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Flashcards in DNA viruses Deck (138):
1

Genomic structure of poxviruses

dsDNA which is covalently closed at either end forming terminal loops, and which has inverted repeats.

2

Poxvirus genomic replication - general

Cytoplasmic gene expression and replication, so viral core has to carry all necessary proteins.
Viral RNAs are not spliced.

3

Poxvirus contribution of host nucleus

Little. Gene expression and genomic replication can occur in enucleated, but not maturation.

4

Poxvirus gene expression patterns

Early genes
Intermediate genes
Late genes

5

Poxvirus early genes

expressed before genomic replication

6

Poxvirus intermediate genes

expressed after DNA replication, but before late genes. Includes late transcriptional activators.

7

Poxvirus late genes

require DNA rep/intermediate gene products. Include factors for packaging.

8

Poxvirus - examples of early genes

RNA polymerase, TK, genes for DNA replication, viral growth factors, immune evasion factors and transcription activators for intermediate genes.

9

Poxvirus - transcription of early genes

Promoters
Transcription factors
Terminal sequence

10

Poxvirus - early gene promoters

A/T rich motif 30 bp upstreatm of mRNA start site. Not the same as a TATA box.

11

Poxvirus - early gene transcription factors

Come in with the virion.

12

Poxvirus - early gene termination signal

TTTTTNT on non-coding strand 50 bp upstream of start site.

13

DNA replication basics

DNA pol is fast, accurate and semiconservative.
Problems with replication - unwinding creates tension, directionality requires lagging strand, and how to prime.

14

Differences between eukaryotic and viral origins of replication.

Cellular origins fire once, but viral origins fire multiple times.

15

DNA replication forks general

DNA polymerase adds dNTPs onto a short primer of RNA using a second DNA strand as a templates ONLY 5' to 3'.

16

dsDNA viruses - examples I should know for DNA replication (5)

SV40, adenovirus, HSV, poxvirus, parvovirus.

17

DNA replication SV40 - key points

Bidirectional replication
Origins of replication and ORI function.
Large T antigen.
Cellular factors

18

DNA replication SV40 - bidirectional replication.

Circular DNA. Strand growing towards fork is constructed by continuous replication, strand growing away is constructed of ligated together Okazaki fragments.
Replication stops when the forks meet.

19

SV40 origins of replication.

Mapped near large T antigen binding sites, has 64 bp core sequence. Key palindromic inverted repeat forms hairpin loop important for function.
Like Murine polyomavirus ORI, but latter requires other cis-acting factors.

20

SV40 ORI function

Stimulated by enhancer and SP1 sites. Probably enhancer used to open up chromatin.

21

Viruses using enhancers to improve ORI function.

SV40, adenovirus and EBV.

22

SV40 Large T antigen in replication

Binds ORI for initiation.
ATPase (if ablated, replication incompetant) and helicase activity for unwinding.
Recruits cellular DNA polymerase

23

Cellular factors for SV40 DNA replication

Cellular DNA polymerase alpha
Topoisomerases
ssDNA binding proteins
Proliferating cell nuclar antigen - stimulated polymerase.

24

Adenovirus DNA genome

35kb dsDNA genome. 90% of replicating DNA in infected cell is viral. Can be replicated in a cell free system.
Terminal ends are inverted repeats, so single strands can form pan-handles. Covalently attached to TP.

25

Adenovirus DNA replication - general

Mechanism - continuous replication of both strands.
Factors

26

Adenovirus DNA replication - mechanism

2 stage replication.
Stage 1. Pre-terminal protein covalently linked to CTP acts as a primer, replication proceeds continuously.
Stage 2. the other template forms panhandle structures. Pre-TP covalently links and etc...
General;
SSDB binds ends of viral genome. Recruits NFI which binds ORI. NFIII also recognises end. They all recruit pre-TP and NFIII.

27

Adenovirus replication - factors

Both viral and host factors are needed

28

Adenovirus replication - viral factors

pre-TP
Single stranded DNA binding protein (SSDB)
DNA pol

29

Adenovirus replication - host factors

NFI, NFIII, NFII/Topoisomerase I. ORPA.

