Novel Insights into the Roles of Bcl-2 Homolog Nr-13 (vNr-13) Encoded by Herpesvirus of Turkeys in the Virus Replication Cycle, Mitochondrial Networks, and Apoptosis Inhibition

Abstract

The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-ΔvNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-ΔvNr-13 showed similar growth kinetics; however, at early time points, HVT-ΔvNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-ΔvNr-13-infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells. IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek’s disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.

Ubiquitination of SARS-CoV-2 ORF7a Prevents Cell Death Induced by Recruiting BclXL To Activate ER Stress

“…Some members of the Bcl-2 family are prosurvival, such as BclXL, whose overexpression inhibits apoptosis induced by viral infection ( 12 , 13 ). For example, some viruses encode proteins, such as herpesvirus-encoded Nr-13, that are functional homologs of cellular Bcl-2 proteins and inhibit apoptosis and persist in host cells for the benefit of viral replication ( 14 , 15 ). Bcl-2 inhibits SARS-CoV infection-induced caspase-dependent apoptosis without affecting viral replication kinetics ( 16 ).…”

V5 and GFP Tagging of Viral Gene pp38 of Marek’s Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing

“…The role of a majority of MDV-encoded proteins and host-virus interactions contributing to the MDV-induced oncogenesis are still to be explored. Following the successful application of the CRISPR/Cas9 system on HVT and MDV genome editing for gene function and virus-host interaction studies [39][40][41][42]50,51] and recombinant vaccine development [37,38,52] with a highly efficient NHEJ repair pathway, we report here the first use of the HDR repair pathway to tag the MDV viral gene pp38 of the vaccine strain CVI988 with V5 and GFP as an alternative of the antibody generation approach. The rapid and efficient tagging of the MDV viral gene provides huge opportunities for gene function studies without the need for generating specific antibodies against the viral proteins.…”

“…Previously, a transfection/infection methodology that involves transfection of Cas9/ gRNA expression plasmids into CEF followed by infection of the parental virus, plaque purification, and characterization of the mutant viruses was used for gene deletion of replicating MDV/HVT in CEF cells with NHEJ repair [41,42,51]. Considering the likely high background of the wild-type virus with low efficient HDR-based editing of this approach, we developed a new pipeline for generating mutant MDV with precise mutation using co-electroporation of genomic DNA extracted from MDV-infected CEF, together with a Cas9/gRNA expression plasmid and the donor DNA, followed by an analysis of the formed plaques.…”

Mastering Death: The Roles of Viral Bcl-2 in dsDNA Viruses

“…These mammalian and zebrafish counterparts were shown to have a conserved Bcl-2 fold [7]. A recent study showed that vNR13/HVT079 localized to mitochondria and the endoplasmic reticulum [148]. In a functional study, vNR13/HVT079 expression inhibited apoptosis, a vNR13/HVT079 deletion mutant affected the viral replication in vitro, and the expression of vNR13/HVT079 disrupted mitochondrial morphology in HVT-infected cells [148].…”