Viruses: Informational parasites
Last revised: Wednesday, March 12, 2003
Reading: Ch. 14 in text

• Every virus has 2 stages:
  
  1. dormant, particulate, transmissible stage called the virion stage
  2. an active, intracellular stage called the infectious stage

Virion Stage

• Virions are the transmissible state of a virus. Metabolically inert.
• Virion = "a piece of bad news wrapped up in a protein coat" (and/or a membrane)
• The "bad news" can be either DNA (double-stranded (ds) or single-stranded (ss)) or RNA (ds or ss); never both.
• The coat (also called viral shell or capsid) can be icosahedron (20-sided regular geometric shape common in many bacterial, animal, and plant viruses), sphere, cylinder, bullet-shaped, or amorphous shaped particle.
• Virions must be able to adhere and allow entry into some host cell(s). Also to survive outside of host cell environment.
• Some virions more hardy than others (e.g., hepatitis virus can withstand short periods of boiling; most virions are destroyed by this).

Infectious Stage

• When virus infects a cell, nucleic acid must be uncoated and gain access to metabolic machinery of cell.
• Virus life cycle is characterized by:
  1. attachment
  2. penetration, with entry of nucleic acid into cell
  3. early expression of virus genes (either directly by translation, if virus contains "+" RNA, or indirectly after transription and then translation)
  4. replication of virus nucleic acid
  5. synthesis of new virion components
  6. packaging and assembly of new virions
  7. exit from cell
• But note: some viruses can have variations on this theme: e.g., lysogeny (see below), retroviruses (see below), etc.

Measurement of viral growth

• Must grow virus on host cells to see anything. Can't grow virus without cells.
• To quantify viruses, need some way to get flat surface of growing cells, allow virus-infected cells to spread radially where present = plaque.
  
  
  
  
• In bacterial cells this is easy. Spread "lawn" of bacteria on plate, add diluted phage suspension or culture infected with phages. After 6-8 hours can see plaques in E. coli.
• In plant cells, can be easy. Example: Tobacco Mosaic Virus (TMV), make virus dilution, rub over surface of tobacco leaf. After leaf growth, can observe plaque areas.
• In animal cells, not so easy. In 1960's, standard assay was to inoculate chicken egg membranes of developing chick embryos, incubate for a week, cut open shell and count plaques on membrane in the air sac. Lots of work to get statistically reliable data!
• In 1970's tissue culture became a viable alternative. Animal cells are cultured as microbes in glass or plastic, use special medium that contains most of nutrients present in blood. Cells will spread as monolayer on surface, can count plaques after staining.
• Note: When counting plaques, report results as Plaque Forming Units, or PFU/ml,not as viruses/ml. Why? When counting viruses per unit volume under EM, often see far more than will cause plaques. (For some viruses only 1 in 100 or even 1000 particles will actually be detectable by plaque assay).
• Viral growth curve (in lab last week). Look at PFU/ml over time. See latent phase, then increase in PFU during viral release.
• View a short history of virology

Taxonomy of viruses

• Based mainly on Virion and Kingdom of host
• Use Host cell type (Animal viruses, plant viruses, etc.)
• Use Nucleic Acid type (ds DNA, ss DNA, ds RNA, ss RNA)
• Use + or - polarity of RNA. "+" is able to serve as mRNA. "-" is the complement of +, must function as template to make a complementary strand of + RNA before any translation can occur.
• Use virus coat morphology. Enveloped vs. non-enveloped viruses.
• Explore virus taxonomy

Virion Structure

1. "Naked" viruses
   1. Helical viruses
      • Tobacco mosaic virus (TMV) is an example of a virus with helical symmetry.
      • A helical array of identical protein subunits surrounds an RNA molecule
        
        TMV electron micrograph: image from University of Leicester
        
   2. Icosahedral viruses
      • built from icosahedral (20-sided) assemblies of protein subunits.
      • View animation of icosahedron shape (from Tulane University)
      • Icosahedral shape is the minimum free energy structure for producing a shell of equivalently bonded identical structures.
      • The simplest icosahedral capsids are built up by using 3 identical subunits to form each triangular face, thereby requiring 60 identical subunits to form a complete capsid. A few simple virus particles are constructed in this way, e.g. bacteriophage ØX174.
      • Most icosahedral viruses have more than 60 subunits, usually some multiple N times 60. N (called the triangulation number) can have values of 1, 3, 4, 7, 9, 12, and more.
      • Visual example: adenovirus diagram
2. "Enveloped" viruses
   • "Naked" viruses require host death so viruses can be released. This may be wasteful, and may cause premature death of host cell.
   • Alternative strategy: shed virus particles by budding out, continued release from cell membrane. Cell does not die (immediately), continues to serve as factory for virus assembly and release. Virus typically acquires a coating of host cell membrane, modified to include virus-specific proteins. This is the "envelope". Virus may have additional protein coats (often icosahedral) inside the envelope.
     
     Enveloped Herpes virus, by Linda Stannard
     
   • Eventually host cell is too depleted to survive. Can see evidence of this as "cytopathic effect" (CPE). Cell then dies.
   • Examples of enveloped viruses include:
     • Retrovirus, including HIV
     • Paramyxovirus, including influenza
     • Rhabdovirus, including rabies
     • Filovirus. Although very "hot" in the news, these viruses are very poorly characterized because of their extreme pathogenicity. They are class IV pathogens, meaning they can only be cultured in total containment facilities, of which there are only two in the U. S. They are thought to be enveloped viruses with - RNA genomes.
       
       
       Ebola virus image by Dr. Frederick A. Murphy, University of California, Davis
       

Virus Genomes

• Rule of Thumb: to estimate # of virus proteins, look at size of viral DNA or RNA. For each 1000 base pairs, can guess the existence of 1 protein
  • "typical" gene has 300-400 amino acids = ~ 1000 base pairs = 1 kbp (= 1 protein)
  • small virus: SV40 => 5000 base pairs = 5 kbp ~ 5 proteins
  • large virus: T4 => 200 kbp ~ 100-200 proteins
  • by comparison, E. coli: 4000 kbp
• For more detailed information, visit the structure and complexity of viral genomes from the University of Leicester

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Bacterial Viruses = Phages

• For further information beyond the three phages studied here, visit Bacteriophages, from University of Leicester, U. K.

Case study #1: phage T4 lytic infection

• View EM of phage T4
• Virion has ds DNA (170 kbp), over 135 genes described. Icosahedral head, tail with contractile sheath, lysozyme at tip of tail, fibers, base plate, etc. Very complex morphology.
• View T4 morphology.
• View QT movie of T4 killing E. coli cells.
• Virus DNA is injected, easily enters host. DNA has promoter sites that look just like host ("TATA" boxes, etc.)
• RNA polymerase of host sees DNA, transcribes mRNA. This gets translated "early proteins".
• Genes grouped into early (uses host sigma) and late (uses new T4 sigma). One of the early proteins specifically blocks host sigma.
• Examples of "early gene" function
  1. endonuclease attacks host DNA
  2. new sigma factor to recognize only phage specific promoters (Not TATA; something altogether different)
  3. enzymes to produce new kind of nucleotide found in virus DNA: hydroxymethyl cytosine. (T4 DNA does not contain normal cytosine)
  4. enzymes for DNA replication. T4 phages use different origin than on host chromosome; don't produce theta structures; instead, produce long strings of repeated phage DNA = "concatomers". DNA replication occurs on linear molecule. Results in concatamer of repeated genes. At packaging time, enzyme cuts DNA into head sized units (includes some repeat at each end of DNA)
  5. new t-RNAs
  6. new ribosomal proteins (work better on T4 messages)
• After early genes have been expressed, cell switches to "late genes"
• Examples of "late gene" function:
  1. phage head and tail assembly
  2. packaging of phage DNA into phage heads
  3. production of T4 lysozyme, and lysis of cell

