VIRUS PROPERTIES

VirusesBradley HillmanDept. of Plant Biology and Pathology339 Foran Hall, Cook932-9375 X 334hillman@aesop.rutgers.edu

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, virusesevolve 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

Sizes of microscopic and submicroscopic biological entities and their ability to be examined using various technologies Flint et al., 2004 Principles of Virology Fig. 1.8

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 naked Families Species 0 5 9 12 9 14 2 5 0 100 200 300 200 600 10 300 Host type Vertebrate Invertebrate Plant Fungus Bacteria - + ++ ++ ++ ++ - ++ - + ++ - ++ ++ - ++ - ++ - + + +++ - + - - - + + + + +++ - + + +++ - + + -

Animal RNA – 5-30 kb DNA: 5-350 kb Many enveloped Range of complexity Range of morphologies Some 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 Overview of Virus Properties • Prokaryote • RNA – 5-8 kb • DNA – 10-200 kb • Few enveloped • Range of complexity • Range of morphologies • Few 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 Raoult et al., Science Express 10/14/04

Mimiviruses (arrowed) can bee seen inside their amoeba host, Acanthamoeba polyphaga using a light microscope Raoult et al., Science Express 10/14/04

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 amoeba Raoult 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 cycle • Virus enters • host cell 5. New virus released from host cell 2. RNA released; translates 3. Replication in cytoplasm 4. New virus assembled

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

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

There are many well-studied DNA phages. These were the cornerstones of much of modern molecular genetics. From 7th Report of the ICTV (Academic Press, 2000)

Bacterial virus architecture is often complex Each plaque on a “lawn” of bacterial cells originates from a single virus particle

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

dsRNA-containing viruses are most prevalent in filamentous fungi From 7th Report of the ICTV (Academic Press, 2000)

Many large, DNA-containing viruses have been identified in algae. From 7th Report of the ICTV (Academic Press, 2000)

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. Uninfected with Reovirus with Hypovirus

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 of whole virus particles. Plasmodesma

Plant viruses usually have simple architecture. Often the genome is divided among multiple particles, all of which are required for infection. From 7th Report of the ICTV (Academic Press, 2000)

Relatively few different kinds of DNA viruses infect plants. From 7th Report of the ICTV (Academic Press, 2000)

Tobacco mosaic virus is one of the most important plant viruses historically Symptoms on tobacco Particles 18 X 300nm

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

Viruses of many different groups represent important pathogens of vertebrates, including humans From 7th Report of the ICTV (Academic Press, 2000)

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

Relative range of particle sizes is immense: greater than 10-fold difference in diameter (~1000-fold difference in volume) From N. Olson web site

Helical symmetry • Tobacco mosaic virusis 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

TBSV icosahedron is 35.4 nm in diameter Cubic (icosahedral) symmetry • Tomato bushy stunt virusis 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 T= 3 Lattice C N Protein Subunits Capsomeres

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

Genome organization of a typical +sense RNA plant virus COAT PROTEIN MOVEMENT REPLICASE Genomic RNA Subgenomic RNAs

Hepatitis B virus structure and genome organization. • Mature, enveloped virions and incomplete particles. • 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.

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

VIRUS PROPERTIES