Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest, Hungary1
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada2
Author for correspondence: Éva Nagy. Fax +1 519 824 5930. e-mail enagy{at}ovc.uoguelph.ca
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Abstract |
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PAdVs have so far been classified into six serotypes (Haig et al., 1964 ; Kasza, 1966
; Clarke et al., 1967
; Hirahara et al., 1990
; Kadoi et al., 1995
). The complete nucleotide sequence of PAdV-3 (Reddy et al., 1998a
) and parts of the PAdV-5 (Reddy et al., 1995
; Tuboly & Nagy, 2000
; Tuboly et al., 2000
) sequence have been published. The aim of this study was to determine the complete nucleotide sequence of the PAdV-5 HNF-70 strain and to identify the locations of putative virus genes and transcriptional elements needed for further work.
The source and propagation of the PAdV-5 HNF-70 strain and extraction and cloning of the viral DNA fragments have been described previously (Tuboly et al., 1995 ). Nested deletions were generated by exonuclease III and S1 nuclease digestions (Henikoff, 1984
) and the clones were sequenced in both directions by the dideoxynucleotide chain-termination technique with SP6 and T7 promoter-specific primers and primers based on the sequence data obtained. The contiguous sequence was assembled from overlapping sequences using the Lasergene software package.
A homology search of the GenBank database for the deduced amino acid sequence of each ORF was done with the aid of the BLAST program (Altschul et al., 1990 ). Promoters and splice sites were predicted at the Berkeley Drosophila Genome Project website (Ohler & Reese, 1998
; Reese et al., 1997
). Polyadenylation site prediction was done with the POLYAH program (Salamov & Solovyev, 1997
). Protein sequence alignments were generated with CLUSTAL W (Thompson et al., 1994
). Distance matrix analysis was carried out with the PHYLIP (version 3.5) package (Felsenstein, 1989
).
The genome of PAdV-5 was 32621 bp in length, the G+C content was 50·5 mol% and the genome structure and arrangement (Fig. 1) were similar to those of published mastadenoviruses.
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The E1A proteins were located between nt 418 and 1084. E1A 173R (19·88 kDa) and E1A 67R (7·76 kDa), encoded by two overlapping ORFs, were similar in length to the canine adenovirus serotype 1 (CAdV-1) E1A proteins, although these ORFs are not overlapping in CAdV-1 (Morrison et al., 1997 ). The retinoblastoma-susceptibility protein (pRb)-binding motif LXCXE (Defeo-Jones et al., 1991
), with a slight difference (97LDYPE), and the zinc finger motif (107CX2CX13CX2C; Culp et al., 1988
) were present in the E1A 173R protein, as in PAdV-3 (Reddy et al., 1998a
) and human adenovirus (HAdV) E1A proteins (Culp et al., 1988
). The E1B region (nt 11802787) encoded two proteins in two non-overlapping ORFs, 163R (19 kDa, small T antigen) and 340R (38 kDa, large T antigen). The E1B 163R protein showed the highest homology to the bovine adenovirus serotype 2 (BAdV-2) E1B ORF2 159R protein (47% identity). The E1B 340R protein had 37% amino acid identity to the PAdV-3 E1B 474R protein.
The mRNAs for the DNA-binding protein (DBP) are transcribed from the E2A region (located on the l strand between nt 21166 and 19841). The putative DBP of PAdV-5 contained 441 amino acids (49·8 kDa) and the N-terminal domain contained two nuclear localization signals (NLS) (40PKPKK and 47RRRK), which were similar to those predicted for BAdV-3 (29PRKK and 35RKRR) and PAdV-3 (47RRKR, 77RRK) (Reddy et al., 1998 a , b
). Two conserved zinc-binding motifs were present, one at 199CXHX52CX15C and the other at 311CXCX51CX15C, exactly as in the corresponding proteins of HAdV-2 (Tucker et al., 1994
) and PAdV-3 (Reddy et al., 1998a
).
