Plant Viruses Bradley Hillman Dept

Plant Viruses Bradley Hillman Dept
Plant Viruses Bradley Hillman Dept. of Plant Biology and Pathology 339 Foran Hall, Cook X 334 Comparative Virology course website:

Plant Viruses Introduction and history Symptoms
Why study plant viruses? How do they relate to animal viruses? How has their study impacted virology? Symptoms Composition and structure Taxonomy and nomenclature Four families contain both plant and animal viruses Seven families contain only plant viruses Many plant viruses belong to genera without family affiliation

Plant Viruses (cont’d)
Survey of some major plant viruses Positive strand RNA RdRp supergroup 3 (Sindbis-related) Tobacco mosaic virus (Tobamovirus) Brome mosaic virus (Bromoviridae) RdRp supergroup 2 (Flavivirus-related) Turnip crinkle virus (Tombusviridae) Red clover necrotic mosaic virus (Dianthovirus) RdRp supergroup 1(Picornavirus related) Tobacco etch virus (Potyviridae) Cowpea mosaic virus (Comovirus) Negative strand RNA Sonchus yellow net virus (Rhabdoviridae) Ambisense RNA Tomato spotted wilt virus (Bunyaviridae)

Plant Viruses (cont’d)
Survey of some major plant viruses (cont’d) Double-stranded RNA Wound tumor virus (Reoviridae) Single-stranded DNA Bean golden mosaic virus (Geminiviridae) Double-stranded DNA (pararetroviruses) Cauliflower mosaic virus (Caulimoviridae) Expression strategies of + strand RNA viruses Plant virus infection cycle Cell-to-cell movement and movement within plants Plant-to-plant transmission Brome mosaic virus, a well-studied plant virus Satellites, defective-interfering RNAs, viroids Plant defense response to virus infection Plant viruses and biotechnology

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 in quantity– great bioreactors Plants have rigid 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 Developed 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

CHARACTERISTICS OF PLANT PATHOGENIC VIRUSES
Loss due to plant viruses is often difficult to quantify, but they are often of great importance as plant pathogens (fungi are most economically important ) Viruses do not usually kill plants, and symptoms on plants are often subtle Virus diseases of plants are not subject to chemical control – no effective cure in individual plants Symptoms, serological, electron microscopic, or molecular methods are used to identify plant viruses Plant virus disease cycles often are dependent on vectors or alternate hosts

Selected highlights of plant virology research

Tulipomania – late 16th century
Before it was known to be caused by a virus, tulips with color breaking symptoms were prized and traded for large sums of goods – this led to “tulipomania” in the late 1500’s Traded for 1 Viceroy tulip bulb: 4 tons of wheat 8 tons of rye 4 fat oxen 8 fat pigs 12 fat sheep 2 hogsheads of wine 4 barrels of beer 2 barrels of butter 1000 lbs of cheese 1 bed with accessories 1 full dress suit 1 silver goblet

Adolf Mayer –1886 – showed that Tobacco mosaic virus was transmissible, could not find bacteria or fungi associated with disease TMV

Dmitri Ivanowski – showed that Tobacco mosaic virus was not retained by filters that retained all bacteria known at that time

Martinus Beijerinck – repeated demonstration that Tobacco mosaic virus was not retained by filters that retained all bacteria known at that time Believed results Did extensive dilution experiments Showed diffusion of infectious agent through agar Named “contagium vivum fluidum”, later virus

Wendell Stanley – 1935 At Rockefeller Foundation in Princeton
Crystallized TMV, thought it was only protein TMV Stanley Hall, U.C. Berkeley

Bawden and Pirie Crystallized Tomato bushy stunt virus (TBSV); find that it and TMV contain phosphorous – conclude that it is not a protein, but is a nucleoprotein TMV TBSV

Markham and Smith Two classes of particles in purified Turnip yellow mosaic virus preparations: light ones containing only protein, which were not infectious heavy ones containing protein+nucleic acid, which were infectious empty full