Case Study #2: lysogenic phage Lambda lytic or lysogenic infection

1. View EM of Lambda virions
2. View diagram of Phage Lambda
3. Lambda has ds DNA, injected into cell as linear molecule. Enough DNA for ~ 55 proteins.
4. View map of Lambda genome
5. Phage has two possible outcomes: lytic infection or lysogeny = "silent" partnership with cell, virus DNA becomes integrated into host chromosome and gets duplicated and passed on to all cell offspring.
6. View alternate fates of Lambda infection
7. DNA has "sticky ends". ~20 bases at each end are single stranded. The two ends are complementary, DNA can circularize once in cell. DNA ligase can seal two ends to make covalently closed circle. View diagram showing circularization.
8. Lambda has 3 promoter sites. Two of these allow transcription of lytic genes. Other promoter leads to transcription of a repressor protein (Lambda repressor) that can bind to the two lytic promoters, block all lytic genes. Repressor does not block its own promoter, so cell continues to synthesize small quantity of repressor (~10-20 copies/cell).
   View Lambda decision: lysogeny or lytic phase?.
9. One early lambda protein is Integrase; causes specific recombination event at region where both Lambda DNA and host DNA have same 13 base pairs. (homologous sequence).
10. View diagram showing integration of Lambda DNA into host chromosome
11. If Lambda repressor is expressed before transcription of late lytic pathway genes occurs, then Lambda remains in host DNA indefinitely, gets replicated just like host genes.
12. Induction: under "nasty" environmental conditions where DNA damage occurs (e.g. UV light or certain chemicals), can mutate repressor, make it ineffective. If this occurs, lytic promoters are no longer blocked, lytic genes get transcribed and translated, and cell becomes phage factory, leads to lytic production of lambda viruses.

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Bacterial defenses against infection

Cell surfaces: possibilities of mutation

1. Virus must attach to some specific cell surface protein or polysaccharide. But these are specified by genes, and genes can mutate. In population, will always find some variant strains with slightly different cell surfaces, may not bind virus well.
2. When phage first discovered, thought this could be effective weapon against bacterial disease. But frequency of resistant bacterial strains was too high, any given strain of virus quickly became useless as resistant survivors propagated.

Nucleases: endo- and exo-DNases and RNases

1. All bacteria seem to have nucleases that can attack DNA (called DNases) and RNA (called RNases).
2. Exoenzymes attack free 5' or 3' ends of DNA, RNA molecules. Bacteria are protected since DNA (and plasmids) are always circular. RNases are present, and in fact destroy mRNA eventually (bacteria are always making new RNAs, very responsive to enviroment changes).
3. Endonucleases are potentially lethal weapons. Called restriction enzymes. Attack at specific sequence: e.g., in E. coli, enzyme called EcoRI will attack any sequence with 5' G-A-A-T-T-C 3' (cuts DNA between G and A).
4. View movie of restriction enzyme cutting DNA
5. Why doesn't this kill cell? Because cell also has a second enzyme, called modification enzyme, that protects all host DNA sequences of this type. Typically adds a methyl (-CH₃) group to one base at the cutting site. The methylated base is modified, and protected from the restriction enzyme. When foreign DNA comes into cell (e.g. virus DNA), if restriction site if present it will be cut and ----- requiem for the virus!

The importance of Restriction Enzymes

Restriction enzymes are responsible for the genetic revolution. They make reproducible, specific cuts with surgical precision. Major industry has emerged in biochemical supply companies to harvest bacteria, purify restriction enzymes, and sell these to research and applied industries. Big $$$$$$$.