According to amino acid sequence alignments with known terminal protein precursors (pTP), ORF94407542 encoded the main body of the pTP in the E2B region, with a predicted splice-acceptor site at nt 9348. All known pTPs have the sequence motif YSRLRYT involved in protein-primed DNA replication initiation (Hsieh et al., 1990 ). In PAdV-5, the 86YSRLKYT motif was identified at the same location. The NLS RLPI(R)4PRI of the pTP of PAdV-5 was similar to that of PAdV-3 [RLPL(R)4PRP] (Reddy et al., 1998a
). The serine residue, involved in the initiation of DNA replication, and the flanking residues (NSGD) were also well conserved (Smart & Stillman, 1982
) at 512NSGD in the PAdV-5 pTP.
ORF77104309, with a predicted splice-acceptor site at nt 7690, comprised the main body of the predicted DNA polymerase (pol) gene of PAdV-5. The conserved region I (YGDTDS) and two possible zinc finger motifs, CEYC(X)7HTC(X)10HH and CETRCDKC(X)23CSVC, of PAdV-5 pol were also present in PAdV-3 (Reddy et al., 1998a ).
Based on the available sequence data, PAdV-5 has the largest E3 region so far reported among PAdVs. Moreover, E3 ORF4 was unique to PAdV-5. The E4 region of PAdV-5 was also about 50% larger than that in most human adenoviruses and in PAdV-3. In addition, 8 of the 11 ORFs were unique to PAdV-5. Detailed analyses of the PAdV-5 E3 and E4 regions have been published previously (Tuboly & Nagy, 2000 ; Tuboly et al., 2000
).
In HAdVs, two genes encoding the IX and IVa2 proteins are classified as intermediate genes (Shenk, 1996 ). The minor capsid component (IX) is needed for packaging the viral DNA (Ghosh-Choudhury et al., 1987
) and is involved in activating the major late promoter (MLP) (Lutz et al., 1997
). The putative IX gene of PAdV-5 (126 aa, 13·7 kDa) showed 42% identity to the BAdV-2 118R ORF-4 protein. The IVa2 protein of PAdV-5 (372 aa, 42·5 kDa) had 66% amino acid identity to the IVa2 protein of HAdV-2. The entire potential NTP-binding site, GPTGCGKS (Gorbalenya & Koonin, 1989
), was present in PAdV-5 IVa2.
The late regions of the genome were characterized by their predicted common poly(A) sites and their products are mainly structural proteins (Shenk, 1996 ). Transcription starts from the MLP, and the primary transcript is processed into several late mRNAs. Six late regions (L1L6) were predicted for PAdV-5 (Fig. 1
). The putative MLP of PAdV-5 (nt 50775273) was deduced by promoter prediction and sequence similarity to known adenovirus MLP sequences. The canonical TATA box of the predicted MLP was located at nt 51225128. An inverted CAAT box (nt 50845088), an upstream promoter element (Sawadogo & Roeder, 1985
) (nt 51045109), an initiator element (Lu et al., 1997
) (nt 51505156) and two downstream activating elements (Leong et al., 1990
), DE1 (nt 52255235) and DE2 (nt 52405255), were identified within this region.
The common poly(A) tail addition site of the L1 region was predicted at nt 12173. The putative L1 52 kDa protein (354 aa, 40·4 kDa) was most similar to the 55 kDa protein of HAdV-17 (62% identity) and pIIIa (573 aa, 64·5 kDa) showed the highest identities to the pIIIa of HAdV-40 (61%).
The putative L2 region had a common poly(A) tail addition site at nt 14172. The III (penton base; 471 aa, 52·7 kDa) and pVII (147 aa, 18·9 kDa) proteins were predicted in this region. The RGD motif of protein III, which interacts with surface integrins v
3 and
v
5 (Wickham et al., 1993
), was missing from the predicted penton protein of PAdV-5. However, the entire LDV motif, which interacts with integrin
4
1 (Komoriya et al., 1991
), was present at 264LDV. The fibre-interacting domain is highly conserved in the penton base proteins of adenoviruses (Caillet-Boudin, 1989
). In PAdV-5, the 230SRLNNLLGIRKR motif was identical to the PAdV-3 fibre-interacting domain (Reddy et al., 1998a
). Protein III of PAdV-5 was most similar to that of PAdV-3 (77% identity), and pVII exhibited 54% similarity to pVII of BAdV-2. One putative protease cleavage site was found in pVII, at 20MYGGA, exactly the same position as in PAdV-3 (Reddy et al., 1998a
).
