Viruses Bradley Hillman Dept. of Plant Biology and Pathology 339 Foran Hall, Cook 932-9375 X 334...

Viruses

Bradley HillmanDept. of Plant Biology and Pathology

339 Foran Hall, Cook932-9375 X 334

[email protected]

VIRUS PROPERTIES• Infectious – must be transmissible horizontally• Intracellular – require living cells• RNA or DNA genome, not both*• Most all have protein coat*• May of may not have lipid envelope• May have broad or narrow host range• Replication involves eclipse (breaking apart of

virus particles) and reassembly• Use host factors for to complete replication cycle

SOME CONSEQUENCES AND EFFECTS OF VIRUS INFECTION

• Like other life forms, viruses promote the propagation of their own kind

• Like other life forms, viruses evolve in response to selection pressure

• Viruses are major factors in promoting the evolution of higher organisms

• Viruses help control populations of their hosts, including humans

Virus-like agents classified and studied with viruses

• Viroids– No coat protein, no coding capacity

• Prions– No nucleic acid (?)

• Retrotransposons– No infectivity (?)

Host properties influence the virus types found in that host group

• Vertebrates have broad range of viruses

• Plants have mostly small RNA viruses

• Fungi have mostly dsRNA viruses

• Single-celled organisms have mostly large dsDNA viruses

Flint et al., 2004Principles of VirologyFig. 1.8

Sizes of microscopic and submicroscopic biological entities and their ability to be examined using various technologies

Viruses may be simple or complex

• Genome sizes 0.3 - 1200 kb; average genome sizes vary with host organism types

• Isometric particle sizes vary from ~24 nm to ~400 nm diameter

• May have single-stranded (ss) or double-stranded (ds) RNA or DNA genome

• If ssRNA, may be + or – sense

• May have one or many proteins in particles

• May or may not have lipid envelope

Types of viral genomes• double-stranded (ds) DNA

– Rarely segmented– Often large

• single-stranded (ss) DNA– Rarely large– Less common than dsDNA

• ssRNA, negative sense– Often found in viruses with broad host ranges

• ssRNA, positive sense– Most common overall

• dsRNA– Often segmented– Particle structure often critical

Composition of viruses infecting different hosts

• No “rules” about virus families that may or may not be present in a given kingdom

• Some types of viruses are found more commonly in some kingdoms than in others– Many plant viruses contain ssRNA genomes– Many fungal viruses contain dsRNA genomes– Many bacterial viruses contain dsDNA genomes

• Host properties determine the types of viruses that tend to be found in members of a biological “kingdom”

Virus types by nucleic acid composition

DNA RNA

ss ds ss ds

env naked env naked env naked env naked0 5 9 12 9 14 2 50 100 200 300 200 600 10 300

FamiliesSpecies

Host typeVertebrateInvertebratePlantFungusBacteria

- + ++ ++ ++ ++ - ++ - + ++ - ++ ++ - ++ - ++ - + + +++ - + - - - + + + + +++ - + + +++ - + + -

Overview of Virus Properties

• Animal– RNA – 5-30 kb– DNA: 5-350 kb

– Many enveloped– Range of complexity

– Range of morphologies– Some divided genomes

• Prokaryote– RNA – 5-8 kb

– DNA – 10-200 kb– Few enveloped

– Range of complexity– Range of morphologies– Few divided genomes

• True Fungi– RNA – 2.5-28 kb

– DNA – none – Enveloped ones have no capsid

– Little genome complexity– Little morphological complexity

– Some divided genomes

• Plant– RNA – 0.3-28 kb– DNA – 3-10 kb – Few enveloped

– Little genome complexity– Little morphological complexity

– Many divided genomes

• Lower eukaryote– RNA – 5-10 kb

– DNA – 180-1200 kb– Internal envelope

– Range of complexity– Range of morphologies

– No divided genomes

Tobacco mosaic virus – a typical small RNA virus

• 18X300 nm• Single 6400 nt RNA• 2130 copies of single

17 kDa coat protein• 3 essential genes• Simple regulatory

elements

Poxvirus – a typical large dsDNA virus

• 120X200 nm• Single 180 kb DNA• Complex coat made up

of numerous proteins• >100 essential genes• Complex regulatory

elements

Virus at the edge: Mimivirus• Mimivirus infects Acantamoeba polyphaga• 400 nm particle, 1.2 megabase genome, 1262 putative ORFs

represent the largest virus identified yet• Many genes for normal cellular functions

