Transcript Viruses
Viruses Disease causing agents Viruses • Can multiply only in cells and which, by virtue of their tiny size can pass through filters that hold back even the tiniest bacteria Viruses • Extensive study in an effort to find methods to control virus-induced diseases Virus • Important tool in molecular biology and rDNA applications • rDNA – recombinant DNA Gene Therapy • Some viruses are used in gene therapy • Retro viruses • Adenovirus • Herpes virus • vaccinia Retro virus • AIDS • Leukemia Adenovirus • Sore throat • Respiratory tract infection • Liver infection • Liver cancer Herpes virus • Cold sores • Genital lesions • Mononucleosis • Chicken pox Herpes virus • Central nervous system infection • Developmental abnormalities Vaccinia • Cowpox Question? • What would you want to know if your Doctor recommended gene therapy using adenovirus as a vector? Virus structure • Very small • Can usually only be seen with an electron microscope Virus Structure • Genome – all genetic information contained by an organism • DNA or RNA double stranded or single stranded DNA or RNA Virus Structure • Sometimes double stranded RNA • Capsid – protective coat Capsid • Nucleic acid and a protein is called a nucleocapsid Virus Structure • Envelope – lipid membrane only found in viruses that infect animal cells • Proteins embedded in envelope envelope • These proteins are often sticking outside the virus particle and have sugars attached to them • Are referred to as envelope glycoproteins Capsid Shapes • Icosohedral – 20 sided sphere • Helical - tubular Icosohedron Helical virus Virus size • Range in size from 10 nm to 1000 nm Virion • Complete virus particle • Four general categories of virion structure • Based on shape of the capsid and whether it has an envelope or not Virion structures • Naked icosohedral • Naked helical • Enveloped icosohedral • Enveloped helical • Some viruses have mixed morphologies • Some bacteriophages have capsids that are part helical and part icosohedral • Most complex virus particles are the pox viruses Prion • Protein only, can be reproduced • Scrapie – in sheep, like mad cow disease • Grinding of animals used in animal feed Kuru • Kuru – degenerative brain disease • Custom of eating the brain of dead relatives • Human equivalent of mad cow disease Host Range Categories • Animal – usually species and cell specific • Plants • bacteria Host range specificity • Requirement for a specific interaction between a protein on the outside of the virus - Envelope glycoproteins, capsid proteins • And a cell surface protein – hormone receptor or some other protein important for cell function Example • HIV – only infects helper T lymphocytes because the gp120 protein in the HIV envelope interacts with the CD4 protein on the helper T cell Multiplication • Attachment of virus to cell surface • Penetration – into cell, sometimes entire virus, genome only • Genome replication Multiplication • Genome (protein) translated into virus proteins using host ribosomes • Assemble new virus particles • Release of new virus from cell either by budding or lysing Virus genomes • Must encode any proteins necessary for multiplication which are not provided by the cell • Capsid proteins and special enzymes Special enzyme • One that could synthesize RNA using an RNA genome as a template • Process would not occur in an uninfected cell Retroviruses • Unique group of animal viruses that include HIV • Contain an RNA genome that is reverse transcribed into DNA • Requires the enzyme reverse transcriptase Animal virus w/ DNA genome • Virus DNA is replicated • Genes are transcribed • mRNA is exported to the cytoplasm and translated • Proteins are transported back into the nucleus to combine with new virus DNA to assemble new virions Enveloped Viruses • Obtain lipid membrane by budding from a cell • Taking part of the plasma membrane with them • Membrane remains enclosed around the nucleocapsid • Virus proteins often imbedded into the envelope and are involved in binding of virion to surface of target cell Bacteriophages • Viruses infecting bacteria • (phages) • Bind to surface of bacterial cell and inject their DNA into the cell Phages • leaves the capsid outside the cell • Genome is replicated and translated • New virions are assembled Phages • Bacterial cell bursts open to release the new viruses How do viruses cause disease • Effect they have on cells when they divide and multiply • Many viruses must lyse the cell Lysis • Explode cell • Non eveloped viruses get out of the cell this way • Cell becomes so full of the new virus from replication that it explodes lyse • Infection of many cells may cause extensive damage to tissues • Symptoms of the viral infection are determined by the cell type infected Rhinovirus • Causes the common cold • Infect and damage cells lining the upper respiratory tract • Symptoms result from the body’s efforts to repair damaged tissue • Even if a virus can be released from a cell with lysis, it still causes significant changes within the cell and interferes with cellular functions • Animal viruses whose genomes become inserted into the cellular DNA can cause infected cells to be transformed into tumor cells • Can enter an animal or plant only through a wound or natural opening • Respiratory system • Digestive system • Blood • Wound • Genital openings Interferron • Blocks translation Retro virus • Use reverse transcriptase • Causes higher rate of mutation • Part of the reason retro viruses are hard to create vaccines for also part of the inherent danger! RDNA Technology • New viruses may be constructed with RDNA technology • Yield modified viruses for vaccines • Virus DNA is easily isolated from infected cells • Can be digested with restriction enzymes to remove portions of the genome • Specific virus genes can be cloned • Resulting virus either lacks certain genetic info or contains additional info that alters the behavior of the virus, causing new proteins to be produced Vaccines • Goal is to stimulate the immune system to recognize and respond to an infectious agent without the animal getting sick • Some portions –disease causing portion -of the virus can be eliminated from the material used to make the vaccine • Animal’s immune system will still respond the same way • Vaccinia virus causes only mild symptoms in humans • Engineered to express proteins from other more dangerous viruses • Altered vaccinia virus will multiply in the host and generate an effective immune response Gene cloning and genetic engineering • Bacteriophages can enter bacterial cells and multiply efficiently • A piece of DNA inserted into a phage will be replicated in the cell • Phages are used in this way as vectors • Vector – mechanism for introducing something into a living system • Bacteriophage Lambda • Popular vector • One third of its DNA is not needed for replication and can be replaced by foreign DNA • Lambda vectors can accommodate much larger pieces of DNA than most plasmid vectors • Reverse transcriptase is used to transcribe RNA from cells into corresponding DNA sequences – called cDNA for cloning • Process allows preferential cloning of DNA • Normally less than 1% of the total DNA in an animal cell • Recombinant phages • Produced by joining phage DNA and cellular DNA • Bacteria is mixed with rDNA and spread on the surface of an agar plate • Bacteria grow across the plate as a “lawn” • If a virus infects a cell the released viruses will infect neighboring cells • Creates plaques – clear areas • Plaque hybridization is performed to identify a plaque containing a virus Plaque hybridization • Nitrocellulose membrane is placed on the plate • Pick up a little of the phage from each plaque • Membrane is treated to release DNA from the phage and attach to the membrane • Probe is used that hybridizes only DNA containing a complementary nucleotide sequence • Animal Viruses can be used to transfer cloned pieces of DNA into animal cells so that the gene will be expressed • Process may be used to study function of a protein • Or to correct a defect caused by a missing or incorrect gene • Retroviruses most commonly used • Retro DNA is efficiently inserted into cellular DNA • Becomes part of the cell’s genetic material • Retroviruses can be used to genetically engineer cells or even whole animals Gene Therapy • Virus vectors – retro and adenoviruses • Used in gene therapy applications for immunodeficiency, cancer, Cystic Fibrosis and other diseases