Research Article: Human adenovirus type 5 vectors deleted of early region 1 (E1) undergo limited expression of early replicative E2 proteins and DNA replication in non-permissive cells

Date Published: July 10, 2017

Publisher: Public Library of Science

Author(s): Bratati Saha, Robin J. Parks, Michael Nevels.


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.

Partial Text

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]. Since their identification in the 1950’s, over 100 serotypes of Ad have been isolated from a variety of species. Of the human Ads, serotypes 2 and 5 (Ad5) have been extensively studied to gain a better understanding of virus biology, host cellular processes and virus-cell interactions during infection [2]. The Ad5 genome (36 kb, double-stranded DNA) consists of “early” and “late” regions, which are expressed before and after viral DNA replication, respectively (Fig 1A). The early regions (E1A, E1B, E2, E3 and E4) encode proteins that are involved in activating transcription of other viral regions, altering the host cellular environment to enhance virus replication, or in replication of the viral DNA [3]. The E1A gene products are the first proteins expressed from the infecting virus, and these proteins transactivate other viral coding regions, interact with a multitude of cellular proteins, and have a variety of other functions that ultimately promote infection [4]. The major product from the E1B region, the 55-kDa protein, is required for selective export of viral late mRNAs from the host cell nucleus; it also acts (in cooperation with E1A and E4orf6) to induce degradation of certain cellular proteins and promotes transformation of the infected cells [5,6]. Together, the E1A and E1B coding regions are absolutely required for efficient viral gene expression and replication.

In this study, we have created Ad constructs containing a FLAG-tag in each of the three E2 protein coding regions (Table 1). Determination of the locations for the FLAG-tag insertion was aided by previous biochemical and linker-insertion mutagenesis studies that identified crucial regions of these proteins, and also regions that were tolerant for insertions of small peptides. We showed that all three proteins could be detected in Ad-infected cells by immunoblot (Figs 2 and 3); however, the low quantities of Pol necessitated the use of immunoprecipitation to concentrate the protein to a detectable level. The presence of the FLAG at amino acid position 467 of pTP allowed the tag to be retained in both the immature and mature version of the protein. Indeed, we easily observed only the 80-kDa immature protein in the infected cell lysates, and the cleaved 55-kDa version in mature virions (Fig 3). Since FLAG-directed reagents are readily commercially available, these E2-tagged viral vectors may be an important resource, permitting renewed investigation of these viral proteins.




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