– central translation functions• Aminoacyl tRNA synthetases• Peptide release factor 1• Translation elongation factor EF-TU

– DNA repair enzymes– Many polysaccharide synthesis enzymes

• Lineage suggests connection with eukaryotes, not prokaryotes

Raoult et al., Science Express 10/14/04

Phylogenetic position of Mimivirus

Compared to other similar DNA viruses

Compared to other prokaryotic and eukaryotic life forms


Mimiviruses (arrowed) can bee seen inside their amoeba host, Acanthamoeba polyphaga using a light microscope


By transmission electron microscopy, isolated Mimivirus can be seen as a large icosahedral virus with fibrilar protrusions

Mimivirus (green) seen by fluorescence microscopy in amoeba

Mimivirus in ultrathin section in amoebaRaoult et al., Science Express 10/14/04

At 1.2 megabases (1.2X106 nucleotides), Mimivirus has the largest genome of any known virus, larger than many bacteria

Simple virus replication cycle1. Virus enters host cell

2. RNA released; translates

3. Replication in cytoplasm

4. New virus assembled

5. New virus released from host cell

Virus taxonomy and nomenclature• Modified binomial is used• Taxonomy depends on particle properties, nucleic

acid properties and especially sequence• Family is the highest taxonomic level that is

commonly used; ends in viridae, e.g., Tobamoviridae

• Genus ends in suffix virus, e.g., Tobamovirus • Species is usually the commonly used virus name;

it is italicized in formal usage, e.g., Tobacco mosaic virus

• Small genome sizes, gene shuffling make broad taxonomic schemes difficult

Host Systems: Bacteria 1

• Simplest of host systems• Lawns of cells grown on solid medium• Cells grown in quantity in liquid medium• Virus-infection may result in lysis of cells and

infection of adjoining cells, causing clearing zone• Bacteria have cell walls but do not develop into

complex organisms• Control of transcription and translation is

different in prokaryotes than in eukaryotes

Host Systems: Bacteria 2

• Many properties of bacterial viruses are different from those of eukaryotic viruses

• Bacteria use have no known active defenses against virus attack

• Easy to infect bacteria with purified viruses• Viruses infect by injection of DNA or RNA at cell

wall or pilus• “Competent” bacterial cells made easily• Competent cells regenerate into active cells

From 7th Report of the ICTV(AcademicPress, 2000)

There are very few RNA-containing viruses that infect bacteria. Phage Q and its relatives have been very well studied and were the subjects of early virus evolution studies.


There are many well-studied DNA phages. These were the cornerstones of much of modern molecular genetics.

Each plaque on a “lawn” of bacterial cells originates from a single virus particle

Bacterial virus architecture is often complex

Viruses known to infect archaea are similar to bacterial viruses – DNA containing viruses with moderate sized genomes. They have been characterized from marine environments and thermophilic vent organisms

Host Systems: Fungi 1

• Fungi are the simplest eukaryotes; yeasts are the simplest fungi

• Yeasts:– transcription and translation is eukaryotic– form colonies and grow as individual cells, and can be

plated as lawns– some of the same methods used for studying bacterial

viruses apply to studying yeast viruses– have a true sexual phase and more complex genetics

than bacteria– yeast viruses are not lytic

Host Systems: Fungi 2

• Filamentous fungi:– developmentally more complex than yeasts– simple cell-to-cell connections; flow of cytoplasmic

contents relatively unrestricted– fungi may use RNA silencing for defense against viruses– generally, fungi cannot be infected with purified virus– infection occurs only by hyphal fusion– protoplasts (=spheroplasts) regenerate cell walls and

grow into colonies (totipotent)– transfection of protoplasts is a valuable tool for use with

fungal viruses, but has not allowed for synchronous infections

From 7th Report of the ICTV(Academic Press, 2000)

dsRNA-containing viruses are most prevalent in filamentous fungi


Many large, DNA-containing viruses have been identified in algae.

Uninfectedwith Reovirus with Hypovirus

Infection of Cryphonectria parasitica with different viruses results in distinct phenotypes and developmental disruption of the host fungus, the causal agent of chestnut blight disease. They also dramatically reduce virulence of the fungus, providing a form of transmissible biological control.

