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Potato leafroll luteovirus is an aphid-transmissible virus which has isometric particles and is confined to the phloem tissue of infected plants. Its multiplication was investigated by using plant protoplasts as a model system. In protoplasts, net accumulation of PLRV ceased at approximately 48 his post-inoculation. Virus-specific products were detectable 15 hrs or more post-inoculation and remained detectable at approximately 100 hrs postinoculation. The amount of PLRV accumulated depended on the conditions in which protoplasts were incubated. Incubation at 25°C rather than 20°C and incubation in the dark for a period rather than continuous light resulted in more PLRV accumulation. RNA extracted from PLRV-infected protoplasts was identical on northern blots to that extracted from leaf tissue of PLRV-infected Maris Piper potato plants. Northern blots of RNA from other plants, some resistant, some susceptible to PLRV multiplication, were very similar. Resistant plants appeared to contain smaller quantities of subgenomic RNA. The genes at the 3'-end of the genome are expressed by translation of a subgenomic RNA. This was mapped to position 3376 on the PLRV genome and is therefore 2505 nucleotides long. The untranslated leader sequence of 212 nucleotides contains some putative promoter sequences although not in the same order as described for other viruses. A sequence of 8 nucleotides at the 5'-end of the genomic RNA was found to be repeated at the 5'-end of subgenomic RNA. The complement of this sequence may form part of an internal initiation site for the viral replicase complex in the minus strand RNA. The possibility of the untranslated leader sequence containing several promoters for both subgenomic RNA synthesis and ORF expression is discussed. Protoplast lysates contained a component that sedimented nearer the top of a sucrose gradient than virus particles. This contained subgenomic RNA and was detectable by ELISA but not by electron microscopy. It was not present in extracts of PLRV-infected plant tissue or in preparations of purified virus particles and may therefore be an unstable structure possibly - playing a role in particle assembly.

The La Crosse (LAC) virus is a member of the California encephalitis group of bunyaviridae (Porterfield et al., 1975 and 1976). It is one of an envelope virus and this virus under acidic conditions (below pH 6.3) has been demonstrated to result in cell-to-cell fusion (Gonzalez-Scarano, 1984). The LAC virus is also capable of forming virus-to-virus fusion particles. The focus of this thesis is the analysis of the structure and function of this virus-to-virus fusion by cryo-electron microscopy at different pH and temperatures. The results of this study provide the basis for further study of the structure and function of the LAC virus. The virus-to-virus fusion event shows a dependence on both pH and temperature. The frequency of the fusion event increases with an elevation in temperature (in the range 4 to 37°C) and with a decrease in pH from 7.3 to 5.4. The process of virus-to-virus fusion gives rise to the formation budding to a chain of fused viruses.

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.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy