Plasmids, Viruses

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Plasmids, Viruses Transposable elements

• Mobile Genetic Elements
• a mobile group of genetic elements that act as
  parasites
• some of these elements replicate independently of
  the host genome, but they all require the host
  cell for replication
• exploit host cells metabolism to multiply-
  powerful tools to study normal cell machinery
• Classes of these elements are
• I. Plasmids Self-replicating,
  extra-chromosomal circles usually consisting of
  of DNA(rarely, RNA) they lack a coat and cannot
  move from cell to cell.
• II. Viruses Self-replicating, infectious DNA-
  or RNA-containing elements that possess a coat
  and can move from cell to cell.
• III. Transposable Elements Mobile DNA elements
  that also lack a coat and can insert into the
  host genome.
• These elements appear to be evolutionarily
  related
• Viruses have likely evolved from plasmids and
  transposable elements

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I. Plasmids

• Properties of Plasmids
• Small, self-replicating, circular DNA (rarely,
  RNA) molecules some are very large
• - commonly found in bacteria
• - typically 2 ? 50 copies/cell (i.e., low
  and high copy)
• b. Often encode functions that are dispensable
  to the host, but may be of selective advantage
  e.g. antibiotic resistance
• c. Some can participate in plasmid-mediated
  conjugation
• - transfer of a plasmid from one cell to another
• d. Sometimes they can undergo recombination
  with each other or with host cell chromosome
• e. Replication similar to hosts chromosome
  replication they have one origin of replication
  specific sequence that allows initiation of
  replication

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• Cell Characteristics Can Be Conferred by Plasmid
  Genes
• Drug (antibiotic) Resistance
• - gene for an enzyme that can inactivate a drug
• - gene for a variant protein which is unaffected
  by a drug (i.e. can substitute for host
  equivalent)
• Virulence
• - Plasmid genes that contribute to, or are
  essential for, the virulence of a pathogenic host
  cell
• e.g., encode toxins anthrax - Bacillus
  anthracis toxin
• tetanus - Clostridium tetani toxin
• Metabolic Activities
• - Plasmid genes that affect metabolic pathways
• e.g., Nitrogen Fixation (N2 ? NH3) by Klebsiella
• e.g., degradation of octane by Pseudomonas

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• Chromosome Transfer
• - integration with host chromosome
• - conjugation plasmid chromosomal DNA
  transferred from one cell to another
• 3. Relationship of Plasmids to Viruses
• Major Difference
• - viral DNA encodes virions with coats ?
  liberated ? pass to other cells via the medium
• Many similarities suggest a close evolutionary
  relationship between plasmids bacteriophages
• Note that plasmids are extremely useful vehicles
  for recombinant DNA technology (more later).

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II. Viruses

• 1. Characteristics
• Often disease-causing agents
• Can be extremely small (e.g. less than 100 nm in
  diam..
• Genetic elements enclosed by a coat that allows
  them to move from cell to cell
• Virus multiplication is often lethal to the cell-
  lysis
• Viruses vary in several features
• Type of nucleic acid and structure of chromosome
• Structure of coat
• Mode of entry into or exit from the host cell
• Mechanism of replication
• Bacterial viruses (bacteriophages) have
  prokaryotic-like molecular biology
• Plant and animal viruses have eukaryotic-like
  molecular biology (e.g. introns were first
  discovered in the adenovirus or human
  cold-causing virus)

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• 2. Bacteriophages or bacterial viruses
• - Many different families. T2, T4 T7 are
• typical bacteriophages
• - Made up of a complex of nucleic acids and
  protein
• Only the nucleic acid enters the host cell
• Genome encodes proteins needed for packaging of
  new virus and release from host cell
• Recall that the T2 bacteriophage was used in
  experiments that established that DNA is the
  genetic material

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T4 Bacteriophage
Head
Collar
DNA
Sheath
Figure 24.22
Tail Fiber
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Lytic viral replication cycle
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• 3. Animal Viruses there are many types of
  eukaryotic viruses some are of considerable
  medical importance
• an example of an animal virus family
• Herpes Viruses DNA viruses
• Herpes Simplex Type 1 ? cold sores
• Type II ? genital lesions
• Varicella Zoster ? chicken pox shingles
• Epstein-Barr ?infectious mononucleosis
  
  
  

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4. Genome Types

• RNA Genomes (single and double-stranded genomes)
• ssRNAgenomes
• - e.g., Poliovirus, Rabies virus, HIV retrovirus
• dsRNA
• - e.g., Reovirus
• This virus genome consists of 10-12 linear
  pieces of dsRNA
• b. DNA Genomes ( also single and
  double-stranded genomes)
• i) ssDNA genomes
• - linear ? paroviruses
• - circular ? M13 phage

