Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid.

Anna Koslová,Jiří Hejnar,Markéta Reinišová,Veronika Krchlíková,Viktor Kožich,Jakub Krijt,Daniel Elleder,Pavel Trefil,Jitka Mucksová,Filip Šenigl,Jiří Plachý,Josef Geryk,Dana Kučerová,Jiří Kalina

doi:10.3390/v13122504

Abstract

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva −/− chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/− siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

Highlights

IntroductionThe retrovirus replication cycle starts with the attachment of a retrovirus particle to the host cell and reconformation of retroviral envelope (Env) glycoproteins
We demonstrated that homozygous in vivo knock-out of the chicken tva gene by a frame-shifting deletion in the second exon results in complete resistance to two ALV subgroups, A and K, which share Tva as an entry receptor
Exchanger 1 [30,34], it is the second example of artificial anti-viral resistance conferred by genetic manipulation of the chicken

Summary

Introduction

The retrovirus replication cycle starts with the attachment of a retrovirus particle to the host cell and reconformation of retroviral envelope (Env) glycoproteins. Env molecules expose hydrophobic fusion peptides and insert them into the host cell cytoplasmic membrane. The process continues with fusion of both viral and host cell membranes and results in virus uptake, uncoating, reverse transcription, and trafficking toward the cell nucleus. Whatever details of the virus entry may differ between retrovirus genera, the Env reconformation is always primed by a lock–key interaction with a highly specific host cell receptor. Display of the proper receptor on the cell surface determines the host range and tissue specificity of a given retrovirus, and vice versa, the absence of receptor molecules means that a given cell or animal species is refractory to the virus infection

Methods

Results

Conclusion