Expression

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Chapter 5

• Expression

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Learning Objectives

• Describe a generalized replication cycle for each
  of the seven virus genome types
• Explain how the pattern of gene expression of a
  virus is determined by the structure of the virus
  genome and how it is replicated
• Understand the role of post-transcriptional
  events in controlling virus gene expression

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Expression of Genetic Information

• Require host for nucleic acid and protein
  synthesis
• Virus replication is determined by tight control
  of gene expression
• Fundamental differences in the control of these
  processes in prokaryotic and eukaryotic cells
• Cells have varied and complex mechanisms for
  controlling gene expression
• Viruses have had to achieve highly specific
  quantitative, temporal, and spatial control of
  expression with limited genetic resources

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Viral Genetic Controls

• Powerful /- signals which promote/repress gene
  expression
• Highly compressed genomes in which overlapping
  reading frames are commonplace
• Control signals which are frequently nested
  within other genes
• Several strategies designed to create multiple
  polypeptides from a single mRNA

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cis or trans

• Both are regulatory loops for gene expression
• cis transcription promoters adjacent to gene
  controls
• trans transcription factors bind anywhere on
  the genome

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Control of ProkaryoteGene Expression

• Initiation of transcription is negatively
  regulated by synthesis of trans-acting repressor
  proteins, which bind to operator sequences
  upstream of protein coding sequences
• Collections of metabolically related genes are
  grouped together and co-ordinately controlled as
  'operons', typically producing a polycistronic
  mRNA

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Transcriptional Controls

• Regulated by mechanisms such as
• anti-termination (makes longer transcript)
• modifications of RNA polymerase
• sigma factor apoprotein that helps RNA pol
  recognize different promoters
• some bacteriophages make altered ? factor that
  sequester RNA pol and affect the rate of viral
  genome is transcribed
• phage T4 covalently changes RNA pol to remove the
  need for ? factor ADP-ribosylation of pol

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Post-Transcriptional Level Control

• Secondary structure of ssRNA phage genome
  regulates quantity of different phage proteins
  AND also operates temporal control in ratios
  different proteins in the infected cell

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Gene Expression Control in Bacteriophage l

• When strains of bacteria are irradiated with uv
  light they stop growing and lysed, releasing a
  crop of bacteriophage particles
• Some bacterial strains carry a bacteriophage in a
  dormant form, known as a prophage, and it can be
  made to alternate between the lysogenic
  (non-productive) and lytic (productive) growth
  cycles
• Very elegant genetic control system

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A Simplified Genetic Map of l
We will ignore the tail and head region of the
genome
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Gene Expression in l

• PL is the promoter responsible for transcription
  of the left-hand side of the ? genome, including
  N and cIII
• OL is a short non-coding region between the cI
  and N genes next to PL
• PR is the promoter responsible for transcription
  of the right-hand side of the ? genome, including
  cro, cII, and the genes encoding the structural
  proteins

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Gene Expression in ? (cont)

• OR is a short non-coding region between the cI
  and cro genes next to PR
• cI is transcribed from its own promoter and
  encodes a repressor protein which binds to OR,
  preventing transcription of cro but allowing
  transcription of cI, and to OL, preventing
  transcription of N and the other genes in the
  left-hand end of the genome

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Gene Expression in ? (cont)

• cII and cIII encode activator proteins which bind
  to the genome, enhancing the transcription of the
  cI gene
• cro encodes a small protein which binds to OR,
  blocking binding of the repressor to this site
• N encodes an anti-terminator protein which acts
  as an alternative ? (rho) factor for host cell
  RNA polymerase, modifying its activity and
  permitting extensive transcription from PL and PR
• Q is an anti-terminator similar to N, but only
  permits extended transcription from PR

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New Cell Infection

• N ( regulator) and cro are transcribed
• N allows RNA pol to transcribe genes responsible
  for DNA recombination and integration cII and
  cIII
• cII and cIII build up and cause transcription of
  cI repressor gene from own promoter
• No N, pol stops at sequence in N or Q called nut
  and qut
• N/RNA pol can overcome restriction to make full
  transcript from PL and PR
• Q/RNA pol will extend transcription in PR only

