MIAMI UNIVERSITY the Graduate School CERTIFICATE FOR
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MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation Of Jianli Xue Candidate for the Degree: Doctor of Philosophy _________________________________________ Xiao-Wen Cheng, Ph.D., Director _________________________________________ Mitchell F. Balish, Ph.D., Reader _________________________________________ Eileen K. Bridge, Ph.D., Reader _________________________________________ Gary R. Janssen, Ph.D., Reader _________________________________________ Michael Novak, Ph.D., Graduate School Representative ABSTRACT COMPARISON OF ASCOVIRUS AND BACULOVIRUS GENOMES AND THEIR REPLICATION AND GENE EXPRESSION STRATEGIES by Jianli Xue A new member of the newly discovered insect virus family Ascoviridae, Spodoptera frugiperda ascovirus-1d (SfAV-1d) was identified from South Carolina, USA. The genome size of SfAV-1d is estimated to be about 100 kb which makes SfAV-1d the smallest ascovirus genome so far. SfAV-1d is closely related to the previously reported SfAV-1a with 99% DNA sequence identity to SfAV-1a. A deletion of 14 kb was found in the SfAV-1d genome that corresponds to the inverted repeat region in SfAV-1a. Cloning and sequencing revealed that the deleted region is highly variable in the SfAV-1d genome with different lengths deleted in individual isolates. SfAV-1d has a narrower host-range than SfAV-1a and it can only develop full infections in S. frugiperda but not in S. exigua in which SfAV-1a is highly infectious. In order to understand better ascoviruses, ascoviruses gene transcription and expression strategies were studied and compared with baculoviruses. The DNA polymerase gene of SfAV-1d was demonstrated to be an early gene while the major capsid protein gene was confirmed to be a late gene. Three RNA polymerase homologues were found in the SfAV-1d genome. In vitro transcription assays showed that an early gene of SfAV-1d was transcribed by the nuclear extract from Sf21 insect cells while a late gene of SfAV-1d was not, suggesting that host RNA polymerase transcribes early genes of SfAV-1d while late gene transcription needs viral factors. Therefore, SfAV-1d follows the same transcription patterns as baculoviruses: early genes are transcribed by the host RNA polymerase while late genes transcription needs viral factors. We further characterized specifically a baculovirus late gene gp37 expression to understand the gene expression strategies. The 3’ untranslated region (UTR) of gp37 gene from the well-studied baculovirus Autographa californica Multicapsid NPV (AcMNPV) was studied. It has multiple polyadenylation sites and can reduce polyhedron production at the polyhedrin locus without changing the total amount of protein expressed. COMPARISON OF ASCOVIRUS AND BACULOVIRUS GENOMES AND THEIR REPLICATION AND GENE EXPRESSION STRATEGIES A Dissertation Submitted to the Faculty of Miami University in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Microbiology by Jianli Xue Miami University Oxford, Ohio 2011 Dissertation Director: Xiao-Wen Cheng, Ph.D. © Jianli Xue 2011 Table of Contents General Introduction………………………………………………………….………...1 Chapter One…………………………………………………….……………………….7 Abstract………………………………………………………………………….…....8 Introduction……………………………………………………….………….…….....9 Materials and Methods………………………………………………….…………...12 Insects, insect cells and viruses…………………………….. ………….………..12 Viral DNA purification……………………………….…….…………………...12 Restriction fragment length polymorphism (RFLP) and DNA hybridization analysis……………………………………………………………………..….…12 Pulse-field gel electrophoresis…………………………………….……………..13 Generation of SfAV-1d libraries for genome sequencing………...……………..13 PCR analysis, DNA cloning and sequencing……………………………….……14 Cell cultures and virus infection…...…………………………………………….14 Bioassay …………………………………………………………………………15 Results……………………………………………………………………………….16 Restriction fragment length polymorphism and Southern hybridization analysis of SfAV-1d and SfAV-1a…………………………………………….……………..16 Genomic DNA sequence deletions of SfAV-1d…………………………............19 Cloning and sequencing of the deletion region of SfAV-1d……….…………….19 Comparison of cell infection between SfAV-1d and SfAV-1a………………….26 iii SfAV-1d has a narrower host-range than SfAV-1a………………….………….30 Discussion...……………………………………………………………….………....32 Acknowledgements……………………………………………………….……….....35 Chapter Two………………………………………………………………….………....36 Abstract…………………………………………………………………….………...37 Introduction………………………………….…………………………….…………38 Materials and Methods……………………….………………………….…...……...40 Results…………………………………………..……………………….….………..43 28S rRNA shows lower Ct in qRT-PCR…….…………………………...….…..43 28S rRNA has the least variation during viral infection…..