Human Herpesviruses: An evolutionary wonder…although, not a simple one.

1. Human Herpesviruses: An evolutionary wonder…although, not a simple one. “Anything produced by evolution is bound to be a bit of a mess.” - Sydney Brenner “Around here, it takes all the running you can do just to stay in the same place.” -Lewis Carroll The Red Queen to Alice in Alice in Wonderland

2. Outline: I. General Background Coexisting with viruses: friends or foe in evolution? Outcomes of Infection DNA Virus evolution II. Herpesvirus Background and Human Cytomegalovirus (HCMV) III. Two examples of evolution: HCMV vs. Human host Innate Host Response Adaptive Host Response IV. HCMV Latency: paradigms, trends, and work in progress

3. We live and prosper in a literal sea of viruses Viruses don’t just impact our biology (disease), they are part of it. You will encounter viruses: Viruses are in everything we touch, eat, breathe: they even become part of our genome! Every milliliter of sea water has >1 million virus particles There are 1030 bacteriophage in the world’s water supply, each particle weighing a femtogram. Thus,the planet’s biomass of bacterial viruses alone exceeds that of elephants by more than 1000-fold. There are 1016 HIV particles on the planet. A whale secretes 1013 Calciviruses (whale diarrhea) each day, and they can infect humans! So, now what?!?!

4. So, you encounter a virus (and you are going to), what now?! We encounter billions of virus particles everyday, the outcome is varied: Nothing Infection and clearance Infection and latency Infection and persistence Infection and death Cancer

5. So, you encounter a virus (and you are going to), what now?!

6. Viruses are ideal and fascinating models for studying evolution Every infection results in many new viral genomes Despite a minimal number of genes, viruses exhibit remarkable diversity Phenomenal speed Virus evolution is defined in terms of population of viruses, not an individual virus particle. No individual particle represents an average for any given population. Every individual virus particle is a potential winner. The most rare genotype in a population can become the most abundant after a single selection event.

7. A few points unique to DNA viruses: DNA viruses tend to have narrow host range Persistent or latent infections common -so replication is less Replication of DNA virus genomes is less error-prone than that of RNA viruses. Errors in DNA replication can be corrected, although error rates for viruses are higher than that of cellular DNA replication.

8. Human Herpesviruses Ancient family of viruses with 9 members Relatively large (largest known human pathogen), complex viruses Double-stranded DNA genome Three subfamilies of herpesviridae Alpha (variable host range; short replicative cycle) Herpes Simplex Type 1 and 2 Varicella Zoster Virus Beta (restricted host range; long replicative cycle) Cytomegalovirus (human herpes virus type 5) Human herpes virus type 6 and 7 Gamma (lymphocyte associated) Epstein-Barr Virus Kaposi’s sarcoma-associated virus (human herpesvirus type 8) A hallmark of all herpesviruses is the ability to establish latent infections

9. Why are herpesviruses (and especially CMV) so fascinating from an evolutionary standpoint? They are ancient Latency = highly evolved While many viruses deal with evolution “passively” (i.e. mutate), herpesviruses “actively” target mechanisms

10. Cytomegaloviruses are over 200 million years old...no wonder they are so good at what they do Human CMV (HCMV) has evolved with us since the beginning of our time (prior to invertebrate-vertebrate split). For every defense mechanism we have, HCMV has at least one counter-mechanism…tit for tat Unsuccessful viruses cannot overcome host defenses.

11. Human Cytomegalovirus A complex -herpesvirus Large genome (230kb) Slow replicating Restricted host range Infects 60-90% of the population worldwide, typically asymptomatic infection Infection in immunocompromised individuals life threatening Stem cell and solid organ transplant recipients HIV infected individuals Cancer patients receiving intensive chemotherapy regimens Infection in utero: Leading cause of infectious disease related birth defects 1 in 100 infected; 1 in 1000 present symptoms/pathology Mild to severe hearing loss Cognitive deficits Physical abnormalities

12. Human Cytomegalovirus Latency is a hallmark of all herpesviruses During LATENCY, viral genomes are maintained in the absence of viral replication = No overt disease The virus exits latency by REACTIVATION resulting from poorly characterized stimuli = Disease in immunocompromised host While once considered benign, the latent infection is associated with age- related immune senescence and increased risk of atherosclerosis

13. Human Cytomegalovirus Virion Structure envelope glycoproteins tegument capsid DNA core 200 nm

14. INNATE Host Defense To contain and alert Early defense that takes care of most infections before we know what’s going on--most underrated arm of the immune system Provides early warning to activate the adaptive defenses MAJOR PLAYERS: Toll receptors (recognize microbial macromolecular patterns) cytokines/interferons (soluble messengers) natural killer cells (eliminate infected cells by direct lysis) complement (antibody-activated) Viral infection stimulates the release of interferons and interleukins (cytokines) that establish an antiviral state in the infected cell and neighboring cells

