Microbiology Midterm 1

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Virus

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genetic element that replicates intracellularly and programs the synthesis of particles that transmit its genome from one cell to another

- Genetic element that moves from cell to cell

- Genes enclosed in some kind of shell

- No energy metabolism or ribosomes

- Intracellular parasite

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Viron

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virus particle

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Bacteriophage

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bacterial virus also called phage

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Naked Capsid Virus

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Consists of only genetic material and protein coat

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Enveloped Virus

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consists of genetic material, protein coat, and (lipid) membrane (picked up from host)

often irregular but nucleocapsid is regular

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Host Range

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type of cells a virus can infect

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Steps of Virus Infection of All Infections

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1) adsorption (attachment)

2) penetration (uncoating)

3) uncoating

*2&3: overlap in bacteriophages. viral capsids are shed at the surface and only the nucleic acid genome enters the cell

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Productive Response

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4) component production (new stuff made)

5) assembly (maturation, encapsidation)

6) release

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Possible Outcomes of Virus Release

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Cell Death: lytic infection (cell burst)

cytocidal virus (killer)

Cell Survival: chronic or persistent infection (filamentous bacteriophages and retroviruses)

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Nonproductive Response

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Latent State: characterized by persistence of viral genome (temperate viruses)

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Temperate Viruses

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can enter into either a productive or non-production relationship with the host cell

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Plaque Assay

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quantify # of infectious viruses in a stock

1 virus particle gives rise to 1 plaque

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Virus Titer

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number of virons in stock (pfu/mL)

pfu: plaque forming units

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One-Step Growth Experiment

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- Single cycle of infection

- Infect every cell in the population

- Achieve synchrony for infected cell population

- Used to find out what is going on inside of the cell

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Multiplicity of Infection (M.O.I.)

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ratio of infecting virus particles to cells

avg. pfu

= ---------------

avg. cell

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Poisson

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relationship between m.o.i. and fraction of cells infected

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Pr(r)

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fraction of cells receiving r virus particles: probability

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Poisson Variable: s

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average number of pfu's per cell (m.o.i.)

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Burst Size

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virus yield (pfu)

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number of infected cells

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Infectivity ratio

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number of infectious particles

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total number of particles

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Helical Symmetry

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Cylindrical shape

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Cubic Symmetry

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Spherical shape

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Binal

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Naked DNA virus structure

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Viral Capsids

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composed of many copies of one or at most, a few different kinds of protein molecules

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Protomer

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identical structural subunit

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Capsomere

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morphological subunits

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Coding capacity limitation

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Viral genomes do not have sufficient coding capacity to specify a protein that is large enough, by itself, to enclose the viral genome

Cannot code for their protein to form capsid by itself.

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Crystallographer's Argument

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The most logical way to build a regular symmetrical structure (virion) out of asymmertric subunits (protein molecules) is by the regular aggregation of many identical subunits

1) Viruses are highly regular structures. Naked capsid viruses tend to crystallize easily

2) Protein structural subunits are by their very nature irregular or asymmetric

3) Easiest way to imagine how a virus is constructed is by the regular aggregation of many identical subunits

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Coding Capacity

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The predicted sum of the molecular weights of all the proteins encoded in a viral genome

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Tetrahedron

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4 vertices, 4 faces, and 6 sides

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Octahedron

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6 vertices, 8 faces, and 12 sides

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Icosahedron

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12 vertices, 20 faces, and 30 sides

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Icosadeltahedron

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Derived from icosahedron by triangulating the 20 faces according to specific rules

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Pentamers

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Contained in the capsomeres.

Placed at on original vertices

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Hexamers

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A new vertex that is generated when pentamers are placed

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Virion Attachment Proteins (Antireceptors)

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proteins on the surface of the of the virion

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Receptors

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located on cell surface in multiple copies and are exploited by virus and are so close they all the viron to attach to multiple giving a tight interaction

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Entry by Enveloped Viruses

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Direct Fusion

Receptor-mediated Endocytosis

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Direct Fusion

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Virion connects to receptor on plasma membrane of the cell and the envelope fuses to the plasma membrane and the nucleocapsid is released still intact into the cell

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Receptor-mediated Endocytosis (Viropexis)

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Whole virion is swallowed into the cell and pinches off part of the plasma membrane, creating an endosomal vesicle/lysosome, surrounded by two membranes. Acidification of the vesicle releases the nucleocapsid.

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Acidification

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Proteins pumped into endosomal vesicle

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DNA Viruses

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Replication: DNA polymerases

Transcription: RNA polymerases

If large, codes for its own DNA polymerases

If small, use host DNA synthesis machinery

Homologous Recombination

Cellular based enzymes

Virally-encoded enzymes

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RNA Viruses

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Replication: Replicase (RNA polymerase)

Reverse transcriptase (retroviruses)

Transcription: Transcriptase (RNA polymerase)

Code for own replicases and transcriptases

Segmented genomes

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DNA Polymerases

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-Found in nucleus

-Template strand instructs polymerase regarding the order of nucleotides in new chain

- DNA strands are synthesized by the successive addition of nucleotides onto the 3' end of the nascent chain

-Requires a primer (usually RNA) to initiate a new chain

-Requires the triphosphate form of the nucleotides as substrates.

