1. LATENCY, LYSOGENY and SYMBIOSIS
LIVING WITH THE HOST

2. What is latency?
a period of quiescence (restricted or no replication or symptoms) that follows acute infection (virus replication w/wo symptoms that resolve) and has potential for repeat bouts of reactivation.
host defenses are not effective (reduced or limited)
“repression” of productive cycle genes – restricted gene expression
virus genome maintained intact

3. Lysogeny occurs only in bacteria
Common to all prokaryotes
Reactivate to lytic by UV
All dsDNA viruses
Maintained and reproduce with cell
Integrated or cytoplasmic
Lysogenic conversion - new host phenotype due to expressed genes
Superinfection immunity
Insertional mutagenesis
Cell wall structure
Exotoxins

4. Latency vs Lysogeny
What cells
What factors required
State of the viral genome
What genes are transcribed
What proteins are expressed
What are the conditions for reactivation

5. Phage Lambda Nutrient rich medium goes lytic (lots of hosts)
Nutrient poor medium goes lysogenic
Stages in lysogeny
Establishment
Maintenance
Induction

6. Lambda genome

7. Lytic cycle Transcription from two promotors
IE products are N protein and cro protein
N is an antiterminator for readthrough on the left and right

8. Delayed early on right is needed for DNA replication (O, P)
Q product can turn on late genes
Still uses host RNA polymerase

9. Same events on Pl and Pr
Early termination without N
N binds to transcript (not DNA) with host factors that read through to later termination signal

10. Nut - N utilization site
Forms structure on nascent RNA
Binds with host proteins and RNA-P

11. Late genes made from new promotor Pr’ in presence of Q

12. Establishment of lysogeny
Readthrough from Pr makes CII
Readthrough from Pl makes CIII
CIII stabilizes CII from host protease Hfl
CII binds to Promotor for Repressor Establishment (PRE) and activates CI (repressor) gene
CII also activates genes for integration
pInt

13. CI binds to operators on left and right
Or 1>Or2>Or3
Or1-2 block Pr and activate PRM (promotor for repressor maintenance)
Or3 blocks PRM
Ol blocks Pl

14. Decision depends on Right side
If CRO reaches high concentration before CI it binds to Or3 and blocks PRM
At high CRO blocks Pr

15. Induction results from destruction of CI
DNA damage stimulates SOS response
RecA protease cleaves CI
CRO beats CI

18. What evidence suggests that HSV is a latent virus?
Can elicit HSV outgrowth by culturing neurons with appropriate cells.
In situ hybridization
PCR
Why is latency a good strategy?
Long term survival and immune escape
Makes for an opportunistic pathogen

19. HSV Lytic cycle
Five IE genes - use host enzymes and host and viral transcriptional activators
All IE promoters contain a common cis-acting sequence (TAATGARAT) that reacts with VP16 tegument protein
VP16 must interact with two cellular proteins, Oct-1 and HCF, to efficiently induce IE promoter activity
Cell stimulated towards apoptosis but virus stops events through several IE gene products

20. HSV establishes latency in terminally differentiated nondividing sensory neurons.
Virus infects and replicates in epithelial tissue, enters the neuron, travels via neuronal flow to cell body (regional ganglion)
May have some replication
DNA is circular episome; returns via neuron (against the flow) to epithelial surface.

21. Effective immune system resolves primary infection and enhances establishment of latency. Recurrences diminish with time (Booster affect?)
Reactivation triggers probably lead to permissive conditions – some host factor? or reduced immune response.
Evidence that transmission occurs without symptoms – is there true latency? Is there some reactivation at all times?

22. Latency associated transcript - major latency product
8 kb processed to stable – 2 and 1.5 kb
nontranslated RNA
Promotor for LAT
has neuron specific elements.
is antisense to one of the immediate early proteins, but can still get latency with fragment that does not overlap. Need 5’ 348 bp
Is neuron IE nonpermissive cell?
ICP4 binds and prevents LAT transcript in lytic cycle

23. Latency is thought to be passive – no viral products are needed to maintain.
Reactivation may require transient transcription and at least a few virions produced.This means the signal changes the transcription factors that are present.
Levels of virus DNA in neuron is same in LAT+ and LAT-
Conclusion: Lat needed for reactivation not establishment

24. Recent evidence that LAT – infection leads to increased neurovirulence (death of neurons)
Does LAT protect cell from death during latency establishment or recurrences thus increasing number of infected neurons and allowing viral reactivation?

25. LAT protects neuron from apoptosis
LAT – give “large number of TUNEL + neurons at 7 days
TUNEL – (terminal deoxynucleotidyl transferase-mediated deoxyuridine TP nick-end labeling) fluorescent/chromogenic label bind to ends of DNA so fragmented DNA gets more label.
A and D = uninfected
B and E = WT HSV
C and F = lat- HSV

26. Protection from apoptosis inducers
Infected three different types of cells with a B-gal gene expression plasmid and LAT+ (APALAT) or LAT- (BABE) in the presence and absence of apoptosis inducers.
If LAT reduces apoptosis then more cells with inducer will live and thus there will be more bgal activity than in absence of LAT gene. Compared to control plasmid with baculovirus antiapoptosis gene(CplAP)

27. The three apoptosis inhibitors work at different points in the pathway
Protein kinase
TNF
Topoisomerase
Thus LAT blocks something further in the pathway
Two other HSV genes are antiapoptotic in productive infections.

28. Apoptosis - Programmed cell death
the death receptor-mediated pathway (Fas or TNF receptor)
DNA damage through mitochondrial pathway
ability of LAT to interfere with apoptosis correlates with its ability to promote spontaneous reactivation
LAT enhances neuronal survival because it has antiapoptotic activity

30. The concept of viral symbiosis
Parasitoid wasps - use insect hosts to develop their larvae
PolyDNAviruses (PDVs) needed for success
PDVs produced in wasp ovaries and injected into insect with eggs
Viral gene expression in insect - products manipulate host immune defenses
No viral replication in insect host
Endogenous “provirus” integrated in wasp genome and lost ability to be “independent”
May go back 70 million years ago - vertical transmission

31. First PDV genome sequenced Oct 2004
570,000 bp
Composed of 30 DNA circles
156 coding sequences (white)
Only 27% genome is coding
42% of coding DNA has no known homology
Some known genes
Protein tyrosine phosphatases (signal transduction?)
Transcription factor regulators
Immunosuppressive proteins
Some genes look like modified host genes

32. So what makes this a virus?
Particles in insect
DNA may have been from derived from host
PDV DNA in wasp DNA at different regions
May have been able to encapsidate in viral protein
May be a virus that transferred replicative information to wasp
Lost unneeded functions