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Molecular cloning and sequence analysis of the penton base genes of type II avian adenoviruses
ELSEVIER
Virus Research 39 (1995) 289-297
Virus Research
Molecular cloning and sequence analysis of the penton base genes of type II avian adenoviruses M. Suresh
a,1, S. St. Cyr
b, J.M. Sharma a,*
a Department of Veterinary PathoBiology, Collegeof Veterinary Medicine, University of Minnesota, 1971 Common WealthAvenue, St. Paul, MN55108, USA b Institute of Human Genetics, 4-122 Moos Tower, 515 Delaware St. S.E., University of Minnesota, Minneapolis, MN 55455, USA Received 6 June 1995; revised 13 September 1995; accepted 13 September 1995
Abstract
We describe here the identification of the penton base gene of hemorrhagic enteritis virus (HEV), a type II avian adenovirus, in a 2477-base pair (bp)-EcoRI fragment of the viral DNA by sequence analysis. Identification is based on an extensive amino acid homology between the HEV-open reading frame and the penton base of a fowl adenovirus (FAV-10) and various human adenoviruses. The 1344 bp-penton base gene of HEV encodes a 448-amino acid polypeptide of molecular weight of 50,843 Da. The nucleotide sequences of penton base genes of HEV and marble spleen disease virus (MSDV) are identical. The HEV penton base lacks the RGD motif, present in most human adenoviruses (Ad2, Ad3, Ad4, and Ad 12) suggesting that HEV may not use a v integrins to gain entry into host cells. Further sequence analysis revealed the presence of a L e u - A s p - V a l (LDV) motif in the HEV penton base amino acid sequence similar to most of the human adenoviruses. LDV motif on the fibronectin has been shown to interact with the a4/31 integrins on cells, which includes lymphocytes and monocytes. The presence of LDV motif in the penton base of HEV implicates the involvement of a4/31 integrins in the viral internalization into host cells. Keywords: Type II avian adenovirus; Hemorrhagic enteritis virus; Penton base; Gene; LDV motif
* Corresponding author. Tel.: +1 (612) 625 5276; Fax: +1 (612) 625 5203; e-mail: [email protected]. i Present address: Department of Microbiology and Immunology, Rollins Research Center, Emory University School of Medicine, Atlanta, GA 30322, USA. 0168-1702/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1702(95)00101-8
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M. Suresh et al. / Virus Research 39 (1995) 289-297
I. Introduction
Type II avian adenoviruses constitute an immunologically distinct group of adenoviruses unrelated to the large group of type I avian adenoviruses (Domermuth et al., 1980). The members of the avian type II adenoviruses are: hemorrhagic enteritis virus (HEV) of turkeys; marble spleen disease virus (MSDV) of pheasants; and avian adenovirus group II splenomegaly virus (AASV) of chickens. The HEV, MSDV, and AASV are antigenically indistinguishable but have been differentiated from each other by restriction endonuclease fingerprinting (Zhang and Nagaraja, 1989). Infection of susceptible turkeys with HEV results in hemorrhagic enteritis (HE) accompanied with variable mortality (Domermuth and Gross, 1991). MSDV, being apathogenic to turkeys has been used as a vaccine against infection with HEV (Sharma, 1994). The major structural proteins of the adenovirus virion are the hexon and penton (fiber and penton base) that surrounds a core containing the double stranded DNA genome (Valentine and Periera, 1965). The characteristic feature of the penton base of type II avian adenoviruses is the presence of a single fiber similar to mammalian adenoviruses (Valentine and Periera, 1965; van den Hurk, 1992). In contrast, fowl adenoviruses have double fibers attached to their penton base proteins (Laver et al., 1971). Although hexon protein has been reported to be the protective antigen (van den Hurk and van Drunen Littel-van den Hurk, 1993), the role of penton base protein in type II adenoviral infections has not been examined. The penton base genes of several human adenoviruses have been shown to contain a conserved Arg-Gly-Asp (RGD) sequence that is predicted to lie at the apex of two extended alpha helices (Mathias et al., 1994). Studies have indicated that the penton base RGD domain promotes efficient infection of host ceils by multiple adenovirus serotypes of humans via interactions with av integrins (Mathias et al., 1994). However, the penton base of the fowl adenovirus 10 (FAV-10) lacks an intact RGD sequence (Sheppard and Trist, 1992). Because the penton of type II avian adenoviruses resembles mammalian adenoviruses in having a single fiber, we were interested to examine if they also contained the RGD motif. Also, we sought to compare the penton base genes of HEV and MSDV to identify possible sequence differences to determine the degree of conservation of the penton base sequence among type II avian adenoviruses. Furthermore, HEV and MSDV are assigned to the adenovirus group solely based on morphologic (Itakura and Carlson, 1975) and histological (Fujiwara et al., 1975) criteria and there is no molecular data to support this contention. In this report, we provide molecular evidence for the first time that both HEV and MSDV are adenoviruses. Furthermore, we also show that the penton base genes of HEV and MSDV are identical and contain an LDV motif, but lack the RGD motif that is present in several human adenoviruses. To our knowledge, this is the first gene of type II avian adenoviruses to be cloned and characterized. In an earlier study, we had cloned and sequenced a 564 base pair EcoRI-HindlII fragment of the genomic DNA of HEV (Suresh and Sharma, unpublished data).
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Sequence analyses and comparison with the sequences in the GenBank had suggested that the E c o R I - H i n d l I I fragment of HEV DNA consisted of nucleotide sequences coding for a part of the penton base gene of HEV. We sought to use this 564 bp fragment as a probe to identify the fragment of HEV DNA that encodes the full length penton base protein. HEV was purified from the spleens collected from HEV-infected specific-pathogen-free turkeys on the fourth day post-infection as described previously (Ossa et al., 1983). The viral DNA, extracted from purified HEV (Scott-Taylor and Hammond, 1992) was digested with E c o R I , electrophoresed and analysed on a Southern blot (Aleefs et al., 1990) with a digoxigenin-labeled-564 bp E c o R I - H i n d l I I HEV fragment as a probe. The 564 base pair probe identified a single ~ 2500 base pair E c o R I fragment from HEV DNA, the putative penton base gene-containing fragment. The ~ 2500 bp DNA fragment of HEV was cloned into pGEM-3Z plasmid (Promega, Madison, WI) as per standard protocols (Sambrook et al., 1989) and sequenced by Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The homology, alignment of amino acid sequences, and calculation of pair-wise percentage identities and similarities were performed by the Pileup, FastA, and GAP program of GCG sequence analysis software package (Genetics Computer Group, Madison, WI).
2. Structure of the HEV penton base gene
The nucleotide sequence of the 2477 bp fragment and the deduced amino acid sequence of the open reading frame (ORF) are shown in Fig. 1. Sequence analysis of both strands of the HEV ~ 2500 bp E c o R I fragment revealed a possible reading frame of 1344 bp. The ATG codon at nucleotides 346-348 marks the beginning of the O R F with a stop codon at nucleotides 1690-1692. Comparisons of the nucleotide sequence and the predicted amino acid sequence for the ORF were made using a nonredundant BLAST search and comparison (Altschul et al., 1990) of the EMBL, GenBank and SWlSS-PROT data bases from the National Center for Biotechnology Information BLAST E-Mail server. As shown in Table 1, the deduced amino acid sequence of the putative HEV penton base protein shares significant homology with the penton base proteins of FAV-10 and human adenoviruses-12 (Adl2), -3 (Ad3), -2 (Ad2), -40 (Ad40), and -5 (Ad5) (Table 1). These data suggested that the open reading frame identified in the E c o R I fragment of HEV DNA encoded the penton base gene. The ORF was deduced to encode for a 448-amino acid polypeptide, with a molecular weight of 50,843 Da. The predicted molecular weight of the HEV penton base matches the reported apparent molecular weight (51 kDa) of the HEV penton base protein (van den Hurk, 1992). The HEV penton base protein is shorter than the penton base proteins of several human adenoviruses including FAV-10 (Table 1). As depicted in Fig. 2, the amino acid sequences of N-terminal and C-terminal regions of the penton base protein of
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M. Suresh et al. / l,qrus Research 39 (1995) 289-297
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Table 1 Amino acid sequence homology of hemorrhagic enteritis virus penton base protein with a fowl adenovirus and human adenoviruses Viruses Amino acid sequence % Similarity a % Identity a Length (with HEV) (with HEV) HEV 100 100 448 FAV-10 b 67 49 525 Ad2 66 47 571 Ad3 66 47 544 Ad5 66 47 571 Adl2 65 48 497 The percent similarity and identity was determined as defined by the GAP program of the University of Wisconsin GCG software analysis software. b Fowl adenovirus. a
H E V are very similar to that of Ad2 penton base. The variation in the polypeptide length was not randomly distributed across the molecule but occurred predominantly in the central region (where the R G D motif of Ad2 is located) of the protein through deletions (relative to Ad2) of 19 and 62 residues. In addition, one deletion of 35 residues occurred at the amino-terminal end. This observation is consistent with an earlier finding that the amino acid sequences of N-terminal and C-terminal regions of the penton base proteins of Ad2, Ad3, Ad4, and Ad12 are very similar (Mathias et al., 1994).
3. Comparison of HEV and MSDV penton base genes We sought to identify possible sequence differences between the penton base genes of H E V and MSDV. In order to identify the fragment of M S D V D N A that encodes the penton base, we analysed the E c o R I digested M S D V genomic D N A by Southern blot hybridization (Aleefs et al., 1990) using the O R F of the H E V penton base gene as a probe. As depicted in Fig. 3, H E V probe hybridized to a ~ 2500 bp fragment of M S D V DNA. The fragment of the M S D V D N A that hybridized with the H E V probe was cloned into p G E M - 3 Z vector and sequenced as described earlier. Alignment of the H E V and M S D V sequences revealed that the nucleotide sequences encoding the penton base gene were identical indicating 100% conservation. These data support the reported similarity in apparent molecular weights of the penton base proteins of H E V and M S D V (Nazerian et al.,
Fig. 1. Nucleotide and deduced amino acid sequences of the penton base gene of hemorrhagic enteritis virus. Asterisks denote the LDV motif. The nucleotide sequence has been submitted to the GenBank nucleotide sequence data base and has been assigned the accession number U28139.
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1991: Zhang et al., 1991). HEV causes hemorrhagic enteritis and death while MSDV is apathogenic to turkeys (Domermuth and Gross, 1991). The molecular basis for the differences in pathogenicity is currently not known. Our data Ad2
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Fig. 2. Alignment of the penton base sequences of human adenovirus type 2 (Ad2) and hemo~hagic enteritis v i e s (HEV). Gaps indicated by dotted lines were used to maximie the alignment. The LDV sequences are in bold and are indicated by asterisks. Identical amino acid residues are indicated by ve~ical fines. Similar amino acid residues are indicated by ':' and '.' between the sequence symbols (similariW = ':' > '.'). ':' and '.' indicates that the comparison values are > 0.50 and > 0.10, respectively (Gfibskov and Burges, 1986).
