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Home , Animal virus, Antigenic shift, Plasmid, Prion, Viral replication

Chapter 13

Viruses, Viroides and

1 A GLIMPSE OF HISTORY

. Tobacco mosaic (1890s) • D. M. Iwanowsky, determined caused by “filterable ” too small to be seen with light microscope, passed through filters for • Decade later, F. W. Twort and F. d’Herelle discovered “filterable virus” that destroyed bacteria • Previously, many bacteria, fungi, identified as infectious • Virus means “” • have many features more characteristic of complex chemicals (e.g., still infective following precipitation from ethyl alcohol suspension or ) VIRUSES: OBLIGATE INTRACELLULAR PARASITES

. Viruses simply genetic information: DNA or RNA contained within protective coat • Inert particles: no metabolism, replication, motility • hijacks ’s replication machinery • Inert outside cells; inside, direct activities of cell • Infectious agents, but not alive • Can classify generally based on type of cell they infect: eukaryotic or prokaryotic • (phages) infect • May provide alternative to 13.1. GENERAL CHARACTERISTICS OF VIRUSES

. Most viruses notable for small size • Smallest: ~10 nm

Tobacco mosaic virus ~10 Adenovirus (250 nm) Hepadnavirus (90 nm) Poliovirus (42 nm) (30 nm) T4 • Largest: (225 nm) Mimivirus ~500 nm (800 nm)

Human red blood cell E. coli (10,000 nm diameter) (3,000 × 1,000 nm) 13.1. GENERAL CHARACTERISTICS OF VIRUSES

. Virion (viral particle) is , coat • Protein coat is : protects nucleic acids • Carries required • Composed of identical Capsomere subunits called capsomers subunits Nucleic acid • Capsid plus nucleic acids Nucleocapsid Capsid (entire protein coat) called nucleocapsid Spikes • Enveloped viruses have (a) Naked virus bilayer envelope Spikes

• Matrix protein between Matrix protein

nucleocapsid and envelope Nucleic acid Nucleocapsid Capsid (entire • Naked viruses lack envelope; protein coat) more resistant to disinfectants Envelope

(b) Enveloped virus 13.1. GENERAL CHARACTERISTICS OF VIRUSES

. Viral genome either DNA or RNA, never both • Useful for classification (i.e., DNA or RNA viruses) • Genome linear or circular • Double- or single-stranded • Affects replication strategy . Viruses have protein components for attachment • Phages have tail fibers • Many viruses have spikes • Allow virion to attach to specific receptor sites . Generally three different shapes • Icosahedral, helical, or complex 13.1. GENERAL CHARACTERISTICS OF VIRUSES

Icosahedral . Three

Protein coat shapes: (capsid) Nucleic acid • Icosahedral

• Helical Adenovirus 75 nm (a) • Complex Helical

Nucleic acid

Protein coat (capsid)

Tobacco mosaic virus (TMV) 100 nm

(b)

Complex

Protein coat (capsid) Nucleocapsid Head with nucleic acid (DNA) Tail

Base plate Tail spike

Tail fibers T4 Bacteriophage 100 nm

(c) 13.1. GENERAL CHARACTERISTICS OF VIRUSES

. International Committee on Viral (ICVT) publishes classification of viruses . 2009 report: >6,000 viruses → 2,288 → 348 genera → 87 families → 6 orders 13.1. GENERAL CHARACTERISTICS OF VIRUSES

. Key characteristics include genome structure (nucleic acid and strandedness) and hosts infected . Other characteristics (e.g., viral shape, disease symptoms) also considered 13.1. GENERAL CHARACTERISTICS OF VIRUSES

. Virus families end in suffix -viridae • Names follow no consistent pattern • Some indicate appearance (e.g., from corona, meaning “crown”) • Others named for geographic area from which first isolated (e.g., Bunyaviridae from Bunyamwera in Uganda, Africa) . Genus ends in -virus (e.g., Enterovirus) . Species name often name of disease • E.g., poliovirus causes poliomyelitis • Viruses commonly referred to only by species name 13.1. GENERAL CHARACTERISTICS OF VIRUSES

. Viruses often referred to informally • Groups of unrelated viruses sharing routes of • Oral-fecal route: enteric viruses • Respiratory route: respiratory viruses • Zoonotic viruses cause zoonoses (animal to human) • (from borne) are spread by ; often can infect widely different species • Important diseases: , dengue fever, West Nile encephalitis, La Crosse encephalitis 13.2. BACTERIOPHAGES

. Three general types of bacteriophages based on relationship with host

• Lytic phages Virion

• Temperate phages Infection

• Filamentous phages Host cell

Genetic alteration Disease of host cell of host cell

Productive Infection Latent State more virus produced Virus nucleic acid integrates into host genome or replicates as a .

Release of virions— Release of virions— host cells lyse. host cells do not lyse.

