Demonstration of the Specificity of Poliovirus Encapsidation Using a Novel Replicon which Encodes Enzymatically Active Firefly Luciferase

Abstract

The specificity of poliovirus encapsidation has been studied using a novel chimeric genome in which the gene encoding firefly luciferase has been substituted for the VP2-VP3-VP1 genes of the poliovirus capsid (P1) gene. Transfection of RNA transcribed in vitro from this genome resulted in a VP4-luciferase fusion protein which retained luciferase enzyme activity. Since the detection of enzyme activity was dependent upon replication of the transfected RNA genome, we refer to these genomes as replicons. The replicon encoding luciferase was encapsidated upon transfection of the genomic RNA into cells previously infected with a recombinant vaccinia virus, VV-P1, which encodes the poliovirus type 1 capsid proteins (P1). Infection of cells with each serial passage, followed by analysis of luciferase enzyme activity, revealed that encapsidated replicons could be detected at the first passage with VV-P1. Amplification of the titer of encapsidated replicons occurred upon serial passage with VV-P1, as evidenced by the high expression levels of luciferase enzyme activity following infection. Serial passage of the luciferase replicons with poliovirus type 1, 2, or 3 resulted in the trans encapsidation into the type 1, 2, or 3 capsids, respectively. In contrast, serial passage with bovine enterovirus, Coxsackievirus A21 or B3, or enterovirus 70 did not result in trans encapsidation, even though co-infection of cells with the replicon and different enteroviruses resulted in high-level expression of luciferase. The results of this study highlight the specificity of poliovirus encapsidation and point to the use of encapsidated replicons encoding luciferase as a reagent for dissecting elements of replication and encapsidation.

5′-Terminal Deletions Occur in Coxsackievirus B3 during Replication in Murine Hearts and Cardiac Myocyte Cultures and Correlate with Encapsidation of Negative-Strand Viral RNA

“…The logic of specific viral RNA packaging has dictated a packaging signal, a sequence or RNA structure which initiates the assembly of the capsid pentamers into a complete virion. No such specific signal has yet been identified in the human enteroviruses, although the generation of replicons with substitutions of foreign proteins in the P1 region which can be encapsidated in trans indicates that such a signal cannot be present in the capsid encoding region (47,70,90,91). In PV, only newly synthesized genomes are packaged (79).…”

Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

“…To retain genetic stability, these reporter genes originally replaced the P1 capsid-encoding sequence (Luc-P2-P3) (4,16,73). These replicons, however, cannot be used in studies of virus morphogenesis unless the capsid proteins are supplied in trans (37,54). Reporter viruses that contain a reporter gene fused to the N terminus of the polyprotein have also been developed (25).…”

Identification of a cis -Acting Replication Element within the Poliovirus Coding Region

“…The specificity of this process (5,14,32) can be readily monitored using subgenomic replicons, in which the capsid-encoding P1 region of the genome is replaced with a reporter gene such as that coding for chloramphenicol acetyl transferase (CAT) (30). We have used such replicons to demonstrate that RNA sequences involved in packaging are not located within the P1, 5Ј NCR, 3Ј NCR, or VPg-encoding regions (5,30).…”