CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses

Abstract

Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses. IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

Abrogation of PRRSV infectivity by CRISPR-Cas13b-mediated viral RNA cleavage in mammalian cells

“…Alternatively, by selectively disrupting HIV proviral DNA integrated into the host genome, some groups have demonstrated that targeting HIV long terminal repeats (LTRs) or essential viral genes is an effective approach to profoundly suppress HIV production in vitro [57][58][59][60][61][62] and in vivo 63 . However, there are no dsDNA intermediates in the majority of RNA viruses, and host essential genes are the only choice for CRISPR/ Cas9 targeting, including for TGEV 64 , IAV 65 , CSFV 66 , FMDV 67 and BVDV 68 . A recent study described the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of exon 7, which encodes SRCR5 of the CD163 gene responsible for binding to GP4 to mediate viral entry.…”

Role of Endoplasmic Reticulum-Associated Proteins in Flavivirus Replication and Assembly Complexes

“…It has been reported that endogenous levels of DNAJC14 are vital for YFV replication [57]. A similar role of DNAJC14 has recently been observed in the RNA replication of the bovine viral diarrhea virus (BVDV) [59]. Therefore, DNAJC14 is a key host cell factor for flavivirus replication.…”

Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus

“…The geminivirus-silencing suppressor βC1 was recently shown to activate and be degraded by host autophagy to reduce viral virulence ( Ismayil et al, 2020 ). Cellular peptidylprolyl isomerases or protein chaperones regulate polyprotein cleavage kinetics and the persistence of human and animal viruses, such as the hepatitis C virus, yellow fever virus, and bovine viral diarrhea virus ( Kaul et al, 2009 ; Bozzacco et al, 2016 ; Isken et al, 2019 ). More recently, a functional link between polyprotein processing and host RNA metabolism was reported in alphaviruses.…”

“…Virus-encoded proteases are multifunctional hubs that coordinate virus replication and host–virus interactions to promote infectivity and virulence ( Martínez et al, 2016 ; Lei and Hilgenfeld, 2017 ; Rodamilans et al, 2018 ). The spatial-temporal regulation of protease activity and the processing of viral polyproteins can further determine viral persistence and disease lethality ( Lackner et al, 2004 ; Yost and Marcotrigiano, 2013 ; Isken et al, 2019 ).…”