Immunology and Cell Biology

(2004)

82

, 189–195 doi:10.1046/j.0818-9641.2004.01223.x

© 2004 Australasian Society for Immunology Inc.

Special Feature

Ovine atadenovirus: a review of its biology, biosafety profile and application as a gene delivery vector

G E R A L D W B O T H

CSIRO Molecular Science, North Ryde, NSW 2113, Australia

Summary

The ovine adenovirus isolate OAdV287 is the prototype of the newly recognized genus of atadenovi-ruses. Although not as well studied as human mastadenoviruses, a substantial amount of work has now been carriedout with this virus and an understanding of its interesting and unique properties is beginning to emerge. In this articlethe biology and biosafety profile of the virus is reviewed. This knowledge underpins the exploitation of the virus asa gene delivery vector. Its potential as a vaccine vector and its application to the treatment of prostate cancer issummarized and discussed.

Key words

:

gene delivery, gene function, ovine atadenovirus, prostate cancer, vaccine, vector.

Ovine atadenovirus isolate, genome structure and classification

Viral isolate OAdV287 (serotype 7; OAdV7) was the secondisolate of this type recovered from sheep in Western Aus-tralia.

1

A very similar virus was also recovered from lambs inNorth America

2

and a serologically related virus was isolatedin New Zealand.

3

Viruses of this type are therefore widelydistributed but are not generally recognized as pathogenicunder field conditions. Although the isolate from the USAwas recovered from three dead lambs in a single flock

2

experimental infection with the prototype OAdV7 isolatedid not produce noticeable symptoms, although this wasperformed in older animals.

1,4

OAdV7 was first propagated in a sheep fetal lung(CSL503) cell line, which allowed the viral genome to beisolated and subcloned. The complete nucleotide sequencewas ultimately determined

5,6,7

. The genome structure (Fig. 1)clearly displayed many differences from the mastadenovi-ruses and aviadenoviruses that were known at the time. Benkoand Harrach

8

therefore proposed the existence of a thirdgenus, termed the atadenoviruses, so-called because of thehigh adenine/thymine (A/T) content of the genome. OAdV7was designated as the prototype isolate but members fromseveral other animal species, including cattle, for example,BAdV4, deer, possum, duck (DAdV1) and snake have nowbeen described.

9,10,11

BAdV4 and DAdV1 have also beensequenced. No human isolate has yet been discovered.

The OAdV7 genome has a high A/T and correspondinglylow guanine/cytidine (G/C) content compared with otheradenoviruses. This means that the nucleotide sequencehomology in many genes is low although at the amino acidlevel the genes are clearly related. OAdV7 has a central core

in its genome that encodes the DNA replication enzymes andmost of the structural proteins. This clearly defines it as anadenovirus but there are significant differences betweenOAdV7 and the genomes of prototypic mastadenovirusesAdV2/5. (The updated sequence is available at GenBankaccession number U40839.) OAdV7 lacks capsid proteins Vand IX and instead has p32 and a 42 kDa protein as structuralpolypeptides. The inverted terminal repeat (ITR) of AdV5and OAdV7 differ in length (103 vs 46 bp, respectively) andsequence, and at the left and right ends there are majordifferences in gene content (Fig. 1). The left end of OAdV7contains reading frames LH1, 2 and 3 but lacks a homologueof the important AdV5 E1A gene. LH3 appears to sharehomology with AdV5 E1B 55 kDa protein. This is discussedfurther below. At the opposite end of the genome lies theputative E4 region containing genes E41, E42 and E43. Theregion is so-called because of the presence in E42 and E43 ofan amino acid motif that is highly conserved in E4 34 kDahomologues from all mastadenoviruses.

7

However, there is noother obvious homology between E42 and E43. E41 is absentfrom DAdV1 but present in BAdV4 and OAdV7. At theextreme right end of the genome there are four or morereading frames that show considerable homology within andacross atadenoviruses.

12

OAdV7 genes RH1, 2, 4 and 6display major identity at the amino acid level with each other,with their BAdV4 counterparts and with two genes inDAdV1. They comprise a unique family of proteins thatcontain an F-box motif whose function is discussed furtherbelow. DAdV1 also has additional genes at the right end thatextend the length of its genome.

13,14

These may have hostspecific functions. Overall, OAdV7 is substantially differentfrom all known human mastadenoviruses. This may be bene-ficial from a biosafety viewpoint as discussed below.

