From Replication to Assembly, a Thesis on the Fate of the Alphavirus RNA Genome During Infection

Adriano Mendes, Purdue University


Positive strand RNA viruses employ a similar strategy with regards to their replication cycle. Generally, these viruses insert their RNA genome into a cell, followed by the replication of the genome and then the assembly of a particle. This pathway makes the genomic RNA of the virus, a substrate for at least three different reactions within an infected cell. These being translation, RNA synthesis/ RNA replication and assembly of a virion. This thesis will outline studies, which aimed at understanding how a model RNA virus controls when and where each process is carried out. The alphaviruses are a group of positive sense, single stranded, RNA viruses of 65–70nm in diameter. These viruses encapsidate a single 11kb RNA genome, within a nucleocapsid particle that is itself surrounded by a lipid envelope. Both the nucleocapsid and the envelope maintain a T=4 symmetry. The alphavirus life cycle has been well characterised with regards to the cleavage of the replication proteins, that defines substrate recognition, during RNA replication. In addition, multiple structures of alphavirus particles have been published, which have allowed for an identification of the morphology of a particle. However, the intracellular events that guide the RNA genome between the processes of translation, replication and assembly are not well understood. More specifically, how the RNA is recognized for each of these processes has been neglected. In Chapter 2 of this thesis, a study was initiated to define how the conserved sequence elements (CSE), found on the genomic RNA of all alphaviruses, govern the fate of the RNA during infection. A system was developed in which the CSEs could be manipulated without effecting the translation of the proteins required for infection. This system is referred to as a replicon-helper system as it employs a replicon RNA molecule, which is able to replicate but not make particles, and a helper RNA molecule, which is able to replicate only in the presence of the replicon and make the structural proteins required for a virion. It was concluded that alphaviruses utilise their CSEs in a specific order. In Chapter 3, a set of experiments was carried out with the basic premise of co-transfecting replicating, full length alphaviruses and determining which was packaged into particles with greater efficiency. Unfortunately, the experiments could not be carried out as predicted, owing to a limitation in how efficiently two genomes could be transfected into a single set of cells. However, despite this, it was observed consistently that a full length genomic RNA was able to out-compete a replicon genome for packaging into particles. This observation was to some degree, dependent on the packaging signal, as its mutation, resulted in more replicon particles in the next generation. However, the effect of the packaging signal was transient, since full length virus with a mutant packaging signal excluded the replicon after 2 subsequent generations. In the final data chapter, the specificity with which genomic RNA was incorporated into a particle, was analysed. In this study, we partnered with Dr Andrew Routh at the University of Texas Medical Branch, to do a deep sequencing analysis of purified SINV particles from two sets of tissue cultured cells. To this end, SINV was purified from BHK cells and C6/36 cells at different time points, which approximated to peak virus release kinetics in each cell line. The results showed that the efficiency of packaging SINV genomic RNA was extraordinarily high, when particles were purified from BHK cells. SINV derived from C6/36 cells on the other hand, showed higher specificity early, and became progressively worse as the infection proceeded, in favour of packaging more host cell RNAs. It was concluded that this effect correlated to the kinetics of infection within the C6/36 cell line, which was able to maintain SINV infection without the cells dying. Host cell RNA packaging was found to have a minor but discernible effect on the thermal stability and specific infectivity of the C6/36 SINV particles. These observations are in line with a multitude of differences that have been previously observed during the infection of model mammalian (BHK) vs. insect (C6/36) systems. In addition, the observation that host cell RNA could be consistently detected in SINV particles, suggested that the mechanism of RNA packaging is not necessarily specific for the genomic RNA of the virus. Therefore, the utilisation of a packaging signal as well as a specific location and coordinated kinetics are all under very tight control in the mammalian system, in order to achieve the high degree of specificity that was observed. (Abstract shortened by ProQuest.)




Kuhn, Purdue University.

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