1. Human Viral Disease; Virus Replication Cycle

2. Human-Virus Interaction Virus extinction Clear virus, immunity Large number of deaths Small Population –Favors persistent virus infection –Virus infection with an immunological naïve person – i.e. herpes simplex virus, parent to newborn Large Population –Many susceptible to infection –Virus infected individuals available all the time –Sporadic spread of virus –i.e. common “cold” virus, school class room

3. Asymptomatic infection – no disease symptoms Acute infection – disease symptoms Persistent infection – long term –Chronic: infectious virus –Latent: no virus replication, virus reactivation Transformation – alter cell regulation, tumor production, cancer –No infectious virus –Viral DNA, complete or partial Patterns of Virus Disease

4. DNA Virus Infections

5. RNA Virus Infections

6. Acute Infection: Varicella- zooster virus (VZV) Herpesvirus – one virus, two diseases Varicella virus Chickenpox; common childhood disease Primary acute mucosal/skin infection Resolves in 1-2 weeks Virus infect and latent in nerve cells

7. Persistent Chronic Infection: VZV Zooster virus Shingles Latent in nerve tissue Presence of viral DNA, no infectious virus Virus held in check by host immune defense Later in life, reactivation of virus, replicates, descends down nerve tissue, replicates in skin cells

8. One-Step Virus Replication In Cell Culture High level of virus infection (1-10 virus/cell) Synchronous virus replication in cells All events required for cell infection

9. Virus Replication Cycle Attachment (Adsorption) Entry / Uncoating (Penetration) Gene Expression (Synthesis: Early, Genome, Late) Assembly (Maturation) Release (Lysis, Budding)

10. Virus Attachment (Adsorption) Contact and interaction of virus to host cell Recognition of virus to host cell Virus molecule that binds to host cell called ligand (viral protein or glycoprotein)

11. Attachment: Host Cell Virus binds to host cell molecule - receptor (i.e. cell protein, glycoprotein, lipid) Receptors are molecules that have a role in normal functioning of the cell

12. Attachment: Cell Receptor Virus may bind up to three different cell receptors in succession: –Low affinity receptor - in high abundance, virus contacts cell surface –Primary receptor - in lower concentration –Co-receptor – follows binding of primary receptor

13. Attachment: Specificity Host Range - the organism(s) that the virus is able to infect (narrow or wide) i.e. plant, animal, human Tissue Tropism- the cell type(s) a virus is able to infect i.e. skin, oral, GI, CNS

14. Attachment: Binding 3-D fit between viral ligand and cell receptor Mainly weak electrostatic charges. Evidence for this is interaction may require: –specific pH –specific ionic strength –presence of specific ions i.e. Ca ++, Mg ++

15. Attachment: Nonenveloped Picornavirus Virus ligand - a deep cleft (“canyon”) in triangular face of capsid (viral proteins VP1, VP2, VP3) Binds to cell receptor ICAM –1 (intracellular adhesion molecule 1), normal function is to bind cells i.e. WBC

16. Attachment: Nonenveloped Virus to Host Cell Membrane

17. Attachment: Enveloped HIV Virus Host cell protein in virus envelope (cyclophilin A) initially binds HIV to low affinity receptor (heparin sulfate) of the cell Followed by binding of viral ligand (gp120) to primary receptor (CD4) on T helper cells, macrophages, and glial cells Binding of gp120 to CD4 results in conformational change of gp120, which then binds to chemokine co- receptor CXCR4 on T lymphocytes or CCR5 on macrophages

18. Attachment: Enveloped Virus to the Host Cell Membrane

19. Entry / Uncoating Entry is the mechanism used by the virus to penetrate into the host cell Uncoating is the separation of the nucleic acid from the capsid, and refers to changes that occur to make the viral nucleic acid ready for expression

20. Entry: Nonenveloped Virus Receptor-mediated endocytosis Clathrin coated pits (seen by EM) Invagination, pinch off membrane Forms intracellular endosome, contains the virus Endosome becomes acidified

21. Uncoating: Nonenveloped Virus Acid pH causes conformational changes in capsid protein Hydrophobic region interacts with membrane, forms a pore Viral nucleic acid released

22. Entry / Uncoating: Nonenvelpoed Poliovirus

23. Nonenveloped Virus: Endocytosis

24. Entry / Uncoating: Enveloped Virus Receptor mediated fusion of virus envelope with cell plasma membrane Two modes of entry: –Direct entry (pH independent) –Receptor-mediated endocytosis (pH dependent; for uncoating)

25. Direct Entry / Uncoating: Enveloped Sendai Virus At cell surface by a viral fusion protein (active upon cleavage) Viral capsid released into cytoplasm