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Infection Continued

• When cII and cIII builds up, enhance cI that
  makes cI repressor protein increases
• Repressor binds OR and OL preventing
  transcription of all phage genes (except cI)
• causes lysogeny that is maintained automatically
  by negative feedback
• increased levels also cause binding to left hand
  end of OR and prevents cI transcription

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Critical Event

• cII constantly degrades (cellular protease)
• If it stays below a critical levels,
  transcription will continue from PR and PL
• productive replication and eventual lysis and
  release of phage

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cI Regulation

• Rare incidence of cII building up to cause
  increased transcription of cI
• Binds OR and OL and inhibits transcription with
  the exception of cI
• When cI is really high will bind all operators
  and shuts itself off
• cell is in lysogeny

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How to Become Lytic

• Physiologic stress induces host-cell protein RecA
  that cause cellular gene expression to adapt to
  altered environment
• RecA cleaves the cI repressor protein not
  enough to keep from lysogeny but no cI on OR
  which causes cro from PR
• Cro binds OR but at left hand end and prevents
  transcriptions of cI - -feedback loop
• locked in lytic infection

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Learned from ?

• Proteins from unrelated organisms can recognize
  and bind specific sequences in DNA
• Proteins have
• Independent DNA binding and dimerization domains
• protein cooperativity in DNA binding
• DNA looping allowing for distant sites to interact

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Control of Eukaryote Gene Expression1. Local
Configuration of DNA

• More complex than in prokaryotic cells, a
  'multilayered' approach
• DNA in eukaryotic cells has an elaborate
  structure, forming complicated and dynamic
  complexes with numerous proteins to form
  chromatin
• Transcriptionally active DNA is also
  hypomethylated compared with the frequency of
  methylation in transcriptionally quiescent
  regions of the genome
• inactive DNA is hypermethylated

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Control of Eukaryote Gene Expression2. Process
of Transcription

• Rate of transcription is a ky control point
• Initiation of transcription is influenced by
  sequences upstream of the transcription start
  site
• binding sites for highly specific DNA-binding
  proteins called transcription factors
• Immediately upstream of the transcription start
  site is a relatively short region known as the
  promoter
• Sequences further upstream from the promoter
  influence the efficiency with which transcription
  complexes form
• enhancer regions can be moved about without
  causing an influence on function
• rate of initiation depends on the combination of
  transcription factors bound to enhancers
• Make mostly monocistronic mRNA from own promoter

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Control of Eukaryote Gene Expression3. Influence
of mRNA Structure

• Stability of mRNAs varies considerably
• some having comparatively long half-lives in the
  cell
• transcripts for regulatory proteins may be very
  short
• Dependent on speed of degradation
• 5 methyl cap, 3 polyA tails and 2? structure
• Gene expression is also regulated by differential
  splicing of heterogeneous (heavy) nuclear RNA
  (hnRNA) precursors in the nucleus
• In eukaryotic cells, control is also exercised
  during export of RNA from the nucleus to the
  cytoplasm

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Control of Eukaryote Gene Expression4.
Translational Control

• Efficiency with which different mRNAs are
  translated varies greatly
• Dependent on ribosomes binding of mRNA and
  finding the AUG sequence, some sequences act as
  translational enhancer
• similar to transcriptional activators

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Genome Coding StrategiesClass I
Double-Stranded DNA

• Can be divided into two groups
• replication is exclusively nuclear (e.g.
  Adenoviridae, Polyomaviridae, Herpesviridae)
• replication occurs in the cytoplasm (Poxviridae)
• Genomes resemble ds-cellular DNA and essentially
  transcribed by the same mechanisms as cellular
  genes
• Profound differences in the reliance on the host
  cell machinery

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Polyomaviruses and Papillomaviruses

• Polyomaviruses heavily dependent on cellular
  machinery both for replication and gene
  expression
• Polyomaviruses encode trans-acting factors
  (T-antigens)
• stimulates transcription and genome replication
• Papillomaviruses in particular are dependent on
  the cell for replication, which only occurs in
  terminally differentiated keratinocytes

