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Short Communication |
Vectored Vaccine Program, Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
Correspondence
Suresh K. Tikoo
suresh.tik{at}usask.ca
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ABSTRACT |
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MAIN TEXT |
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). Due to inherent problems with pre-existing neutralizing antibodies impeding their therapeutic use in humans, a number of non-human adenoviruses are being evaluated as potential vectors (Klonjkowski et al., 1997; Xu et al., 1997; Rasmussen et al., 1999). Bovine adenovirus 3 (BAdV-3) has been characterized with the aim of developing it as a vector for delivering vaccine antigens (Reddy et al., 1998). Although the absence of pre-existing antibodies in humans and resistance to neutralization by human sera make BAdV-3 attractive for use as a vector in man (Rasmussen et al., 1999), as yet BAdV-3 has been found to infect non-bovine cells, including human cells, poorly (Wu & Tikoo, 2004).
Inhibition of productive virus infection in non-permissive cells can occur at any stage of the virus life cycle. The abortive infection of hamster cells by HAdV-12 is caused by a defect in nuclear import of NFIII (Hosel et al., 2003), whereas HAdV-2 infection of monkey cells is blocked due to defective splicing, transport (Ross & Ziff, 1994) and reduced translation of the late mRNAs (Silverman & Klessig, 1989). This host restriction in HAdV-2 replication can be overcome by either co-expression of simian virus 40 (SV-40) large T antigen or a mutation in the N terminus of DNA-binding protein (DBP) (Eron et al., 1975; Nicolas et al., 1983). Furthermore, HAdV-41 exhibits a defect in virus assembly in 293 cells (Pieniazek et al., 1990), whereas ovine adenovirus infection can be aborted prior to viral DNA replication, or due to inefficient expression of late proteins, in different human cell types (Kümin et al., 2002). In this report, the biology of BAdV-3 infection of human 293 (ATCC CRL-10852) and 293T (DuBridge et al., 1987; ATCC CRL-11268) cells was analysed. Our results indicate that an early defect in BAdV-3 propagation in 293 cells results in inefficient viral DNA replication, which can be partially complemented in 293T cells expressing SV-40 T antigen.
Studies using a BAdV-3 recombinant encoding green fluorescent protein (GFP) (BAV304) have suggested that the block in infection of non-bovine cells, including human cells, is in the initial stages of virus–cell interaction (Wu & Tikoo, 2004). To determine whether Madin–Darby bovine kidney (MDBK), 293 and 293T cell types could be infected by the recombinant BAV304, cells were infected with BAV304 (m.o.i. of 5 p.f.u. per cell). At 48 h post-infection (p.i.), GFP fluorescence-positive cells were measured by flow cytometry. Interestingly, BAV304 transduces 293 and 293T cells as efficiently as MDBK cells (Fig. 1).
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). Progeny virions were therefore produced in MDBK and 293T cells, but not in 293 cells. The formation of virus particles in MDBK, 293 and 293T cells was also investigated by transmission electron microscopy (EM). Briefly, cells were infected with BAdV-3 (m.o.i. of 20) and cells were harvested at 72 h p.i. and processed as described previously (Kulshreshtha et al., 2004). As seen in Fig. 2(b), formation of empty capsids and mature virions could be detected in BAdV-3-infected MDBK [Fig. 2b(i)] and 293T [Fig. 2b(iii)] cells; however, formation of empty capsids or mature virions was not detected in BAdV-3-infected 293 cells [Fig. 2b(ii)].
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), infected cells were harvested and freeze–thawed five times. Viruses in cell lysates were titrated on MDBK cells by the TCID50 method. A rapid increase in virus titre was noticed in BAdV-3-infected MDBK and 293T cells from 24 to 48 h p.i., with titres of 5x108 and 1x107 TCID50 ml–1, respectively, at 48 h p.i. (Fig. 2c). Again, productive BAdV-3 infection was not detected in 293 cells. These results confirmed our observations that progeny BAdV-3 is produced in 293T, but not in 293, cells.
We were interested in determining whether BAdV-3 progeny virus could be generated following transfection of infectious plasmid DNA into human cells. 293 and 293T cells were transfected with 5 µg PacI-digested pFBAV304 (Reddy et al., 1999) plasmid DNA, digested to release the full-length BAdV-3 genome harbouring the GFP gene cassette inserted in the E3 region, using Lipofectamine 2000. At 48 h p.i., GFP expression could be detected in more than 70 % of the transfected 293T [Fig. 2d(i)] and 293 [Fig. 2d(iii)] cells. At 12 days post-transfection, cells were collected, freeze–thawed five times and cell lysates were used to infect fresh monolayers of MDBK cells. Cells were tested for the expression of GFP at 72 h p.i. by fluorescence microscopy. GFP expression could readily be seen in MDBK cells infected with lysates from pFBAV304-transfected 293T cells [Fig. 2d(ii)], but not with lysates from pFBAV304-transfected 293 cells [Fig. 2d(iv)].