Transcripts and promoters

The genome is decoded as several transcription units thatfunction at early or late times during infection (Fig. 1),

15,16

although not all temporal analyses have been completed in

Correspondence: CSIRO Molecular Science, PO Box 184, NorthRyde, NSW 1670, Australia. Email: gerry.both@csiro.au

Received 1 December 2003; accepted 1 December 2003.

190

GW Both

detail. The left end P2 promoter appears to function early(< 12 h postinfection; p.i.), in concert with another weakerpromoter (P1) that is probably located in the ITR. A third,independent promoter for LH3 lies between LH2 and LH3.

15

Similarly the IVa

2

and p32 genes appear to have independentpromoters,

15

although based on comparisons with other atadeno-viruses the precise position of the IVa

2

promoter is lesscertain.

11

The E2 transcription unit functions early, asexpected because it encodes the DNA replication enzymes.Multiply spliced transcripts for binding protein (DBP), termi-nal protein (TP) and DNA pol were detected by reversetranscription–polymerase chain reaction (RT–PCR) as earlyas 9 h p.i.

15,16

Differential splicing appears to produce atruncated form of the terminal protein at late times duringinfection, as opposed to the alternative of proteolytic cleav-age.

15

The proposed E4 region forms a transcription unit thatis also controlled by two promoters.

15

These produce multiplyspliced transcripts with different 5

′

proximal leader sequences,some of which were detectable at or before 12 h p.i.

15,16

Earlyproduction of E43 gene transcripts is consistent with itsproposed role in activating the viral E2 promoter, as discussedbelow. Sequences at the right end of the genome also com-prise a discrete transcription unit.

12,17

Transcripts from thisregion were detectable by RT–PCR as early as 6 h p.i. and itmay be that the promoter, which is located within the ITR, isconstitutively active. Given the nature of ITR sequences, thisimplies that the left ITR may contain the LHP1 promoter andthat it may also function immediately after infection togenerate LH1 and LH2 gene products.

12,15

Lastly, the OAdV7lacks a VA RNA gene, although such a gene is present at theright end of DAdV1.

13,18

One or two VA RNA genes arecommonly found in mastadenoviruses.

19

This may partlyaccount for the lesser impact of OAdV7 infection on hostprotein synthesis compared with AdV5.

18

Gene function

Many of the non-structural OAdV7 genes are only defined asopen reading frames (orf) and the gene products remain to becharacterized. However, there were certain expectations forOAdV7 gene function based on knowledge derived from themastadenoviruses where strategies to manipulate the host cellcycle for viral replication had been identified. For example,interaction between the E1A protein and cellular retinoblast-oma protein (pRb) releases E2F transcription factors thatcontrol cell cycle progression.

20

Early in the infectious cycle,the E1B 55 kDa protein also counteracts the effects of E1A-induced stability of p53 so that cell cycle arrest and prematureapoptosis is avoided (

21

and references therein). Similarly, anddespite their unrelatedness to E1A, products of the aviadeno-virus Gam1 and orf22 genes cooperate to achieve activationof E2F factors.

22

Although atadenoviruses lack homologuesof E1A, orf22 or Gam1 they may also have a strategy tomanipulate the cell cycle. However, the identity of any suchgenes requires experimental definition.

Left end genes

The roles of LH1, LH2 and LH3 are yet to be determined.LH1 is conserved OAdV7 and BAdV4 but not in DAdV1. Itsrole may therefore be host (ruminant)-specific. Similarly,LH2 shows no significant homology with other proteinsoutside the genus but the orf is conserved in the threesequenced atadenoviruses.

11,14

LH3, although substantiallyshorter, shows homology with the mastadenovirus E1B55 kDa protein that was identified by a BLAST search. Thisprotein has an important role in cellular transformation and inthe nuclear export of mRNA

23

(and references therein). E1B55 kDa and E4 34 kDa proteins associate in a multi-subunit

Figure 1

Map of the OAdV7 genome. Open arrows indicate open reading frames. Hatched arrows are related reading frames. The heavyhorizontal arrow indicates the major late transcription unit with tripartite leader sequence; arrows with dashed lines are early transcriptionunits; the timing of transcription for IVa

2

, LH3 and p32k has not been precisely determined; small arrowheads show promoter regions(e.g. LHP1, P2); vertical arrows indicate gene insertion sites. Markers on the central bar indicate 5 kb intervals in the genome (29 574nucleotides).

Ovine atadenovirus

191

complex that catalyses the ubiquitination and degradation ofp53.

21,24

Whether this occurs for atadenoviruses is presentlyunknown. The overall homology between LH3 and E1B55 kDa is low

7

and a BLAST search shows that there is athree amino acid insertion in LH3 among residues that arehighly conserved in the major p53 binding site of E1B55 kDa,

25,26

as well as other net deletions and insertions. Onthe other hand, there are also differences between ovine andhuman p53 proteins within the amino terminal 123 residuesthat contain the E1B 55 kDa binding site.

25

This suggests thateven if LH3 is a homologue of E1B55 kDa, it maybe incom-patible with human p53. The stability of p53 after OAdV7infection of human and ovine cells should be investigated todetermine whether the outcome is similar to that described forAdV5 infection of human cells.

E4 genes

The OAdV7 E4 region is tentatively named and the study ofE4 gene products is in its infancy. The E4 gene products inAdV5 and OAdV7 differ in number and do not appear to befunctionally equivalent. It was recently discovered that theE43 gene product plays an important role in viral replicationdue to its ability to activate the E2 promoter that controls thesynthesis of the DNA replication proteins.

27

The viral E2 andan E2F-1-dependent synthetic promoter were activated eitherby viral infection or by transfection of a plasmid thatexpressed the E43 gene. Analysis of the OAdV7 E2 promoterrevealed the presence of a non-palindromic E2F binding site.When this site was mutated, E2F-1- and E43-dependentpromoter activation was substantially reduced. In a reciprocalexperiment a virus, in which a stop codon was introduced intothe E43 gene, grew very poorly, underlining the importanceof the gene for replication. In addition, co-precipitation experi-ments suggested a direct interaction between E43 and E2F-1,but not between E43 and the retinoblastoma protein pRb.Thus, in contrast to mastadenoviruses that activate E2Ffactors via an interaction between E1A and pRb (reviewedin

20

) OAdV7 appears to activate E2F-dependent promotersvia its E43 product. The precise details by which this isachieved remain to be determined.

No other information on E4 gene function is available, butduring the initial search for gene homologues an intriguingrelationship between OAdV7 and mastadenovirus proteinswas identified.

7

The amino acid motif HCHCxxPGSLQCwas identified by comparisons between OAdV7 E42 and E43and mastadenovirus E4 34 kDa proteins.

7

Related motifs weresubsequently found in chicken embryo lethal orphan (CELO)strain (avian adenovirus) orf14 and AO7, a mammalian RINGfinger protein that has been demonstrated to mediate bindingand ubiquitination of proteins.

23,28

It is therefore tempting tospeculate that E42 and E43 may form part of complexes thatregulate certain proteins via ubiquitination pathways. How-ever, their identity remains to be determined.

Right end genes

The function of genes at the right hand end of OAdV7 andother atadenoviruses is especially intriguing. The presence ofmultiple copies of a gene in an otherwise compact (29.57 kb)OAdV7 genome seems rather unnecessary, especially as the

transcription map suggests that not all reading frames aremeaningfully translated.

12,17

It is possible that gene duplica-tion serves to maintain the genome at an appropriate sizebecause other AdV genomes that are too short fail to packageefficiently.

29

The RH3 orf is not conserved, even in the closely relatedBAdV4 virus, and thus appears unlikely to be significant. TheRH5 orf is conserved and demonstrates an interesting conser-vation of cysteine and other key residues when compared tothe E3 protein of siadenoviruses.

12

However, the key observa-tion relevant to function is that genes RH1, 2, 4 and 6 (Fig. 1)have an F-box motif at their amino terminus.

12

Such motifsare approximately 40 amino acids in length and common tomany cellular protein families but rare in the virus kingdom.The F-box protein binds a phosphorylated partner protein andassociates with a multi-subunit protein complex that catalysesthe addition of ubiquitin to the partner, resulting in itsdegradation.

30,31,32

Certain components of such complexescan vary.

24

Depending on the usual role of the partner protein,F-box proteins therefore have the potential to activate or shutdown certain cellular or viral functions. However, genesRH2-6 (Fig. 1) can be deleted from OAdV7 without seriouslyaffecting virus growth

in vitro

17

perhaps reflecting theirredundancy. One possibility is that these genes may be moreimportant

in vivo

. In this sense, their role would be similar tothe E3 region of mastadenoviruses that express immunomo-dulatory genes.

Viral receptors cell tropism

The infection of most human AdV is facilitated by attachmentof the cell binding domain of the fibre protein to the primaryreceptor, CAR. A secondary interaction between an Arg-Gly-Asp motif in the AdV penton and selected integrins thenpromotes endocytosis.

