The Human Adenovirus Type 5 E1B 55 kDa Protein Obstructs Inhibition of Viral Replication by Type I Interferon in Normal Human Cells

Princeton University, Department of Molecular Biology, Lewis Thomas Laboratory, Princeton, New Jersey, United States of America.
PLoS Pathogens (Impact Factor: 7.56). 08/2012; 8(8):e1002853. DOI: 10.1371/journal.ppat.1002853
Source: PubMed


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.

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    ABSTRACT: To begin to investigate the mechanism by which the human adenovirus type 5 E1B 55 kDa protein protects against anti-viral effects of type 1 interferon IFN (Chahal, J.S., Qi, J. and S.J. Flint (2012) PLoS Pathogens 8 doi:10:1371), we examined the effects of precise amino acid substitution in this protein on resistance of viral replication to the cytokine. Only substitution by alanine of residues 443-448 (E1B Sub19) specifically impaired production of progeny virus, and resulted in a large defect in viral DNA synthesis in IFN-treated normal human fibroblasts. Untreated or IFN-treated cells infected by this mutant virus (AdEasyE1Sub19) contained much higher steady-state concentrations of IFN-inducible GBP1 and IFIT2 mRNAs than did wild type-infected cells, and of the corresponding newly transcribed pre-mRNAs, isolated exploiting 5-ethynyluridine labeling and click chemistry. These results indicated that the Sub19 mutations impair repression of transcription of IFN-inducible genes by the E1B 55 kDa protein, consistent with their location in a segment required for repression of p53 dependent transcription. However, when synthesized alone, the E1B 55 kDa protein inhibited expression of the p53-regulated genes BAX and MDM2, but had no impact whatsoever on induction of IFIT2 and GBP1 expression by IFN. These observations correlate repression of transcription of IFN-inducible genes by the E1B 55 kDa protein with protection against inhibition of viral genome replication, and indicate that the E1B 55 kDa protein is not sufficient to establish such transcriptional repression.
    Preview · Article · Feb 2013 · Journal of Virology
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    ABSTRACT: Although several adenovirus type 5 (Ad5) proteins prevent deleterious consequences of activation of p53, it has been reported that viral replication proceeds more efficiently when human tumor cells produce wild-type compared to mutant p53. We have now exploited RNA interference and lentiviral vectors to achieve essentially complete knockdown of p53 in normal human cells: no effects on the kinetics or efficiency of viral gene expression or production of infectious particles were observed.
    Preview · Article · Mar 2013 · Journal of Virology
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    ABSTRACT: Adenovirus type 5 encodes two short, highly structured noncoding RNAs, the virus-associated (VA) RNAI and VA RNAII. These RNAs are expressed in large amounts late during a lytic infection. Early studies established an important role for VA RNAI in maintaining efficient translation in late virus-infected cells by blocking activation of the key interferon-induced PKR protein kinase. More recent studies have demonstrated that the VA RNAs also target the RNAi/miRNA pathway. Collectively, available data suggest that the VA RNAs are multifunctional RNAs suppressing the activity of three dsRNA-sensing enzyme systems in human cells. Here, the known functions of the VA RNAs are summarized and the interplay between VA RNA expression and the activity of the interferon and RNAi pathways are discussed in more detail.
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