Virology short essay plans

Question 1

Discuss retroviral gene expression

Answer 1

• Initiation of transcription
o Host transcription machinery
 Mammalian gene promoters recruit TFs
 Cis acting elements in U3 of LTR recruit transcription factors
 Hormone or tissue development stage
o Presence of poly(A) tail: avoiding transcribing
o Why not transcription from downstream LTR?
• Transcriptional pausing
o Tat, Tar and P-TEFb
• Latency: why aren’t genes expressed?
• Increasing coding capacity
o Splicing
 Complex vs simple
 Not fully spliced RNAs – export. CTEs, direct repeat regions, cytoplasmic accumulation signals and Rev and Rex (protein pathway).
o Ribosomal frameshifting and read through
o Polyprotein processing.

Question 2

What mechanisms do viruses emply to affect cellular proliferation.

Answer 2

• Depends on cell cycle – using viral proteins.
o Growth phase – altering pathways. Homologues, receptors, downstream. Mimics, cellular, insertional.
o S phase arrest – pRb, regulating cyclins or pRb. p53 sequester. Own proteins.
o M checkpoint push through.
• Altering cellular – transactivating, histones, insertional.
• Inhibiting apoptosis. Intrinsic and extrinsic.
• Causing generalized inflammation.
How

Question 3

Discuss latency in herpesviridae.

Answer 3

•	Purpose
•	Studying and modeling. 
•	Mechanism
o	Maintaining the episome
o	Block IE expression
	Initially
•	Lack of host factors/lack of VP16
•	Repression (host factors, LATs, host miRNA).
	Maintaining 
•	Epigenetic
o	Histone code
o	DNA methylation
o	Latent DNA form
o	Key promoters
•	LATs
	EBV transcriptional programs. 
•	Reactivation
o	Stimulation
o	Pathway in different viruses
•	Effect
o	Maintenance in population
o	Neoplasias.

Question 4

Discuss mechanisms of HIV immune evasion

Answer 4

•	Hiding
o	Integrates
	Mechanism – brief. 
	Silencing
•	Evasion of recognition
o	Antigenic variation
	Error prone reverse transcriptase
o	Recombination
o	Masking
•	Counteracting effects
o	Complement
o	Interferon response induction and resistance 
o	Resistance factors
	APOBEC and Vif
	Tetherin and Vpu
o	CTL mediated killing
	Lack of T cell help
Nef actions

Question 5

Discuss differences between low and high risk HPV types.

Answer 5

•	Intro
•	High risk vs low risk
o	E6
o	E7
o	E6 and E7 expression
•	Lifecycle

Question 6

Discuss viral modulation of the cell cycle

Answer 6

Discuss viral modulations of the cell cycle.
Intro
• Draw cell cycle
• Different checkpoints
o Entry into G1 – mostly growth factors
o G1-S transition – Rb and p53, if need to be able to replicate DNA.
 Rb pathway - stimulate
 P53/apoptotic pathways - inhibit
o Mitosis checkpoint
• Now consider specific viruses
o Tax in HTLV
• Why don’t all oncogenic virus infections cause cancer?
o Continuous expression of oncogenic proteins is rare.
o Immune clearance
o Usually proteins not sufficient, also need genetic mutations.

Question 7

Discuss retroviral gene expression

Answer 7

• Initiation of transcription
o Host transcription machinery
 Mammalian gene promoters recruit TFs
 Cis acting elements in U3 of LTR recruit transcription factors
 Hormone or tissue development stage
o Presence of poly(A) tail: avoiding transcribing
o Why not transcription from downstream LTR?
• Transcriptional pausing
o Tat, Tar and P-TEFb
• Latency: why aren’t genes expressed?
• Increasing coding capacity
o Splicing
 Complex vs simple
 Not fully spliced RNAs – export. CTEs, direct repeat regions, cytoplasmic accumulation signals and Rev and Rex (protein pathway).
o Ribosomal frameshifting and read through
o Polyprotein processing.

Question 8

HCMV immune evasion

Answer 8

•	Hiding 
o	Latency
o	Immunoprivileged site
o	Molecular mimicry
•	Counteracting effectors
o	Interferon pathway
o	Counteracting antiviral state
o	CTL mediated killing.