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Nascent Chain

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chain in the process of being replicated

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Prokaryotic Replication

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Almost always circular genomes

Has two replication forks beginning at origin

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Eukaryotic Replication

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Linear genomes

Multiple replication forks

Encounters End Problem

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End Problem in DNA Replication

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The lagging strand is incomplete when the RNA primer is removed and leaves a gap, making the 5' end shorter each time

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Replication As Circles

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Prokaryotes can circularize by cohesive ends or through blunt end ligation to avoid the End Problem

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Concatemers

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- consists of end-to-end tandem repeats of the genome

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Solutions to the End Problem

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1) Replicate as circles

2) Form concatemers

3) Use of special DNA ends

4) Use of 'protein' primers

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Synthesis of Viral mRNAs

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1) to program the cell to make proteins, viruses must synthesize mRNAs

2) mRNAs must conform to the rules dictated by the host cell ribosomes

3) Importance of the first mRNA

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Baltimore Classification Scheme

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For each virus, there will be three considerations concerning the relationship between the viral genome and the synthesis of mRNA

1) Is the viral genome itself a mRNA?

2) Can the cell synthesize mRNA from the viral genome?

3) The virus must provide the transcriptase (RNA polymerase) that is carried within the virion

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Infectivity of Viral Nucleic Acids

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Isolated nucleic acid genome NOT infection if:

- essential enzymes are enclosed within capsid

- genome of the virus is segmented

- DNA genome is too large to remain intact during isolation

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Polycistronic

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More than 1 coding region is found on one mRNA

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Monocistronic

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Only 1 coding region is found on mRNA (most mRNAs)

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First AUG Rule

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40S ribosomal subunit binds at the 5' CAP and scans along the mRNA to reach the first AUG codon where translation is initiated

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Interferon Phenomenon

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- part of the innate immune response

- cell knows when its infected and produced and excretes interferon

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Innate Immune Response

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- not virus specific

- 1st line of defense: slows things down for adaptive immunity and rapid response

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Antiviral State

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If infection occurs, cell dies before any virus can be produced to spare other cells from infection

1) Mx1 inhibits transcription of negative strand viruses (influenza)

2) 2',5' oligo A synthetase induced by interferon

3) RNA-activated protein kinase (PKR) induced by interferon

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Cytokines

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small proteins or glycoproteins that are involved in cell-to-cell communication and growth regulation.

bind to cell surface receptors and alter the pattern of gene expression in the target cell

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Type I Interferons

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IFN-α produced by lymphocytes

IFN-β produced primarily by fibroblasts but also by macrophages and epithelial cells

Induced by virus

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Type II Interferons

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IFN-γ produced by antigen-stimulated T-lymphocytes and natural killer (NK) cells

Not induced by virus

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Type III Interferons

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IFN-λ similar in function to Type I IFN

Induced by virus

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Activities of Interferons

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1) Induction of antiviral state

2) Inhibition of cell growth

3) Induction of MHC class I and II molecules

4) Activation of monocytes, macrophages, cytotoxic T-lymphocytes and NK cells

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Fidelity

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accuracy of RNA and DNA replication

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Exonuclease

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trims out wrong nucleotides in post-replicative mismatch repair

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Antigentic Drift

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Selection against existing variants because immunity is developed. Favors new strains

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Viral Quasispecies

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RNA virus populations are composed of a diverse mixture of genetic variants that arise from high mutation rates

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Oncogenic Transformation

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The non-productive response of animal viruses

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Lysogeny

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non-productive response in bacteriophages

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Lytic Viruses

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can only enter into a productive relationship

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Induction

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when temperate viruses are reactivated to leave the latent state and enter into the productive response

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Eclipse Phase

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period of infection in which no infectious viruses are found inside the cell, emphasizing the loss of infectivity soon after entry because the virus particles are dismantled as a prelude to their reproduction

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Latent Period

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length of time from the beginning of infection until progeny virions are found outside the cells

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Early Phase

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production of proteins required for genome replication

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Late Phase

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proteins synthesized for construction of the new virus particles

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Liphophilic Amine

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Methylamine becomes charged inside of the cell and cannot pass the cell membrane and accumulates

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Rolling Circle Replication

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Site-specific cleavage at origin. Template strand rolls along the inside of the parent strand to produce complementary strand.

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Multipartite Genomes

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genomes encapsidated separately and all parts are needed for infection. mostly plant viruses that have high infection rates.

