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HSV-1 latency-associated transcript miR-H3 and miR-H4 target STXBP1 and GABBR2 genes

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Abstract

During latent infection, the HSV-1 virus generates only a single transcript, LAT, which encodes six miRNAs. The GABAergic pathway signaling system is an essential cell signaling pathway influenced by various therapeutic targets and some brain disorders, such as epilepsy. This study found that miRNAs encoding LAT might target the STXBP1 and GABBR2 genes, which are among the significant genes in the GABAergic pathway. Bioinformatic analysis utilizing TargetScan version 5.2 and the RNA22 tools uncovered miRNAs encoding LAT that can influence STXBP1 and GABBR2 transcripts. To evaluate the targeting effect of candidate microRNAs encoding LAT, namely, miR-H3 and miR-H4, LAT constructs were transfected into HEK 293T cells. The expression levels of microRNAs encoding LAT, as well as STXBP1 and GABBR2, were assayed by real-time PCR. Finally, the targeting potential of STXBP1 and GABBR2 3′UTR by LAT-encoded microRNAs was evaluated by the luciferase assay. In the current study, the bioinformatic tool TargetScan demonstrated that miR-H3 has the potential to target the transcripts of the STXBP1 and GABBR2 genes, whereas miR-H4 solely targeted GABBR2. On the other hand, the bioinformatic tool RNA22 validated the potential targeting of STXBP1 and GABBR2 by miR-H3 and miR-H4. Our findings showed that overexpression of miR-H4, miR-H3, or LAT significantly decreased STXBP1 gene expression by an average of 0.0593-fold, 0.237-fold, and 0.84-fold, respectively. Similarly, overexpression of miR-H3 or miR-H4 decreased GABBR2 expression by an average of 0.055- or 0.687-fold, respectively. Notably, targeting the GABBR2 3′UTR with the LAT transcript had no detectable effect. The evaluation of the targeting potential of STXBP1 and GABBR2 3′UTR by microRNAs encoded by LAT was conducted with a luciferase assay. Our results showed that miR-H3 overexpression reduces Renilla expression in psiCHECK2 plasmids with STXBP1 or GABBR2 3′UTR genes by 0.62- and 0.55-fold, respectively. miR-H4 reduced Renilla gene expression regulated by GABBR2’s 3′UTR plasmid but had no effect on the Renilla gene expression regulated by STXBP1’s 3′UTR. When the LAT transcript was overexpressed, there was a decrease in Renilla expression by 0.44-fold because of the regulation of STXBP1’s 3′UTR. However, there was no significant effect observed through the control of GABBR2’s 3′UTR.

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Data availability

The data utilized in this study have been archived and are accessible to the public upon request by the corresponding author. Additionally, the data can be found at the Department of Microbiology, which is located within the School of Biology at the College of Science, University of Tehran.

Code availability

The software used in this study is commercially available. No custom programming has been carried out for this project.

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Acknowledgements

The authors would like to acknowledge Dr. Zahra Fekrirad for providing technical assistance.

Funding

The Iran National Science Foundation (INSF) funded this research (grant number 99019844). The decision to publish this article, as well as the study’s design and data collection, analysis, and interpretation, was all made independently of the funder.

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Authors and Affiliations

  1. Molecular Virology Lab, Department of Microbiology, School of Biology, College of Sciences, University of Tehran, P.O. Box 14155-6455, Tehran, 1417614411, Iran

    Karrar Mahmood Shaker AL-Khfaji & Ehsan Arefian

  2. Department of Biotechnology, College of Sciences, University of Tehran, Tehran, Iran

    Nika Kooshki Zamani

  3. Paediatric Cell and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran, Iran

    Ehsan Arefian

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  1. Karrar Mahmood Shaker AL-Khfaji
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  2. Nika Kooshki Zamani
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Contributions

K. M. AL-Khfaji and Nika K. Zamani performed the experimental procedures, data analysis, and manuscript drafting. Ehsan Arefian conceptualized the study and revised the manuscript. The present study is under the supervision of Dr. Ehsan Arefian, who serves as the guarantor of the work. Dr. Arefian has unrestricted access to all the data collected during the study and is accountable for ensuring the integrity and precision of the data analysis. The authors assert that all data were produced internally and that no external sources were utilized.

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Correspondence to Ehsan Arefian.

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The study was carried out in accordance with approved protocols from the Department of Microbiology, School of Biology, College of Science, University of Tehran.

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Key points

• STXBP1 is a protein expressed in neuronal cells and has a crucial role in docking and fusing synaptic vesicles.

• Gene expression assays and luciferase assays showed that HSV-1 LAT reduced STXBP1 gene expression by 0.84- and 0.44-fold, respectively.

• miR-H3 directly targets the 3′UTR of STXBP1 and GABBR2, resulting in a decrease in transcript levels of these genes.

• Real-time PCR analysis showed that miR-H4 decreased STXBP1 and GABBR2 gene expression. Luciferase assays showed a significant difference only when miR-H4 targeted the 3′UTR of the GABBR2 gene but not when it targeted the STXBP1 gene.

•The miR-H3 and miR-H4 microRNAs from HSV-1 may contribute to the dysfunction in the GABAergic pathway signaling system, which is linked to neurological diseases.

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AL-Khfaji, K.M.S., Zamani, N.K. & Arefian, E. HSV-1 latency-associated transcript miR-H3 and miR-H4 target STXBP1 and GABBR2 genes. J. Neurovirol. 29, 669–677 (2023). https://doi.org/10.1007/s13365-023-01174-8

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