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• dsDNA genomes
• - linear - T4 phage
• - Herpes viruses
• - circular - Simian virus 40 (SV40)
• - sealed ends/closed - Poxvirus
• - terminal protein - Adenovirus

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5. Viral genomes encode

• genes for their own replication (in the case of
  RNA retroviruses, gene for reverse
  transcriptase-copies RNA into DNA)
• genes for taking over the hosts metabolism
  and/or integrating into the hosts genome (in the
  case of retroviruses, integration gene codes for
  integrase), where it is then replicated by the
  host machinery
• genes for capsid proteins/viral coat proteins

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6. Virus Replication

• Viruses encode some or all proteins required to
  replicate the viral genome
• As mentioned, T4 has 30 genes (out of 300) that
  encode products that lead to rapid replication of
  the phage DNA.
• clever strategies
• T4 uses 5-OHMeC (not cytosine) in its DNA
• can selectively degrade just the E. coli genome
  through T4-encoded nucleases (these will not
  attack the marked viral DNA).
• smaller (simpler) DNA viruses must use the hosts
  replication machinery but have special
  replication origins
• encode proteins which selectively promote
  replication at their own origins by recognizing
  specific DNA sequences in viral genome that act
  as origins
• Virus must overcome cellular mechanisms that
  limit replication

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6. Virus Replication (contd)

• RNA viruses - unusual situation
• Must polymerize nucleotides onto an RNA template
• Single Stranded RNA/DNA viruses replicate their
  genomes by making complementary strands
• Minus (-) Strand Viruses
• The infecting single strand is complementary to
  the protein-encoding strand
• Thus before viral proteins can be made, the
  nucleic acid coding strand () must be synthesized

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Complementary strands

• Note that (-) strand RNA virus particles always
  contain
• (-) strand RNA and an RNA-dependent RNA
  polymerase (replicase) packaged into particle
• this is used once the virus infects a cell to
  begin synthesizing new genomes
• without the replicase the (-) strand RNA genome
  cannot be replicated.
• e.g., influenza virus
• But, in the case of () strand viruses
• These viruses already carry the coding strand ie.
• the viral () strand can act directly as the
  mRNA and the replicase is synthesized from it
  right after infection
• e.g., poliovirus

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Other Specific Features of Viral Replication

• Generally, viral RNA polymerases and reverse
  transcriptases are simple proteins which do not
  have proofreading functions
• is proof reading needed in this case?
• error rate 1 in 10,000 (similar to rate seen in
  DNA transcription)
• Not a big problem for such a small genome
• Viral RNA Synthesis
• Begins at the 3-end of the RNA
  template,synthesis of 5 end of new RNA
  progressing in 5-3 direction
• Viral DNA Synthesis
• Begins at a replication origin
• Viral proteins bind at origin and recruit host
  replication enzymes

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Replication of DNA viruses

• Many different replication schemes based on
  genome form
• DNA polymerase requires a primer
• How can a linear viral DNAs be replicated? There
  is a problem for one of the strands, because the
  primer must be removed. Diverse replication
  schemes allow these molecules to be replicated
• A diverse range of replication mechanisms are
  also observed with the other viral types e.g.
  circular, sealed ends/closed, terminal proteins
  (see slide11)

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Example Linear single stranded genomes With
Terminal Repeats
Terminal repeats can form hairpin
structures 5----CTCGTAAATCAGATTTA-OH-3 5----CTC
GTAAATC 3-OH-ATTTAG
A
- the linear DNA genome can be replicated by
extending the hairpin, restricting the coding
strand, and replicating it until the end
5
extend (polymerase)
?
5
cut (endonuclease)
5
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Integration into host genome

• some viruses upon infection go into a latent
  stage
• Do not produce large numbers of progeny
• Genomes get integrated into host genome- provirus
• Bacteriophages that can integrate are called
  temperate bacteriophages best example Lambda-
  infects E. coli.
• free ends of genome join and circularized genome
  integrates via site specific recombination
• Bacterium multiplies normally until it is
  stressed (e.g. can happen during the SOS response
  due to UV damage)
• This induces the provirus to leave host and begin
  lytic cycle- saves itself from the dying
  bacterium

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Viruses and cancer

• DNA viruses that integrate into cells or exist as
  plasmids
• Sometimes result in genetic changes that cause
  the cell to proliferate in an uncontrolled way
• Transform normal cells into cancer cells - called
  DNA tumour viruses
• SV40 and polyoma viruses affect cell cycle
  regulation by making viral proteins that can
  over-ride normal growth control mechanisms of
  host cell.
• RNA tumour viruses
• Infection can lead to permanent genetic change
  in host genome that makes it cancerous
• They are retroviruses
• That reverse part of normal process of
  information flow, since RNA? DNA
• That use the reverse transcriptase enzyme
• DNA polymerase that uses either RNA or DNA as its
  template