Myron Brakke - 1951 Development of density gradient centrifugation
Isopycnic: particles reach position of equal density in gradient Rate-zonal: Particles sediment differentially through medium as a function of size, shape, and density Equilibrium zonal: Combination of the above

Fraenkel-Conrat 1955-56 Virus Aa: protein
RNA A capsid a protein Complete, infectious TMV particles can be reconstituted in vitro from the RNA and protein components RNA alone is infectious RNA can be “transcapsidated” in protein from closely related virus; resulting virus has properties of RNA strain RNA reconstitute in vitro inoculate plants symptoms (A) extract virus virus Aa

Fraenkel-Conrat – 1955-56 Transcapsidation Virus Aa: Virus Bb:
RNA A capsid a Virus Bb: RNA B capsid b Virus Ab: RNA A capsid a RNA RNA RNA protein protein protein inoculate plants inoculate plants inoculate plants no symptoms no symptoms no symptoms symptoms (A) symptoms (B) symptoms (A) extract virus extract virus extract virus virus Aa no virus virus Bb no virus virus Aa no virus

Crick and Watson – 1956 TMV virions are composed of one nucleic acid and many identical protein subunits: RNA does not have the coding capacity to make many different subunits

Casper and Klug – 1962 Structure of Tomato bushy stunt virus solved by X-ray crystallography, the first icosahedral virus so determined

Heinz Sanger – 1978 Complete sequence of Potato spindle tuber viroid
First pathogen sequence to be determined Yielded relatively little information that was immediately useful 1 cggaactaaa ctcgtggttc ctgtggttca cacctgacct cctgagcaaa aaagaaaaaa gataggcggc tcggaggagc gcttcaggga tccccgggga aacctggagc gaactggcaa aaaaggacgg tggggagtgc ccagcggccg acaggagtaa ttcccgccga aacagggttt tcacccttcc tttcttcggg tgtccttcct cgcgcccgca ggaccacccc tcgccccctt tgcgctgtcg cttcggctac tacccggtgg aaacaactga agctcccgag aaccgctttt tctctatctt cttgcttccg gggcgagggt gtttagccct tggaaccgca gttggttcct 359

Paul Ahlquist – 1984 Infectious viral RNA transcribed in vitro from cDNA clones Done with Brome mosaic virus – with 3 RNAs Brought reverse genetics to RNA viruses RNA Inoculate plants RNA cDNA

Roger Beachy – 1986 Transgenic plants expressing TMV coat protein are resistant to virus infection First example of “pathogen-mediated resistance”

Bill Dougherty – 1991 RNA was critical component in resistance in pathogen-mediated resistance All of the hallmarks that later came to be associated with PTGS and RNAi were first observed with Tobacco etch virus (TEV) (1993 Lindbo et al., Plant Cell 5: )

SYMPTOMS

Plant Virus Symptoms Viruses rarely kill plants
Most severe disease usually in least well-adapted host/pathogen systems Levels of tissue specificity differ among plant viruses Some infect all or most tissues Most cause symptoms only in aerial portions Some accumulate only in roots Symptoms in fruit or flowers may be most harmful Systemic symptoms only in developing tissue Local necrotic lesions undetectable in natural infections

Plant Virus Symptoms Stunting - common Mosaics & mottles - common
Ringspots - common Abnormal growth/tumors/enations - rare Blights - rare Wilts – rare Systemic necrotic lesions – relatively rare

Blight

Stem pitting – usually results in loss of woody perennials

Ringspots, oakleaf

Phyllody – tissue destined to develop flower parts instead develops leaves
Deformities

Wilt Necrotic roots

Mosaics – very common Tomato mosaic Alfalfa mosaic

Mosaics on monocots are streaks or stripes
Maize streak Maize mosaic

Necrotic lesions Local Systemic
“Local lesion” or “hypersensitive” response is an apoptotic response. Cells within a short distance of the initially inoculated cell begin to undergo programmed cell death in advance of virus invasion, preventing further virus spread.

Hypersensitive response – an apoptotic reaction to infection
May be viral coat protein or another viral gene product

TAXONOMY AND NOMENCLATURE

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., Bromoviridae Genus ends in suffix virus, e.g., Bromomovirus Species is usually the commonly used virus name; it is italicized in formal usage, e.g., Brome mosaic virus Small genome sizes, gene shuffling make broad taxonomic schemes difficult (above Family level)

COMPOSITION AND STRUCTURE

Virus properties: Plant viruses are often simpler than animal viruses
Genome sizes kb; plant viruses kb May have single-stranded or double-stranded RNA or DNA genome; most plant viruses ssRNA If RNA, may be + or – sense; most plant viruses + sense ssRNA May have one or many proteins in particles; most plant viruses have 1-2 May or may not have lipid envelope; most plant viruses do not

Types of plant virus genomes
double-stranded (ds) DNA (rare) single-stranded (ss) DNA (rare) ssRNA, negative sense (rare) ssRNA, positive sense (common) dsRNA (rare)

Virus types, by nucleic acid
DNA RNA ss ds ss ds env naked env naked env naked env naked Families Species Host type Vertebrate Invertebrate Plant Fungus Bacteria

Plant viruses are diverse, but not as diverse as animal viruses – probably because of size constraints imposed by requirement to move cell-to-cell through plasmodesmata of host plants Plant viruses often contain divided genomes spread among several particles

Basic Plant Virus Structures
Helix (rod) e.g., TMV Icosahedron (sphere) e.g., BMV

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 TMV rod is 18 nanometers (nm) X 300 nm

Cubic (icosahedral) symmetry
TBSV icosahedron is 35.4 nm in diameter 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 Lattice C N Protein Subunits Capsomeres

GENOME ORGANIZATIONS

Plant virus genome organizations
Very compact Most are +sense RNA viruses, so translation regulation very important Use various strategies for genome expression Only a few genes absolutely required: Replicase Coat protein Cell-to-cell movement protein Other genes present in some viruses

Plant viruses have members in all 3 supergroups of + strand RNA viruses
From Principles of Virology, Academic Press 1999

Genome expression of + strand RNA viruses
Most use more than one strategy Polyprotein processing Subgenomic RNA Segmented genome Translational readthrough Frameshift Internal initiation of translation (without scanning) Scanning to alternative start site (truncated product) Alternative reading frame (gene-within-a-gene)

Polyprotein processing
Post-translational cleavage of viral proteins may occur in cis or in trans Some viruses use polyprotein processing exclusively to regulate gene expression Many viruses use polyprotein processing as one of several regulation mechanisms Examples: Potyviruses* Comoviruses* Closteroviruses Carlaviruses

Subgenomic RNA Similar to traditional mRNA, but synthesized from an RNA template Many viruses use polyprotein processing as one of several regulation mechanisms Examples: Tobamoviruses (TMV)* Bromoviruses (BMV)* Tombusviruses (TBSV) Potexviruses (PVX)

Segmented genome Positive sense RNA genomes are usually encapsidated in separate particles Segmented genomes lend themselves to recombination Examples: Bromoviruses (Brome mosaic virus, BMV)* Dianthoviruses (Red clover necrotic mosaic virus, RCNMV)* Hordeiviruses (Barley stripe mosaic virus, BSMV)

Translational readthrough
Usually UAG codon is read through using suppressor tyrosine tRNA Common mechanism in plant viruses Examples: Tobamoviruses (Tobacco mosaic virus, TMV)* Dianthoviruses (Red clover necrotic mosaic virus, RCNMV)* Hordeiviruses (Barley stripe mosaic virus, BSMV)

Translational frameshift
Typically +1 or -1 Common mechanism in plant viruses Examples: Luteoviruses (Barley yellow dwarf virus, BYBV)* Dianthoviruses (Red clover necrotic mosaic virus, RCNMV)* Closteroviruses (Beet yellow vein virus, BYVV)

