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Plant Plus Strand RNA Viruses SMALL RNA VIRUSES OF PLANTS • Majority of plant viruses have genomes that consist of (+) sense single stranded RNA. • A number of them have rod shaped helical morphology, while majority have icosohedral morphology. • Infection by plant viruses differs from animal viruses because plant viruses are normally placed inside cells by vectors or during mechanical injury. • Transmission in nature most commonly results from feeding of insect vectors such as aphids, leafhoppers, beetles, fungi and nematodes. • No receptors have been identified for plant viruses. Classification of Plant Viruses 1)Most plant viruses have single stranded RNA genomes & simple particle morphologies. The plus sense RNA viruses do not have envelopes and have monopartite, bipartite or tripartite genomes. 2)Rhabdovirus & Bunyavirus have negative strand RNA genomes, are enveloped are transmitted by insects and members of both families infect both plants and animals. 3) Ds RNA viruses belong to the Reovirus Family whose members encompass plants & animals. 4) Geminiviruses and Nanoviruses have ssDNA genomes with no close animal virus relatives. 5) The dsDNA viruses replicate via reverse transcription. Little Cherry Plant Viruses Cause many different Symptoms Vein-banding Tissue Deformation necrosis flower breaking Some Plant Virus Particles Rigid Rods Flexuous Rods Bacilliform Enveloped Pleomorphic Enveloped Spherical Particles Geminate Basic Plant Virus Structures Helix (rod) e.g., TMV Icosahedron (sphere) e.g., BMV Some Plant Viruses Induce Characteristic Inclusions TMV Inclusions Citrus Tristeza Inclusions Spherical Virus Inclusions CPMV Wall Inclusions Potyvirus Pinwheels CPMV MP in Wall Experimental Mechanical Transmission of Plant Viruses Agroinfection: Vegetative Propagation of Plant Viruses Many Viruses are Transmitted through Contact, Seed & Pollen Plant Virus Life Cycle • Virus entry into host – no attachment step with plant viruses – by vector, mechanical, etc. – must be forced – Requires a 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 • 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 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 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 Arthropods as Virus Vectors • Carry Virus From Diseased to Healthy Plants. • Very Efficient Methods of Transmission & Movement. • Viruses & Arthropods have very Specific Relationships. Aphids Leafhoppers Thrips Whiteflies Mites Beetles Nonpersistent Insect Transmission • • • • Stylet-borne Viruses (Viruses Adsorb to Stylet) Acquisition Period (Virus Acquired Immediately) Latent Period (None) Ability to Transmit Lost Quickly (Minutes to Hours) Circulative Insect Transmission • Viruses circulate in vectors and enter salivary Glands • Acquisition period (Hours to days of feeding) • Latent Period (Hours to days after feeding) • Ability to transmit lost slowly (over many days) Propogative Insect Transmission • • • • • Viruses replicate in vectors Acquisition period (Variable) Latent Period (days after feeding) Ability to transmit may last for lifetime May be transmitted to progeny. Plant virus genome expression strategies • 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) Cell-to-Cell Movement • 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) • MPs act as host range determinants • MP alone causes expansion of normally constricted plasmodesmata pores; MPs then traffic through rapidly Plant cells are bound by rigid cell walls and are interconnected by plasmodesmata, which are too small to allow passage of whole virus particles. Plasmodesmata Three different mechanisms have been described for cell-to-cell movement of plant viruses: 1. MP complexed with viral RNA moves along microtubules from ER-associated sites of viral replication; actin microfilaments deliver MP– RNA complexes to putative cell wall adhesion sites and plasmodesmata. These viruses do not require CP for cell-to-cell movement. 2. NSP is a nuclear shuttle protein that moves newly replicated viral ssDNA genomes from the nucleus to the cytoplasm. A movement protein, MPB, associated with ER-derived tubules, traps the NSP–ssDNA complexes in the cytoplasm and guides these along the tubules and through the cell wall into adjacent cells. These viruses also do not require CP for cell-to-cell movement. 3. Some plant viruses move as whole particles through highly modified plasmodesmata (tubules). These viruses require CP in addition to MP for cell-to-cell movement. Patterns of Systemic Movement of Viruses in Plants ¾Cell to Cell Movement Plants can restrict movement by hypersensitive resistance, and by resistant genes that form the plasmodesmata. ¾ Both Viral Genomes and Virions are known to move. ¾Systemic Infection Virus moves through Phloem. Some viruses are restricted to the phloem. Cells have 105 to 106 virions. The Meristem usually is free of virus because of the lack of plasmodesmata. Mosaics illustrate variations of virus in leaf tissue. TOBAMOVIRUS GROUP Type member: Tobacco mosaic virus Physical properties: Rigid rod shaped particles 18 X 300 nm, single infectious RNA, single coat protein Transmission: Primarily mechanical Major diseases: Severe mosaic of tobacco (TMV) and tomato (ToMV), mosaic of melons (cucumber green mottle mosaic virus, CGMMV), ringspot, mosaic and necrosis of orchids (odontoglossum ringspot virus, ORSV) Tobacco Mosaic Virus – Important disease of tobacco, tomato & other plants. – Easily mechanically transmitted (only means of transmission). – Very high concentration in plants. – First plant virus disease characterized (1898). – First virus strains demonstrated; – first cross protection shown. – First virus crystallized (1946 Stanley was awarded the Nobel prize). – First demonstration of infectious RNA (1950s). – First virus to be shown to consist of RNA and