Autonomously Replicating RNAs of Bungowannah Pestivirus: E ^RNS Is Not Essential for the Generation of Infectious Particles

Abstract

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These “defective-in-third-cycle” BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors. IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.

Single-Round Infectious Particle Production by DNA-Launched Infectious Clones of Bungowannah Pestivirus

“…When in vitro-transcribed RNA produced from an infectious cDNA clone of BuPV was transfected, the deletion of E RNS still allowed the generation of infectious particles [ 27 ]. However, in this study there were no indications of cell-to-cell spread in pBuPVΔE RNS DNA-transfected cells or production of SRIPs in transfection supernatants ( Figure 3 C, panels c, d, k, and l), while transfection with pBuPV resulted in single NS3 and E RNS positive cells.…”

“…Plasmid pBuPV∆E RNS with a deletion of 448 bases within the E RNS protein (aa 328-483) was generated by fusion PCR using pBuPV as DNA template and primer pair Bungo_dERNS_F and Bungo_2164R. For construction of the split genome plasmid pBuPV∆E RNS /E RNS , plasmid pCAGGS_BuPV-E RNS [27] was digested with SmaI and NotI and the E RNS comprising fragment was ligated into plasmid pBuPV∆E RNS , digested with PmeI and NotI. Further details of the plasmid constructions are available on request.…”

The use of RNA-based treatments in the field of cancer immunotherapy

“…Early investigation of SAM is direct injection of SAM packaged into viral replication particles (VRP) [419]. VRPs are potent vaccines in mice, non-human primates and humans However, the replicated VRP structural proteins may induce non-specific immunogenicity and toxicity [420]. To decrease the infectious concern of viral components, a propagation-defective type of VRPs was generated [421].…”

Bungowannah Pestivirus Chimeras as Novel Double Marker Vaccine Strategy against Bovine Viral Diarrhea Virus

“…To recover virus, the cDNA construct was linearized by SmaI digest and in vitro transcribed with the T7 RiboMax Large Scale RNA Production System (Promega, Walldorf, Germany). The in vitro transcribed RNA was electroporated into SK6 cells [50]. The supernatant of electroporated cells was harvested 72 h later and was tested for the presence of infectious virus.…”