33,34

The primary receptor for OAdV7infection remains to be identified. However, it is not CARbecause AdV5 and OAdV7 do not compete with each otherfor entry into a cell type that both viruses can infect and theyexhibit a differential ability to infect some cell types.

35,36

OAdV7 also exhibits a distinctive tissue distribution

in vivo

that is non-liver-dominant as is observed for AdV5.

37

Further-more, replacing the fibre cell-binding domain with the equiv-alent domain from AdV5 fibre significantly modifies OAdV7cell tropism.

35

The reciprocal replacement renders the uptakeof a chimeric AdV5 virus CAR-independent.

38

OAdV7 infec-tion occurs in the absence of a recognizable integrin-bindingdomain in penton or any other capsid protein.

7

Despite its ovineorigin, OAdV7 infects a wide range of human and animal celltypes, indicating that its receptor is widespread.

16,37,39,40–42

Biosafety profile of OAdV7

Abortive replication

OAdV7 is known to replicate efficiently only in fetal lung(CSL503)

1

and skin (HVO156)

43

cell lines where it grows tohigh titre. In a wide range of other animal and human celllines replication is abortive, with the replication cycle beingblocked at different stages, depending on the cell type, due tothe lack of viral promoter function. For example, in HepG2human liver carcinoma cells none of the viral promoters is

192

GW Both

active. In other human cell types, for example, MCF-7 humanbreast cancer cells, some early promoters are active but thereis minimal DNA replication and the major late promoter isnot active. Lastly, in cells such as IMR90 human lungfibroblasts, DNA replication occurs and late proteins aresynthesized but incorrect protein processing prevents theformation of infectious virus particles.

16,39,41

In a clinical setting, it is possible that a patient to whom anOAdV7 vector has been administered may acquire an adven-titious human adenovirus infection. To reflect such a potentialsituation, human cell lines were co-infected by AdV5 andOAdV7. Although AdV5 replicated efficiently as expected,importantly, OAdV7 failed to replicate even after materialfrom co-infected cell lysates was passaged.

41

No formation ofhybrid genomes was detected. This result is consistent withthe differences in ITR, genome structure and capsid proteincontent as discussed in the preceding sections and furtheremphasizes the incompatibility between adenoviruses in dif-ferent genera.

Transformation

Many adenoviruses carry genes such as E1A and E1B that co-operate to transform cells. As described above, OAdV7 lacksan E1A homologue and its LH3 orf shows only limitedhomology with E1B 55 kDa. The transforming ability ofOAdV7 was examined directly in rodent embryo cells that arethe historical benchmark for such properties. OAdV7 genomesequences failed to induce the formation of transformedcolonies following infection or transfection, whereas AdV5sequences produced transformants as expected.

44

In a subse-quent experiment, the AdV5 E1A gene was introduced intosite II of the OAdV7 genome. This recombinant comple-mented the replication of an E1A-deficient AdV5 mutant

41

supporting the argument that OAdV7 has no gene that is afunctional homologue of E1A.

Taken together, the above properties indicate that OAdV7is likely to be a safe vector for the delivery of genes to humancells.

Vector development

A substantial amount of work has now been carried out todevelop OAdV7 as a gene delivery vector. The first recom-binants made it easier to demonstrate the ability of the vectorto infect human cell types. However, there were severalchallenges to be overcome in creating a vector from anuncharacterized virus. First, it was necessary to devise astrategy to recover an infectious virus from naked DNA. Thiswas achieved by creating plasmids in which the ITR of thecomplete genome were linked by bacterial sequences contain-ing an antibiotic resistance gene and an origin of replicationin such a manner that these could be excised by digestion witha restriction enzyme (

Kpn

I) that did not cut within thegenome. Such plasmids grow in

Escherichia coli

. The lineargenome was then released by

Kpn

I digestion and transfectedinto permissive CSL503 cells to achieve virus rescue.

45

Next,it was necessary to identify suitable sites for gene insertion(sites I-III, Fig. 1), to determine what insert sizes weretolerated without knowing the packaging capacity of the virusand to identify nonessential sequences that could potentially

increase the carrying capacity of the vector. Site I, betweenthe pVIII and fibre genes, was chosen because in mastadeno-viruses the E3 region in this location was nonessential. InOAdV7, the intergenic region is only approximately200 nucleotides long because there is no E3 region in thislocation.

5

A variety of viruses with site I gene cassettes havebeen rescued.

4,17,36,37,39,41,46

However, some cassettes that usedthe human megalovirus (HCMV) promoter were unstableupon passage.

