Download Infection cycle: DNA viruses

DNA VIRUSES
DNA (genome) replication strategies
similar in all and similar to host
Phage T4 replisome
• ssDNA becomes dsDNA
• 5’ to 3’ synthesis; need for
primer
• Variety of enzymes of host
or viral origin : DNA
polymerase
(proofreading), helicases,
ss binding proteins, ligases
• In nucleus except for
poxviruses
Replication Challenges for
DNAViruses
• Access to nucleus
• Competing for nucleotides
• Cell cycle control in
eucaryotes - S phase
dependent materials for
some
• Primer removal and
replacement (completing
ends)
Transcriptional/translational
challenges
• Access to RNA
polymerase
• Monogenic expression
in eukaryotes
• Temporal control of
gene expression
• Competition with host
for ribosomes
Bacteriophages: T4
• Linear dsDNA - ~
1.2 x 10^8 d (>280
genes)
• circular permuted
• terminally
redundant
http://www.brunel.ac.uk/depts/bl/blst/emma/molecgen/virus/lytic/lytfrm.htm
http://www.brunel.ac.uk/depts/bl/blst/emma/molecgen/virus/lytic/lytfrm.htm
Concatemer formation and packaging
of headful genome
What affect does T4 infection have on
macromolecular synthesis in the cell?
• What MOI would you use?
• How would you measure DNA synthesis? RNA synthesis?
Protein synthesis?
• How can you distinguish between phage and host DNA
synthesis?
• How can you distinguish between phage and host RNA
synthesis?
DNA
protein
Rel
conc
RNA
0
time
RNA production in cell
• Temporal control of transcription
– Immediate early: will occur in presence of ps
inhibitor What RNA-P is used?
– Delayed early - needs protein synthesis and
before DNA replication
– Late - after DNA replication begins - structural
proteins
T4 changes host RNA-P
• RNA-P - 4 subunits plus sigma
factor
• IE uses host enzyme but at
promotors that differ from E.
coli (high affinity)
• IE gene products
– modifies (ADPr) RNA-P to
recognize DE promotors
– Antitermination
– Nucleases (host DNA and
tRNA)
– Membrane repair
• DE further changes to RNA-P
– Antisigma factor (ASiA)
– Activator proteins
– Phage tRNAs
– Nucleotide metabolism
– DNA replication
• Late requires different sigma factors
T4 genome - also 127 ORFs of unknown
fucntion
Gene function
Metabolic, essential
(22)
% of known genome
functions
15
Metabolic, unessential
(60)
Structural (34)
39
Assembly,
nonstructural (19)
10
27
T7 control
• Linear dsDNA
– ~ 25 x 10^6d
• Unique with TR - how
is this formed?
• Genes are in order of
entry on chromosome
T7 promotors differ
• IE - host polymerase
• Creation of a new
polymerase/inactivation
of host polymerase
• T7 polymerase
promoter often used in
gene cloning for control
of expression
Papovaviruses
• Papilloma/Polyoma/Vacuo
lating agent
• Bidirectional replication
from single ori (similar to
Bacteria)
• Early to late strategies
– T ags in SV40 enhance
first and then
suppresses early;
– E ag in BPV is an
enhancer for late genes
– Mutations in T or
Eag/transition lead to
tumors
How do DNA Viruses Get cells out of G1
and into S phase
• Inactivate Rb/p53 - cell
cycle regulators
• SV40 uses T ag against
p53
• p53 inactivation probably
stops apoptosis
• Multiple functions for T
ag increases genome
potential
HPV Transcription using host RNA-P
• Multiple promotors
some with overlapping
reading frames
• Alternative splicing more genes for your
genome
Adenovirus - 5’protein primer
• Linear dsDNA
– 20-30 x 10^6 d
• Terminal protein
linked to 5’nucleotide
• Sequential replication
from linear DNA
• No Okazaki fragments
This is now a
template
Inverted terminal repeats
Adenovirus - transcription
• Monogenic proteins with
individual promotors
• Uses host RNA-P
• Multliple splicing of
mRNA yields different
proteins
• E1A is IE gene- activates
at other E promotors
Poxvirus: DNA with a complex morphology
•
•
•
•
•
Large genomes - 130 n- 240 x 10^6d
Denatured genome is ss circle
Replicates in cytoplasm
Brings in RNA-P; mRNA is capped
Makes all replicating enzymes
DNA replication
Herpes Simplex Virus
•Tegument - ~ 18 proteins
•Access to nucleus
–TIF (VP16 /UL48 ) trans
inducing factor
•binds with host factors
to begin transcription
•500 - 1000 copies/virion
•Determines tissue
tropism
–VHS (UL41) degrades
preexisting mRNA but is
stopped so virus can work
Families of Herpes viruses
Temporal expression of genes
Alpha and Beta proteins
• Alpha
• ICP27 - blocks host RNA
splicing
• Immune escape (MHC1
downregulation)
• Turn on Beta genes
• Beta
• DNA replication
(polymerae,binding
proteins, helicase/primase)
• Thymidine kinase
• DNA repair proteins
• Turn on Gamma/off Alpha
• Gamma
• Structural proteins
• Tegument proteins
Herpes virus supplies all DNA
machinery
• No need for cell to
be in S phase
• Model for
replication
– Rolling circle
leads to
concatemers
Thymidine kinase and Ribonucleotide
reductase are early proteins
• Needed for virulence but
not in cell culture WHY?
• TK needed to activate
acyclovir
• DNA polymerase - target
of acyclovir
• Many proteins have some
cellular homolog - stolen
genes?
– Stress response gene counter stress of viral
infection?
Packaging of Herpesviruses
Protection from host are early products
apoptosis
• Prevention of
apoptosis
• Use mutants and see
affects
• Cisplatin is apo
inducer (+ control)
wt cisplatin ICP-
KSHV v-cyclin/v-FLIP gene gives a single
transcript
• Both cell homologs
– Cyclin regulates
cell cycle
– FLIP delays
apoptosis
• How are two proteins
produced from one
message?