15. P STAT1 p48 STAT2 P Type-I IFN Response 2. Receptor Signal Transduction IFNR IFN/ HCMV Jak1 Tyk2 Cytoplasm P STAT1 p48 STAT2 P TLR? 3. IFN-induced Gene Transcription 1. Induction NFκB IRF3 AP1 IFN/ Production ISRE PKR OAs MxA IRF-7 Antiviral Response RIG-1 MAVS Nucleus

16. INNATE Host Defense Interferons induce death of the infected cell and also ensure that surrounding cells will die if they become infected

17. INNATE Host Defense Viruses have evolved complex strategies to by-pass the innate immune response (antiviral state) -Block the trigger: make proteins that bind dsRNA -Cytokine homologues (decoys) -Cytokine receptors (bind up/inactivate IFN) -Inhibition by viral proteins -Inhibition by viral miRNAs…maybe

18. P STAT1 p48 STAT2 P Type-I IFN Response 2. Receptor Signal Transduction IFNR IFN HCMV Jak1 Tyk2 Cytoplasm P STAT1 p48 STAT2 P TLR? 3. IFN-induced Gene Transcription 1. Induction NFκB IRF3 AP1 IE2 IE1 IFN Production ISRE X PKR OAs MxA IRF-7 Antiviral Response RIG-1 MAVS Nucleus

19. ADAPTIVE Host Response T Cells (Cellular Immunity) cytolytic T-cells (CTL) helper T-cells TH-1: activate CTL TH-2: activate antibody production B Cells (Humoral Immunity-Antibodies) viremic infections Cell-mediated defense is essential for clearing most viruses-- downside is a lot of immunopathology

20. ADAPTIVE Host Response Viruses have evolved complex strategies to by-pass the adaptive immune response: -Block MHC class I or II presentation -Bind MHC in secretory pathway -Degrade MHC -Compete with MHC -Prevent antigen loading onto MHC -Block TH1 (B cell) activation with viral IL-10 homologue

21. When T cells attack – CD8 T cells kill virus-infected cells and tumor cells Video microscopy T cell = blue Infected cell = green Red = toxic granules in T cell delivered to infected cell from Zweifach, et al. 2001. Immunity. 15:847 Elapsed time = 400 seconds

22. MHC Class I Assembly/Antigen Presentation Extracellular Matrix Cytosol Golgi ER Lumen ERp57 calreticulin calnexin tapasin b2m Transport class I hetero- trimer class I hetero- dimer TAP1 TAP2 class I H-chain Peptides Protein Proteasome

23. MHC Class I Assembly/Antigen Presentation Extracellular Matrix Cytosol Golgi ER Lumen ERp57 calreticulin US3 US8 calnexin tapasin b2m US6 US10 Transport class I hetero- trimer class I hetero- dimer TAP1 TAP2 class I H-chain UL18 US2 US11 Peptides Protein Proteasome

24. MHC Class I Assembly/Antigen Presentation Extracellular Matrix Cytosol Golgi ER Lumen ERp57 calreticulin US3 US8 calnexin tapasin b2m US6 UL18 US10 Transport class I hetero- trimer class I hetero- dimer TAP1 TAP2 class I H-chain UL18 US2 US11 Peptides Protein Proteasome

25. Virus Evasion of CD8+ T Cell Responses HCMV (US6) Surface HSV (ICP47) Adv (E3-19K) Folded Protein Proteasome TAP TAP TAP TAP Peptide Dislocation c c m t HCMV (US2, US11) ER ERGIC GOLGI MCMV (gp40) KSHV (kK5/3) Lysosome

26. Viral Latency and Persistence The reward for subverting detection and elimination

27. Herpesvirus evolution is a complex and elegant example of molecular biological warfare… It’s just not always clear who’s winning! In healthy individuals, herpetic pathologies are rarely fatal. Herpes is forever…big bonus for the virus! The better the virus, the less disease it causes, the more it is “tolerated” by the host

28. Herpes B Virus 80% of untreated cases are fatal in humans Why?

29. Herpes B Virus 80% of untreated cases are fatal in humans 22 reported cases, 20 developed encephalitis, 15 were fatal …hmmmm, very rare…

30. Herpes B Virus 80% of untreated cases are fatal in humans 22 reported cases, 20 developed encephalitis, 15 were fatal Herpes B virus infection occurs naturally in Macaque monkeys, most with NO obvious signs of infection. Those with signs of infection have small blisters or ulcers on the mouth, face, lips, genitalia. Reactivation/relapse can occur in stressed monkeys