30

Herpesvirus replication - general

Rolling circle mechanism
Discuss ORIs
Factors required

31

Herpesvirus replication - rolling circle replication, mechanism

Linear DNA in virion is circularised in cell.
ORIs start bidirectional replication.
Later in infection, one strand genome is nicked, and the 3' end acts as a primer for continuous replication. The 'tail' of the rolling circle replication is replicated by discontinous replication.

32

Herpesvirus replication - rolling circle replication, evidence

Few free viral ends.

33

Herpesvirus replication - ORIs

Confer on plasmids the ability to replicate in an infected cell.

34

Herpesvirus replication factors

Several. Replication occurs when sufficient levels have built up.
Some are directly necessary like DNA polymerase, DNA binding proteins.
Others increase the deoxyribonucleotide pool.
Others function as repair enzymes for the newly synthesised strands.

35

Poxvirus DNA replication - general

Mechanism
Factors

36

Parvovirus DNA replication - general.

Mechanism
Factors
Dependence on cell stage

37

Poxvirus DNA replication - mechanism

1) Nick one of the two strands, use the 3' end to extend using the other strand as a template.
2) This extension re-anneals to itself due to the inverted terminal repeat, allowing self-priming.
Continuous replication means no Okazaki fragments.
Actually more complex than this.

38

Poxvirus DNA replication - factors

All viral. Include DNA pol, TK, topoisomerase, Uracil DNA glycosylase, protein kinase.

39

Parvovirus DNA replication - dependence on cell stage.

None can advance the cell into S phase.
Some require cell to pass through S phase for replication to occur. (autonomous viruses).
Others require helper viruses for replication (dependent viruses).

40

Parvovirus DNA replication - mechanism

Similar to poxviruses, but without initial nicking.
Terminal hairpin loop is used to prime, continuous replication occurs, nickases regenerate hairpin loops inn copied genome.

41

Parvovirus DNA replication factors

Dependent viruses: various cellular and from helper virus.
Autonosmous viruses NS1 and NS2, as well as cellular polymerase etc.

42

General structure for DNA viruses

Early events
DNA replication
Late events

43

Late events - topics to cover

Late gene transcription
Post-transcriptional maximisation of gene products
Packaging the viral genome
Facilitating the egress of the virions.

44

Late events in DNA viruses - viruses I should know about (6).

SV40, polyoma, papilloma, adeno, herpes, pox.

45

Late events SV40

Switching from early to late expression
Late promoters
Gene products destination

46

Late events SV40 - switching from early to late expression.

Different promoters lead to differential expression
Activity of large T.
Increase in viral genome template number.
miRNAs

47

Late events SV40 - switching from early to late expression. Large T activity.

Large T binds early promoter and inhibits DNA pol, so switches off early expression.
Large T also sequesters cellular factors for early promoter activation.
Large T activates major late promoter.

48

Late events SV40 - switching from early to late expression. Different promoters.

Late promoters lack TATA. Instead stimulated by 21bp and 72bp repeats in presence of large T

49

Late events SV40 - gene destination.

Alternative splicing to give ORF for VP1, and ORF for VP2/3.
Alternative initiation for VP2 or VP3?
Nuclear localisation.

50

Late events SV40 miRNA.

Probably targets early RNA of large T antigen for degradation.

51

Late events polyoma - general

Switch from early to late gene products
Complex splicing

52

Late events polyoma - switch from early to late gene products.

Large T activity the same as for SV40 large T.
(Also late mRNAs bigger than early so more stable. )

53

Late events polyoma - complex splicing.

Major splicing events place different 3' coding regions onto different 5' leaders, which alter stability of mRNAs and hence frequency of translation.

54

Late events papillomavirus

Maintenance in undifferentiatied cells
Differentiation-dependent virus production.

55

Late events in adenovirus infection - general

Switch from early to late gene expression
VA RNAs
Transport of late RNAs
Function of the tripartite leader.

56

Late events in adenovirus infection - switch from early to late gene expression.

