Abstract
The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.
Highlights
Human endogenous retroviruses (HERV) are around 8% of human DNA and are poorly and variably expressed by host cells
To target the human endogenous retrovirus (HERV)-Kenv gene, we focused on the region spanning from 800 to 1500 bp from the HERV-Kenv gene ATG, because an in silico evaluation across the HERV-K genome sequences present in the GenBank® NIH database showed that this region is well conserved
These sequences were screened for the presence of 20 to 22 nucleotides-long target regions, followed by NNGRR protospacer adjacent motifs (PAMs), which are recognized by SaCas9 endonuclease [38]
Summary
Human endogenous retroviruses (HERV) are around 8% of human DNA and are poorly and variably expressed by host cells. They are highly defective, but a handful of complete proviruses has been described, with the classical genome organization of retroviruses [1,2]. Among cells of the same individual, the expression of different HERVs varies, depending on several factors. The HERV-K family gathered the attention of scientists because its HML-2 subgroup is the most recently integrated in the human genome, the least defective
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