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Adenoviruses

• Heavily dependent on the cellular apparatus for
  transcription
• Possess various mechanisms that regulate virus
  gene expression
• trans-acting transcriptional activators E1A
  protein
• post-transcriptional regulation of expression
• Infection of cells is divided into two stages
  early and late
• late commences at the time when genome
  replication occurs

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Herpesviruses

• Less reliant on cellular enzymes than most other
  viruses in this class
• Encode many enzymes involved in DNA metabolism
  (e.g. thymidine kinase) and several trans-acting
  factors that regulate the temporal expression of
  virus genes, controlling the phases of infection
• Transcription of the large, complex genome is
  sequentially regulated in a cascade fashion
• use host cell RNA pol II
• tightly and coordinately regulated

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3 Classes of mRNA

• ? - immediate early 5 transacting regulators of
  viral transcriptions
• ? - early (delayed) non-structural regulatory
  proteins and minor structural proteins
• ? - late major structural proteins in 2
  sub-classes

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Herpesvirus Gene Expression
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Herpes Regulation

• Translation is blocked shortly after infection
• Early mRNAs immediately accumulate but no other
  viral mRNAs
• Early gene products turn off IE genes and
  initiate genome (DNA) replication
• Late structural proteins ?1 are made independent
  of genome replication but ?2 requires genome
  replication
• ? and ? genes required to initiate genome
  replication virus encoded DNA-dependent DNA
  pol DNA-binding proteins and several are
  regulated
• Enzymes from virus can alter cellular
  biochemistry
• Close control

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Poxviruses

• Genome replication and gene expression are almost
  independent of cellular mechanisms
• requires host cell ribosomes
• Genomes encode numerous enzymes involved in DNA
  metabolism, virus gene transcription, and
  post-transcriptional modification of mRNAs
• many of these enzymes are packaged within the
  virus particle (which contains gt100 proteins),
  enabling transcription and replication of the
  genome to occur in the cytoplasm almost totally
  under the control of the virus

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Poxviruses (cont)

• Gene expression is carried out by virus enzymes
  associated with the core of the particle
• Divided into two rather indistinct phases
• early genes about 50 of the poxvirus genome,
  expressed before genome replication inside a
  partially uncoated core particle resulting in the
  production of 5' capped, 3' polyadenylated but
  unspliced mRNAs
• late genes expressed after genome replication
  in the cytoplasm, but their expression is also
  dependent on virus-encoded rather than on
  cellular transcription proteins

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Genome Coding StrategiesClass II
Single-Stranded DNA

• Both the autonomous and the helper
  virus-dependent parvoviruses are highly reliant
  on external assistance for gene expression and
  genome replication
• Members of the replication-defective Dependovirus
  genus of the Parvoviridae are entirely dependent
  on adenovirus or herpesvirus superinfection for
  helper functions essential for replication
• Adenovirus genes required as helpers are the
  early, transcriptional regulatory genes such as
  E1A rather than late structural genes

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Parvo Genome Transcription

• Get series of spliced subgenomic mRNAs that
  encode Rep (involved in genome replication) and
  Cap (the capsid protein)

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Genome Coding StrategiesClass II
Single-Stranded DNA

• Geminiviridae also fall into this class of
  genomes
• Gene expression is quite different from that of
  parvoviruses, but relies heavily on host cell
  functions
• Has open reading frames in both orientations in
  the virus DNA, which means that both () and
  (-)sense strands are transcribed during infection
• May use splicing but is not fully investigated

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Genome Coding StrategiesClass III
Double-Stranded RNA

• All RNA genome viruses differ fundamentally from
  their host cells
• Reoviruses have segmented genomes and replicate
  in the cytoplasm of the host cell
• separate monocistronic mRNA from each segment
• Early in infection, transcription of the dsRNA
  genome segments by virus-specific transcriptase
  activity occurs inside partially uncoated
  sub-virus particles

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Genome Coding StrategiesClass III
Double-Stranded RNA
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Genome Coding StrategiesClass III
Double-Stranded RNA

