LATENCY, LYSOGENY and SYMBIOSIS LIVING WITH THE HOST 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 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 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 Phage Lambda • Nutrient rich medium goes – lytic (lots of hosts) • Nutrient poor medium goes lysogenic • Stages in lysogeny – Establishment – Maintenance – Induction Lambda genome 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 • Delayed early on right is needed for DNA replication (O, P) • Q product can turn on late genes • Still uses host RNA polymerase • 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 Nut - N utilization site • Forms structure on nascent RNA • Binds with host proteins and RNA-P • Late genes made from new promotor Pr’ in presence of Q 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 CI binds to operators on left and right • Or 1-3 • Or 1>Or2>Or3 • Or1-2 block Pr and activate PRM (promotor for repressor maintenance) • Or3 blocks PRM • Ol blocks Pl Decision depends on Right side • If CRO reaches high concentration before CI it binds to Or3 and blocks PRM • At high CRO blocks Pr Induction results from destruction of CI • DNA damage stimulates SOS response • RecA protease cleaves CI • CRO beats CI 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 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 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. • 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? 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 • 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 • 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? LAT protects neuron from apoptosis • • • • • LAT – give “large number of TUNEL + neurons at 7 days TUNEL – (terminal deoxynucleotidyl transferasemediated 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 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) • 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. 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 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 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 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