Flashcards in DNA viruses Deck (138):
Genomic structure of poxviruses
dsDNA which is covalently closed at either end forming terminal loops, and which has inverted repeats.
Poxvirus genomic replication - general
Cytoplasmic gene expression and replication, so viral core has to carry all necessary proteins.
Viral RNAs are not spliced.
Poxvirus contribution of host nucleus
Little. Gene expression and genomic replication can occur in enucleated, but not maturation.
Poxvirus gene expression patterns
Poxvirus early genes
expressed before genomic replication
Poxvirus intermediate genes
expressed after DNA replication, but before late genes. Includes late transcriptional activators.
Poxvirus late genes
require DNA rep/intermediate gene products. Include factors for packaging.
Poxvirus - examples of early genes
RNA polymerase, TK, genes for DNA replication, viral growth factors, immune evasion factors and transcription activators for intermediate genes.
Poxvirus - transcription of early genes
Poxvirus - early gene promoters
A/T rich motif 30 bp upstreatm of mRNA start site. Not the same as a TATA box.
Poxvirus - early gene transcription factors
Come in with the virion.
Poxvirus - early gene termination signal
TTTTTNT on non-coding strand 50 bp upstream of start site.
DNA replication basics
DNA pol is fast, accurate and semiconservative.
Problems with replication - unwinding creates tension, directionality requires lagging strand, and how to prime.
Differences between eukaryotic and viral origins of replication.
Cellular origins fire once, but viral origins fire multiple times.
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'.
dsDNA viruses - examples I should know for DNA replication (5)
SV40, adenovirus, HSV, poxvirus, parvovirus.
DNA replication SV40 - key points
Origins of replication and ORI function.
Large T antigen.
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.
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.
SV40 ORI function
Stimulated by enhancer and SP1 sites. Probably enhancer used to open up chromatin.
Viruses using enhancers to improve ORI function.
SV40, adenovirus and EBV.
SV40 Large T antigen in replication
Binds ORI for initiation.
ATPase (if ablated, replication incompetant) and helicase activity for unwinding.
Recruits cellular DNA polymerase
Cellular factors for SV40 DNA replication
Cellular DNA polymerase alpha
ssDNA binding proteins
Proliferating cell nuclar antigen - stimulated polymerase.
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.
Adenovirus DNA replication - general
Mechanism - continuous replication of both strands.
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...
SSDB binds ends of viral genome. Recruits NFI which binds ORI. NFIII also recognises end. They all recruit pre-TP and NFIII.
Adenovirus replication - factors
Both viral and host factors are needed
Adenovirus replication - viral factors
Single stranded DNA binding protein (SSDB)
Adenovirus replication - host factors
NFI, NFIII, NFII/Topoisomerase I. ORPA.
Herpesvirus replication - general
Rolling circle mechanism
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.
Herpesvirus replication - rolling circle replication, evidence
Few free viral ends.
Herpesvirus replication - ORIs
Confer on plasmids the ability to replicate in an infected cell.
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.
Poxvirus DNA replication - general
Parvovirus DNA replication - general.
Dependence on cell stage
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.
Poxvirus DNA replication - factors
All viral. Include DNA pol, TK, topoisomerase, Uracil DNA glycosylase, protein kinase.
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).
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.
Parvovirus DNA replication factors
Dependent viruses: various cellular and from helper virus.
Autonosmous viruses NS1 and NS2, as well as cellular polymerase etc.
General structure for DNA viruses
Late events - topics to cover
Late gene transcription
Post-transcriptional maximisation of gene products
Packaging the viral genome
Facilitating the egress of the virions.
Late events in DNA viruses - viruses I should know about (6).
SV40, polyoma, papilloma, adeno, herpes, pox.
Late events SV40
Switching from early to late expression
Gene products destination
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.
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.
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
Late events SV40 - gene destination.
Alternative splicing to give ORF for VP1, and ORF for VP2/3.
Alternative initiation for VP2 or VP3?
Late events SV40 miRNA.
Probably targets early RNA of large T antigen for degradation.
Late events polyoma - general
Switch from early to late gene products
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. )
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.
Late events papillomavirus
Maintenance in undifferentiatied cells
Differentiation-dependent virus production.
Late events in adenovirus infection - general
Switch from early to late gene expression
Transport of late RNAs
Function of the tripartite leader.
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.
Late events - adenoviral VA RNAs.
High GC content, high abundance, constitutively expressed, interfere with interferon response.
Late events - adenoviral late RNAs
Transported to cytoplasm.
5 different tripartite leaders spliced to different 3' acceptor sites.
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.
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.
Herpesvirus gene classes
immediate-early (a), early (B), leaky late (By), strict late (y).
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.
Late events in poxvirus infection
2 classes of late genes
Late events in poxvirus infection - 2 classes of late genes.
Immediately after DNA replication.
Some time after DNA replication.
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.
Late events in poxvirus infection - late promoters.
TATA like motif.
May require cellular transcription factors.
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.
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.
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.
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.
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.