Cis-acting control; only newly replicated DNA is used to make late viral transcripts. Single late promoter.
expression of 18 diff mRNAs from major single promotor.
Switch also involves transactivation by E4 of late promotor.
Late products might repress expression of early promotors and late promote competes more efficiently for TFs

IVa2 drives late promoter activity involving a transcriptional transactivator.
Possibly late products repress early transcription.

57

Late events - adenoviral VA RNAs.

High GC content, high abundance, constitutively expressed, interfere with interferon response.

58

Late events - adenoviral late RNAs

Transported to cytoplasm.
5 different tripartite leaders spliced to different 3' acceptor sites.

59

Late events - adenoviral late RNAs tripartite leaders.

Adenoviral infection leads to inactivation of eIF-4F.
eIF-4F facilitates scanning of cellular mRNAs by 40S subunit.
Tripartite leaders have little secondary structure, so less need for eIF-4F, so preferentially translated.

60

Late events herpes virus infection - general

Switch from IE to late gene expression.
Inhibition of interferon mediated protein synthesis shut-off.
Expression of viion associated host shutoff protein which causes the degradation of cellular and viral RNA.

61

Herpesvirus gene classes

immediate-early (a), early (B), leaky late (By), strict late (y).

62

Switch from IE to late gene expression.

Mechanism not fully understood. May involve autorepression by ICP4 and ICP8.
By expressed at low levels prior to DNA replication, y not at all. Both are expressed at high levels after.

63

Late events in poxvirus infection

2 classes of late genes
Late promoters.
Late transcripts.

64

Late events in poxvirus infection - 2 classes of late genes.

Immediately after DNA replication.
Some time after DNA replication.

65

Late events in poxvirus infection - late transcripts

Heterogenous 3' ends
PolyA tails at both ends
Encode structural proteins and transcription factors for the virion core.

66

Late events in poxvirus infection - late promoters.

TATA like motif.
May require cellular transcription factors.

67

Human cytomegalovirus - gene effects

IE - lead to latency
E - entry into the cell cycle, inhibition of apoptosis, virus and cell transcription.
L - synthesis of the virions.

68

Human cytomegalovirus general.

Larges known herpesvirus, a member of the B-herpesvirinae sub-family.
Latency in peripheral blood, but full productive infections in primary fibroblast cells.

69

HCMV carriage in blood

In monocytes (Taylor, 1991) - found by PCR.
Undifferentiated myeloid cells are sites of true latency in vivo - no viral IE gene expression.
Reactivation occurs when these monocytes differentiate into monocyte-derived macrophages.

70

MIEP in cytomegalovirus

The repressive chromatin structure formed at the major immediate early promoter (MIEP) elicits inhibition of IE gene expression and is a major factor involved in maintenance of HCMV latency.
Exposure of this is important in determining latency or lytic activation.

71

Regulation of MIEP (HCMVV)

Multiple cellular factors involved. Especially TF that usually modify chromatin structure. Histone deacetylases are important.
Some groups claim that NFkB binding to this is important - others say it is not.

72

Histone code hypothesis.

Roughly;
methylation silences
demethylated deacetylated could be active
acetylation activates.
Could control MIEP.

73

Common mechanism of reactivation in herpesviruses

Changes in chromatin structure allow transcription of genes causing 'latency breaking'.
Promoters involved:
MIEP in HCMV
BZLF1 in EBV
ICP0 in HSV1.

74

Model for inhibition of MIEP in non-permissive cell systems

Recruitment of SUV39H1 and HDAC1 to the modulator and enhancer lead to inhibition of chromatin acetylation, induction of histone methylation and so silencing.
YinYang1 and ERF known to mediate repression by histone post-translational modification —> recruit HDACs.

75

Mechanism of chromatin remodelling in HCMV

IE86 promiscuously activates cellular gene expression by interacting with basal transcription machinery and chromatin remodelling. IE86 can also prevent H1 mediated repression of txn via interaction with Histone acetylene PCAF

At late times of infection negatively regulates MIEP via repressive chromatin.

76

General cell cycle progression

Controlled by cdks and control proteins.

