Biologi Molekular

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Biologi Molekular

• Virus
• Bacteriophage

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Definition of a Virus

• Viruses are segments of nucleic acid enclosed in
  a protein coat (virion / virus particle
  extracellular state)

Poliovirus
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Definition of a Virus

• Viruses are genetic elements that can replicate
  independently of a cells chromosomes but not
  independently of cells themselves (intracellular
  state)

a host (a place for initiating the intracellular
state)
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Properties of Viruses

• Small sizegtrangegt0.02 - 0.3 micrometers

Picornavirus (little RNA virus) is one of the
smallest viruses, about 20 nanometers in diameter

• Size alone does not differentiate viruses
  bacteria!
• smallest bacteria (e.g. Mycoplasma, Ralstonia
  pickettii)
• are only 200-300 nm long.

Smallpox virus, one of the largest viruses,
about 300 nanometers, near the resolution of the
light microscope
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Properties of Viruses

• Various morphologiespolyhedralhelicalspherical
  filamentouscomplex

Ebola virus
Rabies virus
Poliovirus
Herpes virus
Coronavirus
Lassa virus
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Properties of Viruses

• Obligate intracellular parasites

Bacteriophage T4, a virus that Infects E. coli
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Properties of Viruses

• Lack membranes and a means to generate energy

HIV
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Properties of Viruses

• Lack metabolic and biosynthetic enzymes

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Properties of Viruses

• Lack ribosomes

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Properties of Viruses

• Do not grow in sizeViruses grow by independent
  synthesis and assembly of their components inside
  of a host cell

Human adenoviruses growing in the nucleus of
their host cell
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Structure of Viruses

• The viral genome is DNA or RNA
• Most bacterial viruses contain double-stranded
  DNA
• Many animal viruses contain ds DNA or ssRNA

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Structure of Viruses

• Most common morphologies are polyhedral
  (icosahedral) and helical

Polyhedral virus
Helical virus
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Structure of Viruses

• Some viruses have additional structures animal
  viruses may have envelopes and spikes

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Structure of Viruses
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Structure of Viruses

• bacterial viruses may have tails and related
  structures

T4 virus
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Classification of VirusesCriteria

• Type of nucleic acid
• Size and morphology
• Additional structures such as envelopes and tails
• Host range gt refers to the range of cells that
  can be infected by the virus, most often
  expressed as bacteria, plant and animal hosts

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Classification of Viruses

• Comparative size and shape of various groups of
  viruses representing
• diversity of form and host range

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Some Families of Bacteriophage
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Some Families of Animal Viruses
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Some Families of Animal Viruses (continued)
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Viral genomes
DNA viruses
RNA viruses
RNA ?? DNA viruses
ss RNA (Retroviruses)
ds DNA (hepadnaviruses)
ds DNA
ss RNA
ss RNA
ss DNA

• genome can function as mRNA
• genome is template for mRNA
• genome is template for DNA synthesis
  ("retrovirus")

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The (dsDNA) Virus Life Cycle
Protein capsid
DNA
1

• Virus enters host cell (method is variable,
  involves host receptor molecule on cell surface)
• Viral DNA replicated using the host's DNA
  polymerase, nucleotides, etc.
• DNA transcribed into mRNA using host's RNA
  polymerase, nucleotides
• mRNA translated using host's ribosomes, tRNAs,
  amino acids, GTP, etc.

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3
mRNA
DNA
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capsid proteins
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The dsDNA Virus Life Cycle
Protein capsid
DNA
1

• New DNA and capsid proteins assemble into new
  virus particles, exit the cell (in various ways)

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3
mRNA
DNA
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capsid proteins
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The ssRNA (type V) Virus Life Cycle
1

• Virus enters host cell
• Capsid removed, RNA released
• complementary RNA made from genomic RNA by enzyme
  encoded in viral genome
• new genomic RNA made from complementary strand
• complementary strand is mRNA, transcribed into
  viral proteins
• Virus assembled, exits cell (by various means)

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RNA
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cRNA
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The Retrovirus Life Cycle
1

• Virus enters host cell
• Reverse transcriptase (encoded in viral genome)
  catalyzes synthesis of DNA complementary to the
  viral RNA (cDNA)
• RTase catalyzes synthesis of 2nd strand of DNA
  complementary to the first
• dsDNA incorporated into host genome ("provirus")
• provirus may remain unexpressed for a period of
  latency

RTase
RNA
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cDNA
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Host's DNA
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The Retrovirus Life Cycle
1