Host Systems: Plants 1• Eukaryotic, but fundamentally different from animals• Plants don’t move, so vectors are very important for

moving viruses from one plant to another• Plants are autotrophic and easy to grow – great

bioreactors• Plants have cell walls and very small cell-to-cell

connections (plasmodesmata)• Synchronous infection of many cells can be

achieved using plant protoplasts (primary cell cultures with cell walls removed)

Host Systems: Plants 2

• Plant cells are totipotent• Virus in one part of a plant moves to another

slowly by cell-to-cell connections; more rapidly through vascular system, mostly phloem

• Plant defense response system exists, but is less specific than vertebrate or invertebrate systems

• Plants are developmentally complex; viruses may be excluded from some tissues

Plant cells are bound by rigid cell walls and are interconnected by plasmodesmata, which are too small to allow passage ofwhole virus particles.

Plasmodesma


Plant viruses usually have simple architecture. Often the genome is divided among multiple particles, all of which are required for infection.


Relatively few different kinds of DNA viruses infect plants.

Tobacco mosaic virus is one of the most important plant viruses historically

Particles 18 X 300nm

Symptoms on tobacco

Host Systems: Vertebrates 1

• Very complex structure and biology – many organs and tissues– complicated circulatory system

• Vertebrate immune system is very well-developed and specific

• Vertebrates move, but vectors still important for virus entry

• Viruses may enter through natural or artificial openings, especially by ingestion or through respiratory tract

Host Systems: Vertebrates 2

• Cell cultures are available for many vertebrate species

• For most practical purposes, animal cells are not totipotent

• No direct experimental systems with human viruses; must rely on related systems

• Maintaining whole animals is expensive, and there are animal rights issues especially with primates


Greatest range of diversity exists within vertebrate viruses


Viruses of many different groups represent important pathogens of vertebrates, including humans

VIRUS STRUCTURE• Basic rules of virus architecture, structure, and

assembly are the same for all families• Some structures are more complex than others• The capsid (coat) protein is the basic unit of

structure; functions that may be fulfilled by the capsid protein are to:– Protect viral nucleic acid– Interact specifically with the viral nucleic acid for

packaging– Interact with vector for specific transmission – Interact with host receptors for entry to cell – Allow for release of nucleic acid upon entry into new cell– Assist in processes of gene regulation

From N. Olsonweb site

Relative range of particle sizes is immense: greater than 10-fold difference in diameter (~1000-fold difference in volume)

Helical symmetry• Tobacco mosaic virus is typical,

well-studied example• Each particle contains only a

single molecule of RNA (6395 nucleotide residues) and 2130 copies of the coat protein subunit (158 amino acid residues; 17.3 kilodaltons)– 3 nt/subunit– 16.33 subunits/turn– 49 subunits/3 turns

• TMV protein subunits + nucleic acid will self-assemble in vitro in an energy-independent fashion

• Self-assembly also occurs in the absence of RNA

• Origin of assembly is RNA sequence required to initiate assembly process TMV rod is 18 nanometers

(nm) X 300 nm

Cubic (icosahedral) symmetry

• Tomato bushy stunt virus is typical, well-studied example

• Each particle contains only a single molecule of RNA (4800 nt) and 180 copies of the coat protein subunit (387 aa; 41 kd)

• Viruses similar to TBSV will self-assemble in vitro from protein subunits + nucleic acid in an energy-independent fashion

• T=3 structure requires quasiisometric architecture

TBSV icosahedron is 35.4 nm in diameter

Protein Subunits Capsomeres

T= 3 Lattice

C

N

Virus genome organizations

• Very compact, especially +sense RNA viruses• Eukaryotic viruses must have specific mechanisms for

expression of downstream genes• Translation regulation very important• Use various strategies for genome expression• Various strategies used to compromise host defenses• Only a few genes absolutely required:

– Replicase– Coat protein*/Cell-to-cell movement protein*

• Other genes present in many viruses

REPLICASE

COAT PROTEIN

MOVEMENT

Genome organization ofa typical +sense RNA plant virus

Genomic RNA

Subgenomic RNAs

A. Mature, enveloped virions and incomplete particles.

B. Genome organization. Light blue minus strand DNA has one nick and polymerase bound to 5’ end; dark blue + strand DNA has a large gap. Transcription begins at different sites, but polyadenylation is at one site. Every nucleotide in the 3 kb genome is used in the coding sequence, with the total coding capacity 1.5X the genome size.

Hepatitis B virus structure and genome organization.

Steps in virus infection 1• Entry into cell

– May involve adsorption of virus– May involve injection or forced entry of virus into cell– Requires that cell not be killed during entry

• Uncoating of virus DNA or RNA– May be cell-specific– May be coordinated with other processes

• Transcription/translation– Genes expressed to make viral gene products

• Replication of genome– Specific process associated with virus-encoded gene

products

Steps in virus infection 2

• Packaging of genome for export– May be simple process or multistep process

involving movement across multiple membranes

• Release/movement of virus– Movement of from one cell to another may not

be movement of complete virus particles– May or may not involve cell lysis

Particle structure and genome organization of Poliovirus, a typical simple positive-sense RNA virus of eukaryotes.

From Flint et al., 2000, Principles of Virology, ASM Press


1-4 Virus binds to receptor, enters cell, releases RNA which is translated in cytoplasm.5-10 Individual proteins are released from polyprotein by cis or trans proteolytic cleavage; replication associated proteins (RNA-dependent RNA polymerase complex) copy the RNA genome in a replication complex; 11-13 Viral structural proteins released by proteolytic cleavage, specifically package viral RNA.; virus exits cell by lysis

Single-cell replication cycle of Poliovirus, a simple + sense RNA virus

Human herpesvirus 1 particles and genome organization: genome of ~200 kb encodes ~180 proteins; ~100 in particle


1-4.Membrane fusion, release of tegument and VHS.

5-8. Nucleocapsids transported on microtubules to nuclear membrane, ejects DNA which circularizes.

9-10. Transcription, export, translation of immediate early genes by cellular Pol II

11-13. Transcription, translation of early genes; genome replication in nucleus.

14-18. Transcription, translation of late (structural) protein genes; import to nucleus, nucleocapsid assembled and stuffed with DNA.

19-23. Nucleocapsid/tegument buds into ER lumen; acquires envelope, egress by fusion.

Herpesvirus infection cycle


Influenza A virus structure and genome organization

• A. Micrograph and drawing of Influenza A virus.

– 9 viral proteins are associated with virion

– All 8 negative-sense genome segments are contained in a single particle

• B. Genome organization. – 8 negative-sense RNAs

encode 10 proteins– Segments 7 and 8 each

encode 2 proteins: one by translation stop/start (7) and one by splicing (8)

– 3’-ends of mRNAs are polyadenylated by cellular machinery; caps are added to 5’-ends by “snatching” from cellular mRNAs


Influenza virus variability

• Antigenic shift– Pseudorecombination, or

reassortment– May occur in any multiply-

infected host– Results in immediate

change in antigenic properties

– 256 possible new strains; one may be more fit than either parent and rapidly selected

Influenza epidemiology

• Experiments in Italy demonstrated flu virus recombination in swine

• Pigs were infected with H3N2 (Hong Kong 68) and HswN1 (now called H1N1)

• 256 possible reassortants could result (=28)

• All reassortants of HA and NA identified following double infection

Depiction of experiments from Flint et al., 2004, ASM Press

From Principles of Virology, Academic Press 2004

Cryo EM of purified Poxvirus particles

30 nm-thick surface domain (S)b is noted

Poxviruses

Poxvirus particle is large and complex

100 nm

Poxviridae• Large family of large, complex viruses• Infect vertebrate and invertebrate hosts• Two subfamilies:

– Chordopoxvirus, 8 genera (vertebrate hosts)– Entomopoxvirus, 3 genera (invertebrate hosts)

• All replication steps in cytoplasm, but probably requires host factors late in infection process

• Particles contain enzymes associated with replication, transcription, protein and nucleic acid modification

• One of the few DNA viruses that make their own RNA polymerase; therefore, promoter specificity achieved

• Substantial cross hybridization and cross reactivity within genera

• Relatively narrow host cell specificity

Biology of poxviruses• Cause disease in many vertebrates, including fowl• May cause mild lesion disease or systemic lethal disease• Different strains cause different diseases; different animal

species react differently to a given strain• Myxoma virus of hare used to control feral European

rabbit population in Australia• Transmission

– Aerosol (common for smallpox)– Direct contact– Arthropods - biting vectors, no virus replication– Indirect contact

Poxvirus genome organization

• Linear dsDNA 130-375 kbp; covalently closed termini.• Large hairpin structure at each terminus - up to 10 kb

total at each end is repeat sequence (replication-associated).

