construction-of-adenoviral-vectors-using-dna-assembly-technology

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...

Chinese Center For Disease Control And Prevention

Chinese Center for Disease Control and Prevention-Wuhan Institute of Virology

The fastidious property of human adenovirus type 41 (Ad41) may be resulted from inadequate expression of protein V (pV), the minor core protein of adenovirus, in packaging cells. In this report, we prepared antiserum to pV of Ad41 and for study the mechanism of Ad41 fastidiousness.

[Preparation of antiserum to protein V of human adenovirus type 41].

The human adenovirus (HAdV) early protein E1B55K interacts with E4orf6 to form an E3 ubiquitin ligase complex, which plays key roles in virus replication. To illustrate the reason for the fastidiousness of HAdV-41 in 293 cells, interaction between heterotypic E1B55K and E4orf6 proteins was investigated. HAdV-5 E1B55K could interact with HAdV-41 E4orf6, and vice versa. To form E1B55K/E4orf6 E3 ubiquitin ligase, HAdV-41 E4orf6 recruited Cul2 while HAdV-5 E4orf6 interacted with Cul5. The ligase complex formed by HAdV-5 E1B55K and HAdV-41 E4orf6 could cause the degradation of p53 and Mre11. However, in E1-deleted HAdV-41-infected 293TE7 cells, which expressed HAdV-41 E1B55K, viral late mRNAs were exported from nucleus more efficiently and accumulated to a higher concentration in cytoplasm when compared with that in infected 293 cells. These results suggested that interaction between homotypic E1B55K and E4orf6 was indispensable for efficient export of viral late mRNAs. 

Inefficient export of viral late mRNA contributes to fastidiousness of human adenovirus type 41 (HAdV-41) in 293 cells.

Neutralizing antibody (NAb) can dampen the immunogenicity of adenovirus (Ad) vector-based vaccine. Vector systems based on human adenovirus type 41 (Ad41) have been constructed and used to develop recombinant vaccines. Here, we attempted to study the seroprevalence of NAbs to Ad5 and Ad41 among children and adults in Qinghai province, China. The positive rates (titer⩾40) of Ad5 and Ad41 NAb in adults from Xining city were 75.7% and 94.7%, respectively. The moderate/high-positive rates (titer⩾160) of NAb were quite close between the two viruses in adults (70.4% for Ad5 and 73.5% for Ad41). Age-dependent increase of NAb seroprevalence was observed for both viruses in children. NAb-positive rate of Ad41 reached 50% at 3.3-4.6years of age for children from Chengxi district, Xining city, approximately 1.5years earlier than that of Ad5 did. Interestingly, NAb level was also associated with sanitary conditions among young children. For Ad5, 8-15% children (0.2-3.0years of age) from city or town, where the sanitations were relatively better, had moderate/high-positive NAb, while the same rate was 62% for children from villages. For Ad41, 22% children from city, 47% from town and 88% from villages possessed moderate/high-positive NAb. The possible influence of NAb titer distributions on the application of Ad41-vectored vaccines was discussed in detail. Our results suggested that children from places with poor sanitations should be included for comprehensive Ad NAb seroprevalence studies, and provided insights to the applications of Ad41 vectors.

Prevalence of serum neutralizing antibodies to adenovirus type 5 (Ad5) and 41 (Ad41) in children is associated with age and sanitary conditions.

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes. In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems.

The gene transduction efficiency of adenovirus to hematopoietic cells, especially T lymphocytes, is needed to be improved. The purpose of this study is to improve the transduction efficiency of T lymphocytes by using fiber-modified human adenovirus 5 (HAdV-5) vectors.

Efficient gene transfer into T lymphocytes by fiber-modified human adenovirus 5.

Commonly used and well accepted approaches are lacking for site-directed modification of adenoviral vectors. Here, we attempt to introduce an easy-to-implement strategy for such purpose with an example of establishing a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR products of GFP expression cassette and plasmid backbone were fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA assembly. NdeI-digested fragment of pMDXE3GA was brought back to pKAd5 to form the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension K562 cells were investigated. Expression of target gene was significantly enhanced in both cell lines infected with HAdV5-XE3GA due to virus replication. However, propagation of virus could not sustain in culture of K562 cells. Shuttle plasmid pSh5RC-GFP was constructed to facilitate exchange of transgene. In summary, the strategy of combined DNA assembly and restriction-ligation cloning is functional, cost-effective and suitable for genetic modification of adenovirus.

Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning.

A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.

User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome.