Internal initiation Cap-free translation
Less complex in plant viruses than in animal viruses Examples: Potyviruses (Tobacco etch virus, TEV)* Sobemoviruses (Southern bean mosaic virus, (SBMV)*

Tobacco mosaic virus is a typical positive-sense RNA
plant virus with a 6.4 kilobase genome

INFECTION CYCLE

Plant Virus Life Cycle Virus entry into host
no attachment step with plant viruses by vector, mechanical, etc. – must be forced requires healable wound – delivery into cell Uncoating of viral nucleic acid may be co-translational for + sense RNA viruses poorly understood for many Replication replication is a complex, multistep process viruses encode their own replication enzymes

Plant Virus Life Cycle 2 Cell-to-cell movement
cell-to-cell movement through plasmodesmata move as whole particles or as protein/nucleic acid complex (no coat protein required) Long distance movement in plant through phloem as particles or protein/nucleic acid complex (coat protein required) Transmission from plant to plant requires whole particles

Typical RNA-containing plant virus replication cycle
2. RNA is released; translates using host machinery 1. Virus particle enters first cell through healable wound 3. Replication in cytoplasm 4a. Infectious TMV RNA is shuttled to adjacent cell through plasmodesmata, by virus-coded movement protein 4b. New virus particles are assembled From Shaw, 1996 Ch. 12 in Fundamental Virology (Academic Press)

Cell-to-Cell Movement of Plant Viruses
Plant viruses move cell-to-cell slowly through plasmodesmata Most plant viruses move cell-to-cell as complexes of non-structural protein and genomic RNA The viral protein that facilitates movement is called the “movement protein” (MP) Coat protein is often dispensable for cell-to-cell movement

Cell-to-Cell Movement of Plant Viruses
Several unrelated lineages of MP proteins have been described MPs act as host range determinants MP alone causes expansion of normally constricted plasmodesmata pores; MPs then traffic through rapidly MPs are homologs of proteins that naturally traffic mRNAs between cells MPs may act as suppressors of gene silencing

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

Understanding virus infection and movement through plants requires understanding architecture of dicotyledonous plants and the connections between different cell types. This has been studied extensively with GFP-labeled virus

Some plant viruses radically modify plasmodesmata, allowing for cell-to-cell movement as whole particles

Systemic spread of plant viruses is primarily through vascular tissue, especially phloem

Plant Virus Transmission
Generally, viruses must enter plant through healable wounds - they do not enter through natural openings (no receptors) Insect vectors are most important means of natural spread Type of transmission or vector relationship determines epidemiology Seed transmission is relatively common, but specific for virus and plant

Plant Virus Transmission
Mechanical transmission Deliberate – rub-inoculation Field – farm tools, etc. Greenhouse – cutting tools, plant handling Some viruses transmitted only by mechanical means, others cannot be transmitted mechanically

Plant Virus Transmission by Vectors
Transmission by vectors: general Arthropods most important Most by insects with sucking mouthparts Aphids most important, and most studied Leafhoppers next most important Some by insects with biting mouthparts Nematodes are important vectors “Fungi” (protists) may transmit soilborne viruses Life cycle of vector and virus/vector relationships determine virus epidemiology A given virus species generally has only a single type of vector

Insect transmission (vectors)
Aphids most important Leafhoppers Whiteflies Thrips Mealybugs Beetles Mites (Arachnidae) Ants, grasshoppers, etc. – mechanical Bees, other pollinators – pollen transmission

Types of vector relationships Terms apply mainly, but not exclusively, to aphid transmission
Non-persistent transmission virus acquired quickly, retained short period (hours), transmitted quickly “stylet-borne” transmission virus acquired and transmitted during exploratory probes to epidermis

Types of vector relationships
Persistent transmission virus acquired slowly, retained long period (weeks), transmitted slowly circulative or propagative transmission virus acquired and transmitted during feeding probes to phloem

