 4E-37 P03277 Hexon protein (CP-H) (Protein II) L3 968 P19900 Hexon protein (Late protein

2) PII 919 76% 5294 0 P36849 Hexon protein (Late protein 2)

PII 944 76% 5294 0

P24932

Shutoff protein (100 kDa protein) (p100K) (100K-chaperone protein) (L4-100K) (Shutoff protein 100K) L4

805 P36714 Late 100 kDa protein 782 66% 3118 0 Q2Y0H1 100 kDa hexon-assembly

associated protein L4 828 64% 3167 0

P14269 Pre-core protein X L2 80 P35986

Late L2 mu core protein (11 kDa core protein) (Protein X) (pX) (pMu) PX

72 64% 280 3E-34 Q2Y0H6 L2 protein pX L2 75 69% 280 3E-34

P0DJX1 Packaging protein 2 (Packaging protein

22K) (L4-22K) L4 195 Q2Y0G9 22 kDa protein (L4 22-kDa

protein) L4 199 50% 427 2E-50 X73487.1 hypothetical protein 55% 121 1E-33

P03262 Packaging protein 3 (L1-52/55 kDa

protein) (Packaging protein 52K) L1 415 P36715 Late L1 52 kDa protein 373 75% 1,687 1E-168 Q2Y0I1 L1 52/55-kDa protein L1 385 70% 1,723 1E-172 P03276 Penton protein (Penton base protein)

(Virion component III) (pIII) PIII 571 P36716

Penton protein (Penton base protein) (Virion component III) (pIII) PIII

497 68% 1,415 1E-135 Q2Y0H9 L2 protein III (Penton base) L2 544 70% 2,364 0

P03274

Pre-protein VI (pVI) [Cleaved into:

Endosome lysis protein; Protease cofactor (pVI-C)] L3

250 P35988

Minor capsid protein 6 (Minor capsid protein VI) [Cleaved into: Protease cofactor] PVI

265 64% 814 1E-107 Q2Y0H5 L3 protein pVI (Protein VI) L3 250 63% 823 1E-109

P03272 Packaging protein 1 (Packaging protein

IVa2) IVa2 449 P12540 Maturation protein (Protein

IVa2) PIVA2 452 76% 2,270 0 Q2Y0J0 Maturation protein IVa2 IVa2 448 82% 2,411 0

P03269 DNA terminal protein (Bellett protein)

(pTP protein) PTP 671 P12541

DNA terminal protein (Bellett protein) (pTP protein) PTP

606 77% 2,488 0 Q2Y0I4 E2B pTP E2B 640 80% 2,749 0

P24939 Splicing factor (Splicing factor 33K) (L4-

33K) L4 228 X73487.1 promoter region EII (query

cover44%) 87% 155 2E-35 Q2Y0H0 L4 33-kDa protein L4 274 46% 511 1E-61

P68950

Pre-histone-like nucleoprotein (Pre-core protein VII) (pVII) [Cleaved into: Histone- like nucleoprotein (NP) (Core protein VII)]

L2

198 X73487.1 (query cover57%) 92% 174 3E-35 Q2Y0H8 L2 protein pVII L2 192 69% 656 3E-85

P03267 Core-capsid bridging protein L2 369 P36717 Minor core protein (Protein

V) (pV) PV 347 57% 1,203 1E-116 Q2Y0H7 L2 protein pV (Protein V) L2 349 61% 1058 1E-141

P0DJX2 U exon protein (UXP) 217 No hits found Q2KSJ3 U protein U 53 60% 174 5E-14

P03289 Uncharacterized protein F-112 112 No hits found specie specific gene

P03290 Uncharacterized protein E-115 115 No hits found in Ad12 DQ08646

6.1 (query cover 78%) 41% 53.9 2E-11

P03287 Uncharacterized 11.6 kDa early protein 106 X73487.1 (query cover100%) 55% 144 4E-28 Q2Y0I7 Putative uncharacterized

protein 106 72% 357 1E-42

P03294 Uncharacterized protein F-121 121 No hits found specie specific gene

P03293 Uncharacterized protein B-137 137 X73487.1 (query cover77%) 60% 92 3E-11 no hits found

P03292 Uncharacterized protein C-168 168 No hits found specie specific gene P03285 Uncharacterized protein D-172 172 No hits found specie specific gene

P03291 Uncharacterized protein F-215 215 No hits found in Ad12 or Specie A, hits in specie C, B, D, E Q2KSK5 Hypothetical 12.6 kDa protein

E2B 111 40% 113 4E-04

P03286 Uncharacterized protein E-95 95 No hits found specie specific gene

24

Table 5. The final reciprocal BLAST search results list compiled by manual curation. HAdV-12 searched against HAdV-2 and HAdV-3.