M. Suresh et al. /Virus Research 39 (1995) 289-297
I
2
295
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Fig. 3. Southern blot analysis of the EcoRI digested DNA from hemorrhagicenteritis virus (HEV) and marble spleen disease virus (MSDV) using digoxigenin-labeledpenton base gene of HEV as a probe. Lane 1, MSDV; lane 2, HEV; lane 3, digoxigenin- iabeled-H/ndlII-digested-,~ DNA as molecular weight standard. The sizes of a few DNA fragments are indicated in base pairs (BP). confounds an earlier speculation that the molecular differences between the penton base proteins of H E V and MSDV determined differences in pathogenicity to turkeys (Zhang et al., 1991; van den Hurk, 1992).
4. Virus internalization motif in the deduced amino acid sequence of H E V / M S D V penton base Majority of the studies on the interactions of adenoviruses with the host cells have been performed with the Ad2 of humans (Greber et al., 1993). Initial attachment of Ad2 to cells is mediated via the fiber coat protein to an unidentified receptor (Philipson et al., 1968). Following attachment to cells, Ad2, Ad3, Ad4, and A d l 2 viruses bind to cell surface a v integrins via a pentavalent R G D sequence that is present in the penton base protein (Wickham et al., 1993) suggesting that a v integrins might play a pivotal role in the entry of adenoviruses into host cells. The penton base of type II avian adenoviruses resemble mammalian adenoviruses in having a single fiber attached to each one of them (van den Hurk, 1992). The penton base protein of FAV-10, on the other hand has two fibers
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(Laver et al., 1971) and lacks the R G D motif (Sheppard and Trist, 1992). We were interested to find out if type II avian adenoviruses also resembled the mammalian adenoviruses in having a R G D motif. The deduced amino acid sequences of H E V and MSDV penton base protein lack the R G D motif similar to FAV-10 (Sheppard and Trist, 1992). These data suggested that avian adenoviruses may not interact with otvfl3 and a v f l 5 cell surface integrins. The lack of an R G D motif does not rule out the interaction of H E V / M S D V with other integrins because non-RGD sequences have also been shown to mediate integrin binding (Hynes, 1992). Further analysis of the amino aci d sequence of the penton base of H E V / M S D V revealed the existence of a motif, L e u - A s p - V a l (LDV) at amino acid position 245-247. Similar LDV motifs are conserved between the penton base proteins of Ad2, Ad3, Ad4, and Ad12 (Mathias et al., 1994; Cuzange et al., 1994). Interestingly, this LDV motif is not present in both Ad40 and FAV-10, which incidentally, also do not have the R G D motif (Davison et al., 1993). The LDV motif, present in the extracellular matrix protein fibronectin (FN; Makarem and Humphries, 1991) has been shown to be recognized primarily by the a4fll-integrin receptor expressed on lymphocytes, monocytes, basophils, eosinophils, bone marrow progenitors, fetal myoblasts, and melanoma cells (Hynes, 1987; Hemler, 1990; Rosen et al., 1992). The LDV sequence seems to be important since it is completely conserved in the human, rat, bovine, and avian fibronectins (Komoriya et al., 1991). Studies have shown that the minimum essential sequence for the recognition of fibronectin by the a4/31 integrin receptor is LDV (Komoriya et al., 1991). Since, H E V / M S D V lack the R G D motif, these data implicate the involvement of a4/31-integrins in the viral entry into host cells via interactions with the LDV motif of the viral penton base protein. Studies to inhibit adenoviral internalization with LDV peptides or antibodies to a4/~l should reveal the role of LDV motif on the penton base protein in the viral internalization.
Acknowledgements We wish to thank Drs. Jamil Ahmad, Kemal Karaca, Terence Pertile and Sudhir Reddy for their suggestions and assistance during the study.
References Aleefs, J.J., Salantijn, E.M., Krens, F.A., and Rouwendal, G.J. (1990) Optimization of non-radioactive Southern hybridization: single copy detection and reuse of blots. Nucl. Acids Res. 18, 3099-3100. Altsehul, S.F., Gish, W., Miller, W., Myers, W., and Lipman, D.J. (1990) Basic local alignment tool. J. Mol. Biol. 215, 403-410. : Cuzange, A., Chroboczek,J., and Jacrot, B. (1994)The penton base of human adenovirustype 3 has the RGD motif. Gen¢ 94, 257-259. Davison, A.J., Telford, E.A.R,, Watson, M.S., McBride, K., and Mautner, V. (1993) The DNA sequence of adenovirus type 40. J. MOI.B~I. 234, 1308-1316. Domermuth, C.H. and Gross, W.B. (1991) Hemorrhagic enteritis and related infections.In: B.W. Calnek, H. J, Barnes, C.W. Beard; W.M. Reid, and H.W. Yoder, Jr, (Eds.), Diseases of Poultry, Iowa State UniversityPress, Ames, Iowa, 9th ed.,' pp. 567-572.
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Domermuth, C.H., Weston, C.R., Cowen, B.S., ColweU, W.M., Gross, W.B., and Dubose, R.T. (1980) Incidence and distribution of avian adenovirus group II splenomegaly of chickens. Avian Dis. 24, 591-594. Fujiwara, H., Tanaami, S., Yamaguchi, M., and Yoshino, T. (1975) Histopathology of hemorrhagic enteritis in turkeys. Nat. Inst. Anim. HealthQ (Tokyo) 15, 68-75. Greber, U.F., Willetts, M., Webster, P., and Helenius, A. (1993) Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75, 477-486. Gribskov, M. and Burgess, R.R. (1986) Sigma factors from E. coli, B. subtilis, phage SPO1, and phage 4 are homologous proteins. Nucl. Acids Res. 14, 6745-6763. Hemler, M.E. (1990) VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu. Rev. Immunol. 8, 365-400. Hynes, R.O. (1987) Integrins: a family of cell surface proteins. Cell 48, 549-554. Hynes, R.O. (1992) Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25. Itakura, C. and Carlson, H.C. (1975) Electronic microscopic findings of cells with Inclusion bodies in experimental hemorrhagic enteritis of turkeys. Can. J. Comp. Med. 39, 299-304. Komoriya, A., Green, L.J., Mervic, M., Yamada, S.S., Yamada, K.M., and Humphries, M.J. (1991) The minimum essential sequence for a major cell type-specific adhesion site (CS1) within the the alternatively spliced Type III connecting segment domain of fibronectin is Leucine-Aspartic acid-Valine. J. Biol. Chem. 266, 15075-15079. Laver, W.G., Younghusband, H.B., and Wrigley, N.G. (1971) Purification and properties of chick embryo lethal orphan virus (an avian adenovirus) Virology. 45, 598-614. Makarem, R. and Humphries, M.J. (1991) LDV: a novel cell adhesion motif recognized by the integrin a4/31. Biochem. Soc. Trans. 19, 380S. Mathias, P., Wickham, T., Moore, M., and Nemerow, G. (1994) Multiple adenovirus serotypes use av integrins for infection. J. Virol. 68, 6811-6814. Nazerian K., Lee, L.F., and Payne, W.S. (1991) Structural polypeptides of type II avian adenoviruses analyzed by monoclonal and polyclonal antibodies. Avian Dis. 35, 572-578. Ossa, J.E., Bates, R.C., and Schurig, G.G. (1983) Hemorrhagic enteritis in turkeys: purification and quantification of the virus. Avian Dis. 27, 235-245. Philipson, L., Lonberg-Holm, K., and Petterson, U. (1968) Virus-receptor interaction in an adenovirus system. J. Virol. 2, 1064-1075. Rosen, G.D., Sanes, J.R., LaChance, R., Cunningham, J.M., Roman, J., and Dean, D.C. (1992) Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell 69, 1107-1119. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2nd ed. Scott-Taylor, T.H. and Hammond, G.W. (1992) Conserved sequences of the adenovirus genome for detection af all human adenovirus types by hybridization. J. Clin. Microbiol. 30, 1703-1710. Sharma, J.M. (1994) Response of specific-pathogen-free turkeys to vaccines derived from marble spleen disease virus and hemorrhagic enteritis virus. Avian Dis. 38, 523-530. Sheppard, M. and Trist, H. (1992) Characterization of the avian adenovirus penton base. Virology. 188, 881-886. Valentine, R.C. and Periera, H.G. (1965) Antigens and structure of the adenovirus. J. Mol. Biol. 13, 13-20. van den Hurk, J.V. (1992) Characterization of the structural proteins of hemorrhagic enteritis virus. Arch. Virol. 126, 195-213. van den Hurk, J.V. and van Drunnen Littel-van den Hurk, S. (1993) Protection of turkeys against hemorrhagic enteritis by monoclonal antibody and hexon immunization. Vaccine. 11, 329-335. Wickham, T.J., Mathias, P., Cheresh, D.A., and Nemerow, G.R. (1993) Integrins av/33 and ctvfl5 promote adenovirus internalization but not virus attachment. Cell 73, 309-319. Zhang, C., and Nagaraja, K.V. (1989) Differentiation of avian adenovirus type-II strains by restriction endonuclease fingerprinting. Am.J. Vet. Res. 50, 1466-1470. Zhang, C., Nagaraja, K.V., Sivanandan, V., and Newman, J.A. (1991) Identification and characterization of viral polypeptides from type-II avian adenoviruses. Am. J. Vet. Res. 52, 1137-1141.
Virus Research 39 (1995) 289-297
Virus Research
Molecular cloning and sequence analysis of the penton base genes of type II avian adenoviruses M. Suresh
a,1, S. St. Cyr
b, J.M. Sharma a,*
a Department of Veterinary PathoBiology, Collegeof Veterinary Medicine, University of Minnesota, 1971 Common WealthAvenue, St. Paul, MN55108, USA b Institute of Human Genetics, 4-122 Moos Tower, 515 Delaware St. S.E., University of Minnesota, Minneapolis, MN 55455, USA Received 6 June 1995; revised 13 September 1995; accepted 13 September 1995
Abstract
We describe here the identification of the penton base gene of hemorrhagic enteritis virus (HEV), a type II avian adenovirus, in a 2477-base pair (bp)-EcoRI fragment of the viral DNA by sequence analysis. Identification is based on an extensive amino acid homology between the HEV-open reading frame and the penton base of a fowl adenovirus (FAV-10) and various human adenoviruses. The 1344 bp-penton base gene of HEV encodes a 448-amino acid polypeptide of molecular weight of 50,843 Da. The nucleotide sequences of penton base genes of HEV and marble spleen disease virus (MSDV) are identical. The HEV penton base lacks the RGD motif, present in most human adenoviruses (Ad2, Ad3, Ad4, and Ad 12) suggesting that HEV may not use a v integrins to gain entry into host cells. Further sequence analysis revealed the presence of a L e u - A s p - V a l (LDV) motif in the HEV penton base amino acid sequence similar to most of the human adenoviruses. LDV motif on the fibronectin has been shown to interact with the a4/31 integrins on cells, which includes lymphocytes and monocytes. The presence of LDV motif in the penton base of HEV implicates the involvement of a4/31 integrins in the viral internalization into host cells. Keywords: Type II avian adenovirus; Hemorrhagic enteritis virus; Penton base; Gene; LDV motif
* Corresponding author. Tel.: +1 (612) 625 5276; Fax: +1 (612) 625 5203; e-mail: [email protected]. i Present address: Department of Microbiology and Immunology, Rollins Research Center, Emory University School of Medicine, Atlanta, GA 30322, USA. 0168-1702/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1702(95)00101-8
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M. Suresh et al. / Virus Research 39 (1995) 289-297
I. Introduction
Type II avian adenoviruses constitute an immunologically distinct group of adenoviruses unrelated to the large group of type I avian adenoviruses (Domermuth et al., 1980). The members of the avian type II adenoviruses are: hemorrhagic enteritis virus (HEV) of turkeys; marble spleen disease virus (MSDV) of pheasants; and avian adenovirus group II splenomegaly virus (AASV) of chickens. The HEV, MSDV, and AASV are antigenically indistinguishable but have been differentiated from each other by restriction endonuclease fingerprinting (Zhang and Nagaraja, 1989). Infection of susceptible turkeys with HEV results in hemorrhagic enteritis (HE) accompanied with variable mortality (Domermuth and Gross, 1991). MSDV, being apathogenic to turkeys has been used as a vaccine against infection with HEV (Sharma, 1994). The major structural proteins of the adenovirus virion are the hexon and penton (fiber and penton base) that surrounds a core containing the double stranded DNA genome (Valentine and Periera, 1965). The characteristic feature of the penton base of type II avian adenoviruses is the presence of a single fiber similar to mammalian adenoviruses (Valentine and Periera, 1965; van den Hurk, 1992). In contrast, fowl adenoviruses have double fibers attached to their penton base proteins (Laver et al., 1971). Although hexon protein has been reported to be the protective antigen (van den Hurk and van Drunen Littel-van den Hurk, 1993), the role of penton base protein in type II adenoviral infections has not been examined. The penton base genes of several human adenoviruses have been shown to contain a conserved Arg-Gly-Asp (RGD) sequence that is predicted to lie at the apex of two extended alpha helices (Mathias et al., 1994). Studies have indicated that the penton base RGD domain promotes efficient infection of host ceils by multiple adenovirus serotypes of humans via interactions with av integrins (Mathias et al., 1994). However, the penton base of the fowl adenovirus 10 (FAV-10) lacks an intact RGD sequence (Sheppard and Trist, 1992). Because the penton of type II avian adenoviruses resembles mammalian adenoviruses in having a single fiber, we were interested to examine if they also contained the RGD motif. Also, we sought to compare the penton base genes of HEV and MSDV to identify possible sequence differences to determine the degree of conservation of the penton base sequence among type II avian adenoviruses. Furthermore, HEV and MSDV are assigned to the adenovirus group solely based on morphologic (Itakura and Carlson, 1975) and histological (Fujiwara et al., 1975) criteria and there is no molecular data to support this contention. In this report, we provide molecular evidence for the first time that both HEV and MSDV are adenoviruses. Furthermore, we also show that the penton base genes of HEV and MSDV are identical and contain an LDV motif, but lack the RGD motif that is present in several human adenoviruses. To our knowledge, this is the first gene of type II avian adenoviruses to be cloned and characterized. In an earlier study, we had cloned and sequenced a 564 base pair EcoRI-HindlII fragment of the genomic DNA of HEV (Suresh and Sharma, unpublished data).
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Sequence analyses and comparison with the sequences in the GenBank had suggested that the E c o R I - H i n d l I I fragment of HEV DNA consisted of nucleotide sequences coding for a part of the penton base gene of HEV. We sought to use this 564 bp fragment as a probe to identify the fragment of HEV DNA that encodes the full length penton base protein. HEV was purified from the spleens collected from HEV-infected specific-pathogen-free turkeys on the fourth day post-infection as described previously (Ossa et al., 1983). The viral DNA, extracted from purified HEV (Scott-Taylor and Hammond, 1992) was digested with E c o R I , electrophoresed and analysed on a Southern blot (Aleefs et al., 1990) with a digoxigenin-labeled-564 bp E c o R I - H i n d l I I HEV fragment as a probe. The 564 base pair probe identified a single ~ 2500 base pair E c o R I fragment from HEV DNA, the putative penton base gene-containing fragment. The ~ 2500 bp DNA fragment of HEV was cloned into pGEM-3Z plasmid (Promega, Madison, WI) as per standard protocols (Sambrook et al., 1989) and sequenced by Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The homology, alignment of amino acid sequences, and calculation of pair-wise percentage identities and similarities were performed by the Pileup, FastA, and GAP program of GCG sequence analysis software package (Genetics Computer Group, Madison, WI).
