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

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.

Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Suppresses Human Adenovirus Gene Expression and Replication

“…The transcription factors E2F1 and SP1 both play a role in transcriptional control of E1A (75,76). E2F1 and SP1 also bind to the E2 promoter and the MLP, respectively, and binding is required for the maximum expression of proteins from those regions (17,58,(77)(78)(79). Although we were unable to rescue HAdV gene expression via knockdown of p21 ( Fig.…”

“…It is deleted of the E3 region. Ad(E1 -)-CMV/RFP (AdRP2619) and Ad(E1 ϩ )TP-F (AdRP3000) constructs have been described previously (17,88,89). All viruses were propagated in 293 cells and purified by cesium chloride buoyant density centrifugation using standard procedures (90).…”

“…All viruses were propagated in 293 cells and purified by cesium chloride buoyant density centrifugation using standard procedures (90). Viral titers were determined by plaque assay on 293 cells as previously reported (17).…”

“…HAdV serotypes 2 and 5 (species C) have been the most extensively studied to gain insights on virus biology, cellular processes, and host-pathogen interactions. The 36-kbp HAdV-5 genome consists of "early" and "late" genes which are expressed before and after viral DNA replication, respectively (16,17). Of the early genes (E1A, E1B, E2, E3, and E4), the E1A gene product is absolutely necessary for efficient virus replication, and it is the first protein expressed from the infecting virus.…”

“…The E2A region encodes the HAdV DNA-binding protein (DBP), while the E2B region codes for the precursor terminal protein (pTP) and the viral DNA polymerase. All three E2 proteins play crucial roles during HAdV DNA replication (17,21,22). The E3 and E4 gene products modulate the host immune system and the cellular environment to make it more conducive to virus replication (23,24).…”