To determine whether the production of progeny virus in 293T cells was due to the expression of SV-40 T antigen, DNA fragments of 2·127 kb, encoding SV-40 large T antigen [primers AP1 (5'-ATGGATAAAGTTTTAAACAG-3') and AP2 (5'-TTATGTTTCAGGTTCAGG-3')], and 0·525 kb, encoding SV-40 small T antigen [primers AP1 and AP3 (5'-TTAGAGCTTTAAATCTCTGTAG-3')], amplified by RT-PCR of RNA isolated from 293T cells, were ligated individually to EcoRV-digested pcDNA3, creating plasmids pLT and pST, respectively. The 293 cells were transfected with 2 µg individual plasmid DNA and, 48 h later, infected with BAdV-3 (m.o.i. of 20). Cells were harvested at 60 h p.i., freeze–thawed five times and cell lysates were titrated on MDBK cells by the TCID50 method. As seen in Fig. 2(e), compared with 293 cells, there were increases in the titres of BAdV-3 in 293 cells expressing small and large T antigens. Co-transfection of small and large T antigens resulted in the greatest increase in titre.
Synthesis of viral proteins in MDBK, 293 and 293T cells was then analysed by Western blotting using BAdV-3 protein-specific rabbit polyclonal antisera (Fig. 3a). Production and characterization of BAdV-3 DBP-specific (Zhou et al., 2001) and BAdV-3 fiber-specific (Wu & Tikoo, 2004) antibodies have been described. Rabbit polyclonal antibodies generated against BAdV-3 hexon, penton and 100K proteins recognize proteins of 98, 62 and 97/130 kDa, respectively. Briefly, MDBK, 293 and 293T cells (5x105 cells) infected with BAdV-3 (m.o.i. of 20) were harvested at 24, 48 and 96 h p.i. Cell extracts were prepared, separated on a 10 % SDS-PAGE gel and transferred to a nitrocellulose membrane. Viral antigens immobilized on the nitrocellulose sheets were probed with BAdV-3 protein-specific antiserum as described previously (Kulshreshtha et al., 2004). As seen in Fig. 3(a), expression of early protein (DBP) was greater in 293T cells than in 293 cells. However, the expression of late proteins (52K, 100K, penton, hexon and fiber) was observed in MDBK and 293T cells, but not in 293 cells. Apart from DBP, expression of viral proteins was observed later in 293T cells than in MDBK cells. In addition, the expression of fiber and hexon proteins was limited in 293T cells compared with MDBK cells. Interestingly, unlike MDBK cells, the expression of viral 100K protein could be detected until 96 h p.i. in 293T cells.
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). The dry membrane was scanned with a phosphorimager (Personal Molecular Imager FX; Bio-Rad) (Fig. 3b) and the signal was quantified by densitometric analysis using AlphaEase software (Alpha Innotech) (Fig. 3c). In 293T and MDBK cells, the amount of viral DNA in the virus-infected cells reached a peak at 48 h p.i. and was slightly reduced at 96 h p.i. However, DNA replication in 293 cells was negligible even at 96 h p.i. Viral DNA replication was observed in MDBK cells at 24 h p.i., suggesting early onset compared with 293 and 293T cells. The delayed onset in viral DNA replication is consistent with the late onset of late protein synthesis in 293T cells. Based on densitometric data, the efficiency of viral DNA replication was approximately three and 20 times higher in MDBK cells than in 293T and 293 cells, respectively. The reduced expression of late proteins in the 293T cells compared with MDBK cells may in part be due to the reduced synthesis of viral DNA in these cells. The stimulation of viral DNA replication by DBP (van Breukelen et al., 2003) and transactivation of the E2A promoter by SV-40 small T antigen (Loeken, 1992) may explain the possible complementation by T antigen in 293T cells.
This study demonstrates that a major barrier to the production of progeny BAdV-3 in 293 cells is inefficient replication of viral DNA. It is possible that MDBK cells contain a cellular factor(s) that is lacking in 293 cells. Alternatively, an inhibitory factor that is counteracted by SV-40 T antigen may be present in 293 cells. The SV-40 T antigen is a multifunctional viral protein that has been shown to be involved in both viral and cellular transcriptional regulation, virion assembly, viral DNA replication and alteration of the cell cycle (Sullivan & Pipas, 2002). Although the successful production of progeny BAdV-3 in 293T cells may be primarily due to the enabling of BAdV-3 DNA replication, the SV-40 T antigen may also promote other stages in the BAdV-3 life cycle. Despite the expression of E1 proteins of HAdV-5 in 293 cells (Graham et al., 1977), which may help to overcome some of the species-specific cellular barriers by targeting cell-cycle regulators early in virus infection, BAdV-3 infection in 293 cells could not successfully proceed to viral DNA replication.
It has previously been suggested that BAdV-3 infection of non-bovine cells, including some human cells, is primarily restricted at the cell-binding stage (Wu & Tikoo, 2004). The present findings demonstrate that BAdV-3 replication in efficiently transduced human 293 cells appears to be blocked early in virus infection. These results further support the development of BAdV-3 vectors as an alternative or supplement to HAdV-5 vectors (Bangari et al., 2005; Wu & Tikoo, 2004).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 June 2005;
accepted 16 December 2005.
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