The L3 region had a predicted common poly(A) site at nt 15356. Protein V of PAdV-5 (374 aa, 42·4 kDa) was most closely related to the corresponding protein of BAdV-2 (57% identity).
The common poly(A) tail addition site of the L4 region was located at nt 19790. The predicted pX protein (70 aa, 7·8 kDa) had 79% amino acid identity to pX of BAdV-2. There was only one protease cleavage site, at 38MSGGF (Weber & Anderson, 1988 ). The pVI protein of PAdV-5 (233 aa, 25·2 kDa) showed the highest similarity to pVI of HAdV-40 (55% identity) and contained two sequence motifs (30MNGGAFNW and 219IVGLGVRS) that corresponded to consensus protease cleavage site sequences (Russell & Kemp, 1995
). In HAdV-2, the protease requires a peptide (GVQSLKRRRCF) that is derived from the C terminus of pVI as a cofactor for its activity (Mangel et al., 1993
; Webster et al., 1993
). In pVI of PAdV-5, this peptide sequence was well conserved, at 222LGVRSVKRRRCF.
The predicted L5 region was characterized by a poly(A) tail addition site at nt 26455. The 100 kDa protein (722 aa) and the 33 kDa protein (219 aa) showed the highest similarity to the corresponding BAdV-3 proteins (59 and 27% identity, respectively). The pVIII gene (222 aa, 24·1 kDa) has been described previously (Tuboly & Nagy, 2000 ). After alignment to the corresponding BAdV-3 protein (Reddy et al., 1998b
), two putative protease cleavage sites were found in the pVIII protein, at 108LAGGGRTT and at 148LAGGSRSS. Fig. 2
shows a phylogenetic analysis of the putative pVIII protein compared with representative adenoviruses. PAdV-1, -2 and -3 had an inferred common ancestor, whereas PAdV-4 and PAdV-5 were in two additional, separate lineages. Similar relationships were noted for the hexon proteins. It seemed that PAdV-5 was phylogenetically closer to certain bovine adenoviruses, specifically to BAdV-1 (based on pVIII) and BAdV-2 (based on hexon, sequences provided by D. Ojkic, Guelph, Canada; no BAdV-1 sequences were available), than to other described porcine adenoviruses. All these findings underline the recent classification of PAdVs (Benkö et al., 2000
).
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Some adenoviruses encode low molecular mass virus-associated RNAs (VA RNAs), transcribed by RNA polymerase III, which are required for the efficient translation of viral mRNAs late after infection (Larsson et al., 1986 ). In the mammalian adenoviruses studied by Ma & Mathews (1996)
, there are either one or two genes for VA RNA, known as VA RNAI and VA RNAII, located between the pTP and the 52 kDa ORFs. Based on sequence analysis and RNA secondary structure prediction only, the presence of VA RNAs in this region could not be predicted for PAdV-5, as has also been observed for non-primate mastadenoviruses.
The analysis of the PAdV-5 genome summarized in this paper indicated that the size and genome organization of this adenovirus were similar to those of mastadenoviruses. However, unique characteristics of PAdV-5 were also identified. Most importantly, the RGD motif of the penton base protein and the TLWT motif of the fibre protein were not present. Only one protease cleavage site was found in pX. Phylogenetic analysis of pVIII and hexon proteins showed that PAdV-5 was well separated from the other PAdVs but was more closely related to BAdV-1 and BAdV-2. The sequence data for PAdV-5 will contribute to the overall understanding of mammalian adenoviruses and specifically will be essential in the improvement of PAdV-5-based vectors (Tuboly & Nagy, 2001 ).
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Acknowledgments |
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Footnotes |
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References |
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Received 31 August 2000;
accepted 29 November 2000.