2. Structure of the HEV penton base gene
The nucleotide sequence of the 2477 bp fragment and the deduced amino acid sequence of the open reading frame (ORF) are shown in Fig. 1. Sequence analysis of both strands of the HEV ~ 2500 bp E c o R I fragment revealed a possible reading frame of 1344 bp. The ATG codon at nucleotides 346-348 marks the beginning of the O R F with a stop codon at nucleotides 1690-1692. Comparisons of the nucleotide sequence and the predicted amino acid sequence for the ORF were made using a nonredundant BLAST search and comparison (Altschul et al., 1990) of the EMBL, GenBank and SWlSS-PROT data bases from the National Center for Biotechnology Information BLAST E-Mail server. As shown in Table 1, the deduced amino acid sequence of the putative HEV penton base protein shares significant homology with the penton base proteins of FAV-10 and human adenoviruses-12 (Adl2), -3 (Ad3), -2 (Ad2), -40 (Ad40), and -5 (Ad5) (Table 1). These data suggested that the open reading frame identified in the E c o R I fragment of HEV DNA encoded the penton base gene. The ORF was deduced to encode for a 448-amino acid polypeptide, with a molecular weight of 50,843 Da. The predicted molecular weight of the HEV penton base matches the reported apparent molecular weight (51 kDa) of the HEV penton base protein (van den Hurk, 1992). The HEV penton base protein is shorter than the penton base proteins of several human adenoviruses including FAV-10 (Table 1). As depicted in Fig. 2, the amino acid sequences of N-terminal and C-terminal regions of the penton base protein of
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attccagatcctaatatagttcaggttcctgtttctaatgttactgttccagttccagcg cccaggactaaatttaaaatgccacaacctgtgtccaggccttcaaaaacagcctatagg tctaaatatcagtatcctagtgaatctgatacagatactgactcagaaattgaggtattt ggtaagccttacggaccaataaaaccagctacaatcgacattgacaacttgtctgctcaa nttaaaagactgaaaggaaagggtttagatatttctaattatatgagaagaaaagcaaga aatgttaatgttagaccatactgaccaattcctggttcattttagatggaatcttcgaac M E S actgccactagaatttttgctccaacggaagggagaaacagtataatttacagcaacttg T A T R I F A P T E G R N S I I Y S cctcctgttcaagatacaaccaaaatattttatatagataacaaggccattgatatagag P P V Q D T T K I F Y I D N K A I D tcatataatcaagagaaagatcattctaattattatactaatataattcaaacacagaac S Y N Q E K D H S N Y Y T N I I Q T atttcaactattgattcaagtatacagcaaattcagttagatgaaaggtctagatgggga I S T I D S S I Q Q I Q L D E R S R ggagaactacatacaagcttagtaacatctgttatgaattgtactaaacattttaattca G E L H T S L V T S V M N C T K H F gataggtgtttagtgaaaattcagactattaagagtccacctacatttgaatggaaagaa D R C L V K I Q T I K S P P T F E W ttgaaaatacctgagggaaactatgttttaaatgagtttattgatttattaaatgaaqqt L K I P E G N Y V L N E F I D L L N attacttctttataccttcagtatggcaggcaacagggtgtacttgaagaagacatagga I T S L Y L Q Y G R Q Q G V L E E D ataaaatttgatactcgcaattttgaaattggtaaagatccaactactaatcttgttact I K F D T R N F E I G K D P T T N L cctggtaaatacttgtttaagggttatcatgctgatataatacttcttcctggttgggct P G K Y L F K G Y H A D I I L L P G W A attgatttttctttttctagattgggtaacattttaggtattagaaaacgtgagacttat I D F S F S R L G N I L G I R K R E aaagctggctttttgattgaatatgatgacttgacaaatggtaatattccaccactgttg K A G F L I E Y D D L T N G N I P P gatgttgctaactataagtctacaagtcaagctaaaccattattacaggatccatctg~c D* V* A N Y K S T S Q A K P L L Q D P agatcttaccacgttatggatagtgattctaacagacctgtgactgcatataggtctttt R S Y H V M D S D S N R P V T A Y R S F gttttgtcatataacaatgaaggtgctgcaaaattaaagtttttgatgtgtatgagtgat V L S Y N N E G A A K L K F L M C M ataacggggggtctcaatcagctgtattggtgtttgcctgattcttataaaccgccagta I T G G L N Q L Y W C L P D S Y K P P V tcttttaagcaagaaacgcaagtagataaactgcctgttgttggtatgcaactttttcct S F K Q E T Q V D K L P V V G M Q L F P tttgtttctaaatctgtgtattctggtgctgctgtttacacacagttaattgaacagcag F V S K S V Y S G A A V Y T Q L I E Q Q actaatttgacacaaatttttaacagatttcatgataatgaaattttaaaacaagctcca T N L T Q I F N R F H D N E I L K Q A P tatgtgaatcaagttttattggctgaaaatgtgcccataaatgttaatcagggaacaata Y V N Q v L L A E N V P I N V N Q G ccaatattttcaactcttccaggagtacagagagtggttgtggaagacgataggagaaga P I F S T L P G V Q R V V V E D D R R R actgtaccctacgttaccaagtcacttgctacagtatatccgaaggttttgtctagcaaa T V P Y V T K S L A T V Y P K V L S actttgcaataatgcattctgttgtttattctccaggggacagtagaggatggggtattg T L Q * gtaattcaagtatgcgagattattatttgataggtggcgctttgcaaccgtctgatattt atactgttagggttcgtgaacattggagacgtaaaaggaggccaactgctcaaactggaa attctgctgtaaccccacgacgtagaagacggagaacaattgcaattcaagtaccagctc caactagagtactaagaaatagaatagttacacctgttgtgcctgcagttcctgtacctg ctcctacagtttctgctgtaccagtacctgctgctcctgtagctgtagctgctaagagac gtagagtaggttagaaatgtttgaaaatttagcacccagaaaaggtctaaaaaccgaaac acggaatgtaaagtttagtaatgaattgagaggtggttttgttgtctctgttttagttcc tttgctttcttctttaataggcgcagctcctgccattgctggaactgtaattgcagctag
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M. Suresh et al. / l,qrus Research 39 (1995) 289-297
293
Table 1 Amino acid sequence homology of hemorrhagic enteritis virus penton base protein with a fowl adenovirus and human adenoviruses Viruses Amino acid sequence % Similarity a % Identity a Length (with HEV) (with HEV) HEV 100 100 448 FAV-10 b 67 49 525 Ad2 66 47 571 Ad3 66 47 544 Ad5 66 47 571 Adl2 65 48 497 The percent similarity and identity was determined as defined by the GAP program of the University of Wisconsin GCG software analysis software. b Fowl adenovirus. a
H E V are very similar to that of Ad2 penton base. The variation in the polypeptide length was not randomly distributed across the molecule but occurred predominantly in the central region (where the R G D motif of Ad2 is located) of the protein through deletions (relative to Ad2) of 19 and 62 residues. In addition, one deletion of 35 residues occurred at the amino-terminal end. This observation is consistent with an earlier finding that the amino acid sequences of N-terminal and C-terminal regions of the penton base proteins of Ad2, Ad3, Ad4, and Ad12 are very similar (Mathias et al., 1994).
