# The snakehead retrovirus promoter functions independently of the 3’ORF protein and its products are maternally inherited in transgenic zebrafish

## TL;DR

The SnRV retrovirus promoter works without its 3’ORF protein and can drive strong gene expression in zebrafish, making it a useful tool for biotechnology.

## Contribution

The SnRV promoter functions independently of the 3’ORF protein and is maternally inherited in transgenic zebrafish.

## Key findings

- The 3’ORF protein is not essential for SnRV replication, indicating it does not act as a transcriptional transactivator.
- The SnRV promoter is active in both fish and mammalian cells and is functional as early as the blastula stage in zebrafish embryos.
- In transgenic zebrafish, the SnRV promoter drives strong expression in sensory organs and gonads, with products maternally inherited.

## Abstract

The exogenous snakehead retrovirus (SnRV) is an unclassified member of the Orthoretrovirinae subfamily, discovered in cell lines derived from several fish species. SnRV resembles complex lentiviruses and potentially encodes accessory proteins, including the product of the 3’ open reading frame (3’ORF). The 3’ORF protein was suggested to function as a transactivator of transcription (Tat). Here, we constructed an infectious molecular clone for SnRV and tested the effects of 3’ORF mutations on SnRV transcription. Although replacing 3’ORF with foreign sequences strongly reduced virus expression and production, an out-of-frame point mutation in 3’ORF had only a minimal effect on SnRV replication. This latter result suggests that the 3’ORF protein does not function as Tat and that SnRV transcription is largely independent of the product of this ORF. We also show that in vitro, the SnRV promoter is versatile and robustly functioning in both fish and mammalian cultured cells. Finally, the SnRV promoter was transiently active in injected zebrafish embryos as early as the blastula stage. In transgenic zebrafish, this promoter drives enhanced expression in sensory organs and gonads, and its generated products are maternally inherited. Considering these characteristics, the SnRV promoter emerges as a promising candidate for developing versatile expression vectors applicable to research and biotechnological applications.

Retroviruses are genetic elements that can insert themselves into the DNA of host cells, and they play important roles in biology and biotechnology. The fish snakehead retrovirus (SnRV) was suggested to enhance its own gene transcription via a protein encoded by its 3’ open reading frame (3’ORF) in a similar way to the Tat protein in other complex retroviruses (e.g., the human immunodeficiency virus - HIV). To test this hypothesis, we created lab clones of SnRV and studied how disrupting the 3’ORF affects the gene expression and replication of the virus. We found that the 3’ORF protein is not essential for SnRV replication, showing that viral transcription occurs independently of this protein. We also discovered that the SnRV’s promoter functions robustly in both fish and mammalian cells. In injected zebrafish embryos, it is active as early as the blastula stage, and in transgenic zebrafish, it drives strong gene expression in sensory organs and gonads. Moreover, the promoter’s expression products in oocytes are maternally inherited to the offspring. These findings have implications for the field of retroviral evolution, and highlight the SnRV promoter’s potential as a versatile tool for scientific research and biotechnology.