Autonomously Replicating RNAs of Bungowannah Pestivirus: ERNS 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, E^RNS, 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-E^RNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of E^RNS still allowed the generation of infectious particles. However, those ΔE^RNS 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 ΔE^RNS 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 E^RNS 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 ΔE^RNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins N^PRO and E^RNS are unique for the genus Pestivirus, but only N^PRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for E^RNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins E^RNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV E^RNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The E^RNS-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.

Keywords: Bungowannah virus; ERNS; foreign gene expression; pestivirus; replicon; viral vector; virus replicon particles.