3. Comparison of HEV and MSDV penton base genes We sought to identify possible sequence differences between the penton base genes of H E V and MSDV. In order to identify the fragment of M S D V D N A that encodes the penton base, we analysed the E c o R I digested M S D V genomic D N A by Southern blot hybridization (Aleefs et al., 1990) using the O R F of the H E V penton base gene as a probe. As depicted in Fig. 3, H E V probe hybridized to a ~ 2500 bp fragment of M S D V DNA. The fragment of the M S D V D N A that hybridized with the H E V probe was cloned into p G E M - 3 Z vector and sequenced as described earlier. Alignment of the H E V and M S D V sequences revealed that the nucleotide sequences encoding the penton base gene were identical indicating 100% conservation. These data support the reported similarity in apparent molecular weights of the penton base proteins of H E V and M S D V (Nazerian et al.,
Fig. 1. Nucleotide and deduced amino acid sequences of the penton base gene of hemorrhagic enteritis virus. Asterisks denote the LDV motif. The nucleotide sequence has been submitted to the GenBank nucleotide sequence data base and has been assigned the accession number U28139.
M. Suresh et al. / ~4n~ Research 39 (1995) 289-297
294
1991: Zhang et al., 1991). HEV causes hemorrhagic enteritis and death while MSDV is apathogenic to turkeys (Domermuth and Gross, 1991). The molecular basis for the differences in pathogenicity is currently not known. Our data Ad2
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Fig. 2. Alignment of the penton base sequences of human adenovirus type 2 (Ad2) and hemo~hagic enteritis v i e s (HEV). Gaps indicated by dotted lines were used to maximie the alignment. The LDV sequences are in bold and are indicated by asterisks. Identical amino acid residues are indicated by ve~ical fines. Similar amino acid residues are indicated by ':' and '.' between the sequence symbols (similariW = ':' > '.'). ':' and '.' indicates that the comparison values are > 0.50 and > 0.10, respectively (Gfibskov and Burges, 1986).
M. Suresh et al. /Virus Research 39 (1995) 289-297
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295
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Fig. 3. Southern blot analysis of the EcoRI digested DNA from hemorrhagicenteritis virus (HEV) and marble spleen disease virus (MSDV) using digoxigenin-labeledpenton base gene of HEV as a probe. Lane 1, MSDV; lane 2, HEV; lane 3, digoxigenin- iabeled-H/ndlII-digested-,~ DNA as molecular weight standard. The sizes of a few DNA fragments are indicated in base pairs (BP). confounds an earlier speculation that the molecular differences between the penton base proteins of H E V and MSDV determined differences in pathogenicity to turkeys (Zhang et al., 1991; van den Hurk, 1992).
4. Virus internalization motif in the deduced amino acid sequence of H E V / M S D V penton base Majority of the studies on the interactions of adenoviruses with the host cells have been performed with the Ad2 of humans (Greber et al., 1993). Initial attachment of Ad2 to cells is mediated via the fiber coat protein to an unidentified receptor (Philipson et al., 1968). Following attachment to cells, Ad2, Ad3, Ad4, and A d l 2 viruses bind to cell surface a v integrins via a pentavalent R G D sequence that is present in the penton base protein (Wickham et al., 1993) suggesting that a v integrins might play a pivotal role in the entry of adenoviruses into host cells. The penton base of type II avian adenoviruses resemble mammalian adenoviruses in having a single fiber attached to each one of them (van den Hurk, 1992). The penton base protein of FAV-10, on the other hand has two fibers
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(Laver et al., 1971) and lacks the R G D motif (Sheppard and Trist, 1992). We were interested to find out if type II avian adenoviruses also resembled the mammalian adenoviruses in having a R G D motif. The deduced amino acid sequences of H E V and MSDV penton base protein lack the R G D motif similar to FAV-10 (Sheppard and Trist, 1992). These data suggested that avian adenoviruses may not interact with otvfl3 and a v f l 5 cell surface integrins. The lack of an R G D motif does not rule out the interaction of H E V / M S D V with other integrins because non-RGD sequences have also been shown to mediate integrin binding (Hynes, 1992). Further analysis of the amino aci d sequence of the penton base of H E V / M S D V revealed the existence of a motif, L e u - A s p - V a l (LDV) at amino acid position 245-247. Similar LDV motifs are conserved between the penton base proteins of Ad2, Ad3, Ad4, and Ad12 (Mathias et al., 1994; Cuzange et al., 1994). Interestingly, this LDV motif is not present in both Ad40 and FAV-10, which incidentally, also do not have the R G D motif (Davison et al., 1993). The LDV motif, present in the extracellular matrix protein fibronectin (FN; Makarem and Humphries, 1991) has been shown to be recognized primarily by the a4fll-integrin receptor expressed on lymphocytes, monocytes, basophils, eosinophils, bone marrow progenitors, fetal myoblasts, and melanoma cells (Hynes, 1987; Hemler, 1990; Rosen et al., 1992). The LDV sequence seems to be important since it is completely conserved in the human, rat, bovine, and avian fibronectins (Komoriya et al., 1991). Studies have shown that the minimum essential sequence for the recognition of fibronectin by the a4/31 integrin receptor is LDV (Komoriya et al., 1991). Since, H E V / M S D V lack the R G D motif, these data implicate the involvement of a4/31-integrins in the viral entry into host cells via interactions with the LDV motif of the viral penton base protein. Studies to inhibit adenoviral internalization with LDV peptides or antibodies to a4/~l should reveal the role of LDV motif on the penton base protein in the viral internalization.
Acknowledgements We wish to thank Drs. Jamil Ahmad, Kemal Karaca, Terence Pertile and Sudhir Reddy for their suggestions and assistance during the study.