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Retroviruses RNA viruses that integrate DNA
copies of their genome into the hosts genome -
how do you make DNA from RNA?
Simplified View
RNA
Reverse Transcriptase
DNA
RNA
DNA
Virus Particle Assembly
DNA
Integrase
integrated DNA
Transcription
Translation
host genome
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Retrovirus Replication
Reverse Transcriptase is a DNA Polymerase so it
needs a primer
Transformation of cells occurs because viral DNA
in the genome leads to production of new proteins
that alter host cell proliferation ?
oncogenes RNA tumour viruses (retroviruses)-
encode altered host proteins (often replace viral
genome sequences) that can cause cancer. DNA
tumour viruses- encode viral proteins that can
cause cancer
-transcription -translation of viral capsid
proteins and RT -assembly of new virus
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Structure of Eukaryotic Viruses
-Nucleic Acid (RNA or DNA) - Capsid ? Viral
proteins is a container for RNA or DNA
(sometimes associated with an envelope) -
Envelope ? contains Lipid Bilayer (not in present
bacteriophages) - Viral Proteins Host membrane
lipids - Envelope obtained by budding from the
plasma membrane (this can allow viruses to exit
host cell without destroying it).
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Acquisition of a Viral Envelope
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Basic Viral Life Cycle
a. Enters Host Cell - binds to host cell membrane
protein e.g., HIV ? CD4 of T-Cells e.g., ? phage
? maltose receptor - fuses with host cell
membrane (HIV) - or injects genome (T4)
b. Replicates Genome
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• Produces Viral Proteins
• Whereas transcription of DNA ? RNA is
  accomplished by host and/or viral machinery,
• translation of proteins is accomplished entirely
  by the hosts cell machinery
• Exits Host Cell
• Cell Lysis (T4)
• Budding

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Retrovirus Life Cycle
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HIV is a retrovirus

• Acquired immune deficiency syndrome (AIDS)
• Disease associated with severe defect in immune
  system due to infection of T lymphocytes (vitally
  important to defending against infections)
• Researchers isolated a retrovirus from T
  lymphocytes of infected individuals
• Virus enters cells by binding cell surface
  receptor, CD4
• eventually kills host cell (unlike most
  retroviruses)
• Stays integrated and latent for long periods-
  makes it difficult to treat with antiviral drugs
• Current research is aimed at understanding its
  life cycle to develop drugs to inhibit its
  enzymes, especially Reverse Transcriptase (RT)

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Anti-Viral Drug Therapyan Example
HIV-1 DNA replication is a target for the
anti-viral drug acyclovir (a nucleoside
analogue)
O
AC-MP Acyclovir monophosphate (trapped in
infected cells)
H
N
ACYCLOVIR
N
G
Viral thymidine kinase ? adds a phosphate group
N
H2N
N
cellular enzymes
HO
O

• AC-TP
• Acyclovir Triphosphate
• Good substrate for DNA polymerase function of
  viral RT thus, it will interfere with viral
  replication.
• Not a good substrate for host DNA polymerase

guanosine has this
OH

• soluble
• relatively non-toxic
• can penetrate the cell membrane

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Uninfected Cell
Ac-MP
ACYCLOVIR
ACYCLOVIR
Infected Cell
Ac-MP
Ac-TP
ACYCLOVIR
Viral DNA
Host DNA
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III. Transposable elements

• mobile DNA particles that cannot leave the host
  cell
• Range in size few hundred to 104 bps
• Present in multiple copies per cell
• Often encode transposase
• Catalyzes transposition from one site to another
  random site in the genome
• Retro-transposons
• Use a mechanism that is identical to retroviral
  life cycle
• (RNA copy of element?RT to make ds copy
  ?integrase to insert randomly into genome)
• But- does not encode a protein coat, can only
  move within a host cell and to progeny cells of
  host

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Direct movement of elements
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Origins of viruses

• retroviruses most likely arose from
  retrotransposons
• Plasmids were probably precursor to viruses
• Can replicate indefinitely outside of host
  chromosome
• Occur in both RNA and DNA forms
• Contain special origins of replication
• first virus probably arose when an RNA plasmid
  acquired a host gene encoding a protein that
  could be used to make a capsid
• Viral genomes have to be small- limits number of
  genes that can be encoded
• Viruses certainly have played an important role
  in evolution because of their ability to pick up
  gene sequences and carry them to different cells
  or organisms