Virology - Genome PDF

VIROLOGY Biology 65-A GENOME  Type– DNA or RNA Number of strands ss Positive sense (mRNA) Negative sense ambisense ds Conformation – linear, circular, segmented Size – 2500 nucleotides (ssDNA) to 1.2 M bp (dsDNA) Mimivirus = 2,400,000 nucleotides (nt) Smallest = b...

VIROLOGY Biology 65-A GENOME  Type– DNA or RNA Number of strands ss Positive sense (mRNA) Negative sense ambisense ds Conformation – linear, circular, segmented Size – 2500 nucleotides (ssDNA) to 1.2 M bp (dsDNA) Mimivirus = 2,400,000 nucleotides (nt) Smallest = bacteriophage Virology - Genome  Some DNA Complexed with cellular histones to form chromatin-like structure Non-coding introns Protein coding exons Spliced mRNA in adenovirus (1977) Prokaryotic introns in bacteriophage T4 (160 kbp dsDNA -1984); compact have to keep up with fast dt of host  Characteristics Composition sizeand number of segments Nucleotide sequence Terminal structures Coding capacity Regulatory signals: transcription enhancers, promoters and terminators  Molecular analysis Physical analysis of structure and nucleotide sequence – in vitro Molecular cloning by PCR Electron microscopy Gel electrophoresis To initiate infection: purified virus proteins mixed with RNA transcribed from cloned cellular DNA to form complexes --- then introduced into susceptible cells to initiate infection  Biologicalapproach Structure – function relationships of intact virus genomes and individual genetic elements; analysis of virus phenotype – in vivo Functional genomics Enables transfection – infection with nucleic acid Reverse genetics – manipulation of a virus via cloned intermediate + sense RNA=infectious; - sense = non- infectious; - sense have to be copied with polymerase to form + sense or mRNA In vitro Ribonucleoprotein into cells infected with a helper virus strain Virology- genome  Viral genetics Biochemical analysis Use of inhibitors of translation – decipher regulatory mechanisms - to construct genetic maps Focal immunoassay Replication visualized by immune staining (HIV) Physical analysis Electrophoresis to ID genetic polymorphisms of virus proteins or nucleic acids Transformed foci Used for non-cytopathic focus-forming viruses : DNA and RNA tumor viruses  Types of genetic maps Recombination maps for non-segmented genomes Reassortment maps or groups For segmented genomes Others: Physical maps Restriction maps Transcription maps Translation maps  Recombination Exchange of DNA from one source with that from another Results to genetic variability  Virus mutants Mutant Strain Different lines or isolates of the same virus – from different geographical locations or patients Type Different serotypes of the same virus – various antibody neutralization phenotypes Variant A virus whose phenotype differs from the original wild-type strain but the genetic basis for the difference is not known – a new clinical isolate from a patient Parental, wild-type, street  Mutations Spontaneous mutations 10-3 to 10-4 (HIV) to as low as 10-8 to 10-11 (herpes virus) Induced by mutagens In vitro: Chemical – nitrous acid, hydroxylamine and alkylating agents, nitrosoguanidine In vivo: analogues of 5-bromouracil – acridine dyes; UV irradiation – form pyrimidine dimers  Types of mutant viruses Biochemical markers Drug resistant mutation Deletions Hostrange Nonsense Stop codons Plaque morphology Temperature sensitive Cold-sensitive Revertants suppression  “mimicking microbe” particles resembling Gram-positive bacteria residing within amoebae isolated from a water-cooling tower Pneumonia causing, like Legionella pneumophila? No! GIANT VIRUS! capsid diameter of 400–500 nanometers (nm) and a total particle diameter, including fibers extending out from the capsid, of approximately 750 nm (Xiao et al. 2005). A second strain of APMV, currently referred to as "mamavirus," may be even larger At approximately 1.2 million base pairs (Raoult et al. 2004), the linear, double-stranded DNA genome of Mimivirus far exceeds the size of any other known virus and a number of bacteria. Again, as a point of comparison, poliovirus has a genome of only 7,500 nucleotides, and the smallpox virus genome is about 200,000 nucleotides long. Virology - genome REPLICATION  Replication cycle Attachment Penetration Uncoating Genome replication and gene expression Assembly Maturation release