References Aleefs, J.J., Salantijn, E.M., Krens, F.A., and Rouwendal, G.J. (1990) Optimization of non-radioactive Southern hybridization: single copy detection and reuse of blots. Nucl. Acids Res. 18, 3099-3100. Altsehul, S.F., Gish, W., Miller, W., Myers, W., and Lipman, D.J. (1990) Basic local alignment tool. J. Mol. Biol. 215, 403-410. : Cuzange, A., Chroboczek,J., and Jacrot, B. (1994)The penton base of human adenovirustype 3 has the RGD motif. Gen¢ 94, 257-259. Davison, A.J., Telford, E.A.R,, Watson, M.S., McBride, K., and Mautner, V. (1993) The DNA sequence of adenovirus type 40. J. MOI.B~I. 234, 1308-1316. Domermuth, C.H. and Gross, W.B. (1991) Hemorrhagic enteritis and related infections.In: B.W. Calnek, H. J, Barnes, C.W. Beard; W.M. Reid, and H.W. Yoder, Jr, (Eds.), Diseases of Poultry, Iowa State UniversityPress, Ames, Iowa, 9th ed.,' pp. 567-572.
M. Suresh et al. / l,qrus Research 39 (1995) 289-297
297
Domermuth, C.H., Weston, C.R., Cowen, B.S., ColweU, W.M., Gross, W.B., and Dubose, R.T. (1980) Incidence and distribution of avian adenovirus group II splenomegaly of chickens. Avian Dis. 24, 591-594. Fujiwara, H., Tanaami, S., Yamaguchi, M., and Yoshino, T. (1975) Histopathology of hemorrhagic enteritis in turkeys. Nat. Inst. Anim. HealthQ (Tokyo) 15, 68-75. Greber, U.F., Willetts, M., Webster, P., and Helenius, A. (1993) Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75, 477-486. Gribskov, M. and Burgess, R.R. (1986) Sigma factors from E. coli, B. subtilis, phage SPO1, and phage 4 are homologous proteins. Nucl. Acids Res. 14, 6745-6763. Hemler, M.E. (1990) VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu. Rev. Immunol. 8, 365-400. Hynes, R.O. (1987) Integrins: a family of cell surface proteins. Cell 48, 549-554. Hynes, R.O. (1992) Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25. Itakura, C. and Carlson, H.C. (1975) Electronic microscopic findings of cells with Inclusion bodies in experimental hemorrhagic enteritis of turkeys. Can. J. Comp. Med. 39, 299-304. Komoriya, A., Green, L.J., Mervic, M., Yamada, S.S., Yamada, K.M., and Humphries, M.J. (1991) The minimum essential sequence for a major cell type-specific adhesion site (CS1) within the the alternatively spliced Type III connecting segment domain of fibronectin is Leucine-Aspartic acid-Valine. J. Biol. Chem. 266, 15075-15079. Laver, W.G., Younghusband, H.B., and Wrigley, N.G. (1971) Purification and properties of chick embryo lethal orphan virus (an avian adenovirus) Virology. 45, 598-614. Makarem, R. and Humphries, M.J. (1991) LDV: a novel cell adhesion motif recognized by the integrin a4/31. Biochem. Soc. Trans. 19, 380S. Mathias, P., Wickham, T., Moore, M., and Nemerow, G. (1994) Multiple adenovirus serotypes use av integrins for infection. J. Virol. 68, 6811-6814. Nazerian K., Lee, L.F., and Payne, W.S. (1991) Structural polypeptides of type II avian adenoviruses analyzed by monoclonal and polyclonal antibodies. Avian Dis. 35, 572-578. Ossa, J.E., Bates, R.C., and Schurig, G.G. (1983) Hemorrhagic enteritis in turkeys: purification and quantification of the virus. Avian Dis. 27, 235-245. Philipson, L., Lonberg-Holm, K., and Petterson, U. (1968) Virus-receptor interaction in an adenovirus system. J. Virol. 2, 1064-1075. Rosen, G.D., Sanes, J.R., LaChance, R., Cunningham, J.M., Roman, J., and Dean, D.C. (1992) Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell 69, 1107-1119. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2nd ed. Scott-Taylor, T.H. and Hammond, G.W. (1992) Conserved sequences of the adenovirus genome for detection af all human adenovirus types by hybridization. J. Clin. Microbiol. 30, 1703-1710. Sharma, J.M. (1994) Response of specific-pathogen-free turkeys to vaccines derived from marble spleen disease virus and hemorrhagic enteritis virus. Avian Dis. 38, 523-530. Sheppard, M. and Trist, H. (1992) Characterization of the avian adenovirus penton base. Virology. 188, 881-886. Valentine, R.C. and Periera, H.G. (1965) Antigens and structure of the adenovirus. J. Mol. Biol. 13, 13-20. van den Hurk, J.V. (1992) Characterization of the structural proteins of hemorrhagic enteritis virus. Arch. Virol. 126, 195-213. van den Hurk, J.V. and van Drunnen Littel-van den Hurk, S. (1993) Protection of turkeys against hemorrhagic enteritis by monoclonal antibody and hexon immunization. Vaccine. 11, 329-335. Wickham, T.J., Mathias, P., Cheresh, D.A., and Nemerow, G.R. (1993) Integrins av/33 and ctvfl5 promote adenovirus internalization but not virus attachment. Cell 73, 309-319. Zhang, C., and Nagaraja, K.V. (1989) Differentiation of avian adenovirus type-II strains by restriction endonuclease fingerprinting. Am.J. Vet. Res. 50, 1466-1470. Zhang, C., Nagaraja, K.V., Sivanandan, V., and Newman, J.A. (1991) Identification and characterization of viral polypeptides from type-II avian adenoviruses. Am. J. Vet. Res. 52, 1137-1141.