AKT is implicated in neurological disorders. AKT has three isoforms, AKT1/AKT2/AKT3, with brain cell type-specific expression that may differentially influence behavior. Therefore, we examined single Akt isoform, conditional brain-specific Akt1, and double Akt1/3 mutant mice in behaviors relevant to neuropsychiatric disorders. Because sex is a determinant of these disorders but poorly understood, sex was an experimental variable in our design. Our studies revealed AKT isoform- and sex-specific effects on anxiety, spatial and contextual memory, and fear extinction. In Akt1 mutant males, viral-mediated AKT1 restoration in the prefrontal cortex rescued extinction phenotypes. We identified a novel role for AKT2 and overlapping roles for AKT1 and AKT3 in long-term memory. Finally, we found that sex-specific behavior effects were not mediated by AKT expression or activation differences between sexes. These results highlight sex as a biological variable and isoform- or cell type-specific AKT signaling as potential targets for improving treatment of neuropsychiatric disorders.

Programmed −1 ribosomal frameshifts (−1 PRFs) are commonly used by viruses to regulate their enzymatic and structural protein levels. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that uses two independent −1 PRFs to express viral enzymes critical to establishing new HTLV-1 infections. How the cis-acting RNA elements in this viral transcript function to induce frameshifting is unknown. The objective of this work was to conclusively define the 3′ boundary of and the RNA elements within the HTLV-1 pro-pol frameshift site. We hypothesized that the frameshift site structure was a pseudoknot and that its 3′ boundary would be defined by the pseudoknot's 3′ end. To test these hypotheses, the in vitro frameshift efficiencies of three HTLV-1 pro-pol frameshift sites with different 3′ boundaries were quantified. The results indicated that nucleotides included in the longest construct were essential to highly efficient frameshift stimulation. Interestingly, only this construct could form the putative frameshift site pseudoknot. Next, the secondary structure of this frameshift site was determined. The dominant structure was an H-type pseudoknot which, together with the slippery sequence, stimulated frameshifting to 19.4(±0.3)%. The pseudoknot's critical role in frameshift stimulation was directly revealed by examining the impact of structural changes on HTLV-1 pro-pol −1 PRF. As predicted, mutations that occluded pseudoknot formation drastically reduced the frameshift efficiency. These results are significant because they demonstrate that a pseudoknot is important to HTLV-1 pro-pol −1 PRF and define the frameshift site's 3′ boundary.

The Human T‐cell Lymphotropic Virus Type 1 (HTLV‐1) encodes two ‐1 programmed ribosomal frameshift (PRF) sites, which allow for the translation of enzymes critical to viral replication. The frequency, or frameshift efficiency, of this event is also thought to be important in the viral life cycle. Each PRF site includes a heptanucleotide “slippery” sequence, a spacer, and a downstream structure. The downstream structure in the HTLV‐1 pro‐pol frameshift site is predicted to be a pseudoknot. The importance of this structure to the efficiency of frameshifting has not yet been established. To improve our understanding of the role this structure has in frameshift stimulation, we designed frameshift site variants that disrupt the pseudoknot structure. The frameshift efficiency for these sites was then measured using an in vitro dual‐luciferase assay. Preliminary data suggests that the removal or mutation of sequences important to pseudoknot formation significantly reduce the HTLV‐1 pro‐pol frameshift efficiency. These findings are significant because they suggest that the HTLV‐1 pro‐pol frameshift site pseudoknot structure plays a criticial role in frameshift stimulation.Support or Funding InformationIH NIGMS SCORE SC2 Award (1SC2GM121197‐01), Research Corporation for Science Advancement Cottrell Scholar Award (23983), Fort Lewis College Undergraduate Scholarly and Creative Activities AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Programmed ribosomal frameshifting (PRF) is a common viral mechanism used to regulate the levels of viral enzymatic and structural proteins. PRF events are stimulated by cis‐acting RNA elements within the viral transcript, occur during translation, and change the ribosomal reading frame. Each PRF occurs at a frameshift site that includes a “slippery” sequence, a spacer, and a downstream structure. The human T‐cell Lymphotropic Virus Type‐1 (HTLV‐1) retrovirus uses two, independent ‐1 PRF sites to express three of its viral enzymes. While the structure was previously predicted to be a pseudoknot, the importance of this structure to frameshifting has not been established. In this work, we are examining the importance of the frameshift site structure to HTLV‐1 pro‐pol frameshift stimulation. Specifically, we examined how mutations that change the RNA structure impact ‐1 PRF efficiency. We hypothesized that mutations that disrupted the pseudoknot structure would decrease the frameshift efficiency. Likewise, mutations that restored the pseudoknot structure should have no impact on frameshift efficiency. To test these hypotheses, we measured the frameshift efficiencies for several variant frameshift sites using an in vitro dual‐luciferase frameshift assay. Preliminary data suggests that when the pseudoknot structure is disrupted, there is a significant decrease in frameshift efficiency. Surprisingly, a mutation that restored pseudoknot formation increased the frameshift efficiency. These results are significant because they suggest that the pseudoknot structure plays a critical role in frameshift stimulation.Support or Funding InformationNIH NIGMS SCORE SC2 Award (1SC2GM121197‐01) and Research Corporation for Science Advancement Cottrell Scholar Award (23983)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.