Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 3 uppsatser.

Thesis (doctoral)--Freie Universität, Berlin.

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and movement in host plants. The positive-sense single-stranded genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions. Previous studies showed that SPMV CP has multiple functions during infection including encapsidation, symptom exacerbation, inhibiting the accumulation of SPMV DIs, and facilitating systemic movement. This dissertation confirms and extends the results of our previous reports with new biological and biochemical evidence. For example, the dosage effect of SPMV CP on symptom severity supports its function as a pathogenicity factor. Biological assays also demonstrate compensatory effects of SPMV CP on virus mutants defective in systemic movement. In addition, it is shown for the first time that SPMV CP is involved in cellto- cell movement of SPMV RNA and associated with the cell wall and membranes, a signature property of plant virus movement proteins. However, SPMV CP in the cytosol exists exclusively as virions and is dispensable for symptom exacerbation. SPMV CP contains a distinctive N-terminal arginine-rich motif (N-ARM), which is required for the in vitro binding of SPMV and PMV genomic RNAs by SPMV CP. Mutations of this region impair all known functions of SPMV CP. Interestingly, manipulation of the C-terminus of SPMV CP resulted in the same phenotypes as alterations in the N-ARM except that this does not affect the RNA binding activity of SPMV CP. Biological experiments demonstrate that virions are not required for the properties of SPMV CP to facilitate local and systemic movement and inhibit the accumulation of SPMV DIs, suggesting that SPMV CP and RNA form alternative complexes for these purposes. This dissertation study reveals the nucleolar localization of SPMV CP and its interaction with PMV CP in the form of virions. The identification of distinct functional domains of SPMV CP and its complex subcellular localization profile resulted in the proposal of a tentative model on how the functions of SPMV CP are coordinated for a robust infection. This dissertation provides a foundation for further understanding of the complex interactions among host plants, helper viruses, and satellites.

Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (p. 157-170). Also available online through Digital Dissertations.

Thesis (Ph. D.)--Cornell University, August, 2007. / Vita. Includes bibliographical references (leaves 152-175).

Abstract Taiwan marine fish suffers the epidemic infection of iridovirus. In this research, infected fish of Epinephelus. lanceolatus was diagnozed by polymerase chain reaction. Transmission electron microscopy showed the morphology of viral particles from tissue lysate. The nucleotide sequence of major capsid protein (MCP) was compared to OSGIV, thereafter the novel virus from Epinephelus lanceolatus is named OSGIV-like iridovirus. By cloning expression of the MCP of TGIV and OSGIV-like, the MCP of 51 kDa was insoluable although minor virus-like substance was observed in TEM. Triton X-100 of 2.5% improved the solubility of protein to 30.95 £gg/£gl. Chloroform treatment enhanced the antibody-detection for the MCP of virus in the tissue. We inference that iridovirus MCP is associated with the envelope because of its hydrophobicity.

Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references (leaves 132-147).

Following human immunodeficiency virus (HIV-1) entry into the host cell, the viral capsid gradually disassembles in a process called uncoating. A proper rate of uncoating is important for reverse transcription of the HIV-1 genome. Host restriction factors such as TRIM5alpha; and TRIMCyp bind retroviral capsids and cause premature disassembly, leading to blocks in reverse transcription. Other host factors, such as cyclophilin A, stabilize the HIV-1 capsid and are required for efficient infection in some cell types. To identify additional cellular factors that alter retroviral core uncoating, we developed a novel in vitro assay of HIV-1 capsid stability. Using this assay, we have shown that a factor in the cytoplasm of cells from multiple vertebrate species slows the spontaneous disassembly of HIV-1 capsid-nucleocapsid (CA-NC) complexes in vitro. We identified the PDZ-Domain-containing protein 8 (PDZD8) as a critical component of the capsid-stabilizing activity in the cytoplasmic extracts. PDZD8 has been previously reported to bind the HIV-1 Gag polyprotein and to make a positive contribution to the efficiency of HIV-1 infection. PDZD8 knockdown accelerated the disassembly of HIV-1 capsids in infected cells, resulting in decreased reverse transcription. The PDZD8 coiled-coil domain is sufficient for HIV-1 capsid binding, but other parts of the protein, including the PDZ domain, are apparently required for stabilizing the capsid and supporting HIV-1 infection. In summary, PDZD8 interacts with and stabilizes the HIV-1 capsid and thus represents a potentially targetable host cofactor for HIV-1 infection.

Parvoviruses cause dreadful enteritis in waterfowls and lead to tremendous financial losses. This study aims at developing effective way to prevent waterfowl parvoviral infection. Duck parvoviruses (DPVs) and goose parvoviruses (GPVs) were isolated from organs of infected waterfowls. The presence of virus in the specimens was identified using polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis. To reveal the genetic variation of viral capsid proteins (VPs), full length VPs gene were amplified and sequenced. The sequence data indicated the sequences diverge 4.1 to 4.4% among viral strains isolated during 1990 to 1999. The variant amino acids cluster in the common regions of VP3 at residues 203-266 and 482-534, which overlaps with the regions proposed to expose on the outer surfaces of parvoviral particles. These data implying that selective pressure from host immune system might play a part. The nucleotide sequences of VPs also reveal that DPV and GPV share 77 % similarity at the DNA, and 84.6% at the protein level. The most variable regions reside in the N-terminal of VP2 before the initiation codon of VP3 with 35% (19/54) amino acids divergence. This study also reveals the presence of conserved strain-specific residues in VPs and these residues seldom vary among different isolates of the same virus, suggesting that they might be important in maintaining viral structure or host specificity which worth further investigation. To investigate the antigenicity of VPs, the GPV genomic DNA encoding common region of VPs was fused in frame with glutathione S-transferase (GST) gene for the expression of GST-GPV (248-516) fusion protein in bacterial cells. Purified fusion protein was used as immunogen for the generation of rabbit anti-GPV (248-516) antiserum. The potential diagnostic usage was confirmed by the fact that this antiserum was able to differentiate between viral infected and uninfected primary embryonic fibroblast cells by immunocytochemical analysis. In addition, VPs in purified DPV and GPV virions were analyzed by Western blotting. This antiserum detected two prominent proteins bands with the molecule weight of 80 and 70 kilodaltons, which correspond to the sizes of VP1 and VP2 reported in the literature. The fact that VP1 of DPV reacts weakly with this antiserum suggests the existence of antigenic discrepancy between DPV and GPV. For the purpose of developing subunit vaccine for the control of Derzy's disease, recombinant full length VPs were expressed using both prokaryotic, GST and histidine-tagged fusion proteins, and eukaryotic, baculovirus and mammalian vero cell, expression systems. After large- scale production and purification, same amount of 4 recombinant VPs were individually used to immunize 1-week-old geese. The antibodies induced after immunization were then evaluated by enzyme-linked immunosorbent assay (ELISA). All four recombinant proteins stimulate approximately 7 to 8 folds increases of ELISA antibodies titers, and together with preliminary data of safety tests suggest a potential usage as subunit vaccine for the control of parvoviral infection.