Ad 12

Gene ID Protein Names Gene names Leng th Ad2

Gene ID Protein names Gene names Leng th Ident

ity Score E-value Ad3 Gene ID Protein Names Gene

names Length Identity Score E-value P06538 DNA polymerase (EC 2.7.7.7) POL 1,061 P03261 DNA polymerase (EC 2.7.7.7) POL 1,056 77% 4,472 0 Q2Y0I9 DNA polymerase E2B 1,122 75% 4,354 0

P36712 Peripentonal hexon-associated

protein (Protein IIIa) PIIIA 582 P03279

Pre-capsid vertex protein (Capsid vertex-specific component IIIa) (CVSC) (Protein IIIa) (pIIIa) [Cleaved into: Capsid vertex protein] L1

585 73% 2,122 0 Q2KSK0 Protein IIIa L1 588 73% 2,169 0

P19900 Hexon protein (Late protein 2) PII 919 P03277 Hexon protein (CP-H) (Protein II)

L3 968 76% 3,923 0 P36849 Hexon protein (Late protein

2) PII 944 78% 3,922 0

P36714 Late 100 kDa protein 782 P24932

Shutoff protein (100 kDa protein) (p100K) (100K-chaperone protein) (L4-100K) (Shutoff protein 100K) L4

805 65% 2,563 0 Q2KSI3 100 kDa hexon-assembly

associated protein L4 824 64% 2,609 0

P36715 Late L1 52 kDa protein 373 P03262

Packaging protein 3 (L1-52/55 kDa protein) (Packaging protein 52K) L1

415 70% 1,401 0 Q2KSK1 55 kDa protein L1 385 73% 1,412 0

P36716 Penton protein (Penton base protein)

(Virion component III) (pIII) PIII 497 P03276 Penton protein (CP-P) (Penton

base protein) (Protein III) L2 571 69% 1,971 0 Q2Y0H9 L2 protein III (Penton base) L2 544 73% 2,046 0 P12540 Maturation protein (Protein IVa2)

PIVA2 452 P03272 Packaging protein 1 (Packaging

protein IVa2) IVa2 449 77% 1,869 0 Q2Y0J0 Maturation protein IVa2 IVa2 448 78% 1,875 0 P12541 DNA terminal protein (Bellett protein)

(pTP protein) PTP 606 P03269 DNA terminal protein (Bellett

protein) (pTP protein) PTP 653 77% 2,488 0 Q2Y0I4 E2B pTP E2B 640 77% 2,466 0

P04498 Early E2A DNA-binding protein DBP 484 P03264 Early E2A DNA-binding protein

DBP 529 50% 1,328 1E-177 Q2Y0H2 DNA binding protein E2A 517 50% 1,312 1.E-175

P04491 E1B protein, large T-antigen (E1B 55

kDa protein) (E1B-55K) 482 P03244 E1B protein, large T-antigen E1B 495 48% 1,224 1E-162 Q2Y0J2 E1B 55-kDa (Large T antigen)

E1B 492 47% 1,198 1.E-158

P36717 Minor core protein (Protein V) (pV) PV 347 P03267 Core-capsid bridging protein

(Core protein V) L2 369 57% 998 1E-132 Q2Y0H7 L2 protein pV L2 349 59% 975 1E-129

P36713 Hexon-associated protein (Protein

VIII) (pVIII) PVIII 233 P03280

Pre-hexon-linking protein (Pre- protein VIII) (pVIII) [Cleaved into:

Hexon-linking protein-N (12.1 kDa protein VIII) (Protein VIII-N);

Hexon-linking protein-C (7.6 kDa protein VIII) (Protein VIII-C)] L4

227 75% 931 1E-126 P11324 Hexon-associated protein

(Protein VIII) (pVIII) PVIII 135 74% 523 2E-65

P36711 Fiber protein (pIV) PIV 587 P03275 Fiber protein (SPIKE) (Protein IV)

L5 582 35% 932 1E-116 P04501 Fiber protein (pIV) PIV 319 37% 190 2E-13

P09569

Adenain (EC 3.4.22.39) (Endoprotease) (Late L3 23 kDa protein)

206 P03252

Protease (EC 3.4.22.39) (Adenain) (Adenovirus protease) (AVP) (Adenovirus proteinase) (Endoprotease) L3

204 75% 857 1E-115 P10381

Adenain (EC 3.4.22.39) (Endoprotease) (Late L3 23 kDa protein)

209 80% 871 1E-117

P36710 Early E4 34 kDa protein 291 P03239 Early E4 34 kDa protein 294 55% 873 1E-115 Q2KRZ1 34 kDa protein E4 294 55% 873 1E-115 P35988

Minor capsid protein 6 (Minor capsid protein VI) [Cleaved into: Protease cofactor] PVI

265 P03274

Pre-protein VI (pVI) [Cleaved into:

Endosome lysis protein; Protease cofactor (pVI-C)] L3

250 64% 814 1E-107 Q2Y0H5 L3 protein pVI L3 250 64% 818 1E-108

YP_0026

40218.1 core protein precursor pVII L2 188 P68950

Pre-histone-like nucleoprotein (Pre-core protein VII) (pVII) [Cleaved into: Histone-like nucleoprotein (NP) (Core protein VII)] L2

198 68% 613 8E-79 Q2KSJ8 Protein VII L2 192 74% 670 1E-87

25

P03259 Early E1A 29.5 kDa protein 266 P03254 Early E1A 32 kDa protein 289 40% 470 9E-55 Q8JSK4 29.1 kDa protein E1A 261 42% 478 3.E-56 NP_5977

83.2 protein 33K (RNA splicing) L4 205 P24939 Splicing factor (Splicing factor

33K) (L4-33K) L4 228 48% 423 2E-49 Q2KSI2 33 kDa protein L4 230 49% 416 3E-48

P04492 E1B protein, small T-antigen 163 P03247

E1B protein, small T-antigen (E1B 19 kDa protein) (E1B-19K) (E1B- 175R) E1B

175 47% 376 1E-43 Q2Y0J3 19 kDa small T antigen (E1B

21-kDa) E1B 178 41% 352 6.E-40

P36706 Early E3A 12.1 kDa protein 105 P27311 Early E3A 12.5 kDa protein 107 61% 360 5E-43 P11319 Probable early E3 12.1 kDa

glycoprotein 106 67% 395 2E-48

P03284 Hexon-associated protein (Protein IX)

(pIX) PIX 144 P03282 Hexon-interlacing protein (Protein

IX) IX 140 56% 365 1E-42 P68970 Hexon-associated protein

(Protein IX) (pIX) PIX 138 58% 352 1E-40 YP_0026

40223.1 control protein E4orf2 (unknown) E4 131 P03242-2 Isoform early 14 kDa protein-1 of

Early 31 kDa protein 136 48% 344 9E-40 Q2KSI6 14.3 kDa protein E4 129 31% 154 4E-11

P03259-3 Isoform Early E1A 22 kDa protein of

Early E1A 29.5 kDa protein 222 P03254 Early E1A 32 kDa protein 289 42% 364 2E-39 Q8JSK4

29.1 kDa protein (E1A 13S protein) (E1A 13s 28 kDa protein) E1A

261 44% 380 3.E-42

P35986 Late L2 mu core protein (11 kDa core

protein) (Protein X) (pX) (pMu) PX 72 P14269

Pre-core protein X (pX) (11 kDa core protein) (Protein mu) (pMu) [Cleaved into: Core protein X] L2

80 64% 280 3E-34 Q2Y0H6 L2 protein pX L2 75 63% 255 3E-30

YP_0026 40224.1

control protein E4orf1 (unknown,

transformation) E4 127 P03242-3 Isoform early 14 kDa protein-2 of

Early 31 kDa protein 128 53% 299 4E-33 Q2Y0F1 13.9 kDa protein (E4 ORF1) E4 125 50% 319 4E-36 Q65288 E3B 14.7 KD protein; 128 P68976 Early E3B 14 kDa protein 128 48% 293 4E-32 P11315 Early E3 15.3 kDa protein 136 56% 354 2E-41 P03259-2 Isoform Early E1A 26 kDa protein of

Early E1A 29.5 kDa protein 235 P03254 Early E1A 32 kDa protein 289 34% 310 2E-31 Q8JSK3