• Encode 150-300 proteins.• Coding regions are closely spaced, no introns.• Coding regions are on both strands of genome, and

are not tightly clustered with respect to time of expression or function.

1. Entry and release of core (mechanism unknown).

2-5. Early mRNA synthesis products release core, cause cell proliferation, and local immune suppression.

6-9. DNA synthesized for packaging and as template for intermediate gene expression – products include transcription specificity factors for late gene expression.

10-18. Transcription and translation of late (structural) protein genes; particles assembled at Golgi; particles released on cell lysis or directly infect adjoining cell.

Poxvirus infection cycle

Smallpox

• Epidemic for more than 2 centuries.• Two main strains:

– Variola major - general lethality >20%– Variola minor - lethality < 5%

• “Variolation” first used for protection.• Subject of first vaccine with related cowpox.• Survives for years in desiccated state.• Human-to-human transmission the only normal

infection route.• Declared eradicated in 1980 by WHO.• Duration of vaccine unclear – some level of

protection appears long-lived.

Sources of poxvirus gene acquisition

• AMV-EPB_034 – inhibitor of apoptosis from Amsacta moorei entomopoxvirus (AMV-EPB)

• GenBank sequence – inhibitor of apoptosis from Bombyx mori (silkworm) BLAST e-value 9e-81

• Bombyx and Amsacta both Order Lepidoptera • 62% of best non-viral GenBank hits are from same

taxonomic Class as viral host

• Duplication/extensive sequence divergence

• Recombination

• Horizontal transfer

Poxvirus Phylogeny

Poxvirus conserved gene order and spacing

Invertebrate pox viruses were the precursors of vertebrate pox viruses. They are more diverse in genome structure and organization. After entering the vertebrate population, gene order an number became relatively fixed. Other major gene clusters then were acquired and became fixed in the population.

Virus evolution• Virus evolution is contemporary and observable• Large numbers of progeny contribute to potential high

rate of evolution of viruses• Mutation rate is higher for RNA than for DNA• Evolution rate does not necessarily reflect mutation rate• Mutation rate for a particular virus may be different in

different tissues• Different parts of viral genomes evolve at different rates• Ability to generate large amounts of sequence data

enhances ability to study evolution

Positive-strand RNA virus evolution

• RNA viruses evolve rapidly; only important functional domains are conserved

• RNA viruses are made up of a limited number of building blocks; only RdRp is required and is the ultimate basis of rational phylogenies

• Evolution of RNA viruses involves conservation of required genes and recombination/shuffling of gene blocks

• Widespread recombination of relatively few of genes makes it impossible to generate single phylogenetic trees

• Basic RNA virus replication machinery likely evolved more than once

Potyviridae - Plants

Picornaviridae - Animals

Genome organizations of three families of Supergroup 1 positive-strand RNA viruses

P-Pro A

Hypoviridae - Fungi

P-Pro B Pol1 Hel2

S-Pro P-Pro Pol1Hel2

AAA

AAA

CP-filC-Pro VPg

Pol1Hel3

AAA

CP-i C-Pro VPg CP-i CP-i

Means of RNA virus evolution

• Minor replication error (substitution, single base change) – no error correction by RdRp

• Major replication error (deletion)

• Intragenomic gene duplication

• Intergenomic gene duplication

• Virus evolution may be accelerated by co-infection

Tobamo

RubiTymo

Carmo

PestiFlaviQ /Narna

ArteriHypoPotyPicorna

supergroup

3

2

1

Evolutionary relationshipsof positive-strand RNA viruses: viruses in different hosts may be more closely related to each other than viruses within a given host

RNA polymerase

Hosts:BacteriaFungiPlantsAnimals

Origin of viruses

• All viruses did not originate from a common progenitor

• Several theories for virus origins - more than one may be right

• Regressive theory - viruses evolved from intracellular parasites

• Cellular origin theory - viruses arose from cellular components that gained autonomy

• Virus/cell coevolution - viruses and cells evolved together from the beginning

Viruses Bradley Hillman Dept. of Plant Biology and Pathology 339 Foran Hall, Cook 932-9375 X 334...

Documents

Transcript of Viruses Bradley Hillman Dept. of Plant Biology and Pathology 339 Foran Hall, Cook 932-9375 X 334...