Packaging of Genomic RNA in Positive-Sense Single-Stranded RNA Viruses: A Complex Story

Figure 1. PS-mediated packaging mechanisms. (A) High-affinity binding mechanism. Selective packaging of the genomic RNA (gRNA) is mediated by high-affinity binding between the capsid protein (CP) and the packaging signal (PS). In this scenario the PS is one small RNA element (and/or sequence) present only in the gRNA. (B) PS allosteric mechanism. Binding of the CP to the PS is an allosteric switch that changes the conformation of the CP from one with weak CP-CP interactions (CPi) to one strong interactions (CPa). (C) Multiple PS-like elements. There is one PS and a series of PS-like elements distributed across the gRNA. The interaction between the PS and the CP (a) are stronger than between the PS-like elements and the CP (b). Unlike the first two scenarios, all these clusters contribute to the nucleation of virion assembly. In all cases the unstructured tail represents the CP N-terminal domain. For most (+)ssRNA viruses the N-terminal domain of the CP is extremely basic and is required for RNA/CP interactions and RNA packaging.

Figure 2. RNA replication for (+)ssRNA viruses and PS-mediated packaging. (A) Insertion of turnip crinkle virus (TCV) PS into tomato bushy stunt virus (TBSV) gRNA is sufficient for TCV capsid protein to package the TBSV RNA in protoplast and in the presence of the wild-type TCV gRNA. (B) In viruses such as Togaviruses, the sgRNA and the (−)ssRNA, which are in excess with respect to the gRNA, are not packaged. This is most commonly attributed to presence of a PS. For example, insertion of the Venezuelan equine encephalitis PS in its sgRNA is sufficient to package this RNA molecule. ORF refers to open reading frame.

Figure 3. The role of RNA replication during RNA packaging. (A) Most RNA-dependent RNA polymerases (RdRp) of (+)ssRNA viruses interact with organelle membranes, creating viral assembly sites or replication factories. Sometimes these sites are very close to the ER. (B) There are viruses in which selective packaging seems to depend only on the ability of an RNA to be replicated by the RdRp (e.g., poliovirus). There is evidence that the ability of an RNA to act as a template for translation contributes to packaging selectivity (e.g., flock house virus). (C) In some other cases, the contribution of RNA replication is not sufficient, and the presence of a PS (red element with an *) is required for selective packaging. For example, in alphaviruses the presence of this signal is key to package only the genomic (blue with the PS in red with an *) but not the subgenomic RNA (orange).