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

Article (PDF Available)inPLoS Pathogens 8(8):e1002853 · August 2012with34 Reads
DOI: 10.1371/journal.ppat.1002853 · Source: PubMed
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.

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    • "The mechanism of IFNα activity was not determined. We attribute the moderate effects observed in these assays to the profound effects of IFNs in our studies to potential differences in cell populations and/or the amount of IFNα used to treat the cells (250U/ml in [12], 500 U/ml here). PML-NBs have established intrinsic and IFN-induced activity against many herpesviruses [49,50] which prompted us to examine if resident PML-NB proteins PML, Daxx or Sp100 inhibited Ad replication in HDF-TERT cells plus or minus IFN signaling. "
    [Show abstract] [Hide abstract] ABSTRACT: Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.
    Full-text · Article · Jan 2016
    • "The initial activation of type I interferon synthesis occurs 12 h postinfection through sensing of different PAMPs, of which VA RNA future science group is one [50]. To counteract the cellular innate immune response, viral early proteins, such as E1A, E4orf3 and E1B-55K, can antagonize the negative effect of interferon pathways on viral DNA synthesis555657 . For example, the adenovirus E1A protein inhibits the transcriptional coactivator proteins p300/CBP and PCAF [58], both of which are essential for the transcriptional activation of the interferon genes. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Apr 2013
  • [Show abstract] [Hide abstract] 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.
    Article · Feb 2013
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