Influence of RNA on icosahedral virus particle structure

Viruses are extremely successful pathogens that infect organisms of every type, including plants, animals (including humans), fungi, and bacteria of all types. They are essentially intracellular parasites that introduce their genetic material into host cells and subvert the normal cellular functions to make more copies of themselves. They are normally present in the environment in the form of virus particles in which the viral nucleic acid, either DNA or RNA, is surrounded by a shell made up of multiple copies of one or more type of protein subunit encoded by the virus genome. In some cases (enveloped viruses) they are further surrounded by a membrane of host origin. The purpose of the protein shell is to protect the delicate genetic information from the, often harsh, environment outside the cell and to enable the virus to successfully spread to other hosts. This often involves uptake and transmission by vectors, such as insects or fungi, and the nucleic acid must be able to survive this process.

Once inside a susceptible host, the virus makes more copies of itself by undergoing a "replication cycle" that includes several stages: uncoating of the particles to release the viral genome, expression of viral genes, replication of the viral nucleic acid and the encapsidation of the newly synthesised nucleic acid into particles that are then released to infect further hosts. This is all a carefully choreographed process, with replication and encapsidation of the viral genome usually closely linked. The specific encapsidation of viral, as opposed to host, nucleic acid into infectious particles is a vital step in the replication cycle of viruses. The process must result in the formation of virus particles that protect the labile genetic material effectively. This has led to the identification of defined RNA sequence elements or "packaging signals" in RNA viruses. Such packaging signals have been envisaged as labelling the viral RNA so that it is effectively "barcoded" in such a way that it is specifically recognised from a mixture of cellular molecules by the coat protein subunits, thereby conferring encapsidation selectivity. However, our recent work has shown that selectivity of packaging is determined by replication of an RNA molecule within infected cells, with synthesis of the coat protein being tightly coupled to RNA replication. In such "replication factories" the viral RNA genome is not in competition with other, non-replicating RNAs, suggesting that "barcoding" may not be required selective packaging. Rather, they may be present to ensure that the incorporation of RNA into particles proceeds efficiently to produce fully infectious virions. We have also obtained evidence that the length of replicating RNA may control the architecture of the resulting particles.
To determine the role of potential packaging signals in the context of a replicating RNA, we will use a vector, pEff, based on the plant virus, potato virus X (PVX), that simultaneously produces replicating RNA and the coat proteins within plants. This closely mimics the situation that occurs in vivo during a viral infection. We have recently shown that the replicating RNA from pEff can be encapsidated by the coat protein from different, unrelated viruses. Thus, pEff is an ideal system for examining the role of packaging signals in the context of viral replication. The effect of elimination, duplication or mutation of the packaging signals on the assembly and morphology of the particles will be examined to obtain a more complete understanding of the mechanism of virus assembly. These studies will not only lead to a greater understanding of a critical stage of the viral replication cycle but will also enable specific RNAs to be deliberately packaged in capsids of defined architecture for use in bionanotechnology and the creation of novel RNA delivery systems.

The process by which viral RNA is specifically encapsidated within particles of isometric viruses is still a subject of debate. In vitro studies have indicated that selectivity may be conferred by the presence of defined sequences (packaging signals) on the RNA. The presence of such signals would enable viral RNA to be distinguished from host RNA during infection and ensure that the RNA is folded in a manner compatible with efficient capsid formation. However, we have recently obtained evidence that the coupling of RNA replication and coat protein synthesis within "replication factories" in infected cells obviates the need for a selection mechanism, since only the replicating RNA is able to interact with the coat protein.

This project will build on highly successful previous work at JIC on the production of virus-like particles (VLPs) in plants using transient expression. In the absence of a replicating RNA encapsidate VLPs of isometric viruses contain either undetectable levels of RNA or a heterogeneous mixture of RNAs of host origin. We propose using a vector based on a potato virus X (PVX) replicon to examine the role of packaging signals on RNA which is replicating. This vector enables both replicating RNA and coat protein to be produced within the same "factory" within a cell, closely mimicking the situation in vivo. Using this system, we will investigate the effect of eliminating, duplicating or mutating potential packaging signals on the efficiency of formation of isometric particles, and the nature of the RNA that can be packaged. We will assess the structures of any unusual or aberrant particles that may form using electron microscopy and image reconstruction to understand the influence of the RNA length and sequence incorporated on the architecture of virus particles. The ability to encapsidate and stabilise RNA within a variety of viral structural will have implications for bionanotechnology, particularly in regard to RNA delivery to cells.