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