26. Fusion at the Cell Membrane: Enveloped Virus

27. Entry By Receptor- Mediated Endocytosis: Enveloped Influenza Virus Lower pH in endosome Conformational change in HA of influenza exposes a fusion peptide Fusion of viral envelope with endosomal envelope Release capsid into cytoplasm

28. Influenza Virus Envelope : Cell Membrane

29. Endocytosis: Enveloped Virus

30. Receptor-Mediated Endocytosis: Enveloped Virus

31. Synthesis: “Early” Gene Expression Release of viral genome into cell (cytoplasm or nucleus) Virus regulates host cell metabolic machinery Only some viral genes expressed (“early” transcription & translation) Viral regulatory proteins and enzymes for initial synthetic events

32. Synthesis: Genome Replication Replication of viral nucleic acid Cellular or viral polymerase New genome synthesis “signals” for additional viral synthetic events

33. Synthesis: “Late” Gene Expression Further expression of viral genome “late” transcription and translation Some regulatory proteins Mainly structural (capsid, envelope) proteins for progeny virus

34. Assembly (Maturation) This phase of viral replication is FUNDAMENTALLY DIFFERENT from organisms Viruses assembled from component parts, not from division of a pre-existing virus i.e. not exponential growth kinetics, but “burst” of new virions

35. Self Assembly Concentration of viral structural proteins and genomes (“reactants”) adequate Self forming process (recognition between viral components) Assembly follows basic laws of thermodynamics

36. Virion Assembly Assembly requires protein-protein interactions and protein-nucleic acid interactions The order of assembly occurs two ways: –The genome serves as a focus for assembly of the capsid surrounding it (helical viruses) –A hollow capsid formed and then filled with the genome (icosahedral virus)

37. Assembly – Helical Virus: TMV Rigid helical virus Composed of RNA plus identical capsomers arranged in a helix TMV capsid proteins only recognize TMV RNA This means that the protein-nucleic acid interactions are very specific

38. TMV Assembly: Proteins First, 34 capsid proteins assemble into a pair of disks The outer portions interact to hold the two disks together, while the inner portion has a gap where RNA binds When the RNA enters, the gap is closed to hold the RNA in place

39. TMV Assembly: Genome RNA interacts with the disks beginning at the “pac” (packaging signal) site, which is about 1000 bases from the 3’ end of the genome The “pac” site consists of ~ 500 bases that can form a series of hairpin loops

40. Summary: TMV Assembly Capsomers Disc Multiple helical disc RNA binds to disc Helix elongation of RNA through central hole

41. Assembly: Icosahedral Virus Has 20 triangular faces and each face is composed of 3 subunits (or multiples of 3). The subunits may be identical or different

42. Assembly: Poliovirus Protomer is made with Vp0, VP1, and VP3 Five protomers combine to form a pentamer Twelve pentamers combine to form an empty procapsid (60 protomers) RNA enters the procapsid A maturation cleavage converts VP0 into VP2 and VP4 to form intact virion

43. Cell Lysis Virus lytic infections cause distinct changes of infected cell Changes called cytopathic effect (CPE) and include: –Inclusion body –Nuclear pyknosis (shrinking) –Vacuole –Apoptosis –Syncytia (multinucleated cells)

44. Inclusion (Negri) Body - Rabies Virus

45. Syncytia (“giant” cell) Formation - Herpesvirus

46. Virus Release: Cell Lysis CPE usually secondary result of changes in host cell metabolism by viral replication Virus may halt or alter host cell DNA synthesis, transcription, and/or protein synthesis (translation) Results in disintegration of infected cell and release of progeny virus

47. Virus Release: Budding (Exocytosis) Synthesis and insertion of viral glycoproteins in host cell membrane (nuclear, ER, Golgi, plasma membrane) Assembly of viral nucleocapsid Nucleocapsid and virus modified membrane brought together (capsid protein may interact directly with viral glycoprotein or via a viral matrix protein) Exocytosis, or budding - may or may not kill the cell

48. Virus Budding Through Cell Plasma Membrane

49. Polarized Cell Plasma Membrane Exit Viral envelope proteins contain apical or basolateral plasma membrane transport signals Virus that bud apically tend to cause localized infections (release via surface) Virus that bud basolaterally tend to cause systemic infections (release via interior)

50. Reading & Questions Chapter 4: Patterns of Some Viral Diseases of Humans Chapter 6: The Beginning and End of the Virus Replication Cycle (omit Questions 3, 4)

51. QUESTIONS???

52. Class Discussion – Lecture 3 1. How does an acute virus infection differ from a persistent (chronic, latent) infection? 2. Is virus attachment/entry similar to a normal cell process? 3. How is the capsid of a helical virus (TMV) assembled? 4. How is the capsid of a spherical virus (poliovirus) assembled? 5. Non-enveloped virus are able to self- assemble in vitro, but not enveloped viruses. Why?