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Animal Viruses

• Animal viruses are different in many respects from bacterial viruses. The host cells are more complex, with multiple compartments and more complex regulation of replication, transcription, and translation. Animal cells are not bounded by cell walls.
• Explore EM atlas of animal virus structure
• Not surprisingly, animal viruses have evolved to overcome these problems. They attach and enter by different mechanisms than phages, and their intracellular activities include the ability to move between different compartments as needed.
• Viral entry and exit from cells is very different from bacteriophages. Animal viruses must enter through cell membrane, either by triggering endocytosis pathway or by fusing viral envelope with the cell envelope.
  
• View animal virus fusing with host cell membrane
• Modifications are needed in both cellular and viral mRNA to allow recognition and movement from nucleus to cytoplasm. For example:
  1. 3' tail of poly A
  2. 5' cap of methyl Guanosine triphosphate

Types of infection

Viruses in animal cells show a variety of infection patterns:

• lytic infection: destroys host cells.
• persistent infection: host cell continues to shed virus over long time. Cell gradually becomes recognizably poorer (recognized as cytopathic effect, or CPE), eventually "crumps out".
• transformation: infection by certain viruses causes cells to change, become cancerous. Responsible genes are called oncogenes (tumor-producing genes). Viral oncogenes have also been found in uninfected cells. These are genes involved in regulation of cell cycle; when defective, normal regulatory control is lost and cell can become cancerous.
• latent infection: virus genes may not be expressed for long time (ex. many Herpes infections). Not the same as lysogeny -- genes are not integrated into host chromosome.

Case Study #3: small +RNA Poliovirus

1. + RNA virus, 7500 bases long (code for 2500 AAs max)
2. pico-RNA virus, very small
3. See Web information on picornaviruses
4. simple icosahedral shell
5. RNA makes single polyprotein, then cleaved to ca. 20 smaller proteins: include 4 virion proteins, RNA polymerase (replicase), protease
6. replication is cytoplasmic
7. Replicase makes "-" strand from "+" template, uses these to make more "+" strands
8. RNA gets covalently linked to VPg protein (22 AA)
9. Host RNA & protein synth. inhibited by destruction of cap-binding protein requd. for translation.
10. Medical: epidemic in late 1940's, early 1950's. 1.5 cases/100 people. Salk vaccine licensed 1951, immediate impact. By early 60's disease gone.
11. Virus was waterborne disease, transmitted by drinking or swimming. Can be eliminated by proper chlorine treatment.

Case Study #4: -RNA Influenza virus

1. member or orthomyxoviruses (myxo = interact with mucus in respiratory tract)
2. most lethal pathogen of 20th century. Epidemic of 1918-19 killed 20 million people!
3. Transmitted by person-to-person contact, mainly by droplets from coughing and sneezing.
4. Virus attacks mucous membranes of upper respiratory tract, sometimes lungs.
5. Symptoms: 3-7 days fever, chills, fatigue, headache, muscular aches. Most serious problems due to bacteria that invade while defences are weak. Death may occur in infants, elderly.
6. enveloped virus, not icosahedral. Virion contains 8 pieces of ss "-" RNA, each codes for separate protein.
7. View influenza virus electron micrographs
8. View QT movie of influenza virus model
9. Virus membrane has several protein: Hemagluttinin, Neuraminidase.
10. Virion also carries RNA-dependendent RNA polymerase and RNA endonuclease
11. Entry: by endocytosis. Then RNA replicates in nucleus. New "+" RNA migrates to cytoplasm, is translated. from 8 RNAs get 10 proteins (2 proteins are cleaved).
12. Exit by budding out. Viruses shed for long time.
13. Antigenic shift occurs every few years. Thought to result from mixing of different virus strains in same cell, recombination of surface protein genes to give new arrangements.
14. Vaccines not good for more than a few years because of new strains. Usually only recommend for those at most risk
15. Aspirin not good for flu; possible link with Reye's syndrome in children especially.

