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DNA viruses Flashcards

1
Q

What are the major families of DNA viruses?

A 
Polyomaviridae
Papillomaviridaie
Parvovirdae
Adenoviridae
Herpesviridae
Poxviridae (cytoplasmic replication)
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2
Q

What are the similarities between DNA viruses?

A

Purified DNA is often infectious
Viral transcripts may be present in the vision (purpose unknown)
Phases of viral gene expression
Gene expression through cellular RNA pol II (apart from pox viruses)

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3
Q

What is the genome structure of SV40?

A

Small, dsDNA, circular
Viral DNA is supercoiled and associated with cellular histones in the virion
6 genes with coding on both strands, overlapping genes

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4
Q

What are some examples of polyomaviridae?

A

SV40
Mouse polyomavirus
Merkel cell polyomavirus

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5
Q

Why are polyomaviridae useful?

A

Models for DNA replication (viral and cellular) as the genome is small and there is a robust in vitro culture system

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6
Q

What is SV40? What are the characteristics of infection?

A

A polyomavirus isolated from monkey kidney cells
Causes persistant lytic infection in monkeys with no overt effects
In vitro, get lytic cycle in monkey kidney cells (with low levels of early protein expression) and an abortive infection and transformation of rodent cells (high levels of antibody against the T (for tumour) antigen)
Causes tumours in rodents

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7
Q

What is the structure of SV40?

A

Small, non-enveloped icosahedral particle with 3 capsid proteins

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8
Q

What are the entry receptors used by SV40?

A

c-myc and c-fos

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9
Q

What is the hierarchy of gene expression in SV40?

A

Large and small T antigen are expressed early

VP1,2,3 and Agno protein are late

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10
Q

What is the large T antigen of SV40?

A

A nuclear phosphoprotein
Multiple domain protein (multifunctional)
Regulates viral gene expression - turns off early and turns on late gene expression
Initiates DNA replication by binding the origin
Phosphorylation decreases DNA binding
Interacts with cellular proteins to stimulate replication
Has ATPase and helices activity
Also regulates the cell cycle through interactions with p53 and Rb

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11
Q

What is the small T antigen of SV40?

A 
Nuclear and cytoplasmic non-phosphorylated protein
Binds PP2A (protein phosphatase 2A), activates MAP kinase pathway
Transactivates cell cycle genes to stimulate growth in quiescent cells - cells are in pseudo S phase for DNA replication
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12
Q

What is the middle T antigen?

A

Only found in murine polyoma, is a membrane associated phosphoprotein
Found at the plasma membrane, increases transformation by mimicking and activated growth factor receptor
Acts as a constitutively active tyrosine kinase and associates with c-src and PP2A (protein phosphatase 2A)

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13
Q

Describe the early promoter of SV40

A

Well defined as it is a model for DNA replication
TATA box for RNA pol II binding
Upstream enhancer to modulate the core promoter - binds cellular transcription factors e.g. AP1, AP2, NFkB as well as tissue specific transcription factors
T antigen binding inhibits the early promoter to switch to late expression

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14
Q

What are the consequences of infection with polyomaviridae for humans?

A

Merkel cell polymavirus can cause merkel skin cancer
Normally have no symptoms of infection (infected early in life and virus persists)
In immunocompromised hosts e.g. AIDs or after transplantation, get disease

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15
Q

What are the consequences of infection with mouse polyomavirus for mice?

A

Cause solid tumours if the mouse is also infected with murine leukaemia virus

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16
Q

What is the genome structure of papillomaviridae?

A

8kb, dsDNA, cirucular
Associated with cellular histones to form a chromatin like structure
7 early genes and 2 late genes

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17
Q

What are the characteristics of Papillomaviridae?

A

Small, non enveloped icosahedral particles, 2 capsid proteins (1 major 1 minor), strains are highly species specific
Bind squamous epithelium
Normal infection gives a wart
Some are highly oncogenic

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18
Q

What is the structure of the HPV16 genome?

A

1 promoter for early genes - p97. Early genes are polyadenylated at pAE
1 promoter for late genes, p670. Only active in differentiated epithelial cells. Are polyadenlyated at pAL

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19
Q

How do papillomaviridae control their gene expression?

A

Virus infects basal cells of skin, get early gene expression.
Viral genome is maintained in dividing cells by plasmid like DNA replication (E1 and E2)
E6 and E7 are expressed as cells differentiate
Late gene expression is restricted to terminally differentiated cells and results in virion production

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20
Q

What are the functions of E1, E2, E5, E6 and E7 in papillomaviridae?