…………………….43 Inclusion of the 28S rRNA-R primer into oligo-dT-primed cDNA synthesis decreases Ct and variation of 28S rRNA…………………..………….…………50 Inclusion of the 28S rRNA-R primer into oligo-dT-primed cDNA synthesis does not interfere with the detection of other gene transcription.……..…….………...50 Discussion……………………………………………………...………….…..……..57 Acknowledgements……………………………………………………….…..……...59 Chapter Three…………………………………………………………………..……....60 Introduction………………………………….………………………………...….….61 Materials and Methods……………………….………………………………………64 DNA sequence analysis……………………………………………..………..….64 Phylogenetic analysis……………….…………………………….……..........….64 iv Plasmids, cloning and sequencing…………………………………….…..….….64 Viral replication inhibition analysis……….…………………………………......64 In vitro transcription assay……………………………………………………….65 RT-PCR analysis………………………………………………………………....66 Results…………………………………………….………………………………….68 Partial genome annotation…………………….…………………....….…………68 Phylogenetic analysis of ascovirus………………………………………………68 Identification of early and late genes…………………………………………….68 Dependence of viral genes transcription on the host RNA polymerase…………72 Discussion……………………………………………………………………………77 Chapter Four……………………………………………………………………………80 Introduction…………………………………………………………………………..81 Materials and Methods……………………………………………………………….84 Cell lines and viruses…………………………….………………………………84 Comparison of 3’ downstream sequences of AcMNPV gp37, polyhedrin gene and CfDEFNPV spindlin gene……..…………………………………….………84 Construction of viruses…………………….………………..…………………...84 Quantitative analysis of polyhedron production and size measurement of polyhedra……………………………………………….……….………………..86 Protein yield assay……………………………………………………………….86 Results………………………………………………………………………………..88 v 3’ UTR sequence analysis…………………………………….……….…….…..88 Construction of two sets of viruses………………………….……………….….88 Polyhedron production and their sizes comparison between AcpolUTR and Acgp37UTR...……………………………….…….…....………………………..88 Comparison of total polyhedrin protein production…...…………….……..…....97 Discussion …………………………………………………………………………..102 General Conclusions…….…………………………………………………………….105 References…………………………………………………….…………..…………...110 vi List of Tables Table 1-1. Summary of the variants of the SfAV-1d deletion region……………..…….27 Table 1-2. Susceptibility of noctuid larvae to SfAV-1a and SfAV-1d (% mortality)…...31 Table 2-1. A list of primers used in this study…………………………………………..42 Table 2-2. Comparison of average Ct of the possible reference genes…………………..46 Table 2-3. Comparison of SD [±Ct] by Bestkeeper analysis……………………….…...49 Table 2-4. Comparison of ΔΔCt of the possible reference genes………………….…….51 Table 3-1. Oligonucleotide primer sequences used in real-time PCR and in vitro transcription………………………………………………………………………….......67 Table 3-2. Partial genome annotation of the SfAV-1d genome………….……….……..69 vii List of Figures Figure 1-1. RFLP and Southern hybridization analysis of the new ascovirus isolate SfAV-1d………………………………………………………………………………...17 Figure 1-2. Assembly of the SfAV-1d genome from the shotgun library sequences with SfAV-1a as the reference using Sequencher……………….……………………...……20 Figure 1-3. Confirmation of the major 14kb deletion of SfAV-1d comparing to SfAV-1a….……………………………………………………………….…………….22 Figure 1-4. Characterization of the variable sequence in the deletion region of SfAV-1d….……………………………………….…………………………………….24 Figure 1-5. Infection of the insect cell line, IOZCAS-Spex-II by SfAV-1a or SfA-1d..28 Figure 2-1. Melting curve analysis of housekeeping gene amplification using gene specific primers by qRT-PCR…………………………………………………………..44 Figure 2-2. Comparison of cycle threshold (Ct) fluctuation of housekeeping genes in Sf21 cells infected with viruses at different time points post infection………………...47 Figure 2-3. Comparison of cycle threshold (Ct) in qRT-PCR using mixture of oligo-dT/28S-R and oligo-dT in cDNA synthesis for 28S gene amplification…………52 Figure 2-4. Comparison of Cts of PPI gene amplification in qRT-PCR between oligo-dT/28S-R and oligo-dT synthesized cDNA for interference detection..…………55 Figure 3-1. Evolutional relationship between baculovirus and ascovirus………...……70 viii Figure 3-2. Determination of early or late gene in the SfAV-1d genome……………..73 Figure 3-3. In vitro transcription analysis of different baculovirus and ascovirus promoters with insect cells nuclear extract……………………………………………..75 Figure 4-1. 3’ downstream sequence analysis of AcMNPV gp37 (A), polyhedrin (B) genes and the spindling gene from CfDEFNPV (C)……………...…………………….89 Figure 4-2. A schematic presentation of four constructed viruses……...………...…....92 Figure 4-3. The insect cell lines were infected by AcpolUTR or Acgp37UTR…...…...94 Figure 4-4. Bio-Rad protein assay of polyhedrin amount in Sf21 cells infected by AcpolUTR or Acgp37UTR……………………………………………………………..98