Host cell multiplies— Host cell multiplies Host cell dies. continuous release but often of virions. changed because of viral genes. 13.2. BACTERIOPHAGES

. Lytic Phage 1 Attachment Phage attaches to specific receptors on • Lytic or virulent phages exit host the E. coli cell wall. • Cell is lysed 2 Genome Entry

The tail contracts and • Productive infection: new particles phage DNA is injected into the bacterial cell, leaving the phage formed coat outside.

3 Synthesis • T4 phage (dsDNA) as model; entire Phage genome is trans- cribed and phage synthesized. Phage DNA is replicated, other virion process takes ~30 minutes components are made, and host DNA is degraded. • Five step process 4 Assembly

Phage components are assembled into mature • Attachment virions.

DNA Empty inside head • Genome entry head + + + • Synthesis

• Assembly 5 Release The bacterial cell lyses and many new infectious • Release virions are released. 13.2. BACTERIOPHAGES

. Lytic Phage Infections (cont...) 1 Attachment Phage attaches to specific receptors on • Attachment the E. coli cell wall. • Phage exploits bacterial receptors 2 Genome Entry

The tail contracts and • Genome entry phage DNA is injected into the bacterial cell, leaving the phage coat outside. • T4 lysozyme degrades cell wall

3 Synthesis • Tail contracts, injects genome Phage genome is trans- cribed and phage proteins synthesized. Phage DNA is replicated, other virion through cell wall and membrane components are made, and host DNA is degraded.

• Synthesis of proteins and genome 4 Assembly

Phage components are assembled into mature • Early proteins translated within minutes; virions.

DNA Empty inside nuclease degrades host DNA; protein head head + modifies host’s RNA to not + + recognize its own promoters 5 Release

The bacterial cell lyses and many new infectious virions are released. 13.2. BACTERIOPHAGES

. Lytic Phage Infections (cont...) 1 Attachment Phage attaches to specific receptors on • Late proteins are structural proteins the E. coli cell wall. (capsid, tail); produced toward end of cycle 2 Genome Entry The tail contracts and phage DNA is injected into the bacterial cell, leaving the phage • Assembly (maturation) coat outside. • Some components spontaneously 3 Synthesis Phage genome is trans- cribed and phage proteins assemble, others require protein synthesized. Phage DNA is replicated, other virion components are made, scaffolds and host DNA is degraded.

• Release 4 Assembly Phage components are assembled into mature virions. • Lysozyme produced late in infection; DNA Empty inside head head digests cell wall + + + • Cell lyses, releases phage

5 Release • Burst size of T4 is ~200 The bacterial cell lyses and many new infectious virions are released. 13.4. BACTERIAL DEFENSES AGAINST PHAGES

. Several approaches bacteria can take . Preventing Phage Attachment • Alter or cover specific receptors on cell surface • May have other benefits to bacteria • E.g., produces protein A, which masks phage receptors; also protects against certain human host defenses • Surface polymers (e.g., biofilms) also mask receptor 13.5. METHODS USED TO STUDY BACTERIOPHAGES

. Viruses multiply only inside living cells • Must cultivate suitable host cells to grow viruses • Bacterial cells easier than animal cells • Plaque assays used to quantitate phage particles in samples: sewage, seawater, soil • Soft agar inoculated with bacterial host and specimen, poured over surface of agar in Petri dish • Bacterial lawn forms • Zones of clearing from bacterial are plaques Bacteriophage plaques in lawn • Counting plaque forming of bacterial cells units (PFU) yields titer 13.6. REPLICATION

. Five-step infection cycle • Attachment • Viruses bind to receptors • Usually glycoproteins on plasma membrane • Often more than one required (e.g., HIV binds to two) • Normal function unrelated to viral infection • Specific receptors required; limits range of virus • E.g., dogs do not contract measles from humans 13.6. ANIMAL VIRUS REPLICATION

. Five-step infection cycle (continued...) • Penetration and uncoating: fusion or

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. • Naked Nucleocapsid released Membrane fusion into Adsorption Envelope of virion fuses remains part Spikes of virion attach to Uncoating with plasma membrane. of plasma membrane. viruses specific host cell receptors. Nucleic acid separates from capsid.

Protein spikes Fusion of virion and cannot host Envelope

Receptors fuse Nucleocapsid Capsid Host cell plasma membrane Nucleic acid

(a) Entry by membrane fusion

Endocytosis Plasma membrane surrounds the virion, forming an endo- cytic vesicle. Release from vesicle Envelope of virion fuses with Adsorption Uncoating the endosomal membrane. Attachment to receptors Nucleic acid separates triggers endocytosis. from capsid.