Question 9

Virion assembly essay

Answer 9

Intro: budding most common. Lysis very rare (vaccinia), cell associated rare (measles). 
•	Can be either with or before budding
•	Encapsidation of genome
o	Concerted assembly – some icosahedral, some helical. Nucleation site. 
o	Empty shell – icosahedral.
o	Self-assembly or chaperones. 
•	Selection of genome
o	Specific signal
o	Non-specific
o	Segmented viruses – influenza, reo. 
•	Localisation of components
o	Cytoplasmic assembly
	Virus factories
	Host pathway
o	Nuclear assembly
	Nuclear localization signals
o	Export of core particles from nucleus (although many that are made in nucleus are non-enveloped)
•	Acquiring tegument
•	Acquiring the envelope.
o	At plasma membrane
	Bud during assembly
	Bud after assembly
o	Vesicular pathways
	Bud into vesicles – vaccinia, herpes. 
o	Membrane scission
•	Escaping the cell. 
•	Maturation events.

Question 10

How do viruses enter? Effect on understanding of disease and on control.

Answer 10

Viral entry: general importance – cell specificity, druggable target, virus-cell interactions. 
Section 1: Enveloped vs non-enveloped
•	Enveloped: overcoming energy barrier to membrane fusion. Binding of common attachment factors followed by binding of receptor. Conformational change. 
•	Non-enveloped: poorly understood. 
Section 2: mechanisms
•	Conformational changes.
o	1
o	2
o	3
•	Druggable targets
Section 3: Receptor specificity
•	Disease tropism
•	Manipulation for vector therapies
•	Druggable targets
•	Constraint on evolution – influenza
Section 4: beyond the plasma membrane
•	Immune effects of entry – binding, PAMPs
•	Delivery to the correct compartment.

Question 11

How does HSV establish and maintain latency?

Answer 11

• Establishment of latency in lifecycle (amplification in mucosa etc)
• Invasion of neurons – pH indep, tegument proteins, retrograde transport complex.
• Prevention of transcription
• Latent DNA structure etc
• Maintenance of genome – LATs. Importance, function, distinct ones.
• Reactivation. ICP0 and VP16

Question 12

Poliovirus co-ordination and control of rep cycle

Answer 12

• Lifecycle (brief)
• Poliovirus genome
• Translation
o IRES control in initiation
o Host cell shut off – control in maximizing.
o Circularisation and efficiency.
• Replication
o Host nuclear factors - nuclear pore cleavage
o Location
o Uridylated VpG primer
o Opposing polarity to translation: control of switch.

Question 13

Discuss the regulation of DNA virus transcription

Answer 13

Intro – hierarchy, describe different gene groups. E.g. early , DNA rep, late. Also early, intermediate, late. 
Regulation of different groups. 
•	Promoters
o	1 or many
o	Sequence or cis-acting control
o	Sequence details – TATA, enhancers.
•	Transcription factors
o	Cascade of TFs e.g. immediate early and early in herpes. 
o	Cellular 
	tissue type allows or doesn’t
	tissue differentiation stage. 
o	Viral
	Transactivating
	Acting as TFs. 
	Large T. 
•	Regulation of silencing
o	Chromatin.
•	Other controls of mRNA

Question 14

Compare and contrast persistent infections caused by lymphocytic choriomeningitis virus and by hepatitis B virus.

Answer 14

1) Importance of diseases
2) Mechanisms of persistence.
a. Immune response
b. Avoidance of immune response (evasion rather than sabotage: both have small genomes. Site, tolerance, escape)
c. Overwhelming immune response
3) Different outcomes of persistence

Question 15

Mechanisms by which viruses cause neoplasia.

Answer 15

1. Acutely transforming
2. Slowly transforming
3. Viral proteins – cell cycle checkpoints, alteration of cellular transcription.
4. Viral RNAs.
5. Increased turnover due to chronic inflammation
6. Immunosuppression.