• Primary transcription results in capped
  transcripts that are not polyadenylated
• Various genome segments are transcribed/translated
  at different frequencies advantage for the
  virus
• RNA is transcribed conservatively using the
  (-)sense strand, resulting in synthesis of
  ()sense mRNAs, which are capped inside the core
• Secondary transcription occurs later in infection
  after genome replication, inside new particles
  produced in infected cells and results in
  uncapped, non-polyadenylated transcripts
• The genome is replicated in a conservative
  fashion
• strand is used as template to make (-)sense
  strand that then can make sense strands, not 1
  to 1 as in eukaryotic semi-conservative
  replication

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Genome Coding StrategiesClass
IVSingle-Stranded ()Sense RNA

• Viral genomes act as messenger RNAs and are
  themselves translated immediately after infection
  of the host cell
• plant and animal viruses
• Class displays a very diverse range of strategies
  for controlling gene expression and genome
  replication
• There are two main strategies of gene expression
• 1 polyprotein
• 2 produce sub-genomic mRNAs in at least 2
  rounds of transcription
• both can regulate ratio of different virally
  encoded proteins and stage of replication
  relatively independent of host

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Strategy 1

• Production of a polyprotein encompassing the
  whole of the virus, which is cleaved by proteases
  to produce precursor and mature polypeptides
• picornavirus does this
• Plants with multipartite genomes make a separate
  polyprotein from each segment
• comovirus cowpea mosaic virus

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Strategy 2

• Produce sub-genomic mRNAs that will separate into
  early and late transcription
• Early stage is for non-structural proteins such
  as viral replicase
• Late stage is for structural proteins that make
  up capsid
• Can have proteolytic cleavage of a polyprotein
  but it is made from only part of the genome
• allows for regulation of ratio of the different
  polypeptides as seen in togavirus (rubella)
• produce alternative polypeptides from subgenomic
  mRNA thru stop codons and deliberate ribosome
  frame-shifting

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Genome Coding StrategiesClass V
Single-Stranded (-)Sense RNA

• The genomes of these viruses may be either
  segmented or non-segmented
• First step in the replication of segmented
  orthomyxovirus genomes is transcription of the
  () sense vRNA by the virion-associated
  RNA-dependent RNA polymerase to produce
  monocistronic mRNAs, which also serve as the
  template for genome replication
• Packaging of a virus-specific transcriptase/replic
  ase within the virus nucleocapsid is essential

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Genome Coding StrategiesClass V
Single-Stranded (-)Sense RNA
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Genome Coding StrategiesClass V
Single-Stranded (-)Sense RNA

• Viruses with non-segmented genomes also produce
  monocistronic mRNAs
• These are produced individually by a stop and
  start mechanism of transcription regulated by the
  conserved intergenic sequences present between
  each of the virus genes
• Splicing mechanisms cannot be used because these
  viruses replicate in the cytoplasm
• Ratio of different proteins is regulated both
  during transcription and afterwards

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Paramyxovirus Gene Expression

• Transcription is more at the 3 end as it is the
  structural genes and less at the 5 end where the
  non-structural genes are
• Monocistronic mRNA have advantage of having
  varying translational efficiency

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Genome Coding StrategiesClass VI
Single-Stranded ()Sense RNA with a DNA
Intermediate

• Retrovirus RNA genome forms a template for
  reverse transcription to DNA
• only ()sense RNA viruses whose genome does not
  serve as mRNA on entering the host cell
• DNA provirus is under the control of the host
  cell and is transcribed like cellular genes
• Some retroviruses have evolved a number of
  transcriptional and post-transcriptional
  mechanisms that allow them to control the
  expression of their genetic information

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Genome Coding Strategies Class VII
Double-Stranded DNA with an RNA Intermediate

• Expression of Hepadnavirus genomes is complex and
  relatively poorly understood
• Contain a number of overlapping reading frames
  clearly designed to squeeze as much coding
  information as possible into a compact genome
• The X gene encodes a transcriptional
  trans-activator believed to be analogous to the
  HTLV tax protein
• At least two mRNAs are produced from independent
  promoters, each of which encodes several proteins
  and the larger of which is also the template for
  reverse transcription during the formation of the
  virus particle

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Genome Coding Strategies Class VII
Double-Stranded DNA with an RNA Intermediate

• Cauliomavirus is similar
• 2 major transcripts 35S and 19S
• each encodes several proteins
• 35S is template for RT when forming new viruses