Case Study #5: ds DNA Herpes viruses

1. large group of ds DNA viruses.
2. Diseases include: cold sores, venereal herpes, chickenpox, shingles, infectious mononucleosis, and cancer
3. Able to become latent for long time; e.g. in neurons.
4. Virion contains DNA (150 kbp), icosahedral shell, and envelope. Enough DNA to code for about 70 proteins.
5. View EM of herpesvirus
6. View model of herpesvirus virion. View QT movie of herpesvirus model.
7. Infection: attach to cell receptors, fuse virus membrane with cell membrane, release inner particle, travel to nucleus. Then, uncoat virus DNA. DNA synthesis occurs in nucleus. Proteins synthesized in cytoplasm, travel back to nucleus for assembly.
8. Virions bud through inner membrane of nucleus to outside of cell.

Case Study #6: Retroviruses

1. See Section 8.21.
2. HIV virus as example: Virion contains 2 identical "+" RNAs, also enzyme reverse transcriptase
3. View 3-D diagram of HIV virion
4. RNA is about 9 kb in length, could code for 3000 AA, about 10 proteins.
5. RNA could serve as mRNA but doesn't. Instead, travels to nucleus. One of two RNA's is copied by reverse transcriptase into ss DNA, then into ds DNA (again by reverse transcriptase, which now acts as DNA replicase)
6. View life cycle of HIV
7. View animated movie showing HIV virus uncoating and replication
8. Primer for DNA synthesis is special tRNA brought by virion from last host.
9. DNA integrates into host chromosome
10. Virus DNA then transcribed & translated into viral proteins. RNA is packaged at cell membrane into new viruses by budding from cell
11. View TEM of HIV particles budding out of host cell
12. Medical: some retroviruses cause cancer; e.g. Rous sarcoma virus

Human Immunodefiency Virus (HIV) and AIDS

1. AIDS first recognized in 1981. Over 300,000 cases reported in U.S., over 8 million in Africa, over 12 million infected world-wide.
2. View AIDS in perspective: a PBS website with current data on the AIDS epidemic.
3. Transmission: sexual activity, especially with multiple sex partners. Also contaminated blood, needles, hospitals. Not just a disease of homosexuals! In Africa (most # cases) about equal # of male and female victims.
4. AIDS lowers immune system's ability to respond to other infections, allows opportunistic pathogens to invade body. Most common infection is pneumonia (lung infection) caused by Pneumocystis (2/3 of all AIDS patients get this at some point).
5. Host cell for the virus is CD4 (T-helper) cell, needed to activate antibody production. In normal human, CD4 cells account for 70% of total T cells -- in AIDS, number decreases, may reach 0% of T cell pool.
6. Progress of HIV infection only recently understood. Formerly thought that virus became latent. Now discover that virus is anythig but latent: during infected period (which can last 10 years), body is destroying ~ billion virions/day, and virus is killing about 100 million CD4 T cells a day. HIV virus continues replicating, and body rapidly replenishes lost T cells. Only when lymph nodes wear out does virus gain the upper hand. See handout in class titled "Huge HIV turnover helps explain drug resistance, pathogenicity".
7. Prognosis: with carefully selected treatments, better than before. Virtually every infected person dies sooner of later, usually within 10 years of infection. No cure known, no vaccine yet available. Virus mutates rapidly, many strain variations. Vaccines being tried, results mixed but preliminary.
8. Drugs: some types of drugs offer limited success.
   1. AZT (azidothymidine) is analog of thymidine, but is blocked at 3'-position, so no further chain growth possible. These target viral reverse transcriptase enzyme. Should reduce DNA synthesis in treated cells. But eventually, viral mutants resistant to drug arise. Also, long term use of drug can cause toxic side effects.
   2. protease inhibitors. Like many viruses, HIV needs to cleave large protein product into smaller products, using viral protease protein. By inhibiting this enzyme, should block necessary stage in viral replication cycle. Still under development, but resistant viral mutants to these type of drugs have already been found. Still, drug offers promise. See handout article for more details. Safe sex! Caution with sharps. Extra caution in clinical settings!