46

Site II, which is located within a nonessentialregion,

17

has only rarely been used.

41

Site III, which is locatedbetween the E4 and RHE transcription units,

15

is preferredbecause inserts at this site propagate stably over at least10–15 passages.

47,48

Plasmids that facilitate vector construc-tion by recombination in

E. coli

have also been constructed.The inclusion of a cosmid packaging signal also facilitates theselection of large recombinant genomes,

49

thus avoiding thecommon problem of large plasmid instability.

As mentioned before, genes RH2-6 (

∼

2 kb in length) canbe deleted from the genome without loss of virus viability andapproximately 4.3 kb of ‘stuffer’ DNA can be inserted.

35

Therefore, the theoretical packaging capacity of OAdV7 is atleast 6.3 kb. However, there has so far been no attempt torescue a virus with such a large functional insert.

Applications in gene delivery

Because adenoviruses do not integrate their genomes, OAdV7vectors are considered to be of most utility for applicationssuch as vaccination and cancer treatment where transient genedelivery may produce the desired outcome. While there arenow numerous demonstrations that OAdV7 can deliver genes

in vivo

, the most advanced application of the vector is in thesetwo areas as discussed below.

Gene delivery

in vivo

Most individuals are infected with adenoviruses early in theirlifetime and develop immunity as a result. All human sera thathave been examined lacked neutralizing activity againstOAdV7 but, almost without exception, they neutralizedAdV5.

37

This potentially presents problems for the use ofAdV5-based vectors in humans. It was demonstrated in micethat pre-immunity to AdV5 compromised the delivery ofgenes by AdV5-based vectors administered i.v., but genedelivery by OAdV7 was unaffected.

37,47

Although immunityto OAdV7 was induced, these vectors should provide aninitial advantage in a clinical setting and may be given morethan once, depending on how the vector is administered.

40

Following i.m. injection in mice, very high level reportergene expression was achieved with an OAdV vector. Expres-sion of the reporter gene from the same cassette delivered byAdV5 or a plasmid was approximately 1.5 and 2 logs lower,respectively. By careful adjustment of the initial dose, asecond efficacious dose of OAdV7 could be given.

40

Thismay be useful in prime/boost vaccination strategies. In addi-tion, a single i.m. injection of an OAdV7 recombinantexpressing the hepatitis C virus NS3 protein elicited a strong,sustained, antigen-specific T-cell response in mice, even inthe face of immunity to AdV5.

47

Lastly, an OAdV/GFPrecombinant was used to examine whether the vector wascapable of infecting human dendritic cells. Although this

Ovine atadenovirus

193

vector preparation retained very little of its reporter gene (byPCR analysis <5% of the gene; it was one of the unstablevectors)

46

at a multiplicity of infection (moi) of 10

3

particlesper cell, approximately 5–10% of dendritic cells showedfluorescence at 72 h p.i. (this was undoubtedly a significantunderestimate) and viability was comparable to uninfectedcells (D Healey, A Irvine and G Both, unpubl. data). Thesefindings demonstrate in principle that OAdV7 has potential asa vaccine vector for humans.

Gene therapy for prostate cancer

The most advanced application of OAdV7 as a gene deliveryvector is in its use for gene-directed enzyme pro-drug therapy(GDEPT) for the treatment of prostate cancer. The strategyis to generate local chemotherapy to treat a tumour in theprostate without systemic side-effects that usually occurbecause of anti-neoplastic drug-induced toxicity. An OAdVvector that expresses the

E. coli deoD

gene (that encodespurine nucleoside phosphorylase, PNP) is injected directlyinto a tumour. After allowing time for expression, the pro-drug fludarabine is administered systemically over days 1–5.The drug is metabolized by PNP to 2-fluoradenine, a toxicproduct. The pro-drug and toxin can inhibit DNA, RNA andprotein synthesis in infected cells, and because 2FA candiffuse freely across membranes, a ‘bystander’ killing effectis also generated.

50

Two vectors, OAdV220 and OAdV623,have been used to assess GDEPT efficacy in several animalmodels of prostate cancer where tumours growing s.c. or inthe prostate were injected directly.

36,48,51

OAdV220 carries asite I insertion where the gene cassette comprises the RSVpromoter, PNP gene and BGH polyadenylation signal. InOAdV623, PNP is controlled by the enhancer from prostate-specific membrane antigen gene

52

coupled to the rat probasingene promoter. This provides prostate-specific expression ofthe PNP gene, which adds a further safety factor if the vectorshould escape from the injection site. OAdV623 is alsoformulated with cationic lipid to improve its ability to infectcells that have a low number of receptors on the surface.

