Identification and Characterization of the Red Clover Necrotic Mosaic Virus Origin of Assembly

Abstract

Red clover necrotic mosaic virus (RCNMV) is a single-stranded positive-sense RNA plant virus of the Dianthovirus genus, family Tombusviridae. Each RCNMV virion contains 180 subunits of the 37 kDa capsid protein (CP) forming a non-enveloped, isometric particle of T=3 quasi symmetry, 30-35 nm in diameter. The RCNMV genome consists of two RNAs, RNA-1 and RNA-2. RNA-1 codes for three proteins: i) p88, the RNA-dependent RNA polymerase, ii) p27, the replicase related protein and iii) the CP. RNA-2 is monocistronic and codes for the movement protein (MP).

Currently, the RNA complement within RCNMV virions has not been determined. Density gradient centrifugation data has suggested the probability of a single type of particle, whereas the viral RNA profile from virions suggests otherwise. My thesis research has determined the RNA content within RCNMV virions after exposure to either heat or UV-irradiation. Both treatments result in the formation of a stable RNA-1: RNA-2 heterodimer. This leads to the conclusion that RCNMV virions co-package RNA-1 and RNA-2. Upon either treatment, RNA-2 multimers are also observed, suggesting the presence of particles packaging RNA-2 exclusively. These observations suggest that the RCNMV virion population consists of two distinct types of particles having similar densities.

Based on the above RNA complement findings, I proceeded to delineate the specific viral sequences that determine the RNA content of an RCNMV virion. During virion assembly, the RCNMV CP must be able to distinguish and package both RNA-1 and RNA-2 while excluding heterologous host RNAs present in the cell. Viral CP subunits recognize specific cognate genomic sequences and/or structures that are unique to each viral genome. These are designated as origin of assembly sequences or packaging signals. To elucidate the assembly mechanism of RCNMV, I searched for the presence of distinct packaging signals on each of the genomic RNAs. Various constructs of RNA-1 and RNA-2 were tested for their assembly efficiencies in vivo using a plant assay system. While it has been previously demonstrated that RNA-1: RNA-2 base pairing directs the synthesis of the CP subgenomic RNA (sgRNA) from RNA-1 via the trans-activating (TA) element on RNA-2, it was not determined whether this interaction had a role in assembly. I have found that RNA-1 does not have the ability to package by itself given sufficient amounts of CP. Also, the CP sgRNA is not encapsidated into RCNMV virions. RNA-2 appears to play the major role in directing RCNMV assembly. My results indicate that a 209 nt sequence within the MP open reading frame on RNA-2 (containing the TA element) directs RCNMV assembly. Deletion mutagenesis of RNA-2 to delimit the packaging signal proved that the 34-nucleotide TA element was the origin of assembly. As further proof, expression of the TA element from a Tomato bushy stunt virus vector directed the co-packaging of RCNMV RNA-1 into virions proving it to be essential for RCNMV assembly. Deletion mutagenesis also revealed that RCNMV has an RNA packaging size requirement for production of stable virions.

Based on all of the above observations, I propose the following model for the RCNMV packaging mechanism: the base pairing interaction of the TA element with RNA-1 initiates CP production and enables the formation of an RNA-1: RNA-2 heterodimer . This dimer formation allows the co-packaging of both RNAs into a single virion via the packaging signal on RNA-2. The discreet packaging signal on RNA-2 also allows the formation of some virions containing solely RNA-2.