A

E1 - Initiation of DNA replication (helicase)
E2 - transcriptional regulation and DNA replication (attachment to host genome)
Together, E1 and E2 do plasmid maintenance and recruit cellular DNA pol to other early genes
E5 - transforming protein, interacts with growth factor receptors
E6 - transforming protein, binds p53 for degradation
E7 - transforming protein, binds Rb

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21
Q

What are parvovirdae?

A

Single stranded linear DNA viruses, such as adeno-associated virus (dependent)

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22
Q

What is the structure of adenoviridae?

A

Double stranded linear DNA viruses, 35kb, with a protein attached to the 5’ end of each strand (the TP protein, 55kDa)
Terminal sequence of each strand is inverted repeats (denatured single strands can form a pan handle)
Non-enveloped

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23
Q

What are the consequences of an adenovirus infection?

A

Upper respiratory tract infection

Can cause tumours in rodents

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24
Q

How do adenoviruses infect cells

A

Virus attaches to cell by a fibre projection

Enters phagocytic vacuoles, uncoats, moves to nucleus where it replicates

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25
Q

How do adenoviruses regulate transcription?

A

3 phases
Immediate early (including E1A which is a trans acting transcriptional regulator for other early genes)
Early (including E1B, E2A, E2B, E3 and E4)
Late (including vision proteins
Early genes are expressed from at least 6 genome regions with complex splicing patterns

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26
Q

What does E1A do in adenoviruses?

A

Trans acting transcriptional regulator needed for activation of transcription of early genes and some cellular genes
Different splicing gives 2 proteins (13S and 12S)
13S has 3 conserved regions (CR1-3); 12S doesn’t have CR3
CR3 stimulates early transcription and interacts with cell transcription initiation complex
CR1 and CR2 interact with Rb, releasing E2F and DP to get transcription from E2 promoters (up regulate cellular S phase gene expression)
Doesn’t bind DNA directly - binds cellular transcription factors and interacts with TBP
Links upstream transcription factors with the basal transcription complex

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27
Q

What is the effect of E1B and E1A from adenoviruses?

A

Transforming.
E1A interacts with Rb to release E2F and up regulate S phase gene expression
E1B stops apoptotic signals through p53

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28
Q

What do E2A and E2B do in adenovirus infection?

A

Involved in DNA replication. E2B is the polymerase and also the precursor to the terminal protein TP which is covalently attached to the 5’ end of the protein

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29
Q

What is the structure of Herpesviridae?

A

Double stranded linear DNA that circularises upon entry
Terminal repeats and internal repeats around unique regions
3 virus types (alpha, beta, gamma) depending on genome structure

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30
Q

What are the characteristics of alpha herpesviridae?

A

Variable host range, latency in neuronal cells

Includes HSV and VZV

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31
Q

What are the characteristics of beta herpesviridae?

A

Restricted host range, infected cells are enlarged. Latency in myeloid cells
HCMV, HHV6, HHV7

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32
Q

What are the characteristics of gamma herpesviridae?

A

EBV, HHV8 - Kaposi associated herpesvirus

Infect T and B cells and are latent in lymphoid cell types

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33
Q

How does gene expression work in HSV-1?

A

Immediate early - 5 genes expressed. ICP0 and ICP4 interact with the viral genome to make nuclear complexes. ICP47 modulates expression of viral antigens to the immune system. Promoter has a motif which binds cellular Oct1 which is bound by VP16. VP16 interacts with the basal transcription complex through TBP
Early - encodes genes associated with DNA replication
Late - structural protein

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34
Q

What is the structure of Poxviridae?

A

Double stranded linear DNA with covalently closed ends
Cytoplasmic replication
Large genome - 300kb
Oval particles, 200-400nm long
Contain many proteins
Note: viral DNA is not infectious - it needs incoming virion proteins for DNA replication and gene expression (doesn’t hijack host)

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35
Q

What do early genes in DNA viruses do?

A

Create and environment which supports high levels of DNA replication
Expression of viral genes required for replication of viral DNA

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36
Q

What is the genome structure of poxviridae?

A

Double stranded linear DNA, with ends covalently closed (so when denatured get a single strand loop)
Inverted terminal repeats
Tandem repeats in the genome

37
Q

How do poxviridae enter cells?

A

Binds undefined receptor (seems to be HA mediated)
Virus uncoats in 2 stages - outer membrane removed on entry, particle further uncoated as the core passes into the cytoplasm
Viral DNA replication and gene expression is cytoplasmic

38
Q

How do poxviridae express their genes?

A

Uses viral proteins (as not in the nucleus) that are associated with the core
3 phases
Early - before DNA replication, 50% genome
Intermediate - after DNA replication, including late transcriptional activators
Late - after DNA replication, dependent on DNA replication activity, includes factors to be packaged to initiate transcription in a new cell

39
Q

What does the viral core of pox viruses contain?

A

RNA polymerase, capping enzymes, transcription factors for early gene expression
Everything the virus needs for early gene expression

40
Q

What are the early genes expressed in pox virus infections?

A

RNA polymerase (has 9 subunits) - needed as original RNA pol is degraded/might not be able to do intermediate/late gene expression
Transcription factors for intermediate gene expression
PolyA polymerase
Capping enzymes
Thymidine kinase
Viral growth factor
DNA replication enzymes (e.g. nicking, joining, DNA topoisomerase, DNA polymerase)

41
Q

What is the structure of the early promoter in pox viruses?

A

An A/T rich motif, not the same as a TATA box
Early transcription factors bind to initiate transcription
Early mRNAs have a termination signal on the non coding strand

42
Q

How does DNA replication of SV40 work?

A

Bi-directional from a single origin, so same as cellular (makes it a good model)
Leading and lagging strand with okazaki fragments
2 replication forks that go in either direction until they meet

43
Q

What does the origin of replication for SV40 DNA look like?

A

64bp minimum functions by itself
Enhancer regions of 72bp repeats and an SP1 site (21bp repeat)
Thought that enhancer opens up the chromatin

44
Q

What are the roles of the large T antigen in SV40 replication?

A

Binds ORI to initiate replication
Has ATPase activity and helicase activity for unwinding DNA
Unwinding provides access for DNA pol
May help recruit DNA pol by direct interaction

45
Q

What cellular factors are required for SV40 DNA replication?

A

DNA pol alpha
Topoisomerase I (relieves superhelical tension to prevent knotting)
Topoisomerase II (mediates separation of daughter genomes)
ssDNA binding protein
PCNA

46
Q

What is the mechanism for adenovirus DNA replication

A

Pre-terminal protein (Pre-TP) acts as a primer for DNA replication
Pre-TP is covalently linked to CTP (which recruits the viral polymerase)
Get continuous replication on both strands, 5’ -> 3’
Lower strand is displaced once DNA synthesis begins, forming a pan handle structure

47
Q

What viral factors are required for adenovirus DNA replication

A

Pre-terminal protein (the primer)
Single stranded DNA finding protein
DNA polymerase
All encoded in E2 transcription unit

48
Q

What cellular factors are required for adenovirus DNA replication?

A

NFI (CTF)
NFIII (Oct I)
NFII/Topoisomerase eI
ORPA (not essential)

49
Q

What is the process of adenovirus DNA replication?

A

Ends of the viral genome bind single stranded DNA binding protein
ss DNA BP recruits cellular transcription factor NFI
NFI and NFIII bind to specific site in the origin
NFI interacts with viral DNA pol
NFIII allows recruitment of pre-TP and polymerase into the pre initiation complex
Covalent linkage between pre-TP and dCMP primes for initiation
Get DNA synthesis
Genome is packaged into pre-foremen capsomere using a packaging signal
Pre-TP is processed to TP

50
Q

Where are the origins of replication in the HSV I genome?

A

3 origins - 1 ORI(l) and 2 ORI(s)
Both are AT rich imperfect palindromes
Are all in transcriptionally active areas
Virus only needs one

51
Q

What is the mechanism of genome replication of HSV I?

A

Linear genome circularises
DNA is denatured by the viral origin binding protein (UL9)
Helicase/primase complex and single stranded binding proteins (UL5,8,52 and UL290
Genome is nicked at point of joining when circularisation happens
3’ end acts as a primer
Get continuous replication - many genomes joined together
Get discontinuous replication (okazaki fragments)
Genomes are cleaved to separate - cleavage point can differ, given different genome isomers

52
Q

What viral factors are required for HSV I replication?

A

7 viral factors
DNA polymerase
DNA binding proteins x2
ORI binding protein
Helices/primase complex (3 proteins)
DNA replication when enough of these proteins have been made
Other early proteins increase the dNTP pool, these are not essential for virus replication

53
Q

How does pox virus DNA replication work?

A

The linear double stranded genome with covalently closed ends is nicked on one strand just before the loop
The short remaining bit of double stranded DNA acts as the primer for continuous DNA synthesis
The strand synthesised then folds back and acts as a primer for more continuous DNA synthesis
The process is repeated

54
Q

What are the viral factors required for poxvirus DNA replication?