48

Asthe tissue-specific gene switch is androgen independent, for-mulated OAdV623 is active against human prostate cancercells that are androgen sensitive (LN3 cells) and insensitive(PC3 cells), thus potentially allowing early and late stagedisease to be treated. Moreover, evidence is accumulatingfrom our work and from other GDEPT systems that animmune response that is effective at sites remote from theprimary treatment site can be generated.

53–55

Thus, GDEPTmay be most effective if early diagnosis allows treatmentwhen the metastatic tumour burden is low.

During the course of this work, a cell bank was laid downusing good manufacturing practice and procedures for scalingup vector production were developed. Assays to monitor thequality and reproducibility of vector batches that were pro-duced for preclinical studies were established and docu-mented. Formal toxicology studies have also been carried out.This work has provided a sound basis for seeking regulatoryapproval to conduct a phase I clinical trial. The testing of theOAdV7 vector in humans will complete the journey in whichthis virus has progressed from being a novel and scientificallyinteresting isolate to a vector that may eventually be useful ina variety of gene delivery applications.

Acknowledgements

The author offers his sincere thanks to the many colleagueswho contributed to this work over the years for their willing-ness to share information, ideas and data. Their cooperationand contributions are acknowledged by co-authorship in thereferences cited in this review. In addition, the contributionsof Mr Keith Smith, Dr Anne Collins, Dr K Setiawan and DrA Piekarska of FH Faulding & Co Limited and later MaynePharma Pty Ltd, who supported the prostate cancer work, aregratefully acknowledged.

References

1 Boyle DB, Pye AD, Kockerhans R, Adair BM, Vrati S, Both GW.Characterisation of Australian ovine adenovirus isolates.

Vet.Microbiol.

1994;

41

: 281–91.2 De Bey BM, Lehmkuhl HD, Chard-Bergstrom C, Hobbs LA.

Ovine adenovirus serotype 7-associated mortality in lambs in theUnited States.

Vet. Pathol.

2001;

38

: 644–8.3 Davies D, Humphreys S. Characterisation of two strains of

adenovirus isolated from New Zealand sheep.

Vet. Microbiol.

1977;

2

: 97–107.4 Rothel JS, Boyle DB, Both GW

et al.

Sequential nucleic acidand recombinant adenovirus vaccination induces host-protectiveimmune responses against

Taenia ovis

infection in sheep.

Para-site Immunol.

1997;

19

: 221–7.5 Vrati S, Boyle D, Kocherhans R, Both GW. Sequence of ovine

adenovirus homologs for 100K hexon assembly, 33K, pVIII, andfiber genes: Early region E3 is not in the expected location.

Virology

1995;

209

: 400–8.6 Vrati S, Brookes DE, Boyle DB, Both GW. Nucleotide sequence

of ovine adenovirus tripartite leader sequence and homologues ofthe IVa2, DNA polymerase and terminal proteins.

Gene

1996;

177

: 35–41.7 Vrati S, Brookes DE, Strike P, Khatri A, Boyle DB, Both GW.

Unique genome arrangement of an ovine adenovirus: Identifica-tion of new proteins and proteinase cleavage sites.

Virology

1996;

220

: 186–99.8 Benko M, Harrach B. A proposal for a new (third) genus within

the family Adenoviridae.

Arch. Virol.

1998;

143

: 829–37.9 Both G. Atadenoviruses. In: Tidona C, Darai G (eds).

TheSpringer Index of Viruses

, Berlin: Springer-Verlag, 2001; 11–17.10 Benko M, Elo P, Ursu K

et al.

First molecular evidence for theexistence of distinct fish and snake adenoviruses.

J. Virol.

2002,2003;

76

: 10056–9.11 Davison A, Benko M, Harrach B. Genetic content and evolution

of adenoviruses.

J. Gen. Virol.

2003;

84

: 2895–908.12 Both G. Identification of a unique family of F-box proteins in

atadenoviruses. Virology 2002; 304: 425–33.13 Hess M, Blocker H, Brandt P. The complete nucleotide sequence

of the egg drop syndrome virus. An intermediate betweenmastadenoviruses and aviadenoviruses. Virology 1997; 238:145–56.

14 Both G. Xenogenic adenoviruses. In: Curiel D, Douglas J (eds)Adenoviral Vectors for Gene Therapy, San Diego: AcademicPress 2002; 447–79.

15 Khatri A, Both GW. Identification of transcripts and promoterregions of ovine adenovirus OAV287. Virology 1998; 245:128–41.