(b) Entry by endocytosis 13.6. ANIMAL VIRUS REPLICATION

. Five-step infection cycle (continued...) • Synthesis • Expression of viral genes to produce viral structural and catalytic genes (e.g., capsid proteins, enzymes required for replication) • Synthesis of multiple copies of genome • Most DNA viruses multiply in nucleus • Enter through nuclear pores following penetration • Three general replication strategies depending on type of genome of virus – DNA viruses – RNA viruses – Reverse transcribing viruses 13.6. ANIMAL VIRUS REPLICATION

. Five-step infection cycle (continued...) • Replication of DNA viruses • Usually in nucleus • Poxviruses are exception: replicate in cytoplasm, encode all enzymes for DNA, RNA synthesis • dsDNA replication straightforward • ssDNA similar except complement first synthesized

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA viruses

ss (–) ss (+) DNA DNA

ds (±) DNA ds (±) DNA ds (±) DNA

ss (+) RNA ss (+) RNA ss (+) RNA (mRNA) (mRNA) (mRNA)

protein protein protein

(a) 13.6. ANIMAL VIRUS REPLICATION

. Five-step infection cycle (continued...) • Replication of RNA viruses • Majority single-stranded; replicate in cytoplasm • Require virally encoded RNA polymerase (replicase), which lacks proofreading, allows – E.g., viruses Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA viruses • ss (+) RNA used as mRNA ss (+) RNA ss(–)RNA • ss (–) RNA, dsRNA viruses (mRNA) carry replicase to protein

ss (–) ss (+) RNA synthesize (+) strand RNA (mRNA)

• Some RNA viruses protein

segmented; ds (±) ss (+) RNA RNA (mRNA)

results in protein

(b) 13.6. ANIMAL VIRUS REPLICATION

. Five-step infection cycle (continued...) • Replication of reverse-transcribing viruses • Encode : makes DNA from RNA • have ss (+) RNA genome (e.g., HIV) • Reverse transcriptase synthesizes single DNA strand • Complementary strand synthesized; dsDNA integrated into host cell • Can direct productive infection or remain latent Reverse transcribing viruses

• Cannot be eliminated ss (–) DNA ds (±) DNA

ss (+) RNA (mRNA)

protein

(c) 13.6. ANIMAL VIRUS REPLICATION

. Five-step infection cycle (continued...) • Assembly • Protein capsid forms; genome, enzymes packaged • Takes place in nucleus or in of cytoplasm • Release • Most via budding • spikes insert into host cell membrane; matrix proteins accumulate; nucleocapsids extruded • Covered with matrix protein and lipid envelope • Some obtain envelope from organelles • Naked viruses released when host cell dies, often by apoptosis initiated by virus or host 13.6. ANIMAL VIRUS REPLICATION

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

3 Nucleocapsid extrudes from the host cell, becoming coated with matrix proteins 2 Viral matrix protein and envelope with protein spikes. coats inside of 1 Viral proteins that will plasma membrane. 4 New virus is become envelope spikes released. insert into host plasma membrane. Enveloped virus Viral proteins

Host plasma Matrix membrane protein Intact host Nucleic acid Capsid membrane

(a)

(b) b: © Dr. Dennis Kunkel/Visuals Unlimited 13.7. CATEGORIES OF ANIMAL VIRUS INFECTIONS

. Acute and Persistent Infections • Acute: Acute infection (influenza) State of Virus • Rapid onset Infectious virions Virus disappears Influenza Disease after disease ends.

• Short duration

Appearance of Appearance symptoms and symptoms • Persistent: virions infectious Time (days) (a)

• Continue for Chronic infection ()

State of Virus years or lifetime After initial infection with Hepatitis B Release of virus or without disease symptoms, infectious virus is released from host with

• May or may not no symptoms.

Appearance of Appearance

symptoms and symptoms infectious virions infectious have symptoms Days Time Years (b)

• Some viruses Latent infection (cold sores)

State of Virus Cold Cold After initial infection, virus exhibit both sores Virus sores is maintained in activation in non-infectious state. Virus (e.g., HIV) Non-infectious activated to produce new

disease symptoms.

Appearance of Appearance

symptoms and symptoms infectious virions infectious Days Time Years (c) 13.7. CATEGORIES OF ANIMAL VIRUS INFECTIONS

. Acute and Persistent Infections (continued…) • Persistent infections chronic or latent • Chronic infections: continuous production of low levels of virus particles • Latent infections: viral genome () remains silent in host cell; can reactivate 13.7. CATEGORIES OF ANIMAL VIRUS INFECTIONS

. Acute and Persistent Infections (continued…)

• Latent infections: (cont...) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. • Latent Cranial nerve Pons Provirus integrated into viral DNA host chromosome or

Virus moves up replicates separately, cranial nerve.

The initial infection in children causes much like plasmid cold sores and sometimes sore throat. The virus then moves along a sensory cranial nerve to the cell body near the stem • Cannot be eliminated brain, where it becomes latent.