25 kDa protein (E1A 12S protein) (E1A 12s 25 kDa protein) E1A

230 39% 363 7.E-40

P36704 Probable DNA-binding protein

(Agnoprotein) 205 P03263 I-leader protein 145 43% 290 2E-30 Q2Y0I5 Probable DNA binding

agnoprotein 198 39% 288 2E-29

P36708 Probable early E4 11 kDa protein 116 P03241 Probable early E4 11 kDa protein 116 45% 275 9E-30 Q2Y0F3 11 kDa protein E4 117 61% 378 2E-45 P36709 Probable early E4 13 kDa protein 120 P03240 Probable early E4 13 kDa protein 114 40% 247 2E-25 Q2Y0F5 13.6 kDa protein E4 122 37% 221 2E-21 NP_5977

84.2

control protein E4orf6/7 (gene

regulation, cell cycle regulation) E4 125 P03238

Probable early E4 17 kDa protein [Cleaved into: Early E4 10 kDa protein]

153 41% 248 4E-25 Q2Y0F7 E4 ORF6/7 E4 141 42% 239 7E-24

P36705 Early E3B 10.4 kDa protein 91 P15133 Early E3B 10.4 kDa protein 91 44% 210 1E-20 P11318 Early E3B 10.4 kDa protein 91 52% 218 8E-22 P36707 Early E3B 12.7 kDa protein 110 P03250 Early E3B 14.5 kDa protein 130 33% 158 5E-12 P11316 Early E3B 14.5 kDa protein 134 29% 132 4E-08 YP_0026

40222.1 protein U (unknown) U 52 Q2KSJ3 U protein U 53 43% 131 1E-09 ref|AP_0

00189.1| U exon 54 46% 50 2E-12

YP_0026 40219.1

encapsidation protein 22K (DNA encapsidation, capsid morphogenesis, transcriptional control) L4

182 P24939 Splicing factor (Splicing factor

33K) (L4-33K) L4 228 41% 133 6E-07 Q2Y0G9 22 kDa protein (L4 22-kDa

protein) L4 199 43% 328 9E-36

P03259-4 Isoform Early E1A 6 kDa protein of

Early E1A 29.5 kDa protein 52 No hits found, specie specific gene No hits found, specie specific gene

YP_0026 40220.1

membrane glycoprotein E3 CR1-alpha

(unknown) E3 264 No hits found, specie specific gene No hits found, specie specific gene

YP_0026 40221.1

membrane glycoprotein E3 CR1-beta

(unknown) E3 252 No hits found, specie specific gene No hits found, specie specific gene

26

Table 6. The final reciprocal BLAST search results list compiled by manual curation. HAdV-3 searched against HAdV-12 and HAdV-2

Ad3 Gene ID Protein Names Gene

names Len

gth Ad12 Gene

ID Protein names Gene names Leng th Iden

tity Score E- value

Ad2 Gen

e ID Protein Names Gene names Leng th Ident

ity Score E- value YP_002213830.1 encapsidation protein IVa2

IVa2 448 P12540 Packaging protein 1 (Packaging protein

IVa2) IVa2 452 78% 1875 0 P032

72

Packaging protein 1 (Packaging protein

IVa2) IVa2 449 82% 1,991 0

YP_002213831.1 DNA polymerase E2B 1193 P06538 DNA polymerase (EC 2.7.7.7) POL 1,061 75% 4354 0 P032

61 DNA polymerase (Pol) (EC 2.7.7.7) 1,198 74% 4,896 0

YP_002213832.1 terminal protein precursor

pTP E2B 658 P12541 DNA terminal protein (Bellett protein)

(pTP protein) PTP 606 77% 2466 0 P032

69

Preterminal protein (pTP) (Bellett protein) (Precursor terminal protein) [Cleaved into: Intermediate terminal protein (iTP); Terminal protein (TP)] PTP

671 80% 2,749 0

YP_002213772.1 encapsidation protein 52K

L1 385 P36715 Packaging protein 3 (L1-52/55 kDa

protein) (Packaging protein 52K) L1 373 73% 1409 0 P032 62

Packaging protein 3 (L1-52/55 kDa

protein) (Packaging protein 52K) L1 415 70% 1,433 0

YP_002213773.1 capsid protein precursor

pIIIa L1 588 P36712

Pre-capsid vertex protein (Capsid vertex-specific component IIIa) (CVSC) (Protein IIIa) (pIIIa) [Cleaved into:

Capsid vertex protein] L1

582 73% 2167 0 P032

79

Pre-capsid vertex protein (Capsid vertex- specific component IIIa) (CVSC) (Protein IIIa) (pIIIa) [Cleaved into: Capsid vertex protein] L1