Viroids and Prions

• Viroids = very small ss RNA genomes (~300 nucleotides). No coat, and RNA does not encode protein. Known viroids cause diseases in plants because host cells replicate the RNA.
• Prions (protein infectious agent) do not have a nucleic acid genome. Prion diseases are often called spongiform encephalopathies because of the post mortem appearance of the brain with large vacuoles in the cortex and cerebellum.
• View pathology of brains infected with prion diseases Examples:
  1. Scrapie (sheep)
  2. bovine spongiform encephalopathy (cows) = "mad cow disease"
  3. Creutzfeldt-Jakob Disease (humans)
• Prion diseases in humans are probably primarily a genetic neurotoxic disorder. Transmission of the disease to humans via infectious prions is likely to be rare.
• The prion is a modified form of a normal cellular protein known as PrPc (for cellular), found predominantly on the surface of neurons and thought to be involved in synaptic function.
• The modified form of PrPc (= prion) is known as PrPsc (for scrapie) which is relatively resistant to proteases and accumulates in cytoplasmic vesicles of diseased individuals. Prion protein may cause normal protein to fold abnormally.

Creutzfeldt-Jakob disease occurs worldwide and usually becomes evident as dementia. Very rare; afflicts one person in a million. 10 to 15 percent of cases are inherited. Typically begins in 60's with a loss of memory. Over several weeks the mental deterioration progresses to dementia, abnormalities of vision or coordination, rigidity, and involuntary. Death usually occurs within six months, and at necropsy the brain shows a "spongy" texture View section of brain tissue from Creutzfeldt-Jakob autopsy

Mad Cow Disease Cows becomen uncoordinated and unusually apprehensive. Disease traced to use of sheep tissue as food supplement for cows over long period of time in United Kingdom. Practice was begun in late 1970s. Small number of younger humans (mostly in 20s) came down with Creutzfeldt-Jakob disease in mid-1980s in Britain. Never before seen in humans younger than 50s. Suspected cause was eating beef from cattle fed with sheep products. British government banned animal-derived feed supplements in 1988

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Animal defenses against viral infection

Unlike bacteria, animal cells do not make restriction (and modification) enzymes. More complex defenses are used.

Fever: fever is a non-specific response to infection, and can inhibit or significantly slow growth of many infectious agents, including some bacteria and some viruses.

Interferons:
IF's are low molecular weight proteins, produced by cells in very tiny amounts in response to certain stimuli (e.g. double-stranded RNA). Very species specific; mouse IF won't work in humans. IF genes in cell are usually inhibited, but activated after viral infection, cause cell to produce IF. IF then acts on other cells by binding to specific membrane receptors, triggering activation of genes that help make cell more virus-resistant. Two specific inhibitors:

(1) Oliogo(A) synthetase: after stimulation by ds RNA, leads to activation of endoRNase activity, which can cleave viral RNA
(2) Inactive protein kinase: after stimulation by ds RNA, leads to activation of inihibitor of elongation factor 2 (eF-2) needed for protein synthesis; blocks translation of viral proteins.

Specific Immunity. (Details to be discussed when immune system is discussed). Normally requires 1-2 weeks after initial exposure before significant response appears.
(1) Antibodies produced against viruses can clump virus particles, lead to phagocytic uptake and destruction.
(2) T-cells can identify virus-infected cells by presence of new viral antigens, and T-killer cells can destroy these cells, prevent infection from spreading.

Antiviral chemotherapy. In general, antiviral agents much more difficult to find, much more limited in scope, than antibacterial agents. Why? Bacteria have unique targets not found in animal cell: peptidoglycan, 70S ribosomes. By contrast, animal virus infects human cell, any treatment which harms virus probably harms host as well.

A few successful antiviral drugs. Ex 1: amantadine treats influenza A infections (if given early in infection); blocks penetration and uncoating of virions. Ex 2: acyclovir, prevents herpes infections; after phosphorylation, resembles dGTP and blocks activity of DNA polymerase.

But, drug-resistant virus strains can develop.

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