The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.Retroviruses require an interaction between the viral glycoproteins and a specific cell surface protein (receptor) to initiate entry into a cell (reviewed in references 32 and 56). The envelope glycoproteins of retroviruses are composed of trimers of two glycoproteins: the surface glycoprotein (SU), which contains the domains responsible for interaction with the host receptor, and the transmembrane glycoprotein (TM), which anchors SU to the membrane and mediates fusion of the viral and host membranes. The interaction of the SU glycoprotein with the host receptor usually involves multiple, noncontiguous determinants in both proteins that specify receptor choice and binding affinity and trigger a conformational change in the envelope glycoproteins that initiates the fusion process. Despite the complexity and specificity of the interaction between the viral glycoproteins and host receptors, closely related retroviruses carry envelope glycoproteins with mutations that alter receptor usage. The natural selection of retroviral subgroups with altered receptor usage may help the virus overcome host resistance and promote coinfection and may lead to heterotransmission.The five highly related envelope subgroups of...

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

Subgroup J avian leukosis virus (ALV-J) is unique among the avian sarcoma and leukosis viruses in using the multimembranespanning cell surface protein Na ؉ /H ؉ exchanger type 1 (NHE1) as a receptor. The precise localization of amino acids critical for NHE1 receptor activity is key in understanding the virus-receptor interaction and potential interference with virus entry. Because no resistant chicken lines have been described until now, we compared the NHE1 amino acid sequences from permissive and resistant galliform species. In all resistant species, the deletion or substitution of W38 within the first extracellular loop was observed either alone or in the presence of other incidental amino acid changes. Using the ectopic expression of wild-type or mutated chicken NHE1 in resistant cells and infection with a reporter recombinant retrovirus of subgroup J specificity, we studied the effect of individual mutations on the NHE1 receptor capacity. We suggest that the absence of W38 abrogates binding of the subgroup J envelope glycoprotein to ALV-J-resistant cells. Altogether, we describe the functional importance of W38 for virus entry and conclude that natural polymorphisms in NHE1 can be a source of host resistance to ALV-J.T he first step in retrovirus infection is attachment of the virus envelope glycoproteins to the specific cell surface receptor. Consequently, the susceptibility of each cell strictly depends on the expression and display of proper receptor molecules. This attachment, as well as the following phases of virus entry, requires a perfect match of receptors and envelope glycoproteins (1). Structural alterations within variable and hypervariable envelope glycoprotein regions easily abrogate the infectivity or even change the receptor usage and broaden the host range. This is best exemplified by avian sarcoma and leukosis viruses (ASLVs), a closely related group of retroviruses which evolved into five subgroups, A to E, that utilize three different receptors encoded by three genetic loci, tva, tvb, and tvc (1, 2). The tva locus encodes a protein belonging to the family of low-density lipoprotein receptors and determines susceptibility to the subgroup A ASLVs (3, 4). The tumor necrosis factor receptor-related protein encoded by the tvb locus confers susceptibility to subgroup B, D, and E ASLVs (5-7). Finally, subgroup C ASLVs utilize the Tvc protein of the butyrophilin family with two immunoglobulin-like domains (8). The complete resistance or decreased susceptibility of host chicken cells to a particular ASLV subgroup can then be caused by premature termination or a frameshift in the receptor-encoding loci (8-10), decreased receptor expression and display (11), and even single amino acid substitutions in the receptor sequence (9, 12).Subgroup J avian leukosis virus (ALV-J), the prototype virus isolate of which is HPRS-103, is an independent envelope subgroup which does not interfere with subgroups A to E and cannot be neutralized by antisera raised against subgroups A to E (13). ALV-J was originally d...

Highlights d A lentiviral-based assay was developed to map SHMsusceptible regions of the genome d SHM susceptibility and SHM resistance are confined within TADs d Robust transcriptional activity does not explain SHM susceptibility d SHM targeting elements present in the genome likely help explain SHM susceptibility