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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
How do adenoviruses regulate transcription?
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
26
What does E1A do in adenoviruses?
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
27
What is the effect of E1B and E1A from adenoviruses?
Transforming. E1A interacts with Rb to release E2F and up regulate S phase gene expression E1B stops apoptotic signals through p53
28
What do E2A and E2B do in adenovirus infection?
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
29
What is the structure of Herpesviridae?
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
30
What are the characteristics of alpha herpesviridae?
Variable host range, latency in neuronal cells | Includes HSV and VZV
31
What are the characteristics of beta herpesviridae?
Restricted host range, infected cells are enlarged. Latency in myeloid cells HCMV, HHV6, HHV7
32
What are the characteristics of gamma herpesviridae?
EBV, HHV8 - Kaposi associated herpesvirus | Infect T and B cells and are latent in lymphoid cell types
33
How does gene expression work in HSV-1?
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
34
What is the structure of Poxviridae?
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)
35
What do early genes in DNA viruses do?
Create and environment which supports high levels of DNA replication Expression of viral genes required for replication of viral DNA
36
What is the genome structure of poxviridae?
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
How do poxviridae enter cells?
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
How do poxviridae express their genes?
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
What does the viral core of pox viruses contain?
RNA polymerase, capping enzymes, transcription factors for early gene expression Everything the virus needs for early gene expression
40
What are the early genes expressed in pox virus infections?
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
What is the structure of the early promoter in pox viruses?
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
How does DNA replication of SV40 work?
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
What does the origin of replication for SV40 DNA look like?
64bp minimum functions by itself Enhancer regions of 72bp repeats and an SP1 site (21bp repeat) Thought that enhancer opens up the chromatin
44
What are the roles of the large T antigen in SV40 replication?
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
What cellular factors are required for SV40 DNA replication?
DNA pol alpha Topoisomerase I (relieves superhelical tension to prevent knotting) Topoisomerase II (mediates separation of daughter genomes) ssDNA binding protein PCNA
46
What is the mechanism for adenovirus DNA replication
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
What viral factors are required for adenovirus DNA replication
Pre-terminal protein (the primer) Single stranded DNA finding protein DNA polymerase All encoded in E2 transcription unit
48
What cellular factors are required for adenovirus DNA replication?
NFI (CTF) NFIII (Oct I) NFII/Topoisomerase eI ORPA (not essential)
49
What is the process of adenovirus DNA replication?
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
Where are the origins of replication in the HSV I genome?
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
What is the mechanism of genome replication of HSV I?
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
What viral factors are required for HSV I replication?
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
How does pox virus DNA replication work?
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
What are the viral factors required for poxvirus DNA replication?
Thymidine kinase (dNTP production, needed in non-dividing cells) Protein kinase DNA polymerase Topoisomerase Uracil NDA Glycosylase (part of repair and replication complex)
55
What are the basics of parvovirus DNA replication?
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
How does parvovirus DNA replication work?
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
What factors do dependent parvoviruses need for DNA replication?
``` Viral replication gene products Helices activity Cellular DNA pol Other cellular factors A helper virus ```
58
What factors do autonomous parvoviruses need for DNA replication?
``` E.g. B19 NS1 and NS2 viral genes (transcriptional activators) Viral helicase/ATPase Cellular DNA pol Other factors ```
59
What do late events in DNA virus infection do?
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
How are late events in SV40 infection triggered?
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
What is the structure of the late promoter of SV40?
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
What are the late genes made in SV40?
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
How does SV40 assemble?
Viral proteins contain a nuclear localisation signal Assembly is in the nucleus Structure is simple so assembly and maturation are simultaneous
64
How is the shift to late gene production regulated in polyomaviridae?
Same as SV40 large T | Structure of late mRNA aids translation
65
How is splicing involved in late translation of polymavirus genes?
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
How is the conversion to late gene production controlled in papillomaviridae?
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
How is the switch to late gene production controlled in adenoviridae?
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
What is the structure of the adenovirus late promoter?
One major late promoter whose primary transcript is elaborately spliced (and also has multiple polyadenylation signals) Other minor late promoters
69
What is the function of the tripartite leader sequence found on adenovirus late mRNAs?
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
What do adenoviral VA RNAs do?
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
What are the classes of late genes expressed in herpesvirus infection?
``` 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
How is the switch to late gene expression controlled in herpesvirus infections?
DNA replication results in a major shift in control of transcription Viral factors are involved in the inhibition of early promoters
73
What are the classes of late genes expressed in poxvirus infection?
Genes expressed immediately after DNA replication | Genes expressed some time after DNA replication
74
How is the switch to late gene expression controlled in poxviruses?
DNA replication induces a dramatic shift in viral gene expression
75
What is the structure of late promoters in poxviruses?
Have a TATA-like motif, may bind TBP in the cytoplasm | May require cellular transcription factors such as YY1
76
What is the structure of poxvirus late transcripts?
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
What is HCMV?
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
What are the sites of latency of HCMV and where does it reactivate?
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
What are the stages of gene expression in HCMV?
Immediate early - regulatory viral genes targeting viral and cellular transcription and the cell cycle Early - genes for DNA replication Late - packaging genes
80
How is HCMV reactivated from latency?
Latent in undifferentiated myeloid cells Differentiation can induce gene expression (immediate early genes are expressed). Virus needs certain conditions to reactivate infectious virus.
81
How is immediate early gene expression turned on in latent HCMV?
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
What are the immediate early products of HCMV?
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
What do the 2 immediate early products of HCMV do?
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
What does IE86 do in HCMV infection?
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
How does HCMV manipulate the cell cycle?
Many IE and E genes induce cell cycle advance, including IE86 Also encodes anti-apoptotic proteins
86
What pro-apoptotic signals are generated by HCMV infection?
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
What anti-apoptotic genes does HCMV encode?
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
What does IE72 do in HCMV infection?
Targets p107, releasing functional E2F and activating p107/E2F regulated gene expression

Virology (13 decks)

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