585 75% 2,266 0

YP_002213774.1 penton base L2 544 P36716 Penton protein (CP-P) (Penton base

protein) (Protein III) L2 497 73% 2046 0 P032

76

Penton protein (CP-P) (Penton base

protein) (Protein III) L2 571 70% 2,031 0

YP_002213779.1 hexon L3 944 P19900 Hexon protein (CP-H) (Protein II) L3 919 78% 3939 0 P032

77 Hexon protein (CP-H) (Protein II) L3 968 76% 3965 0 YP_002213782.1 hexon assembly protein

100K L4 828 P36714

Shutoff protein (100 kDa protein) (p100K) (100K-chaperone protein) (L4- 100K) (Shutoff protein 100K) L4

782 64% 2601 0 P249

32

Shutoff protein (100 kDa protein) (p100K) (100K-chaperone protein) (L4- 100K) (Shutoff protein 100K) L4

805 64% 2602 0

YP_002213833.1 single-stranded DNA-

binding protein E2A 517 P04498 Early E2A DNA-binding protein DBP 484 50% 1312 1E- 175

P032 64

DNA-binding protein (DBP) (Early 2A

protein) 529 54% 1479 0

YP_002213767.1 control protein E1B 55K

E1B 492 P04491 E1B protein, large T-antigen (E1B 55

kDa protein) (E1B-55K) 482 47% 1198 1E-

158 P032 44

E1B 55 kDa protein (E1B-55K) (E1B

protein, large T-antigen) (E1B-495R) E1B 495 53% 1.492 0 YP_002213776.1 core protein V L2 349 P36717 Core-capsid bridging protein (Core

protein V) L2 347 59% 975 1E-

129 P032 67

Core-capsid bridging protein (Core

protein V) L2 369 61% 1,058 1E-141

YP_002213785.1 capsid protein precursor

pVIII L4 227 P36713

Pre-hexon-linking protein (Pre-protein VIII) (pVIII) [Cleaved into: Hexon-linking protein-N (12.1 kDa protein VIII) (Protein VIII-N); Hexon-linking protein- C (7.6 kDa protein VIII) (Protein VIII-C)]

L4

233 76% 928 1E-

125 P032 80

Pre-hexon-linking protein (Pre-protein VIII) (pVIII) [Cleaved into: Hexon-linking protein-N (12.1 kDa protein VIII) (Protein VIII-N); Hexon-linking protein-C (7.6 kDa protein VIII) (Protein VIII-C)] L4

227 79% 963 1E-131

YP_002213780.1 protease L3 209 P09569

Protease (EC 3.4.22.39) (Adenain) (Adenovirus protease) (AVP) (Adenovirus proteinase) (Endoprotease) L3

206 81% 874 1E-

118 P032 52

Protease (EC 3.4.22.39) (Adenain) (Adenovirus protease) (AVP) (Adenovirus proteinase) (Endoprotease) L3

204 80% 871 1E-117

YP_002213836.1 control protein E4 34K E4 299 P36710 Early E4 34 kDa protein 291 52% 853 1E- 112

P032 39

Early 4 ORF6 protein (E4-ORF6) (Early 4

34 kDa protein) (E4-34k) 294 59% 958 1E-128

YP_002213778.1 capsid protein precursor

pVI L3 250 P35988

Pre-protein VI (pVI) [Cleaved into:

Endosome lysis protein; Protease cofactor (pVI-C) L3

265 64% 818 1E-

108 P032 74

Pre-protein VI (pVI) [Cleaved into:

Endosome lysis protein; Protease cofactor (pVI-C)] L3

250 63% 823 1E-109

YP_002213765.1 control protein E1A E1A 261 P03259 Early E1A 29.5 kDa protein 266 42% 478 3E-56 P032

54 Early E1A 32 kDa protein 289 36% 458 5E-53

YP_002213786.1 control protein E3 12.5K E3 106 P36706 Early E3A 12.1 kDa protein 105 67% 395 2E-48 P273

11 Early E3A 12.5 kDa protein (E3-12,5K) 107 55% 318 1E-36 YP_002213838.1 control protein E4orf3 E4 117 P36708 Probable early E4 11 kDa protein 116 61% 378 2E-45 P032 Early 4 ORF3 protein (E4-ORF3) (E4 ORF3 116 49% 253 2E-26

27

41 control protein) (Early 4 11 kDa protein) (E4-11k)

YP_002213794.1 control protein E3 14.7K E3 136 Q65288 E3B 14.7 KD protein 128 56% 354 3E-41 P689

76 Early 3 14.7 kDa protein (E3-14.7k) 128 49% 324 1E-36 YP_002213768.1 hexon associated protein

IX, capsid protein IX IX 138 P03284 Hexon-interlacing protein (Protein IX)

