Influence of RNA on icosahedral virus particle structure

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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.