16 Kumin D, Hofmann C, Rudolph M, Both GW, Loser P. Biologyof ovine adenovirus infection of nonpermissive cells. J. Virol.2002; 76: 10882–93.

194 GW Both

17 Xu ZZ, Hyatt A, Boyle DB, Both GW. Construction of ovineadenovirus recombinants by gene insertion or deletion of relatedterminal region sequences. Virology 1997; 230: 62–71.

18 Venktesh A, Watt F, Xu ZZ, Both GW. Ovine adenovirus(OAV287) lacks a virus-associated RNA gene. J. General Virol.1998; 79: 509–16.

19 Horwitz MS. Adenoviridae and their replication. In: Fields BN,Knipe DM (eds) Virology, New York: Raven Press 1990;1679–721.

20 Nevins J. Adenovirus E1A: transcription regulation and altera-tion of cell growth control. Curr. Top Microbiol. Immunol. 1995;199: 25–32.

21 Harada JN, Shevchenko A, Shevchenko A, Pallas DC, Berk AJ.Analysis of the adenovirus E1B-55K-anchored proteome revealsits link to ubiquitination machinery. J. Virol. 2002; 76: 9194–206.

22 Lehrmann H, Cotten M. Characterization of CELO virus proteinsthat modulate the pRb/E2F pathway. J. Virol. 1999; 73: 6517–25.

23 Nevels M, Rubenwolf S, Spruss T, Wolf H, Dobner T. Two dis-tinct activities contribute to the oncogenic potential of the adeno-virus type 5, E4orf6 protein. J. Virol. 2000; 74: 5168–81.

24 Querido E, Blanchette P, Yan Q et al. Degradation of p53 byadenovirus E4orf6 and E1B55K proteins occurs via a novelmechanism, involving a Cullin-containing complex. Genes Dev.2001; 15: 3104–17.

25 Kao CC, Yew PR, Berk AJ. Domains required for in vitro associ-ation between the cellular p53 and the adenovirus 2 E1B 55Kproteins. Virology 1990; 179: 806–14.

26 Grand RJA, Parkhill J, Szestak T, Rookes SM, Roberts S,Gallimore PH. Definition of a major p53 binding site onAd2E1B58K protein and a possible nuclear localization signalon the Ad12E1B54K protein. Oncogene 1999; 18: 955–65.

27 Kümin D, Hofmann C, Uckert W, Both G, Löser P. Identifica-tion of an ovine atadenovirus gene whose product activates theviral E2 promoter: possible involvement of E2F-1. Virology2004; 318: 79–89.

28 Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S,Weissman AM. RING fingers mediate ubiquitin-conjugatingenzyme (E2) -dependent ubiquitination. Proc. Natl Acad. Sci.USA 1999; 96: 11 364–9.

29 Parks RJ, Graham FL. A helper-dependent system for adenovirusvector production helps define a lower limit for efficient DNApackaging. J. Virol. 1997; 71: 3293–8.

30 Bai C, Sen P, Hofmann K et al. SKP1 connects cell cycle regula-tors to the ubiquitin proteolysis machinery through a novel motif,the F-box. Cell 1996; 86: 263–74.

31 Spruck C, Strohmaier H, Watson M et al. A CDK-independentfunction of mammalian Cks1: targeting of SCF (Skp2) to theCDK inhibitor p27Kip1. Mol. Cell 2001; 7: 639–50.

32 Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW. F-boxproteins are receptors that recruit phosphorylated substrates tothe SCF ubiquitin-ligase complex. Cell 1997; 91: 209–19.

33 Bergelson JM, Cunningham JA, Droguett G et al. Isolation of acommon receptor for coxsackie B viruses and adenoviruses 2and 5. Science 1997; 275: 1320–3.

34 Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrin-alpha-v-beta-3 and integrin-alpha-v-beta-5 promote adenovirusinternalization but not virus attachment. Cell 1993; 73: 309–19.

35 Xu ZZ, Both GW. Altered tropism of an ovine adenovirus carry-ing the fiber protein cell binding domain of human adenovirustype 5. Virology 1998; 248: 156–63.

36 Voeks D, Martiniello-Wilks R, Madden V et al. Gene therapy forprostate cancer delivered by ovine adenovirus and mediated bypurine nucleoside phosphorylase and fludarabine in mousemodels. Gene Ther. 2002; 9: 759–68.

