Copy of author's previously published article on back end-paper. / Bibliography: leaves 105-158. / xii, 160, [58] leaves, [35] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the early events in the synthesis of HIV RNA and integration if viral DNA using a cell-to-cell transmission of infection model. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology & Immunology, 1998

Beet yellows virus (BYV) is a filamentous, positive-strand RNA virus that belongs to the family Closteroviridae. BYV particles encapsidate a 15.5 kb RNA and posses complex polar architecture. A long virion body is formed by the major capsid protein(CP), whereas the minor capsid protein (CPm) assembles a short tail that encapsidates the 5'-terminal region of BYV RNA. In addition to proteins required for viral RNA replication and encapsidation, BYV encodes four proteins whose role in the virus life cycle was unknown. These proteins include a small, 6-kDa, hydrophobic protein (p6), a homolog of the cellular 70-kDa heat shock proteins (Hsp7Oh), a 64-kDa protein (p64), and a 20-kDa protein (p20). It was found recently that Hsp7Oh, p64, and p20 are incorporated into BYV virions, and that Hsp7Oh is required for the virus movement from cell to cell. In this study, we characterized genetic requirements for BYV assembly and cell-to-cell movement, and determined relationships between these two processes. It was demonstrated that in addition to Hsp7Oh, p6, p64, CP, and CPm are each essential, but not sufficient for virus movement. These results indicated that five-component movement machinery of BYV is the most complex among plant viruses. Extensive mutational analysis of CP and CPm revealed strong correlation between abilities of BYV to assemble tailed virions and to move from cell to cell, suggesting that formation of functional virions is a prerequisite for virus translocation. We have found that CPm, Hsp7Oh, and p64 are necessary for the efficient virion tail formation. Assembly of the virion tails and bodies was shown to occur independent of each other and likely to involve two separate packaging signals within the genomic RNA. Our work demonstrated that BYV encodes one conventional movement protein, p6, whose only known function is to mediate virus movement. The other four movement associated proteins of BYV, CP, CPm, Hsp7Oh, and p64 are the virion components, each of which is required for assembly of the tailed, movement-competent virions. Based on these and other data, we propose that BYV and other closteroviruses evolved virion tails as a specialized device for the directional cell-to-cell movement of large RNA genomes. / Graduation date: 2005 / Best scan available.

Turnip yellow mosaic virus is a monopartite, plus sense RNA virus infecting the Cruciferae, and is a model system for the study of RNA virus replication. A cDNA clone (pTYMC) representing an infectious RNA genome of the European isolate of TYMV was constructed and used to assess the importance of virus genes in virus infectivity. Derivatives of pTYMC with alterations in open reading frame 69 (ORF- 69) were made. The mutations disrupted the expression of ORF-69 in vitro as predicted. Although the ORF-69 mutants were competent for replication in protoplasts, none of the mutants detectably infected turnip or Chinese cabbage plants, except where reversion mutations led to the restoration of an uninterrupted ORF-69. The data suggest a role for ORF-69 expression in the cell-to-cell movement of the virus. Mutant RNAs with a deletion or frameshift in the coat protein ORF infected protoplasts and plant leaves. No systemic infection symptoms were generated by these mutants, and no viral products were detected in young, expanding tissue of infected plants. When the coat protein deletion mutant and an ORF-69 mutant were co-inoculated onto plants, only a virus producing a coat protein of wild type size was detected in symptomatic, systemic tissue in these inoculations, emphasizing a requirement for the expression of native size coat protein for the systemic translocation of TYMV infection. The role of ORF-206 expression in TYMV replication was examined. Three classes of mutants were made in ORF-206: those affecting the synthesis of the 150 kDa protein, those affecting the synthesis of the 70 kDa protein, and those affecting the synthesis of both the 150 and the 70 kDa proteins. All ORF- 206 mutations eliminated RNA infectivity. Protoplast inoculations using mixtures of individual ORF-206 mutant RNAs and a helper genome demonstrated that co-replication of defective genomes could occur. Moreover, inoculations in which individual 150 kDa and 70 kDa protein mutant RNAs were combined showed that complementation between these two classes of mutants was possible. The data indicate that RNAs expressing wild type 150 kDa protein are favored replication substrates in mixed infections, and suggest that the 150 kDa protein functions preferentially in cis. / Graduation date: 1993

Bibliography: leaves 116-117.

Bibliography: leaves 116-117 / viii, 131, [82] leaves, [20] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1983

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease, which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1) of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several techniques were attempted to correct this mutation, but none were successful. Even though the second mutation could not be corrected, in vitro transcriptions were performed on three clones followed by subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA- mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and provided preliminary evidence that the mutated clones were still capable of systemic infection and viral movement. These results are still inconclusive, and several post-infection studies will have to be performed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will be investigated further and post-infection analysis performed to deduce whether the viral progeny are devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable of systemic infection in N. benthamiana plants were constructed in this study and have laid the foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.

Bibliography : leaves 92-99. / v, 99, [37] leaves, [14] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1992