A

Thymidine kinase (dNTP production, needed in non-dividing cells)
Protein kinase
DNA polymerase
Topoisomerase
Uracil NDA Glycosylase (part of repair and replication complex)

55
Q

What are the basics of parvovirus DNA replication?

A

Very small virus, so requires cell to be in S phase (needs many cellular factors). Can’t advance cells into S phase
Autonomous viruses just need S phase
Dependent viruses also need helper viruses to co-infect cells e.g. Adeno-associated virus
Genome has hairpins at the end which are essential for DNA replication (ssDNA normally)

56
Q

How does parvovirus DNA replication work?

A

The 3’ hairpin acts as a primer for continues DNA synthesis

Nickases regenerate the final hairpin loops in the copied genome (nick and elongate from the nick)

57
Q

What factors do dependent parvoviruses need for DNA replication?

A 
Viral replication gene products
Helices activity
Cellular DNA pol
Other cellular factors
A helper virus
58
Q

What factors do autonomous parvoviruses need for DNA replication?

A 
E.g. B19
NS1 and NS2 viral genes (transcriptional activators)
Viral helicase/ATPase
Cellular DNA pol
Other factors
59
Q

What do late events in DNA virus infection do?

A

Drive late gene transcription (activate late promoters, repress early)
Maximise expression of late gene products (often by post transcriptional control mechanisms)
Package viral genomes
Facilitate egress of virions

60
Q

How are late events in SV40 infection triggered?

A

Large T antigen binding to the early promoter region for DNA replication inhibits binding of RNA pol for early gene expression
Large T also sequesters cellular factors necessary for early promoter activity
Therefore get late promoter activation
Also, viral mRNAs expressed from the late promoter target early mRNAs for degradation by RNAi
Get more late gene expression as there are more genomes due to replication

61
Q

What is the structure of the late promoter of SV40?

A

Late genes are on the opposite strand to the T antigens
Has no TATA box (allows difference in regulation)
Stimulated by 21bp repeat elements and 72bp repeats in the presence of large T

62
Q

What are the late genes made in SV40?

A

Agno, VP1, VP2 and VP3
The VP3 gene is encoded within VP2 (genes overlap))
Agno is a small protein which enhances viral assembly and spread

63
Q

How does SV40 assemble?

A

Viral proteins contain a nuclear localisation signal
Assembly is in the nucleus
Structure is simple so assembly and maturation are simultaneous

64
Q

How is the shift to late gene production regulated in polyomaviridae?

A

Same as SV40 large T

Structure of late mRNA aids translation

65
Q

How is splicing involved in late translation of polymavirus genes?

A

Late mRNAs have a leader sequence at the 5’ end. 2 leader sequences are spliced together, then spliced to a coding region (e.g. for VP1). Leader sequences are believed to alter mRNA stability to up regulate translation

66
Q

How is the conversion to late gene production controlled in papillomaviridae?

A

Dependent on cellular differentiation
In differentiated cells, the early promoter is suppressed along with the early polyadenylation signal (likely involving E2)
This drives late gene transcription

67
Q

How is the switch to late gene production controlled in adenoviridae?

A

Only newly replicated DNA is used for late gene transcription (cis acting control switch)
Transcriptional transactivator encoded by E4 aids the switch
IVa2 (late gene) encodes a transcription factor to drive the late promoter
There maybe competition for transcription factors between early and late promoters (late wins)
Late products may repress the early promoters
Viral late mRNAs are selectively transported to the cytoplasm. This involves cellular transport factors and a viral protein complex
Late viral mRNAs have a leader sequence (spliced in from 3 regions of the genome) which ensures preferential translation

68
Q

What is the structure of the adenovirus late promoter?

A

One major late promoter whose primary transcript is elaborately spliced (and also has multiple polyadenylation signals)
Other minor late promoters

69
Q

What is the function of the tripartite leader sequence found on adenovirus late mRNAs?

A

Has little secondary structure, allowing ribosomes to scan through without the helicase activity of eIF4F
The virus inactivates eIF4F so only mRNAs which don’t require eIF4F are translated - cellular mRNAs are not, and neither are early viral mRNAs (which resemble cellular mRNAs)

70
Q

What do adenoviral VA RNAs do?

A

Transcribed using RNA pol III, they are 160p in length

They block action of protein kinase R which responds to interferon and dsRNAs to stop translation

71
Q

What are the classes of late genes expressed in herpesvirus infection?

