Human Viral Disease; Virus Replication Cycle

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. Patterns of Virus Disease • 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

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 gp120to 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)