The infection of human transplant recipients by porcine endogenous retrovirus (PERV) is a safety issue for xenotransplantation (XTx). CRISPR/Cas9 technology has enabled the generation of pigs free of functional PERVs, and the susceptibility of these animals to reinfection by PERVs remains unclear. To assess virological safety, we characterized a cell line in which PERVs have been inactivated by CRISPR/Cas9 (PK15 clone 15) for its susceptibility to infectious PERV. First, basal expression of PERV pol, the porcine PERV-A receptor (POPAR), and reverse transcriptase (RT) activity of PERV were determined. PK15 clone 15 cells were inoculated with PERV and monitored post infection for virus expression and RT activity. Particles were visualized by electron microscopy. Our data show that PK15 clone 15 cells still produce viral proteins that assemble to produce impaired viral particles. These virions have an irregular morphology that diverges from that of mature wild type. The particles are no longer infectious when tested in a downstream infection assay using supernatants of PK15 clone 15 cells to infect susceptible swine testis-IOWA (ST-IOWA) cells. The expression of POPAR was quantified to exclude the possibility that lack of susceptibility to reinfection, for PERV-A, is caused by absence of viral host receptor(s). PK15 and PK15 clone 15 cells do, in fact, express POPAR equally. PERV RT inactivation mediated by CRISPR/Cas9 does not compromise virus assembly but affects virion structure and proviral integration. The constitutive virion production seems to maintain cellular resistance to superinfection and possibly indicates a protective side effect of this specific CRISPR/Cas9 mediated RT inactivation.

Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. As a dNTP triphosphate triphosphohydrolase (dNTPase), SAMHD1 is proposed to limit cellular dNTP levels correlating with inhibition of HIV-1 reverse transcription. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is also not completely understood. We use BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1. Using CRISPR/Cas9-mediated homologous recombination, we introduced specific mutations into the genomic SAMHD1 locus. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. However, T592E KI cells retain wild type dNTP levels, suggesting the antiviral state might not only rely on dNTP depletion. In conclusion, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled.