Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2006. / Includes facsimiles of articles previously published (leaves 37-79). Includes bibliographical references.

The purpose of this investigation was to examine the structure of the murine cytomegalovirus (MCMV) genome and to study its expression during productive and non-productive infections caused by the virus. The kinetic complexity of MCMV DNA was not less than its molecular weight implying the absence of major reiterations. The restriction endonuclease EcoR^ cleaved this molecule into twenty-five fragments, which were present in the digest in equimolar amounts and ranged in molecular weights from 20 to 1 million. The sum of the molecular weights of the fragments was 136 million. The genomes of the 'K 181' and 'Smith' strains of MCMV appeared to share more than 99 percent of their sequences, although the DNAs exhibited slightly different fragmentation patterns when treated with EcoR^ and Hind III endonucleases. Control was exerted on the transcription of the MCMV genome at temporal, quantitative, and processing levels. During productive infections, approximately 25 percent of the genome was represented as stable transcripts in the cell at 6 hours post infection, i.e., before the onset of viral DNA synthesis, whereas RNA transcribed from 35 to 40 percent of the DNA was present in the cells in the later stages of infection. RNA sequences corresponding to 6 h (early) transcripts would be detected in the cell throughout the infectious cycle. Both 'early' and 'late' RNA comprised two RNA classes differing about 7 to 10 fold in concentration. Viral DNA synthesis in the host cell was required for the expression of 'late' genes since in the presence of inhibitors of protein and DNA synthesis only 'early' transcription occurred. Control was also exerted on the transport of transcripts from the nucleus to the cytoplasm of infected cells. Although RNA extracted from the nuclei of infected cells arose from 25 (early) and 35 (late) percent of the viral genome, transcripts from only 11 (early) and 15 (late) percent of the DNA were detected in the cytoplasm. Cells of mouse origin (3T3.cells), arrested in the G^ phase of the cell cycle, retained the viral genome in a non-replicating state, but could be induced to enter the lytic cycle by serum activation. Transcripts from 19 percent of the genome were observed in G^ arrested, MCMV-infected cells. Viral RNA in these cells comprised only one abundance class, which was similar to the scarce class in 'early' RNA from infected exponentially growing cells. Some evidence was also obtained for the transmission of latent MCMV genomes from mother to progeny. Cells cultured from embryos of infected mice did not normally produce infectious virus. However, the presence of the virus, at least in some of these cells, could be demonstrated by immunofluorescence, and by in-situ hybridization, using iodinated MCMV DNA as probe. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The interaction between Murine Cytomegalovirus (MCMV) and the cell cycle has been investigated in synchronized murine cells. Based on the following evidence it was concluded that MCMV replication depends upon the host S phase: (1) the normal latent period of viral growth in exponentially growing 3T3 cells (12 h). was protracted until the host S phase (ca. 20 to 24 h) in synchronized cells infected in early G—l; (2) G-l arrested 3T3 cells failed to support viral replication; (3) entry of the virus was equally efficient in G-l, S and exponential cells; (4) in exponential 3T3 cells viral DNA synthesis began at 10 h post infection, and in synchronized cells it began approximately 16 to 18 h after infection, or early S phase. Therefore, the replication of viral DNA requires host S phase events. Another herpesvirus, Herpes Simplex Virus type-1 (HSV-1) replicated independently of S phase. However, a mutant of HSV-1, deficient in its ability to induce thymidine kinase, demonstrated a dependency upon S phase similar to MCMV. These data indicate a key role of thymidine kinase in the ability of HSV-1 to replicate outside of S phase. However, MCMV induced neither a cellular nor a viral thymidine kinase, and thymidine kinase was not essential for normal viral replication. When G-l arrested 3T3 cells were infected with MCMV, viral DNA synthesis did not initiate and the lytic cycle was reversibly blocked. The non-replicating viral genome remained viable in G-l cells and could be activated at any time by stimulating the cells to enter S phase. The G-l non-permissive system was studied to help ascertain the cell cycle requirements of MCMV. Specifically, two approaches were pursued. In vitro endogenous MCMV DNA synthesis was first studied in G-l, S phase, and exponential 3T3 cells. Under the appropriate conditions, nuclei from infected cells synthesized viral DNA when they had the capacity to dp so in vivo. Nuclei from G—1 phase cells synthesized cell DNA only and not viral DNA. Infected G-l and S phase cells contained a new DNA polymerase which was distinguished from the host enzyme by the high salt requirement for maximal activity. The putative viral DNA polymerase was inhibited by antiserum prepared against infected cell proteins. Therefore, the novel DNA polymerase present in infected G-l and S phase cells was a viral gene product. The second approach involved a comparison of viral transcription in permissive and non-permissive 3T3 cells. The kinetics of hybridization in solution were analyzed by a computer program which evaluated the number of viral RNA classes and the fraction of the viral genome coding for each class. This study revealed the following: (1) in permissive cells by 6 h post infection (early, i.e. before viral DNA synthesis), two classes of viral transcripts were detected, differing by 7 fold in concentration. The abundant class was transcribed from approximately 7% of the viral DNA and the scarce class from approximately 20%. (2) in permissive cells at 24 h post infection (late), abundant and scarce classes (differing by 6 fold in concentration) were transcribed from approximately 10 and 33% of the viral DNA respectively. (3) in non-permissive cells at 6 h, only one class of RNA was present, representing approximately 15% of the viral DNA. (4) in non-permissive cells at 24 h post infection, a single RNA class was observed which was transcribed from approximately 24% of the viral DNA. Summation hybridization experiments indicated that in non-permissive (G-l) cells, only those regions of the DNA which code for early RNA are transcribed. A model has been proposed to describe cell cycle dependent replication of MCMV. It is concluded that MCMV does not replicate in G—l cells due to the absence of specific S phase 'helper-functions' which are required either for the initiation of viral DNA synthesis directly, or for the transcription of viral DNA sequences. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

The family Bunyaviridae is the largest known taxonomic group of arboviruses. Four of the five genera possess members which are responsible for serious human and livestock disease. The worldwide distribution of these viruses justify studies which will allow understanding of the replication and transcription cycles within permissive cells. The bunyaviruses have been shown to possess a tripartite single strand RNA genome of negative polarity. Replication is confined to the cytoplasm and the virion envelope is acquired when the genome ribonucleoproteins bud into the golgi. Virus release is presumed to be through exocytosis and ultimately cell lysis. The messenger RNA species of all five genera do not possess a poly-A tail of sufficient length to bind to an oligo(dT) cellulose column. This has made separation of viral transcripts from replicating RNAs difficult. In an effort to achieve this separation, infected cell extracts were centrifuged over 20-40% CsCl gradients which permitted replicating RNA structures to band at a density of 1.32 while cellular and viral mRNAs pellet. Recovery of viral transcripts from the CsCl pelleted RNA required synthesis of a cDNA copy of the virus genome to use as a probe. This was done by an unusual method which employs both genome and antigenomic RNA as templates for reverse transcriptase in a first strand synthesis reaction. Recombinant viral clones were then used in a hybrid selection scheme to recover virus mRNA from pelleted material. After recovery, the messages were visualized on acid urea agarose gels pH 3.5, or used to program an in vitro translation reaction. Using these methods, it was established that each genome segment codes for a single messenger RNA which is most likely capped, and that for at least the mid sized segment, proteins with molecular weights which exceed the coding capacity of the genome are translated from the single message.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Viruses infecting the Archaea exhibit a tremendous amount of morphological and genetic diversity. This is especially true for crenarchaeal viruses from the family Fuselloviridae, which possess spindle-shaped capsids and genomes that harbor a great number of uncharacterized genes. The functions of these unidentified gene products are of interest as they have the potential to provide valuable insights into the fusellovirus infection cycle and archaeal viruses in general. In an effort to better characterize the genetic requirements of the Fuselloviridae, we have performed genetic and biochemical experiments using the best studied fusellovirus, Sulfolobus spindle-shaped virus 1 (SSV1). A comprehensive genetic analysis of SSV1 was conducted using long inverse PCR and transposon mutagenesis. The results of this work illustrate that SSV1 is highly tolerant of mutagenesis. A robust protocol for the purification of recombinant VP2 protein from E. coli was developed and should be useful for future studies aimed at characterizing the biochemical and structural characteristics of this SSV1 structural protein. Finally, the first insights into a fusellovirus infection are presented and provide the framework for a detailed characterization of the fusellovirus infection cycle. The results and significance of this work are presented in the chapters that follow.