Fibre is the viral protein that mediates the attachment and infection of adenovirus to the host cell. Fowl adenovirus 4 (FAdV-4) possesses two different fibre trimers on each penton capsomere, and roles of the separate fibres remain elusive. Here, we attempted to investigate the function of FAdV-4 fibres by using reverse genetics approaches. Adenoviral plasmids carrying fiber1 or fiber2 mutant genes were constructed and used to transfect chicken LMH cells. Fiber1-mutated recombinant virus could not be rescued. Such defective phenotype was complemented when a fiber1-bearing helper plasmid was included for co-transfection. The infection of fiber-intact FAdV-4 (FAdV4-GFP) to LMH cells could be blocked with purified fiber1 knob protein in a dose-dependent manner, while purifed fiber2 knob had no such function. On the contrary, fiber2-mutated FAdV-4, FAdV4XF2-GFP, was successfully rescued. The results of one-step growth curves showed that proliferative capacity of FAdV4XF2-GFP was 10 times lower than that of the control FAdV4-GFP. FAdV4XF2-GFP also caused fewer deaths of infected chicken embryos than FAdV4-GFP did, which resulted from poorer virus replication . These data illustrated that fiber1 mediated virus adsorption and was essential for FAdV-4, while fiber2 was dispensable although it significantly contributed to the virulence.

Fiber1, but not fiber2, is the essential fiber gene for fowl adenovirus 4 (FAdV-4).

Pre-existing immunities hamper the application of human adenovirus (HAdV) vectors in gene therapy or vaccine development. Fowl adenovirus (FAdV)-based vector might represent an alternative.

Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.

Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector.

Essential genus-specific genes have not been discovered for fowl adenovirus (FAdV), which hampers the development of FAdV-based vectors and attenuated FAdV vaccines. Reverse genetics approaches were employed to construct FAdV-4 mutants carrying deletions or frameshift mutations covering the whole left and right ends of the viral genome. The results of virus rescue and plaque forming experiments illustrated that all the 22 designated ORFs (open reading frames) were dispensable for the replication of FAdV-4 in chicken hepatoma Leghorn male hepatoma (LMH) cells and primary embryo hepatocytes. RNA-seq data demonstrated that ORF28 and ORF29 were not protein-encoding genes, and suggested a promoter (RP1) and an intron in these regions, respectively. The promoter activity of RP1 was further confirmed by reporter gene expression experiments. GAM-1-deleted FAdV-4 formed small plaques, while deletion of GAM-1 together with ORF22 resulted in even smaller ones in LMH cells. Simultaneous deletion of ORF28, ORF29, and GAM-1 led to growth defect of FAdV-4. These facts implied that genus-specific genes contributed to and synergistically affected viral replication, although no single one was essential. Notably, replication of FAdV-4 mutants could be different  and . XGAM1-CX19A, a GAM-1-deleted FAdV-4 that replicated efficiently in LMH cells, did not kill chicken embryos because virus propagation took place at a very low level . This work laid a solid foundation for FAdV-4 vector construction as well as vaccine development, and would benefit viral gene function study.  Identification of viral essential genes is important for adenoviral vector construction. Deletion of nonessential genes enlarges cloning capacity, deletion of essential genes makes a replication-defective vector, and expression of essential genes in  generates a virus packaging cell line. However, the genus-specific essential genes in FAdV have not been identified. We constructed adenoviral plasmid carrying deletions covering all 22 genus-specific ORFs of FAdV-4, and found that all virus mutants could be rescued and amplified in chicken LMH cells except those that had defects in key promoter activity. These genus-specific genes affected virus growth, but no single one was indispensable. Dysfunction of several genus-specific genes at the same time could make FAdV-4 vectors replication-defective. In addition, the growth of FAdV-4 mutants could be different in LMH cells and in chicken embryos, suggesting the possibility of constructing attenuated FAdV-4 vaccines.

Article	Year
[Preparation of antiserum to protein V of human adenovirus type 41]. Wei sheng wu xue bao = Acta microbiologica Sinica\| PubMed ID: 19637578	2009
Inefficient export of viral late mRNA contributes to fastidiousness of human adenovirus type 41 (HAdV-41) in 293 cells. Virology\| PubMed ID: 25240325	2014
Prevalence of serum neutralizing antibodies to adenovirus type 5 (Ad5) and 41 (Ad41) in children is associated with age and sanitary conditions. Vaccine\| PubMed ID: 27682509	2016
DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus. Journal of virological methods\| PubMed ID: 29703616	2018
Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems. Human gene therapy\| PubMed ID: 30793964	2019
Efficient gene transfer into T lymphocytes by fiber-modified human adenovirus 5. BMC biotechnology\| PubMed ID: 31014302	2019
Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning. Journal of biotechnology\| PubMed ID: 31751597	2020
User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome. Viruses\| PubMed ID: 32168853	2020
Fiber1, but not fiber2, is the essential fiber gene for fowl adenovirus 4 (FAdV-4). The Journal of general virology\| PubMed ID: 33625352	2021
Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells. The journal of gene medicine\| PubMed ID: 34050587	2021
Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector. Viruses\| PubMed ID: 35336953	2022
No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells. Microbiology spectrum\| PubMed ID: 35638786	2022

Zhuozhuang Lu

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