Types of vector relationships
Semi-persistent transmission virus acquired fairly quickly, retained moderate period (days), transmitted fairly quickly virus acquired and transmitted during exploratory probes

Brome mosaic virus Relatively little studied prior to 1980
Relatively narrow host range Causes no important disease Mechanically transmitted, probably not vectored Similar to Alfalfa mosaic virus and Cucumber mosaic virus, two important plant pathogens Now most thoroughly understood plant virus at RNA level

BMV structure Rigid isometric particles 27 nm
RNA1 (3.2 kb) and RNA2 (2.9 kb) packaged alone; RNA3 (2.1 kb) and RNA4 (1.2 kb) packaged together Particle is held together primarily by protein/RNA interactions With RNA, 180-subunit, T=3 particles predominate; without RNA, 120-subunit, T=1, particles

Brome mosaic virus genome organization
3 genomic RNAs, one subgenomic RNA Only RNAs 1 and 2 required for replication in protoplasts 3’-terminal 200 bases of segments nearly identical All three genomic and subgenomic RNA are capped Non-templated C and A at 3’-ends Capping Helicase Polymerase Movement Capsid

Why is BMV such a powerful system
3 RNAs only 1 and 2 required for replication RNAs can be studied independently All three promoter types (+, -, sg) found on RNA 3, which is not required for infection Replication is fast in plant protoplasts Infects monocot and dicot host plants Replicates in yeast, best eukaryotic genetic system Efficient transcriptase/replicase complex has been isolated for in vitro studies Structurally stable particles allow for encapsidation studies

Brome mosaic virus contributions
1980 studies of 3’-terminal pseudoknot (Ahlquist/Kaesburg) 1984 complete BMV sequence (Ahlquist/Kaesburg) 3’-terminal replicase recognition site (- strand promoter) identified (Ahlquist/Hall) Nonstructural proteins of BMV, TMV, and alfaviruses are similar (Haseloff; also Ahlquist) First infectious transcripts from a cloned RNA virus genome (Ahlquist) 1985 Continued dissection of 3’-end functions

1986 CAT gene substituted for CP (French/Ahlquist) 1987 Requirement of intercistronic region of RNA 3 for replication (French/Ahlquist) 1988 Identification of subgenomic RNA promoter (French/Ahlquist) 1990 Rescue of CCMV deletion mutants by recombination (Allison/Ahlquist)

1992 Determination of 5’-terminal sequences involved in transcription and replication (+ strand promoter (Pogue/Hall) TMV movement protein can substitute for BMV MP (DeJong/Ahlquist) 1993 BMV replication in yeast (Janda/Ahlquist) BMV transcription and replication requires compatibility between polymerase and helicase proteins (Dinant/Ahlquist) 1994 Recombination between viral RNA and transgenic plant transcripts (Green/Allison) 1995 Formation of RdRp in yeast requires coexpression of RNA 1 and 2 proteins (Quadt/Ahlquist)

1997 Inducible expression of active RNA 3 replicons in yeast from DNA plasmids (Ishjkawa/Ahlquist) Yeast mutations in multiple complementation groups inhibit BMV RNA replication and transcription (Ishjkawa/Ahlquist) 1998 Specific residues critical for recognition of the 33 nt subgenomic RNA promoter by the BMV RdRp identified, demonstrating functional homology of RNA and DNA promoters (Siegel/Kao) 1999 BMV 1a protein functions in in vitro, in yeast, and in plants in methylation of GTP and cap analogs (Ahola/Ahlquist; Kong/Kao) BMV RdRp can use RNA, DNA, or hybrid to initiate RNA synthesis, suggesting that transition from RNA to DNA world may have been relatively easy (Siegel/Kao)

2000 Host protein associated with efficient RNA template selection identified (Diez/Ahlquist) BMV protein 2a (RdRp) is directed to ER by capping/helicase-like 1a protein (Chen/Ahlquist) 2001 Factors regulating template switching during RNA synthesis by viral RdRps identified using in vitro assays (Kiml/Kao) 2002 3’-terminal tRNA-like structure of BMV RNAs mediate particle assembly (Choi/Dreher/Rao) Crystallographic structure of BMV solved (Lucas/McPherson)