Geminiviruses ( family Geminiviridae ) have circular single - stranded genomes encapsidated in twinned quasi - isometric particles and are responsible for major crop losses worldwide. The largest genus, Begomovirus, comprises viruses transmitted by the whitefly Bemisia tabaci. Most begomoviruses have bipartite genomes, termed DNA A and DNA B. The DNA A component encodes proteins required for viral DNA replication and encapsidation whereas the DNA B encodes two proteins that are essential for systemic movement. A small number of begomoviruses have a monopartite DNA genome that resembles the DNA A of bipartite begomoviruses. This DNA carries all gene functions for replication and pathogenesis. Specific small circular single - stranded DNA satellites containing a single open reading frame ( ORF ), termed DNA β, have recently been found in association with certain monopartite begomovirus infections. They comprise about 1350 nucleotides and require a helper begomovirus for replication and encapsidation. DNA β contributes to the production of symptoms and enhanced helper virus accumulation in certain hosts. This study examines the role of DNA β satellite in viral pathogenicity and movement in the host plant. Infectivity analysis of Tomato leaf curl virus and DNA β having mutation in the C1 and V1 ORF indicated that the complementary - sense ORF, βC1, is responsible for inducing disease symptoms in Nicotiana tabacum. An ORF present on the plus strand, βV1, appeared to have no role in pathogenesis. Tobacco plants transformed with the βC1 ORF under the control of the Cauliflower mosaic virus 35S promoter, or with a dimeric DNA β exhibited severe disease - like phenotypes, while plants transformed with a mutated version of βC1 appeared normal. Northern blot analysis of RNA from the transgenic plants using strand - specific probes identified a single complementary - sense transcript. The transcript carried the full βC1 ORF encoding a 118 amino acids product. It mapped to the DNA β nucleotide ( nt ) position 186 - 563 and contained a polyadenylation signal 18 nt upstream of the stop codon. A TATA box was located 43 nt upstream of the start codon. These results indicate that βC1 protein is responsible for DNA β induced disease symptoms. Tomato leaf curl New Delhi virus ( ToLCNDV ) is a bipartite begomovirus in which both DNA A and DNA B are required for systemic infection. Inoculation of tomato plants with ToLCNDV DNA A alone induced local but not systemic infection whereas co - inoculation with DNA A and the DNA β resulted in systemic infection. The presence of both DNA A and the DNA β in systemically infected tissues and the absence of DNA B was confirmed by probe hybridization. DNA β containing a disrupted βC1 ORF did not mobilize the DNA A for systemic infection. Co - inoculation of plants with DNA A and a construct of βC1 ORF, under the control of the Cauliflower mosaic virus 35S promoter, resulted in the systemic movement of the DNA A. βC1 fused to GFP accumulated around and inside the nucleus, at the periphery of tobacco and onion epidermis cells and co - localized with the endoplasmic reticulum. This distribution would be consistent with βC1 mediating intra cellular transport from the nucleus to the plasma membrane. These results showed that the βC1 protein can replace the functions of DNA B in allowing the systemic movement of a bipartite geminivirus DNA A. / Thesis (Ph.D.)--School of Agriculture, Food and Wine, 2006.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.