The PKR-binding domain of adenovirus VA RNAI exists as a mixture of two functionally non-equivalent structures

VA RNAI is a non-coding adenoviral transcript that counteracts the host cell anti-viral defenses such as immune responses mediated via PKR. We investigated potential alternate secondary structure conformations within the PKR-binding domain of VA RNAI using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNAI apical stem can adopt two different conformations and that it exists as a mixed population of these two structures. In contrast, in two sequence variants we designed to eliminate one of the possible structures, while leaving the other intact, each formed a unique secondary structure. This clarification of the apical stem pairing also suggests a small alteration to the apical stem–loop secondary structure. The relative ability of the two apical stem conformations to bind PKR and inhibit kinase activity was measured by isothermal titration calorimetry and PKR autophosphorylation inhibition assay. We found that the two sequence variants displayed markedly different activities, with one being a significantly poorer binder and inhibitor of PKR. Whether the presence of the VA RNAI conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation

Promotion of exon 6 inclusion in HuD pre-mRNA by Hu protein family members

The Hu RNA-binding protein family consists of four members: HuR/A, HuB, HuC and HuD. HuR expression is widespread. The other three neuron-specific Hu proteins play an important role in neuronal differentiation through modulating multiple processes of RNA metabolism. In the splicing events examined previously, Hu proteins promote skipping of the alternative exons. Here, we report the first example where Hu proteins promote inclusion of an alternative exon, exon 6 of the HuD pre-mRNA. Sequence alignment analysis indicates the presence of several conserved AU-rich sequences both upstream and downstream to this alternatively spliced exon. We generated a human HuD exon 6 mini-gene reporter construct that includes these conserved sequences. Hu protein over-expression led to significantly increased exon 6 inclusion from this reporter and endogenous HuD. Studies using truncated and mutant HuD exon 6 reporters demonstrate that two AU-rich sequences located downstream of exon 6 are important. RNAi knockdown of Hu proteins decreased exon 6 inclusion. An in vitro splicing assay indicates that Hu proteins promote HuD exon 6 inclusion directly at the level of splicing. Our studies demonstrate that Hu proteins can function as splicing enhancers and expand the functional role of Hu proteins as splicing regulators

Preparation of soluble extracts from adenovirus-infected cells for studies of RNA splicing

Here we describe a collection of methods that have been adapted to produce highly efficient nuclear and cytoplasmic extracts from adenovirus-infected HeLa cells. We describe how to produce extracts from virus-infected cells and how to analyze RNA splicing in vitro using T7 RNA polymerase-derived splicing substrate RNAs

Characterization of Inducible Transcription and Translation-Competent HIV-1 Using the RNAscope ISH Technology at a Single-Cell Resolution

Identifying the source and dynamics of persistent HIV-1 at single-cell resolution during cART is crucial for the design of strategies to eliminate the latent HIV-1 reservoir. An assay to measure latent HIV-1 that can distinguish inducible from defective proviruses with high precision is essential to evaluate the efficacy of HIV-1 cure efforts but is presently lacking. The primary aim of this study was therefore to identify transcription and translation competent latently infected cells through detection of biomolecules that are dependent on transcriptional activation of the provirus. We investigated the applicability of two commercially available assays; PrimeFlow (TM) RNA Assay (RNAflow) and RNAscope (R) ISH (RNAscope) for evaluation of the efficacy of latency reversal agents (LRAs) to reactivate the HIV-1 latent reservoir. The J-Lat cell model (clones 6.3, 9.3, and 10.6) and four LRAs was used to evaluate the sensitivity, specificity, and lower detection limit of the RNAflow and RNAscope assays for the detection and description of the translation-competent HIV-1 reservoir. We also checked for HIV-1 subtype specificity of the RNAscope assay using patient-derived subtype A1, B, C, and CRFOLAE recombinant plasmids following transfection in 293T cells and the applicability of the method in patient-derived peripheral blood mononuclear cells (PBMCs). The lower detection limit of RNAflow was 575 HIV-1 infected cells/million and 45 cells/million for RNAscope. The RNAscope probes, designed for HIV-1B, also detected other subtypes (A1, B, C, and CRF&lt;b&gt;01 _AE). RNAscope was applicable for the detection of in patient-derived PBMCs following LRA activation. In conclusion, our study showed that RNAscope can be used to quantify the number of directly observed individual cells expressing HIV-1 mRNA following LRA activation. Therefore, it can be a useful tool for characterization of translation-competent HIV-1 in latently infected cell at single-cell resolution in the fields of HIV-1 pathogenesis and viral persistence.QC 20181015</p