• Can later be reactivated (a) Virus becomes latent after initial infection

Activation of virus in

Virions move down cranial nerve.

The latent virus is activated, moves back along the sensory nerve to the face and causes cold sores again.

(b) Activation of latent virus 13.8. VIRUSES AND HUMAN TUMORS

. Tumor is abnormal growth • Cancerous or malignant can metastasize; benign do not • Proto- and tumor suppressor genes work together to stimulate, inhibit growth and • cause abnormal and/or uncontrolled growth • Usually multiple changes at different sites required • Viral oncogenes similar to host proto-oncogenes; can interfere with host control mechanisms, induce tumors 13.8. VIRUSES AND HUMAN TUMORS

. Productive infections, latent infections, tumors • Virus-induced tumors rare; most result from mutations

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Viral DNA integrates into Viral DNA Virus replicates host DNA forms plasmid. in host. Viral DNA as a plasmid

or or or

Tumor Latent infection Tumor Latent infection Productive infection Productive infection Normal cells are Viral DNA replicates Normal cells are Viral DNA replicates New virions released New virions released transformed into as part of the transformed into as a plasmid without when host cell lyses. by budding. tumor cells. chromosome without tumor cells. harming the host. harming the host. 13.9. CULTIVATING AND QUANTITATING ANIMAL VIRUSES

. Viruses must be grown in appropriate host • Historically done by inoculating live • Embryonated (fertilized) chicken eggs later used • or culture now commonly used • Can process animal tissues to obtain primary cultures • Drawback is cells divide only

limited number of times Tissue

1 Cut tissue into small pieces and incubate with a • Tumor cells often used, (trypsin) to separate cells. Single cells

2 Place cells into flask multiply indefinitely with growth medium.

Monolayer 3 Allow cells to settle on bottom of flask and grow into a single layer (a monolayer).

100 µm Stained cells in monolayer 13.9. CULTIVATING AND QUANTITATING ANIMAL VIRUSES

. Effects of of Cell Cultures • Many viruses cause distinct morphological alterations called • Cells may change shape, fuse, detach from surface, lyse, fuse into giant multinuclear cell (), or form inclusion body (site of viral replication)

(a) 0.5 μm

Dead cells

100 µm Stained cells in monolayer

(b) 0.5 μm 13.9. CULTIVATING AND QUANTITATING ANIMAL VIRUSES

. Quantitating Animal Viruses • Plaque assays using monolayer of cells • Direct counts via EM • Quantal assay: dilution yielding

ID50 or LD50 • Hemagglutination: relative

Red blood Virions Hemagglutination concentration cells (a) Controls

Dilution

Empty capsid Patient Titer A 256

B 32

C 512

D 8

E 32

F 128

G 64

H >2

100 nm (b) Hemagglutination No hemagglutination 13.10. VIRUSES

. Plant viruses very common • Do not attach to cell receptors; enter via wounds in cell wall, spread through cell openings (plasmodesmata) (a) • rarely recover, lack specific immunity • Many viruses extremely hardy

• Transmitted by soil; (b) humans; insects; contaminated seeds, tubers, pollen; grafting (c) 13.11. OTHER INFECTIOUS AGENTS: AND PRIONS

. Viroids are small single-stranded RNA molecules • 246–375 nucleotides, about 1/10th smallest RNA virus • Forms closed ring; hydrogen bonding gives ds look • Thus far only found in plants; enter through wound sites • Many questions remain: • How do they replicate? • How do they cause disease? • How did they originate? • Do they have counterparts in animals? 13.11. OTHER INFECTIOUS AGENTS: VIROIDS AND PRIONS

. Prions are proteinaceous infectious agents • Composed solely of protein; no nucleic acids • Linked to slow, fatal human diseases; animal diseases • Usually transmissible only within species • Mad cow disease in England killed >170 people 13.11. OTHER INFECTIOUS AGENTS: VIROIDS AND PRIONS

. Prions (continued...)

• proteins accumulate in Brain tissue neural tissue • Neurons die • Tissues develop holes • Brain function deteriorates

• Characteristic appearance (a)

Spongiform Brain tissue gives rise to general term for lesions all prion diseases: transmissible spongiform

(b) 13.11. OTHER INFECTIOUS AGENTS: VIROIDS AND PRIONS

SC C PrP PrP 1 Both normal (PrPC) and . Prions (continued...) abnormal (PrPSC) proteins Neuron are present. • Cells produce normal form C • PrP (prion protein, cellular) 2 PrPSC interacts with PrPC. • readily destroy • Infectious prion proteins c 3 PrP is SC converted • PrP (prion protein, ) into PrPSC. • Resistant to proteases; become

insoluble, aggregate 4 Conversion continues and PrPSC • Unusually resistant to heat, accumulates. chemical treatments • Hypothesized that PrPSC converts PrPC folding to PrPSC