A 
Leaky-late (beta gamma) - expressed at low levels prior to DNA replication, peaks after replication is initiated
Strict late (gamma) - require DNA replication to be activated
72
Q

How is the switch to late gene expression controlled in herpesvirus infections?

A

DNA replication results in a major shift in control of transcription
Viral factors are involved in the inhibition of early promoters

73
Q

What are the classes of late genes expressed in poxvirus infection?

A

Genes expressed immediately after DNA replication

Genes expressed some time after DNA replication

74
Q

How is the switch to late gene expression controlled in poxviruses?

A

DNA replication induces a dramatic shift in viral gene expression

75
Q

What is the structure of late promoters in poxviruses?

A

Have a TATA-like motif, may bind TBP in the cytoplasm

May require cellular transcription factors such as YY1

76
Q

What is the structure of poxvirus late transcripts?

A

Have heterogeneous 3’ ends that can self anneal
Late mRNAs have no termination signal (unlike early mRNAs)
Have polyA tail at 5’ and 3’ end
Encode structural proteins and transcription factors packaged in the virion to initiate viral replication

77
Q

What is HCMV?

A

Human cytomegalovirus
Beta herpesvirus
Rarely causes disease in primary infection unless immunonaive or immunosuppressed
Infection persists for life, reactivation can be fatal in patients with AIDs or after transplantation
Species specific infection

78
Q

What are the sites of latency of HCMV and where does it reactivate?

A

Latent in the peripheral blood - monocytes and their precursors. Genome exists as an episome
Reactivation results in infection in many cell types (endothelial, vascular smooth muscle etc). High level production is seen in primary fibroblast cells

79
Q

What are the stages of gene expression in HCMV?

A

Immediate early - regulatory viral genes targeting viral and cellular transcription and the cell cycle
Early - genes for DNA replication
Late - packaging genes

80
Q

How is HCMV reactivated from latency?

A

Latent in undifferentiated myeloid cells
Differentiation can induce gene expression (immediate early genes are expressed). Virus needs certain conditions to reactivate infectious virus.

81
Q

How is immediate early gene expression turned on in latent HCMV?

A

Cellular factors control IE gene expression through a major promoter enhancer (MIEP)
Yin Yang 1 (YY1) and ERF mediate repression by histone post-translational modification - recruit HDACs (inhibit promoter)
Reactivation occurs by changes in the chromatin structure

82
Q

What are the immediate early products of HCMV?

A

Get differential splicing of the RNA transcript to give 2 major products
Regions 2 and 3 are in both, one product has region 4, the other has region 5
234 makes the nuclear phosphoprotein
235 also makes a nuclear phosphoprotein

83
Q

What do the 2 immediate early products of HCMV do?

A

They are both nuclear phosphoproteins
234 is a weak positive auto regulator. It associates loosely with chromatin and is a week independent activator of some cellular genes. Activates the TATA-less DNA polymerase alpha promoter. Increases activation of viral early and late promoters. interacts with some cellular factors, but not general transcription factors
235 is a negative auto regulator and can directly bind DNA. Activates early and late genes and interacts with a number of cellular transcription factors and cell cycle regulatory proteins. Advances the cell cycle through G1/S block. Interacts with general transcription factors such as TBP

84
Q

What does IE86 do in HCMV infection?

A

Promiscuously activates cellular gene expression by interacting with basal transcription machinery and chromatin remodelling factors
Negatively regulates MIEP (enhancer of IE genes) by inducing repressive chromatin
Interacts with PCAF (a HAT) to open up chromatin
Also interacts with Rb to aid progression of cell cycle into S phase (E2F is released). IE86 also binds to MCM3-AP (a cellular licensing factor) to inhibit cellular DNA synthesis

85
Q

How does HCMV manipulate the cell cycle?

A

Many IE and E genes induce cell cycle advance, including IE86
Also encodes anti-apoptotic proteins

86
Q

What pro-apoptotic signals are generated by HCMV infection?

A

Chemokine and cytokines are released (and detected)
Viral replication centres aggresomes, triggering unfolded protein response and ER stress
Viral DNA replication
Cell cycle advance

87
Q

What anti-apoptotic genes does HCMV encode?

A

Many. Has genes that act on death receptors (extrinsic and intrinsic), has genes that activate cellular anti-apoptotic genes e.g. FLIP, has genes that manipulate mitochondria permeability, has genes that inhibit intrinsic apoptotic pathway

88
Q

What does IE72 do in HCMV infection?

A

Targets p107, releasing functional E2F and activating p107/E2F regulated gene expression

Virology (13 decks)

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