2003 The BMV 3’-terminal core promoter element, stem-loop C (SLC), functions at different positions on the template, can initiate RNA synthesis internally, and can potentiate RNA synthesis from a cellular tRNA template (Ranjith-Kumarl/Kao) Systematic, genome-wide identification of host genes affecting replication of BMV (Kushner/Ahlquist) 2004 Two distinct types of homologous RNA recombination in BMV replication (Bujarski) 2005 Gold nanoparticles encapsidated in BMV coat protein form normal, solid particles (Rao)

Some major BMV contributors
Ahlquist – most prolific, many aspects Hall – Cis-acting RNA elements Dreher – 3’-terminal structure Kao – recent dissection of cis-acting RNA elements Bujarski – Intra-strand and inter-strand RNA recombination Rao – RNA packaging

BMV promoters for + strand RNA synthesis
Promoter sequence for genome-length plus strand synthesis is on 3’-end of minus strand; corresponding sequences on plus strand are important for efficient transcription 5’-terminal regions are less highly conserved than 3’-terminal regions Internal poly(A) tract of variable length precedes subgenomic promoter and is required for efficient sgRNA transcription Core promoter elements

Three classes of 3’ termini among + sense RNA viruses
A. Full phylogeny, Supergroups 1-3 B. Supergroup 3 Type of 3’-terminal structure cannot be predicted based on polymerase phylogeny alone, supporting the role of interviral recombination in selection of 3’-end. TLS = tRNA-like structure Het = Heteropolymeric, non-tRNA- like sequence An = poly(A) tail From Dreher 1999, Annu. Rev. Phytopathol. 37:

Brome mosaic virus 3’-terminal tRNA-like structure is a multifunctional domain
Promoter for – strand RNA synthesis RNA protection against nuclease Can be charged with tyrosine in vitro and in vivo (ATP, CTP) tRNA nucleotidyl transferase adds terminal C and A residues Involved in RNA packaging into virions – intact tRNA-like fold is required Relatively little involvement of 3-end of BMV RNA in translation regulation

Infection of yeast by Brome mosaic virus constructs

Brome mosaic virus replication
1a protein (capping & helicase) localizes to ER 1a protein recruits 2a protein (RdRp) during active translation of 2a Viral RNA templates are recruited to nascent replication complex by 1a protein RNA replication occurs in membrane-bound, capsid-like spherules

Identification of host genes involved in BMV replication (Kushner/Ahlquist 2003, PNAS 100:15764)
Used yeast – genetics easy, many mutants available Provides information about replication, not systemic infection Transform with inducible two plasmid system requiring replication for reporter gene expression Screen for replication-associated genes by monitoring luciferase expression 4500 yeast mutants examined (~80% or yeast genes) ~100 yeast genes whose deletion altered BMV-directed expression of luciferase by > 3 fold

Identification of host genes involved in BMV replication (Kushner/Ahlquist 2003, PNAS 100:15764)
Mutants that were positive in the luciferase screen were examined further by northern and western blot

Tomato bushy stunt virus genome

Comparative properties of BMV and TBSV
A T=3 icosahedral virus with a 20 kDa capsid protein subunit and tripartite RNA genome of 8 kb RdRp supergroup 3 No known helicase domain 5’ end of RNA capped; 3’ end has tRNA-like structure aminoacylated with tyrosine Expression of 4 gene products via normal cap-dependent translation (3) and subgenomic RNA (1) Defective-interfering (DI) RNAs present, not prevalent TBSV A T=3 icosahedral virus with a 40 kDa capsid subunit and monopartite RNA genome of 5 kb RdRp supergroup 2 No known helicase domain 5’ end of RNA uncapped; 3’ end has no poly(A) or tRNA-like structure Expression of 5 gene products via cap-independent translation (1) readthrough (1), subgenomic RNAs (2), and internal initiation (1) Defective-interfering (DI) RNAs replicate to high levels in permissive plant host; equally high in yeast

