Identification of sites in adenovirus hexon for foreign peptide incorporation.
Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.
Wu
Hongju
H
Division of Human Gene Therapy, Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Han
Tie
T
Belousova
Natalya
N
Krasnykh
Victor
V
Kashentseva
Elena
E
Dmitriev
Igor
I
Kataram
Manjula
M
Mahasreshti
Parameshwar J
PJ
Curiel
David T
DT
D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development; D015153:Blotting, Western; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D018014:Gene Transfer Techniques; D005814:Genes, Viral; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D036022:Capsid Proteins; D000939:Epitopes; D009841:Oligonucleotides; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C018315:hexon capsid protein, Adenovirus
J Virol. 1999 Aug;73(8):6729-42
10400771
J Mol Biol. 1994 Sep 30;242(4):430-55
7932702
J Virol. 2002 Jul;76(14):6893-9
12072490
Trends Microbiol. 2000 Sep;8(9):391-4
10989301
J Virol. 1993 Sep;67(9):5198-205
8350395
Nat Biotechnol. 1999 May;17(5):470-5
10331807
Eur J Cancer. 2002 Sep;38(14):1917-26
12204675
J Virol. 2002 Jan;76(1):127-35
11739678
Oncologist. 2002;7(1):46-59
11854546
Gene Ther. 1999 Sep;6(9):1558-64
10490765
Cancer Gene Ther. 2000 Oct;7(10):1329-35
11059690
J Virol. 1998 Dec;72(12):10260-4
9811772
BioDrugs. 2002;16(1):3-10
11908997
J Virol. 1994 Jun;68(6):4104-6
8189552
J Mol Biol. 1987 Nov 5;198(1):73-89
3430605
J Virol. 2002 May;76(9):4580-90
11932423
Cell. 1993 Apr 23;73(2):309-19
8477447
J Virol. 1998 Mar;72(3):2388-97
9499099
J Gen Virol. 1999 Jun;80 ( Pt 6):1519-1527
10374971
Science. 1986 May 30;232(4754):1148-51
3704642
Biochim Biophys Acta. 1997 Nov 13;1330(1):8-16
9375808
Hum Mol Genet. 1996 Nov;5(11):1703-12
8922997
Science. 1997 Feb 28;275(5304):1320-3
9036860
J Virol. 2002 Dec;76(24):12775-82
12438602
Microbiol Mol Biol Rev. 1999 Sep;63(3):725-34
10477314
Mol Ther. 2000 Jan;1(1):18-30
10933908
Curr Top Microbiol Immunol. 1995;199 ( Pt 1):47-66
7555060
Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1401-5
7877990
J Gene Med. 2003 Apr;5(4):267-76
12692861
J Virol. 1996 Apr;70(4):2116-23
8642632
Gene Ther. 2002 Aug;9(16):1101-8
12140738
Gene Ther. 1996 Jun;3(6):496-502
8789799
J Virol. 1988 Jul;62(7):2321-8
3373570
J Virol. 1997 Nov;71(11):8221-9
9343173
Virology. 2002 Feb 15;293(2):210-6
11886240
J Virol. 1998 Aug;72(8):6875-9
9658137
J Virol. 1995 Apr;69(4):2004-15
7884845
Curr Top Microbiol Immunol. 1995;199 ( Pt 1):25-38
7555058
J Virol. 1998 Dec;72(12):9706-13
9811704
J Gen Virol. 1994 Jan;75 ( Pt 1):133-9
7509367
J Virol. 1996 Mar;70(3):1836-44
8627708
J Virol. 2003 Sep;77(17):9553-66
12915569
Mol Imaging. 2004 Apr;3(2):105-16
15296675
Hum Gene Ther. 2002 Sep 1;13(13):1647-53
12228019
Hum Gene Ther. 1992 Apr;3(2):147-54
1391034
Nat Biotechnol. 1996 Nov;14(11):1570-3
9634823
Hum Gene Ther. 1999 May 20;10(8):1349-58
10365665
Hum Gene Ther. 2002 Jan 20;13(2):311-20
11812286
Biochim Biophys Acta. 2002 May 3;1575(1-3):1-14
12020813
J Virol. 1999 Jun;73(6):5156-61
10233980
Virology. 1973 Mar;52(1):130-47
4139804