Abstract
Adenovirus (Ad) vectors deleted of the early region 1 (E1) are widely used for transgene delivery in preclinical and clinical gene therapy studies. Although proteins encoded within the E1 region are required for efficient virus replication, previous studies have suggested that certain viral or cellular proteins can functionally compensate for E1, leading to expression of the early region 2 (E2)-encoded replicative proteins and subsequent virus replication. We have generated a series of E1-encoding and E1-deficient Ad vectors containing a FLAG-epitope tag on each of the E2-encoded proteins: DNA-binding protein (DBP), terminal protein (TP) and DNA polymerase (Pol). Using these constructs, we show that for the replication-competent virus, the expression level of each E2-encoded protein declines with increasing distance from the E2 promoter, with E2A-encoded DBP expression being ~800-fold higher than E2B-encoded TP. Pol was expressed at extremely low levels in infected cells, and immunoprecipitation from cell lysates was required prior to its detection by immunoblot. We further show that DBP was expressed 200- to 400-fold less efficiently from an E1-deficient virus compared to a replication-competent virus in A549 and HepG2 cells, which was accompanied by a very small increase in genome copy number. For the E1-deficient virus, late gene expression (a marker of virus replication) was only observed at very high multiplicities of infection. These data show that E1-deleted Ad gives rise to limited expression of the E2-encoded genes and replication in infected cells, but highlight the importance of considering viral dose-dependent effects in gene therapy studies.
Highlights
Human adenovirus (Ad) mainly causes self-limiting respiratory illnesses and can rapidly spread through confined populations such as day care centers, hospitals, retirement homes and military training venues [1]
A 5544 bp BamHI/SpeI fragment from pRP3118 was cloned into pRP2014, generating pRP3120 which is an Ad5 genomic plasmid lacking early region 1 (E1) and E3, but containing a FLAG-tag on the DNA-binding protein (DBP). pRP3120 was recombined with pRP2483 [36] to produce pRP3121, an E1/E3-deleted Ad5 genomic plasmid containing a monomeric red fluorescent protein (RFP) reporter gene [37] under the regulation of the cytomegalovirus (CMV) immediate early enhancer/promoter and bovine growth hormone polyadenylation sequence (BpA) replacing the E1 region, and the FLAG-tagged DBP. pRP3120 was recombined with pXC1 [38] to generate pRP3122, an E1+/E3- infectious plasmid containing the FLAG-tagged DBP. pRP3121 and pRP3122 were digested with PacI to liberate the inverted terminal repeats, transfected into 293 cells and recovered as viruses, which are designated AdE1-DBP-F and AdE1+DBP-F, respectively, in this study
A 7127 bp XbaI fragment from pBRS103 was cloned into XbaI digested pRP2014ΔXbaI, generating pBRS104, an Ad5 genomic plasmid lacking E1 and E3 but containing a FLAG-tag on the Pol. pBRS104 was recombined with pRP2483, generating pBRS105, an E1/E3-deleted Ad5 genomic plasmid containing a CMV-RFP-BpA expression cassette replacing the E1 region and the FLAG-tagged Pol. pBRS104 was recombined with the pXC1, generating pBRS106, an E1+/E3- infectious plasmid containing the FLAG-tagged Pol. pBRS105 and pBRS106 were digested with PacI to liberate the inverted terminal repeats, transfected into 293 cells and recovered as viruses, which are designated AdE1-Pol-F and AdE1+Pol-F, respectively
Summary
Human adenovirus (Ad) mainly causes self-limiting respiratory illnesses and can rapidly spread through confined populations such as day care centers, hospitals, retirement homes and military training venues [1]. First generation Ad (fgAd) vectors are deleted of the entire E1 region, which is commonly believed to render the virus replication-deficient in most cell lines These vectors are ideal for studies involving short-term gene expression either in vitro or in vivo [23]. DNA replication and late gene expression of fgAd has been observed in many “nonpermissive” cell lines (i.e. cells that do not complement the E1A/E1B deletion in these vectors) at multiplicities of infection (MOI) as low as 10 [26] In such cases, it has been speculated that activation of the E2 promoter by viral or cellular proteins and subsequent expression of the E2 replicative proteins likely led to the observed vector replication [27,28]. E2 proteins were expressed at low levels, removal of E1 severely compromised the ability of the virus to replicate
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