Possible Mechanism of Adenovirus Generation from a Cloned Viral Genome Tagged with Nucleotides at Its Ends

Abstract

The entire cloned human adenovirus type 5 (Ad5) genome is known to be able to generate infectious virus after transfection into 293 cells when the both ends of the genome are exposed by digestion with appropriate restriction enzymes. However, when one or both ends of the genome are tagged with nucleotides and are not intact, whether the tagged end of the viral genome was remained tagged or corrected to be intact during the generation of viral clones has been unclear and, if such oligonucleotide removal occurs, how does the virus remove these tagged sequences and thereby restore its proper structure? Here, we show in our semi‐quantitative study that the generation efficiency of virus clones decreases depending on the length of nucleotide tags at the both ends and that both the oligonucleotide tags were precisely removed during virus generation with restoration of the proper terminal sequences. Interestingly the viral genome of which one end was tagged, while the other was attached about 12‐kb sequences, did generate intact viral clones at a reduced but significant efficiency. From these results, we here propose a possible mechanism whereby the terminal‐protein‐deoxycytidine complex enters from the enzyme‐cleaved end and reaches deoxyguanine at the initiating position of DNA synthesis in vivo. A replication origin at one end, embedded deeply in double‐stranded DNA, can be activated by two cycles of one‐directional full‐length DNA synthesis initiated by the other exposed replication origin about 30 kilobases away. We also describe new cassette cosmids which can use not only PacI but also BstBI for construction of an adenovirus vector, without reducing construction efficiency.

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector

“…AxLR14EL-AC, AxLR16EL-AC, AxLR16EFL, Ax-AC, AxNZ, AxLNZCAL, AxALNZCAL, AxA2AdsR and AxEFdsR are described for the first time in this work. AdVs described here were constructed using cosmid transfection (21). All the aforementioned viruses except for AxA2AdsR and AxEFdsR possessed E3 region with a 2.4-kb deletion, while the latter two viruses bore E3 region with the 1.9-kb deletion described in reference (21) (see ‘Discussion’ section).…”

“…AdVs described here were constructed using cosmid transfection (21). All the aforementioned viruses except for AxA2AdsR and AxEFdsR possessed E3 region with a 2.4-kb deletion, while the latter two viruses bore E3 region with the 1.9-kb deletion described in reference (21) (see ‘Discussion’ section). The switch unit was inserted at the Sna BI site (nt position 35 770), located 165-nt downstream from the right end of the adenovirus-5 (Ad5) genome.…”

Activities of Various FLP Recombinases Expressed by Adenovirus Vectors in Mammalian Cells

“…34 The FLPe-expressing AdV (AxCAFLPe) was generated with the intact-genome transfection method. 39 Briefly, the FLPe recombinase gene was derived from pBSSK-FLPe, which was prepared from pCAGGS-FLPe (Gene Bridges). The FLPe gene used here contained an initiation codon that complies with Kozak's rule 40 and lacked a nuclear localization signal.…”

An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes

“…The vector needs to be digested with the restriction enzyme Csp45I before the introduction into HEK293 cells, in order to produce infectious virus. Linearisation next to replication origin of adenovirus (the ITR; inverted terminal repeat) is thought to favour binding of the precursor terminal protein (pTP) complex, that contains viral DNA polymerase and deoxycytidine needed for DNA replication [5]. Cellular DNA repair mechanisms are thought to generate single stranded DNA at the site of linearisation, which favours binding of the complex.…”

“…pAxCAwtit2 is an adenoviral cosmid vector based on Ad5 [5] which contains the genes required for virus replication but lacks the E1 gene region. The recombinant adenovirus can only proliferate in cells such as HEK293 that express the E1A and E1 B proteins.…”