Question 16

Discuss virus vaccines, old and new

Answer 16

1. History of vaccination
   a. Variolation
   b. Jenner
   c. Earliest forms of vaccination: live vaccines that do not cause such bad disease e.g. variola minor (in variolation), and vaccinia virus.
2. Passive versus active immunisation
3. Active immunisation: live attenuated or dead?
4. Modern vaccine development.
   a. Subunit vaccines
   b. Rational attenuation
   c. Viral vectors
   d. Nucleic acid vaccination
   e. Adjuvants
   i. Controlled release
   ii. Depot delivery
   iii. Immunostimulatory
   iv. Tolerogenic
5. Molecular insights into novel vaccines.
   a. Effective immune correlate?
   b. Challenges:
   i. Mutability - especially RNA viruses.
   ii. Multiple clades – especially RNA viruses.
   iii. Viruses that target the immune system – providing targets. E.g. HIV
   iv. Timing – early stages of infection crucial, but difficult to access.
   v. Incomplete understanding e.g. types of immunity and how to induce.
   vi. Animal models
   vii. Knowledge outstripping ability: HLA downregulation.
   viii. Some viruses: danger (e.g. Ebola)

Question 17

Clear plan - Write an essay on viral RNA and polypeptide synthesis in cells infected with negative strand RNA viruses.

Answer 17

Intro; 
Diagram
Mechanisms
1)	Transcription
2)	Translation
3)	(Post-translational modifications)
4)	Replication
Control
5)	Transcribe/replicate
6)	Packaging vRNA over cRNA

Question 18

Comparing +ssRNA and dsRNA synthesis.

Answer 18

Cellular polymerases don’t use RNA as a template
• Rdrp not same as cellular ones.
• Process of replication
o Template; Is it replicated from parent strand, replicative intermediates, or mRNA?
o Efficient utilisation of template – circularisation, oligomerisation.
o Initiation –
 starts at 3’ end – 3’ cis acting elements? Initiation the same for both sense and anti-sense? Picorna Vpg pUpU, Toga conserved not polyA.
 Subgenomic genomes made for corona or calici
o translation or replication?
 +ssRNA viruses;
• Locational specificity?
• Factor binding to cloverleaf.
• Cleavage of IRES bound PCBP
 dsRNA viruses;
• locational
• pan-handles
o Packaging; Making sure the correct sense gets packaged
• Packaging poorly understood for rotaviruses - the right genome. Channel specialisation  all made. But selection.
• Picornaviruses; massive bias towards production of +mRNA – how?
• Avoid dsRNA recognition
o Hiding – dsRNA viruses.
o Avoid translational repression of equimolar sense and anti-sense (ssRNA) – bias in replication. Limiting replicative intermediates.

Question 19

Describe the HIV replication cycle and discuss the steps currently targeted for antiviral intervention. What natural host resistance factors have been identified?

Answer 19

General intro re lifecycle.
• Entry
o CD4 binding; in development. Fusion inhibitors; enfuvirtide. Inhibitor fuzeon. Co-receptor CCR5 inhibitors maraviroc
• Reverse transcription
o Uncoating and transport. Reverse transcription. Reverse transcriptase inhibitors, vast majority anti-retrovirals
• Integration - Diagram
o Integrase interactor 1? PML? Integrase inhibitors; inhibit raltegravir.
• Transcription
o Without Tat Binding of Tat unique to HIV-1 so good target, but although some small molecule inhibitors of Tat-TAR binding have been identified, none have progressed to approval for human use.
o Transcription. Transcriptional latency maintains important reservoir. Inhibited – in development
• Splicing and export
• Packaging
o Packaging depends on dimerisation. In development.
• Release - BST-2
• Maturation of particle
• Proteolysis of viral polyproteins. Proteases.

Question 20

+ssRNA essay plan.

Answer 20

Translation: initiation, efficiency, host shut off, protein proportions.
Replication: initiation, location, translation replication switch.
Packaging: which RNA?

Question 21

-ssRNA essay plan

Answer 21

Translation: initiation, protein proportions, increasing coding capacity (leaky scanning, frameshifting). Segmented vs non-segmented.
Transcription or replication: priming. Transcription/rep switch segmented vs non-segmented. Capping and polyA. Rdrp (Normal, vs L and P). Increasing coding capacity.
Packaging correct RNA.