Histone code hypothesis.
demethylated deacetylated could be active
Could control MIEP.
Common mechanism of reactivation in herpesviruses
Changes in chromatin structure allow transcription of genes causing 'latency breaking'.
MIEP in HCMV
BZLF1 in EBV
ICP0 in HSV1.
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.
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.
General cell cycle progression
Controlled by cdks and control proteins.
HCMV effect on cell cycle
Induces S phase arrest
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.
Effect of inducing progression in cell cycle
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.
Places HCMV proteins are anti-apoptotic.
Death receptor engagement
TRADD/FADD interaction with caspases
Cellular stress signals
Mitochondrial outer membrane permeability
Less complex DNA viruses
Middlingly complex DNA virus
Very complex DNA viruses
Herpes and pox.
Initial events of infection DNA viruses
Purified DNA is often but not always infectious (not true for pox).
Hierarchy of viral gene expression
In humans most cause few symptoms and persist for life. Include SV40 and Merkel cell polyoma virus.
Doublestranded circular dsDNA genomes.
Monkey polyoma virus causing lytic infection in kidneys with no overt effect.
Abortively infects and transforms rodent cells in tissues.
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.
Maximising use of genome
Use both strands of DNA (polyoma)
Use overlapping genes (polyoma)
Use multifunctional genes.
T antigens of SV40
Large T antigen,
Middle T antigen
Small T antigen.
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.
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.
SV40 Large T antigen role
Determined both by direct DNA binding and by protein-protein interactions.
Affects replication, gene expression, and cell cycle.
SV40 Large T and cell cycle.
Binds p53 and pRB to stimulate S phase entry. In vivo often in terminally differentiated cells.
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.
Middle T antigen (SV40) acting as a constitutively active tyrosine kinase.
Associates with c-src and protein phosphatase-2A (PP2A) and is mitogenic
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.
Polyomavirus (SV40) early protein facilitating VP1 nuclear localisation.
Viruses to know about early events
SV40 early promoter
Core promoter (TATA box). Upsteam enhancer.
These bind cellular transcription factors.
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.
Small, non-enveloped icosahedral. Much knowledge comes from bovine papillomaviruses.
All are tropic for squamous epithelial cells. Are asympotomatic, cause warts or cause cancer.
Complexity of human papilloma viruses.
70 identified, multiple transcriptional regulation profiles.
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.
Papilloma genome replication.
Maintenance by plasmid like DNA replication.
Later viral DNA replication leads to virus production.
Cause upper respiratory tract infections.
Tumours in rodents.
Important model system for understanding eukaryotic gene expression.
Adenovirus early infection events
Attachment via fibre projections.
Uncoats and moves to nucleus.
Replication divided into early and late stages.
Adenovirus early gene expression
Early gene expression originates from at least 6 regions.
Immediate early and early genes.
Complex splicing patterns.
Adenovirus immediate early gene.
Adenovirus early genes.
E1B, E2A, E2B, E3, E4, some virion proteins.
Adenovirus immediate early gene. Trans-acting transcription regulatory factor. Differential splicing gives 2 different forms of this.
All forms have 3 highly conserved regions.
E1A - highly conserved regions.
CR1, CR2 interact with pRB.
CR3 stimulates early transcription by interacting with transcription complex.
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.
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
Co-operates to transform cells and inhibit apoptosis.
Essential for DNA replication
DNA pol, precursor to TP
Downregulation of MHC class 1.
With E2F helps activate E2 promoters.
dsDNA linear. Have terminal repeats and internal repeats bounding unique regions
HSV alpha genes
ICP0, ICP4, ICP22, ICP27, ICP47.
ICP0, ICP4, ICP27
Alters transcription, inhibits cellular splicing and is involved in viral export.
Regulation at IE (a) HSV promoters
First: VP16/Oct1/HCF activates.
Transactivates early and late genes via action on TFIIP.
Binds DNA directly to repress IE promoter.
Promiscuous transactivator. Degrades cellular repressors of IE expression.
Regulation of DNA virus transcription.
Promoters, TF, regulation of silencing, other controls of mRNA.
Regulation of DNA virus transcription - promoter
1 or many.
Sequence or cis-acting controls.
Sequence details - TATA.
Constitutively active enhancer (E1A)
Regulation of DNA virus transcription - transcription factors
Cascade of TFs.
Regulation of DNA virus transcription - regulation of silencing.
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.
Regulation of DNA virus transcription - promoter - Sequence/TFs, or cis-acting control?
Adeno and herpes have cis-acting control switch.
Regulation of DNA virus transcription - promoter - sequence of promoters
Some have TATA (SV40), others don't (pox).
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.
Pox virus early transcription factor
VETF binds VACV ealry promoter.
RAP94 gives specificity to early transcription.
No evidence of enhancers.
Poxvirus intermediate transcription.
Possibly due to genome being inaccessible to newly synthesised TFs until replicated.
Maximising late gene expression
Stability - inherent, and due to complex splicing (polyoma)
Preferential translation (tripartite leaders, adeno)
Host shut off herpes.