TBSV replication constructs for yeast assay – based on trans replication of a defective interfering RNA

Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses Tadas Panavas, Elena Serviene, Jeremy Brasher, and Peter D. Nagy* PNAS 102: Replication of both BMV and TBSV is suppressed in about 100 out of 4,800 yeast knock-out mutants (YKOs) Of those 100 mutants, only 4 were common between the two viruses: three genes involved in protein metabolism (ubiquitin pathway), and the fourth a transcription regulator Another 10 mutants affecting replication of one or the other viruses represented genes with known functions in common. These were involved in: i) protein biosynthesis; ii) protein metabolism; iii) transcription/DNA remodeling Genes involved in protein targeting, membrane association, vesicle transport, and lipid metabolism affect TBSV, but none in BMV

Viroids

Viroids Very small, covalently closed, circular RNA molecules capable of autonomous replication and induction of disease Sizes range from nucleotides No coding capacity - do not program their own polymerase Use host-encoded polymerase for replication Mechanically transmitted; often seed transmitted More than 40 viroid species and many variants have been characterized “Classical” viroids have been found only in plants

Viroids are divided into two groups, based on site and
details of replication

Viroid Diseases Potato spindle tuber viroid (PSTVd)
May be limiting to potato growers First viroid characterized Many variants described Control with detection in mother stock, clean seed PSTVd in potato PSTVd in tomato

Viroid Diseases Citrus exocortis viroid (CEVd)
Causes stunting of plants, shelling of bark May result in little yield loss May be useful to promote dwarfing for agronomic advantage Transmitted though stock, graft Control by removal of infected plants, detection, clean stock Citrus exocortis viroid

Healthy Infected Avocado sun blotch viroid Apple crinkle fruit viroid Citrus exocortis viroid

Potato spindle tuber viroid (PSTVd) is the most thoroughly characterized viroid disease
(From R. Owens, USDA, Beltsville)

Viroid structures All are covalently closed circular RNAs fold to tightly base-paired structures Two main groups of viroids: self-cleaving and non-self-cleaving Non-self cleaving viroids replicate in nucleus and fold into “dog bone” or rod-like structure Five domains identifiable in non-self-cleaving Left hand (LH) and right hand (RH) domains are non-base-paired loops Single mutations to pathogenic domain often alter virulence Mutations to conserved central domain are often lethal Mutations to variable domain are often permitted

Minor variations in viroid sequence, and presumably attendant
RNA structure changes, are associated with virulence differences (From R. Owens, USDA, Beltsville)

Viroid replication In nucleus or chloroplasts, depending on class of viroid Chloroplast-associated viroids process into monomers by ribozyme-mediated cleavage; nucleus-associated viroids process into monomers by using host-derived enzyme In both classes, host DNA-dependent RNA polymerase is the performs RNA polymerization on + and – strand RNA templates Ribozyme-mediated Cleavage by host-factor RZ HF

Viroid movement Traffic within cell through nuclear pores using VirP1, a nuclear localization protein that binds viroid RNA Traffic cell-to-cell through plasmodesmata Traffic long distance through phloem All of these processes are associated with host proteins

+ and – viroid strands are differentially localized within the nucleus
Viroid strands of + polarity localized to nucleolus, as well as nucleoplasm Viroid strands of - polarity localized to only to nucleoplasm Qi and Bing, 2003, Plant Cell 15:2566

Hepatitis delta Hepatitis delta virus has many viroid-like properties, but the RNA is larger (1.7 kb), is encapsidated, and encodes a virion-associated protein (hepatitis delta antigen) Intensifies HBV infection HDV requires HBV as helper virus for encapsidation, so it has satellite-like properties (like a “virusoid”) Replicates in nucleus via cellular DNA-dependent RNA polymerase II

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