77

HCMV effect on cell cycle

Induces S phase arrest

78

HCMV induction of S phase arrest

Via many IE/E gene products effects.
Via IE86 interaction with Rb, preventing Rb-mediated repression of E2F promoters.
IE72 targets p107 releasing functional E2F proteins.

79

Effect of inducing progression in cell cycle

Pro-apoptotic signals.

80

Proapototic signals in HCMV infection

1) Inappropriate induction of cell cycle.
2) Viral DNA replication leads to lots of free viral genome ends which are proapoptotic.
3) Viral replication centres lead to unfolded protein response and ER stress.

81

Places HCMV proteins are anti-apoptotic.

Death receptor engagement
TRADD/FADD interaction with caspases
Caspases
Cellular stress signals
Mitochondrial outer membrane permeability

82

Less complex DNA viruses

Papova, parvo.

83

Middlingly complex DNA virus

Adenovirus

84

Very complex DNA viruses

Herpes and pox.

85

Initial events of infection DNA viruses

Purified DNA is often but not always infectious (not true for pox).
Hierarchy of viral gene expression

86

Polyomaviruses.

In humans most cause few symptoms and persist for life. Include SV40 and Merkel cell polyoma virus.
Doublestranded circular dsDNA genomes.

87

SV40 virus

Monkey polyoma virus causing lytic infection in kidneys with no overt effect.
Abortively infects and transforms rodent cells in tissues.

88

SV40 early events

Virus bind stimulates c-myc and c-fos.
Migrates to nucleus and uncoats. Early phase restricted to expresion of viral early genes.
Expression of T antigen.

89

Maximising use of genome

Use both strands of DNA (polyoma)
Use overlapping genes (polyoma)
Use multifunctional genes.

90

T antigens of SV40

Large T antigen,
Middle T antigen
Small T antigen.

91

SV40 early transcripts

Only from one strand of the genome (late transcripts from other strand).
Forms early T antigens, but requires splicing as not enough genomic room.

92

Splicing for SV40 T antigens.

Small T antigen - splice after translation stop codon.
Large T antigen - splice out the translation stop codon, so the rest of the mRNA is removed.
They have a common 3' splice site, so have common amino termini but different carboxy termini.

93

SV40 Large T antigen role

Determined both by direct DNA binding and by protein-protein interactions.
Affects replication, gene expression, and cell cycle.

94

SV40 Large T and cell cycle.

Binds p53 and pRB to stimulate S phase entry. In vivo often in terminally differentiated cells.

95

Middle T antigen (SV40)

Mutants are defective for replication, persistence, transformation and tumour induction in mice.
Acts as a constitutively active tyrosine kinase.
Mimics growth factor receptor.

96

Middle T antigen (SV40) acting as a constitutively active tyrosine kinase.

Associates with c-src and protein phosphatase-2A (PP2A) and is mitogenic

97

Small T antigen activity.

Binds PP2A activating MAPK and resulting in growth stimulation due to transactivation of cyclin A and cyclin D1 promoters.
Activates Akt and telomerase.

98

Agnoprotein

Polyomavirus (SV40) early protein facilitating VP1 nuclear localisation.

99

Viruses to know about early events

SV40, papillomaviruses,

100

SV40 early promoter

Core promoter (TATA box). Upsteam enhancer.
These bind cellular transcription factors.

101

SV40 early promoter - transcription factors.

AP1 (mitogen stim), AP2 (cAMP stim), NFkB, SP1 (constitutive). Act via TBP or TAFs in basal transcription complex.
Also tissue specific transcription factors.
Inhibited by T antigen binding.

102

Papillomaviruses

Small, non-enveloped icosahedral. Much knowledge comes from bovine papillomaviruses.
All are tropic for squamous epithelial cells. Are asympotomatic, cause warts or cause cancer.

103

Complexity of human papilloma viruses.

70 identified, multiple transcriptional regulation profiles.

104

Switch from early to late gene expression - papilloma viruses.

No clearly defined late promoters, but changes in transcription factor milieu with differentiation alters gene expression.

105

Papilloma genome replication.

Maintenance by plasmid like DNA replication.
Later viral DNA replication leads to virus production.

