Abstract
Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN) was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT) virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.
Highlights
A major obstacle to the therapeutic application and efficacy of adenoviral vectors is the induction of powerful innate and proinflammatory immune responses following systemic delivery [1,2,3,4], independently of viral gene expression [5,6,7,8,9]
The most frequently used therapeutic vectors for gene transfer or cancer treatment are derived from human adenovirus type 5 (Ad5)
We have observed previously that the E1B 55 kDa protein encoded by a gene routinely deleted from these vectors represses expression of numerous cellular genes regulated by interferon (IFN) a and b, which are important components of the innate immune response to viral infection
Summary
A major obstacle to the therapeutic application and efficacy of adenoviral vectors is the induction of powerful innate and proinflammatory immune responses following systemic delivery [1,2,3,4], independently of viral gene expression [5,6,7,8,9]. Interferon a and b, designated hereafter IFN, bind to the same heterodimeric receptor to establish a front line of antiviral defense via stimulation of transcription of numerous genes [20,21,22,23]. The products of such interferon-stimulated genes (ISGs) inhibit replication of a wide variety of viruses by multiple direct or indirect mechanisms [22,24,25,26,27]. Proteins encoded by ISGs reinforce synthesis of IFN and other cytokines, promote processing and presentation of antigens, and modulate the activity of important effector cells of the immune system [22,25,26,27,28,29]
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