Manipulation of the invertebrate host cell machinery by dicistroviruses

The dicistroviridae family of viruses is a relatively new family of viruses that primarily infects insects. Recently, the Taura syndrome virus (TSV) was sequenced and was classified as the first member of dicistroviridae to infect a non-insect host. Specifically, TSV primarily infects the Pacific white shrimp though a number of other shrimp species are experimentally susceptible to the virus. Other viruses in the dicistroviridae family have been shown to contain internal ribosome entry sites (IRESs) capable of initiating translation in the absence of initiation factors. Initial studies were undertaken to characterize the TSV IRES and determine if it had a similar function to the IRESs found in the other, insect-infecting viruses of dicistroviridae. Analysis of the TSV IRES suggested the IRES was weaker in directing translation than the well characterized IRES found in the insect infecting dicistrovirus, cricket paralysis virus (CrPV). However, further investigation indicated that the TSV IRES was capable of binding and positioning assembled 80S ribosomes in the absence of initiation factors. These assembled ribosomes were positioned to initiate translation at a non-AUG codon and addition of tRNA specific for that codon induced ribosome translocation, suggesting it was the authentic translation start site. Furthermore, the TSV IRES was able to direct the synthesis of polyproteins in the absence of initiation factors in an in vitro reconstituted translation system. These experiments demonstrated that the TSV IRES functions in a manner similar to its insect infecting relatives and it was able to do so using the translational machinery from a variety of species, suggesting that the dicistrovirus IRESs may be manipulating ribosomes through an evolutionarily conserved mechanism. Another aspect of this research was to examine the host cell response to an infection from dicistroviruses. Using CrPV, we performed preliminary microarray experiments to investigate the steady-state mRNA levels of Drosophila SL2 cells 6 hours post infection. From these preliminary microarrays, we found a number of Drosophila heat shock genes were upregulated late in infection. This upregulation was found to be a general stress response of the cells and not specific to CrPV infection. However, infected SL2 cells that were undergoing heat stress were found to be unable to properly assemble virus and were also unable to produce small heat shock proteins. These data suggest that there is an interplay between CrPV infection and the heat shock pathway and between CrPV infection and the translational machinery. In summary, multiple approaches were taken to understand not only the mechanism of dicistrovirus IRESs, but also to understand the Drosophila innate immune response to infection by these viruses. Taken together, these experiments begin to develop a better understanding of the innate immune response to viral infection and how viruses are able to overcome these responses.