IX 144 58% 352 1E-40 P032

82 Hexon-interlacing protein (Protein IX) IX 140 51% 328 4E-37 YP_002213840.1 control protein E4orf1 E4 125 S10861 hypothetical protein 127 50% 124 5E-40 P032

42

Early 4 ORF1 protein (E4-ORF1) (E4 ORF1

control protein) 128 46% 276 1E-29

YP_002213766.1 control protein E1B 19K

E1B 178 P04492 E1B protein, small T-antigen (E1B 19

kDa protein) (E1B-19K) 163 41% 352 8E-40 Q2Y0

J3 19 kDa small T antigen (E1B 21-kDa) E1B 178 100% 943 1E-129 YP_002213783.1 protein 33K L4 234

emb|X 73487.1

|

336 bp (Query cover 72%) 64% 177 2E-38 P249

39

Splicing factor (Splicing factor 33K) (L4-

33K) L4 228 54% 557 4E-69

YP_002213777.1 core protein precursor pX

L2 75 P35986 Late L2 mu core protein (11 kDa core

protein) (Protein X) (pX) (pMu) PX 72 63% 255 3E-30 P142 69

Pre-core protein X (pX) (11 kDa core protein) (Protein mu) (pMu) [Cleaved into: Core protein X] L2

80 69% 280 3E-34

YP_002213771.1 protein 13.6K L1 139 P36704 Probable DNA-binding protein

(Agnoprotein) 205 44% 275 2E-28 P032

63 I-leader protein 145 48% 340 7E-39

YP_002213784.1 encapsidation protein 22K

L4 199

emb|X 73487.1

|

255 bp (Query cover 67%) 50% 140 6E-27 P249

39

Splicing factor (Splicing factor 33K) (L4-

33K) L4 228 38% 167 2E-11

YP_002213796.1 fiber L5 319 P36711 Fiber protein (SPIKE) (Protein IV) L5 587 33% 271 2E-23 P032

75 Fiber protein (SPIKE) (Protein IV) L5 582 29% 243 9E-20 YP_002213792.1 membrane protein E3 RID-

alpha E3 91 P36705 Early E3B 10.4 kDa protein 91 52% 218 8E-22 P151

33

Pre-early 3 receptor internalization and degradation alpha protein (Pre-E3-RID- alpha protein)

91 51% 259 4E-28

YP_002213837.1 control protein E4orf4 E4 122 P36709 Probable early E4 13 kDa protein 120 37% 221 2E-21 P032

40 Early 4 ORF4 protein 114 45% 275 1E-29

YP_002213839.1 control protein E4orf2 E4 129 S10863 hypothetical protein 131 30% 62 1E-15 P0DJ

X0 Early 4 ORF2 protein (E4-ORF2) 130 32% 177 1E-14 YP_002213834.1 protein U U 52

emb|X 73487.1

|

50% 67.4 9E-12 P0DJ

X2 U exon protein 217 59% 176 3E-16

YP_002213793.1 membrane protein E3 RID-

beta E3 134 P36707 Early E3B 12.7 kDa protein 110 29% 132 5E-08 P032

50

Early 3 receptor internalization and degradation beta protein (E3 RID-beta protein) (Early E3B 14.5 kDa protein) (E3- 14.5k)

130 37% 232 8E-23

YP_002213835.1 control protein E4orf6/7 E4 141 P36710 Early E4 34 kDa protein 291 34% 93 9E-02 P032 38

Early 4 ORF6/7 control protein (E4-

ORF6/7) (Early E4 17 kDa protein) 150 47% 328 6E-37 YP_002213775.1 core protein precursor pVII

L2 192 No hits found in Ad12 P689

50

Pre-histone-like nucleoprotein (Pre-core

protein VII) (pVII) L2 198 69% 656 3E-85

YP_002213787.1 membrane glycoprotein E3

CR1-alpha E3 146 No hits found in Ad12

YP_002213788.1 membrane glycoprotein E3

gp19K E3 172 No hits found in Ad12 P689

78

Early E3 18.5 kDa glycoprotein (E3-19K)

(E3gp 19 kDa) (E19) (GP19K) 159 32% 201 2E-17 YP_002213789.1 membrane glycoprotein E3

CR1-beta E3 179 No hits found

YP_002213790.1 membrane glycoprotein E3

CR1-gamma E3 189 No hits found

YP_002213791.1 membrane protein E3 CR1-

delta E3 77 No hits found