• Proviral genes are transcribed by host's
  transcriptional machinery into RNA
• RNA serves as mRNA for translation into viral
  proteins and as genomic RNA
• New viruses are assembled containing genomic RNA
  and Reverse Transcriptase
• Virus exits cell

RTase
RNA
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cDNA
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Host's DNA
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Bacteriophages

• Viruses that infect bacterial cells
• Two types of infections
• 1. Lytic infection phage replicates its DNA and
  lyses the host cell2. Lysogenic infection
  phage DNA is maintained by the host cell, which
  is only rarely lysed

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Bacteriophage
Prophage can exist in a dormant state for a long
time
Virulent phages only undergo a lytic cycle
Temperate phages can follow both cycles
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Lytic phages

• Clockwise Pseudomonas aeruginosa phage
  Aeromonas
• phage Shigella K II phage Listeria phage

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Life Cycle of a Lytic Phage

• Step 1 Adsorption virus attaches to the cell
  wall surface
• Step 2 Penetration entry of the viral DNA

Phage T4 adsorption to the cell wall of E. coli
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Life Cycle of a Lytic Phage

• Step 3 Synthesis of early viral proteins
• Step 4 Replication of viral DNA

Phage T2 attacks E. coli
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Life Cycle of a Lytic Phage

• Step 5 Synthesis of late viral proteins
• Step 6 Assembly
• Step 7 Lysis and release of mature viruses

Lysis of E. coli cell by Phage T4
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Life Cycle of a Lytic Phage
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Bacteriophage
Prophage can exist in a dormant state for a long
time
Temperate phages can follow both cycles
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Lysogeny

• Lysogenic phages are also called temperate phages
• Lysogenic infection begins like a lytic infection
  with adsorption of the virus and penetration of
  the viral DNA

Lambda phage, adsorbed to the surface of E. coli,
injecting Lambda DNA
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Lysogeny

• After penetration, phage DNA interates into the
  bacterial chromosomal DNA
• Integrated phage DNA is called prophage
• Prophage genes for DNA replication and coat
  proteins are repressed

Phage lambda, a lysogenic phage of E. coli
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Lysogeny

• Bacterial cell containing prophage DNA is
  lysogenized
• Lysogenized bacteria replicate the prophage DNA
• Lysogenized bacteria divide normally and appear
  normal

Phage mu,another lysogenic phage of E. coli
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Lysogeny

• Occasionally (1/10,000 in lambda) prophage
  deintegrates (excises) from the bacterial
  chromosome
• This is called derepression and leads to a lytic
  cycle that reproduces more phage particles

A lambda particle reeling in a headfull of DNA
during an occasional lytic cycle in E. coli
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Bacterial Gene TransferThrough Bacteriophage

• Transduction
• - Generalized
• - Specialized

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Generalized Transduction
Any piece of bacterial DNA can be incorporated
into the phage
Closely spaced genes can be mapped by their
cotransduction frequency.
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Specialized Transduction
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Phages host specificity
Viruses are usually very host-specific one virus
infects only one strain, maybe not even other
members of the same species Why?
Viruses enter cells via specific proteins in the
membrane
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Proteins differ, even within a species
Lipid bilayer (same in all cells) cannot be
penetrated
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• Consequences of viruses attacking specific
  proteins
• A cell cannot be totally immune to all viruses
  because it needs the membrane proteins to
  communicate with outside environment
• Best example lambda phage attacks E.coli via the
  maltose transporter. No transporter, no phage
  problembut no maltose (a sugar) also.
• So, viruses can affect uptake, etc.

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Bacteriophages Quantification

• There are three methods
• Electron Microscopy
• Epifluorescence microscopy
• Plaque Assay

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Electron microscopy Difficult,
expensive More definitiveyoure sure its a
virus More information from morphology Epifluores
cence microscopy Easy, less expensive Less
definitive viral-like particles More
quantitative
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A drop of seawater viewed with an electron
microscope (from Eric Wommack)
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Phage
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One of many phages
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diatom
bacteria
Viruses (smallest particles)
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Virus counts with epifluorescence are higher than
with electron microscopy (TEM). Why?

• Epifluorescence counts things that are not
  viruses.
• TEM misses things that are viruses
• Loss of viruses during preparation of samples for
  TEM.

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Quantification of bacteriophages by plaque assay
Ph2
plaques
host bacterial cells
lawn of host bacteria
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l forms plaques on a lawn of bacteria
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Uses for Bacteriophages

• Phages as vectors in genetic engineering and
  biotechnology designs
• Phage lytic enzymes to control infections
• Phage therapy in animals and other uses of phage
  in agriculture
• Bacteriophage therapy
• Phages for detection of pathogenic bacteria