37 Hofmann C, Loser P, Cichon G, Arnold W, Both GW, Strauss M.Ovine adenovirus vectors overcome preexisting humoralimmunity against human adenoviruses in vivo. J. Virol. 1999; 73:6930–6.

38 Glasgow J, Both G, Krasnykh V, Curiel D. A chimeric adeno-virus vector expressing an ovine adenovirus fiber has CAR-independent tropism. Proceedings of the American Society ofGene Therapy meeting; 2003 June 4–8; Washington DC, USA.Abstract, 498.

39 Khatri A, Xu ZZ, Both GW. Gene expression by atypical recom-binant ovine adenovirus vectors during abortive infection ofhuman and animal cells in vitro. Virology 1997; 239: 226–37.

40 Löser P, Hillgenberg M, Arnold W, Both GW, Hofmann C.Ovine adenovirus vectors mediate efficient gene transfer to skel-etal muscle. Gene Ther. 2000; 7: 1491–8.

41 Lockett LJ, Both GW. Complementation of a defective humanadenovirus by an otherwise incompatible ovine adenovirusrecombinant carrying a functional E1A gene. Virology 2002;294: 333–41.

42 Voeks DJ, Both GW, Cameron FH, Cooke BE, Russell PJ.Transduction of biopsy samples: Bridging gene therapy betweenanimals and humans. Biotechniques 2001; 31: 4649.

43 Löser P, Kümin D, Hillgenberg M, Both G, Hofmann C. Prepa-ration of ovine adenovirus vectors. In: Morgan J (eds). Methodsin Molecular Medicine, Gene Therapy Protocols, 2nd edn.Totowa, NJ: Humana Press, 2001; 415–26.

44 Xu ZZ, Nevels M, MacAvoy ES et al. An ovine adenovirusvector lacks transforming ability in cells that are transformed byAD 5 E1A/B sequences. Virology 2000; 270: 162–72.

45 Vrati S, Macavoy ES, Xu ZZ, Smole C, Boyle DB, Both GW.Construction and transfection of ovine adenovirus genomicclones to rescue modified viruses. Virology 1996; 220: 200–3.

46 Löser P, Cichon G, Jennings G, Both G, Hofmann C. Ovineadenovirus vectors promote efficient gene delivery in vivo. GeneTher. Mol. Biol. 1999; 4: 33–43.

47 Wüest T, Hillgenberg M, Schneeweiss U et al. Recombinantovine atadenovirus induces a strong and sustained T cellresponse against the hepatitis c virus ns3 antigen in mice.Vaccine 2004 (in press).

48 Wang X-Y, Martiniello-Wilks R, Shaw J et al. Preclinical evalu-ation of a prostate targeted gene directed enzyme prodrugtherapy delivered by ovine atadenovirus. 2004 (submitted forpublication).

49 Löser P, Hofmann C, Both G, Hillgenberg M. Construction,rescue and characterization of vectors derived from ovine atade-novirus. J. Virol. 2003; 77: 11941–51.

50 Martiniello-Wilks R, Garcia-Aragon J, Daja MM et al. In vivogene therapy for prostate cancer: Preclinical evaluation of twodifferent enzyme-directed prodrug therapy systems delivered byidentical adenovirus vectors. Hum. Gene Ther. 1998; 9:1617–26.

51 Martiniello-Wilks R, Dane A, Voeks D et al. Gene-directedenzyme prodrug therapy for prostate cancer in a mouse modelthat imitates the development of human disease. J. Gene Med.2004; 6: 43–54.

52 Watt F, Martorana A, Brookes DE et al. A tissue-specificenhancer of the prostate-specific membrane antigen gene,FOLH1. Genomics 2001; 73: 243–54.

53 Kianmanesh AR, Perrin H, Panis Y et al. A ‘distant’ bystandereffect of suicide gene therapy: Regression of nontransducedtumors together with a distant transduced tumor. Hum. GeneTher. 1997; 8: 1807–14.

54 Hall SJ, Mutchnik SE, Chen SH, Woo SLC, Thompson TC.Adenovirus-mediated herpes simplex virus thymidine kinase

Ovine atadenovirus 195

gene and ganciclovir therapy leads to systemic activity againstspontaneous and induced metastasis in an orthotopic mousemodel of prostate cancer. Int. J. Cancer 1997; 70: 183–7.

55 Martiniello-Wilks R, Wang X-Y, Voeks D et al. Purine nucleo-side phosphorylase and fludarabine phosphate gene-directedenzyme prodrug therapy suppresses primary tumour growth andpseudo-metastases in a mouse model of prostate cancer. 2004(submitted for publication).