106

Adenoviruses

Cause upper respiratory tract infections.
Tumours in rodents.
Important model system for understanding eukaryotic gene expression.

107

Adenovirus early infection events

Attachment via fibre projections.
Uncoats and moves to nucleus.
Replication divided into early and late stages.

108

Adenovirus early gene expression

Early gene expression originates from at least 6 regions.
Immediate early and early genes.
Complex splicing patterns.

109

Adenovirus immediate early gene.

E1A.

110

Adenovirus early genes.

E1B, E2A, E2B, E3, E4, some virion proteins.

111

E1A

Adenovirus immediate early gene. Trans-acting transcription regulatory factor. Differential splicing gives 2 different forms of this.
All forms have 3 highly conserved regions.

112

E1A - highly conserved regions.

CR1, CR2 interact with pRB.
CR3 stimulates early transcription by interacting with transcription complex.

113

Functions of E1A (13S) of adenovirus.

Multifunctional. Needed for activation of early genes.
Activation of promoters
Immortalisation and transformation via interactions with pRB, negative regulation of some cellular promoters, and interactions with some cellular TFs.
Does not bind DNA.

114

Way E1A transactivates cellular promoters.

Cannot bind DNA, but believed to bridge between upstream TFs and basal TFs, causing induction of transcription.
Also binds pRB, releasing E2F for action on DNA

115

Adenovirus E1B

Co-operates to transform cells and inhibit apoptosis.

116

Adenovirus E2A

Essential for DNA replication

117

Adenovirus E2B

DNA pol, precursor to TP

118

Adenovirus E3

Downregulation of MHC class 1.

119

Adenovirus E4

With E2F helps activate E2 promoters.

120

Herpesvirus genomes

dsDNA linear. Have terminal repeats and internal repeats bounding unique regions

121

HSV alpha genes

ICP0, ICP4, ICP22, ICP27, ICP47.

122

ICP0, ICP4, ICP27

Alter transcription.

123

ICP27

Alters transcription, inhibits cellular splicing and is involved in viral export.

124

Regulation at IE (a) HSV promoters

First: VP16/Oct1/HCF activates.
ICP4 represses.

125

ICP4 activity

Transactivates early and late genes via action on TFIIP.
Binds DNA directly to repress IE promoter.

126

ICP0

Promiscuous transactivator. Degrades cellular repressors of IE expression.

127

Regulation of DNA virus transcription.

Promoters, TF, regulation of silencing, other controls of mRNA.

128

Regulation of DNA virus transcription - promoter

1 or many.
Sequence or cis-acting controls.
Sequence details - TATA.
Constitutively active enhancer (E1A)

129

Regulation of DNA virus transcription - transcription factors

Cascade of TFs.
Cellular TFs
Viral TFs.

130

Regulation of DNA virus transcription - regulation of silencing.

At MIEP
VP16.

131

Regulation of DNA virus transcription - promoter - 1 promoter or many

Polyomaviruses (SV40) - one promoter for early.
Papilloma = HPV has single early, multiple late. BPV has multiple.
Adeno = one key late, but minor late promoters controlling transcription of TFs.

132

Regulation of DNA virus transcription - promoter - Sequence/TFs, or cis-acting control?

Adeno and herpes have cis-acting control switch.

133

Regulation of DNA virus transcription - promoter - sequence of promoters

Some have TATA (SV40), others don't (pox).

134

Regulation of DNA virus transcription - transcription factors - viral factors

Promiscuous transactivators (E4). Some have both repressive and activation activity (Large T, ICP4).
Some are brought in with virus, others not.

135

Pox virus early transcription factor

VETF binds VACV ealry promoter.
RAP94 gives specificity to early transcription.
No evidence of enhancers.

136

Poxvirus intermediate transcription.

Possibly due to genome being inaccessible to newly synthesised TFs until replicated.

137

Maximising late gene expression

Template number
Stability - inherent, and due to complex splicing (polyoma)
Preferential translation (tripartite leaders, adeno)
Host shut off herpes.

138

Polyoma transcription

Accumulation large T ag.
Splicing