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Baltimore Classification Scheme: Group II

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- ssDNA (+ or -)

- phages φX174, fd, M13, & parvoviruses

- replication occurs in the nucleus to form dsDNA to form +mRNA

- uses host cell to form +mRNA

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Baltimore Classification Scheme: Group III

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- dsRNA

- phage φ6 & reoviruses

- segmented genome

- transcribed separately to produce monocistronic mRNAs

- must carry it's own transcriptase to get mRNA

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Baltimore Classification Scheme: Group IV

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- ssRNA (+)

- RNA phage, picornaviruses, togaviruses, coronaviruses, flaviviruses

- can be translated directly from +RNA to m

- infectious

- can't make it's first mRNA

- translation results in the formation of a polyprotein product

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Baltimore Classification Scheme: Group V

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- ssRNA (-)

- rhabdoviruses (VSV), orthomyxoviruses (influenza), paramyxoviruses (measles)

-carries RNA polymerase in virion

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Baltimore Classification Scheme: Group VI

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- ssRNA (+)

- retroviruses

- template for reverse transcriptase

- +RNA -> -DNA -> +- DNA -> +mRNA

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Baltimore Classification Scheme: Group VII

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- hepatitis B

- starts as dsDNA and relies on reverse transcriptse

- dsDNA -> +RNA -> -DNA -> dsDNA -> +mRNA

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Bacteriophage T7 Replication

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1) Two strands are replicated and each produce a shorten replicated strand

2) Shortened 5' end binds with 3' of original strand, forming a concatemer

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Bacteriophage T4 Recombination

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forms concatemers

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Endonucleases

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Cuts at a specific site

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Hairpin Ends in Parvoviruses

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Ends are self-priming that are complementary base-pairing which uses an endonuclease to cleave and form a template strand.

Produced ss Progeny DNA that can be encapsided or replicated

Can produce many cycles (progeny DNA)

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Continuous Ends in Poxviruses

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Sequence-specific endonuclease cleave followed by displacement synthesis

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Protein Primer in Adenoviruses

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Terminal Protein allows addition of Viral DNA Polymerase to start replication, displacing the 5' end

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Cos

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The site-specific position when bacteriophage λ concatemer is cleaved by endonucleases to produce staggered cuts. Gives unit sized genomes.

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Baltimore Classification Scheme: Group I

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- dsDNA viruses

- phages T4, T7, λ, φ29, SV40, polyoma, adenoviruses, herpesviruses

- replicated using cellular genome to from +mRNA

poxviruses: replicates in cytoplasm, carries its own RNA polymerase

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Prokaryotic mRNAs

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- polycistronic

- ribosomes bind to AUG

- can also be translated at stop, ribosome can keep moving until it finds another ribosome or falls off

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Eukaryotic mRNAs

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- mostly mRNAs

- 40s ribosome binds to a CAP and scans along until AUG to build polypeptide chain (translation)

- when ribosome hits stop, it always falls off

- can't translate at an internal site

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Gene Expression for Eukaryotic DNA Viruses

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- promoter for each gene (adeno, herpes, papova, parvo, poxviruses)

- monocistronic mRNAs

-RNA splicing (adeno, herpes, papova, parvo)

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Gene Expression for Eukaryotic +RNA Viruses

picornaviruses (polioviruses, rhinoviruses) and flavivirues (Hep C, West Nile virus)

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- genome +RNA transcription to give viral polyprotein

- viral polyprotein undergoes proteolysis to for viral proteins

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Gene Expression for Eukaryotic -RNA Viruses

rhabdoviruses (VSV, rabies), paramyxoviruses (measles & sendai) and filoviruses (Ebola)

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- -RNA undergoes Start, Stop, Restart mechanism and makes moncistronic mRNAs (capped with poly A) to form translation

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Gene Expression for -RNA Viruses

orthomyxoviruses (influenza)

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- replicates and splicing in nucleus

- -RNA segments infects nucleus of cell and undergoes virion transcriptase adding monocistronic mRNA poly A tails to get viral proteins

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Initiation of Synthesis of Influenza mRNAs (Cap Snatching)

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- Cellular mRNA is cleaved from 5' end and transcriptase adds it to the 3' end of -RNA segment

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Gene Expression for Ambisense RNA Viruses

bunyaviruses and arenaviruses

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- Segmented ambisense RNA genome undergoes early transcription by virion transcriptase

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Gene Expression for dsRNA Viruses

reoviruses & φ6

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- Segmented dsRNA genome undergoes virion transcriptase to make monocistronic mRNAs which undergoes translation to create viral proteins

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Conservative Gene Expression

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Original RNA is conserved and can be used again

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Semiconservative Gene Expression

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One of the original parent strands is displaced

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Gene Expression for Bacteriophage T4

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- 3 programs of transcription: Early, Middle, Late

- Acts on host to modify it

- Uses T4 promoters for early genes preferentially

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Induction of 2',5' oligo A synthetase

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Interferon is induces the production of inactive gene. In presence of dsRNA it activates and uses ATP to make pppA(2'p5'A) that activate with RNase L

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RNase L

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degrades RNA

protein synthesis is inhibited: cell dies

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Induction and Activation of Protein Kinase (PKR)

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is activated in presence of dsRNA (& ATP) and is phosphoralyated to activate eIF-2α to prevent initiation of translation which leads to cell death

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Reassortment

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Two parental flu viruses combine randomly to make a recombinant flu virus

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Copy Choice Recombination

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- Intact genomes

- Nascent chain combines with opposite parental chain