Diss. (sammanfattning) Stockholm : Univ., 2004. / Härtill 4 uppsatser.

Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.

The Tetraviridae are a family of ss (+) RNA viruses that specifically infect lepidopteran insects. Their icosahedral capsids are non-enveloped and approximately 40 nm in diameter with T=4 quasi-equivalent symmetry. The omegatetraviruses, which are structurally the best characterised in the family, include Helicoverpa armigera stunt virus (HaSV) and Nudaurelia capensis omega virus (NwV). The omegatetravirus procapsid is composed of 240 identical copies of the capsid precursor proteins, which undergo autoproteolytic cleavage at its carboxyl-terminus generating the mature capsid protein (b) and γ-peptide. This process occurs in vitro following a shift from pH 7.6 to pH 6.0. The viral capsid encapsidates two ss genomic RNAs: The larger RNA1 encodes the viral replicase as well as three small ORFs while RNA2 encodes the capsid precursor protein together with an overlapping ORF designated P17. While a wealth of structural data pertaining to the assembly and maturation of omegatetraviruses is available, little is known about how this relates to their lifecycle. The principle aim of the research described in this thesis was to use an experimental system developed in the yeast, Saccharomyces cerevisiae, to investigate the assembly of HaSV and NwV virus-like particles (VLPs) in terms of maturation and encapsidation of viral RNAs, in vivo. The yeast expression system used two promoter systems for expression of capsid precursor protein: in the first, a hybrid promoter (PGADH) was used for high-level expression, while the second, PGAL1, produced substantially lower levels of the virus capsid protein precursors. An increase in the level of HaSV capsid protein precursor (p71) via the PGADH promoter resulted in a dramatic increase in VLP assembly as compared with the PGAL system. A protein equivalent to the mature capsid protein (p64) appeared at later time intervals following induction of transcription. Transmission electron microscopic studies showed that p64 correlated with the presence of mature VLPs as opposed to procapsids in cells containing p71. This confirmed that the presence of p64 denoted maturation of VLPs in vivo. Further investigation indicated that maturation correlated with cell aging and the onset of apoptosis. It was shown that induction of apoptosis resulted in VLP maturation while inhibition of apoptosis prevented maturation. These results suggested that the process of apoptosis might be the trigger for maturation of virus procapsids in their host cells. The increase in the efficiency of VLP assembly observed in the high-level expression system was proposed to be due to an increase in the cellular concentrations of viral RNA. To test this hypothesis, HaSV P71 was co-expressed with either P71 mRNA or full length RNA2. An increase in the solubility of p71 was observed in cells expressing increased levels of both RNAs, but there was no increase in the efficiency of VLP assembly. Northern analysis of encapsidated RNAs revealed that there was no selective encapsidation of either P71 mRNA or viral RNA2. This data indicated that the increase in viral RNA was not the reason for increased efficiency of VLP assembly, but most likely resulted from higher concentrations of p71 itself. It was decided to determine whether a highly efficient nodavirus replication system developed in yeast for heterologous production of proteins, could be used as a method for expressing the capsid protein precursor. The aim of using this system was to determine if VLPs assembled in a replication system specifically encapsidated viral RNA. Transcripts encoding the NwV capsid protein precursor (p70) were generated in yeast cells by replication of a hybrid RNA template by the Nodamura virus (NoV) replicase. Western analysis confirmed the presence of p70 as well as a protein of 62 kDa corresponding to the mature NwV capsid protein. Northern analysis of purified VLPs showed that NoV RNA1 and RNA3 were encapsidated, but no RNA2 was detected. Taken together, the data lead to the conclusion that specific encapsidation of tetraviral RNAs required more than close proximity of the viral RNAs and assembling virus-like particles. Encapsidation specificity in the omegatetraviruses may require additional viral proteins such as p17 during encapsidation or specific viral RNA encapsidation was replication-dependent. Replication-dependent assembly has been shown in the nodaviruses.