Expression Of Type-specific E1B55K Gene Enhanced The Propagation Of Human Adenovirus Type 41 In 293 Cells

Adenoviruses type 40 (Ad40) and 41 (Ad41) are natural pathogens infecting human digestive tract and can cause diarrhea. There have been great interests in developing Ad40 or Ad41 as a gene delivery vector targeting gastrointestinal tract, which is hampered by that their viruses are hard to cultivate in vitro. Many possible reasons contribute to the fastidiousness of Ad40 or Ad41, such as weak expression of the early genes, decreased genome replication, insufficient expression of viral structure proteins, and defects in virus assembling and release. However, the fundamental reason remains unknown.HEK293 cell line, also called as 293, is human embryo kidney cell transformed by the El region of Ad5, which constitutively express E1A, E1B19K and E1B55K protein of Ad5. Some of Ad41 isolates can be passaged on 293 cells. Generally, it is explained as El proteins of Ad5 in 293 can partly remedy the insufficient expression of Ad41 El. However, the growth of Ad41 in 293 cells is far from perfect. The yield of progeny viruses was so low that some isolates were just lost after being consecutively passaged on 293 cells. We assumed that adenovirus E1B55K proteins of different serotypes could not totally complement each other. There are some evidences. During the construction of Ad35 replication-defective vector, for example, Ad35 E1B55K-transduced 293 rather than 293 was used as the packaging cell because El-deleted Ad35 could not be rescued and proliferated in 293 cells. In our previous work, we established Ad41 E1B55K-transduced HEp2 (HEp2-ElB) and 293 (293E12) cell lines. HEp2-E1B and 293E12 can be used to amplify wild-type Ad41 isolated from children's stools. Furthermore,293E12 can be utilized as the packaging cell for E1-deleted Ad41 vectors.The E1 region is the first transcription unit to be activated following Ad infection. They modify the host cell environment to facilitate Ad replication. We assume that poor expression of Ad41 E1 takes the major responsibility for the virus fastidiousness. In this study, we focus on the correlation between the expression of Ad41 E1B55K and the efficiency of Ad41 replication in 293 cells.1. Increased expression of Ad41 E1B55K improved Ad41-packaging ability of 293 cells.We have established Ad41 E1B55K-transduced 293 cell line (293E12), in which the expression of Ad41 E1B55K was controlled by CMV promoter. In this study, we attempt to further enhanced Ad41 E1B55K expression by employing Ad41 tripartite leader sequence (TPL). Adenovirus late mRNAs all share a common 5'non-translated region about 200 nucleotides in length. This sequence is termed as TPL because it is coded by three extrons just following the major late promoter in the viral genome. TPL was well known due to its function of modulating mRNA export from the nucleus as well as facilitating mRNA translation during the late phase of adenovirus infection. Therefore, TPL could enhance the efficiency of gene expression when being inserted downstream of a promoter. Firstly, we cloned TPL fragment. RNA was extracted from Ad41-GFP-infected 293E12 cells at 24 h post infection. Reverse transcription was performed to prepare single-stranded cDNA using a specific primer complemented to the L1 52K gene of Ad41. And then nested PCR was employed to amplify the 5'region of L1 52K mRNA sequence with the cDNA as the template. PCR product was inserted into the cloning site of pMD-18T vector, followed by sequencing. TPL sequence information was obtained after bioinformatics analysis. New primers carrying restriction sites on the 5'ends were designed, synthesized and used to amplify TPL of Ad41. PCR product was digested and then inserted into the downstream of CMV promoter of pcDNA3-E1B plasmid, which was a eukaryotic expression vector carrying Ad41 E1B55K. The generated plasmid, named as pcDNA3-TPL-E1B, was purified and mixed with lipofectamine 2000 to transduce 293 cells. The G418-resistant cell colonies (293TE) were isolated and proliferated. Packaging ability of 293TE cell lines were preliminarily evaluated by the increase of GFP-positive cells after being infected with equivalent Ad41-GFP. Three cell lines,293TE7,293TE12 and 293TE20, were relatively more productive and kept for further study. Ad41 E1B55K was also cloned into pET30A(+) vector and expressed in E. Coli strain BL21(DE3). Antiserums to Ad41 E1B55K were collected from Ad41 E1B55K-immunized mice.293TE7 was assayed for the expression of Ad41 E1B55K with the method of immunofluorescence using antiserum against Ad41 E1B55K. The results showed that more Ad41 E1B55K was expressed in 293TE7 cells than in 293E12 cells, suggesting that TPL could substantially enhance E1B55K expression. To compare the ability of virus rescue, adenovirus plasmid pAd41-GFP was linearized by Pmeâ… and used to transfect 293TE7 or 293E12 cells. The product of rescued viruses in 293TE7 was about twenty times higher than that in 293E12 cells by virus titration assay. The virus-packaging ability was also studied quantitively. The amount of progeny viruses produced in 293TE7 was three to ten times higher than that in 293E12 cells when equivalent seed Ad41-GFP viruses were incubated. Furthermore, Ad41-GFP still possessed a genetically-stable genome after being passaged six times on 293TE cell line based on the restriction digestion analysis.In summary, TPL of Ad41 was cloned and used to construct eukaryotic expression plasmid pcDNA3-TPL-E1B.293TE cell lines with increased Ad41 E1B55K expression were successfully established, which possessed an enhanced Ad41-GFP-Packaging ability.2. Enhanced virus mRNA export partially accounts for the promoted Ad41-GFP-packaging ability of 293E12 or 293TE7.Virus genome synthesis and structural protein expression are two key events before Ad assembly, which were investigated in this study.293,293E and 293TE cells were infected with Ad41-GFP at an MOI of 100 (MOI was calculated with the viral particle titer). At 24 hours post infection, virus genomes were extracted with Hirt's method and analyzed on agarose gels before stained with EB. The amount of virus genomes synthesized in 293E12 was equal to that in 293 TE7, and was 4 times more than that in 293 cells. To determine if the increased E1B55K expression resulted in an increased virus late mRNA concentration in the cytoplasm, viral late cytoplasmic and nucleic RNAs were analyzed by quantitative RT-PCR at 12,16 and 24 h after infection. At all times tested, the L2 and L3 mRNAs were expressed at higher levels in cytoplasm of 293E12 cells than that of 293 cells. We further evaluated the V proteins with Western blot.293TE7 produced more these structure proteins than 293E12, which also produced more than 293 cells after being infected by Ad41-GFP. These data were consistent with the results of Real-time RT-PCR. We therefore concluded that Ad41 E1B55K, which can modulate viral mRNAs export from the nucleus, contributed to improved structure proteins expression during the late phase of adenovirus infection.3. Development of new Ad41 vector systemThe Ad41 E1B55K could not be totally complemented by the corresponding gene of Ad5, and expressing Ad41 E1B55K in packaging cells can improve the replication of Ad41. These findings prompted us to establish a new replication-deficient Ad41 vector system, in which only E1A and E1B19K genes were deleted and replaced with transgene while the E1B55K was preserved. The E1B55K gene of Ad41 as well as the corresponding promoter of Ad5 was inserted downstream of the transgene (GFP) expression cassette of Ad41 shuttle plasmid pSh41-CMV. The generated plamid pSh41ElB-GFP was used to co-transform E. coli BJ5183 together with the backbone plasmid pAdbone41. The recombinant, adenovirus plasmid pAd41E1B-GFP, was linearized by Pmel and then used to transfect 293 cells. We had thought that Ad41 E1B55K saved in the virus genome would make the recombinant vector propagate effectively in 293 cells. Ad41E1B-GFP did be rescued from and could be passaged on 293 cells. However, the yield of progeny viruses was very low, which we attribute to the low expression of Ad41 E1B55K. Further efforts will be made to exchange the promoter of Ad41 E1B55K in the vector system.Conclusively, in this study we investigated the correlation of expression of Ad41 E1B55K and virus replication in 293 cells. The synthesis of virus genomes and structure proteins were remarkably enhanced in virus-infected 293 cells when exogenous Ad41 E1B55K was expressed, which finally resulted in promoted yield of progeny viruses. A more effective packaging cell line (293TE7) was established and could be used to amplify wild-type or recombinant Ad41 viruses. This study also laid a foundation for further improvement of Ad41 vector system.