protein. – First virus characterized by X-ray crystallography. – First plant virus genome to be completely sequenced. – First virus used for coat protein mediated protection. – First virus to have a resistance gene characterized. PARTICLE STRUCTURE • Tobacco mosaic virus is best studied example of a rod. • Each particle contains only a single molecule of RNA (6395 nt) and 2130 copies of the coat protein subunit (158 aa; 17.3 kDa). – 3 nt/subunit – 16.33 subunits/turn – 49 subunits/3 turns • TMV protein subunits + nucleic acid will selfassemble in vitro in an energy-independent fashion. • Coat discs nucleate with TMV RNA at hairpin region. • Self-assembly also occurs in the absence of RNA. 18 nm x 300 nm Tobacco Mosaic Virus Symptoms • Symptoms include mosaic, mottling, necrosis, stunting, leaf curling and yellowing of plant tissues. Some hosts develop resistance by forming necrotic lesions that constrain movement of the virus. • Symptoms are very dependent on the host, the plant age, the environmental conditions, & the genetic background of the host plant and the virus strain. TMV local lesion assay WT GUS V73E-22 V73E-1 L252*-33 L252*-36 Development of local lesions on the leaves of PAP mutant transgenic lines V73E-1, V73E-22, L252*-33 and L252*-36 upon infection with TMV. The untransformed tobacco and a 599-36 gus transgenic line were used as controls. The picture was taken 7 days post-inoculation. TMV Causes Several Crop Diseases Strains of TMV infect tomato and cause poor yield, distorted fruits, delayed fruit ripening and various fruit discoloration problems that affect market values. Tobacco mosaic virus is a typical positive-sense RNA virus with a 6.4 kilobase genome, has a cap at its 5’ end and a tRNA like structure at its 3’ end Tobacco mosaic virus is a positive-sense RNA virus with a 5’ cap & a 3’ tRNA like structure. 6.4 KB Genome Encodes Four ORFs Two sg mRNAs Amounts of RdRp subunits regulated by translational readthrough. MP & CP are late proteins regulated by timing & amounts of mRNA synthesis. The MP mRNA is bicistronic with a silent CP cistron requiring expression from the monocistronic CP mRNA. Three supergroups of positive strand RNA viruses From Principles of Virology, Academic Press 1999 TMV Replication Cycle 1)Virus enters injured cells. 2) Ribosomes strip off coat and begin to translate the RdRp. 3) RdRp binds to the tRNA-like 3’ end & initiates synthesis of minus strands to form RI RNA. 4) Plus-strand genomic RNAs & the movement protein (MP) & coat protein (CP) sgRNAs are synthesized. 5) MP & CP are translated. 6) MP forms complexes with newly synthesized viral RNA & membranes. The complex moves through plasmodesmata to adjacent cells. 7) CP & genomic RNAs accumulate to very high levels and virus crystals accumulate in the cytoplasm. Double-strand replicative form (RF) RNA accumulates in the cell and elicits host gene silencing responses. 8) Replication is very highly regulated but many details are not yet known. Tobamovirus Multiplication mechanical ribosomes drive co-translational disassembly UAG* MP mt/hel Translation 30K 126K 126 kD, 183 tRNAhis 183K CP (RdRp) 17.5K RdRp Replication cap Transcription [from –RNA] ppp ? 30K CP 54K ppp tRNAhis [54 kD] tRNAhis MP (30 kD) tRNAhis CP 17.5K 30K CP ER derived membranes 17.5K vRNA cap virus particles transmission CP 17.5K cell-to-cell movement kD Cell-to-Cell Movement of TMV Viral movement protein/ viral RNA complex 1. 2. 3. 4. 5. Movement protein (MP) binds to TMV RNA to form MP complexes. Host proteins and/or other virus proteins may be in the MP-complex. The MP-complex moves from to adjacent cells through plasmodesmata, In the new cell, the viral RNA is released from the MP-complex. The viral RNA is then translated on host ribosomes and the replication cycle repeats. The Coat Protein Is Required for Vascular Movement!!! MOVEMENT OF TMV IN THE INFECTED PLANT: • TMV uses its movement protein to spread from cell to cell through plasmodesmata. • Normally, the plasmodesmata are too small for passage of intact TMV particles. • The movement protein enlarges the plasmodesmatal openings so that TMV RNA can move to the adjacent cells. • TMV moves through plasmodesmata as a long, thin ribonucleoprotein complex composed of the TMV genomic RNA and the MP. • As the virus moves from cell to cell, it reaches the plant’s vascular system for rapid systemic spread through the phloem to the roots and tips of the growing plant. TMV MP Associations and Function in an Expanding Lesion Leading Edge 10 kD dextran move inward TMV-GFP:MP Late Uninfected 10 kD dextran not move Confocal Microscopy Across Lesion (temporal relationships) Time course of TMV GFP-MP effects on plasmodesmal gating and associations with the cytoskeleton (microtubules and actin filaments) and ER (aggregates) in different cells within an expanding lesion. Since virus is moving outwards from the center, cells at the leading edge are at early stages in infection while those at the center are at late stages in infection. Cartoon based on Heinlein et al. (1998) Plant Cell 10: 1107. Figure from Oparka et al. (1996) Tr. Plant Sci. 1: 412. Laarowitz (2001) in Fields Virology, 4th Ed. (Howley & Knipe, eds) TMV: A cytoplasmically replicating +RNA virus that moves without CP Nucleus cortical ER replication sites MP PD microfilaments ER derived membranes vRNA sites of replication and protein synthesis ER microtubules proteosome degradation, transport away from ER PM anchoring sites Adapted from Lazarowitz (2001) in Fields Virology, 4th Ed. (Howley & Knipe, eds) TMV Assembly: Assembly initiates at the origin of assembly (OAS) sequence near the 3’ end. Loop 1 of OAS is threaded through the center of a 2 layered disk made up of CP The disk assumes lockwasher conformation and elongates in 3’to 5’ direction Infected plants recover during Systemic Virus Invasion Non transgenic plant CP transgenic plant Dark Green Islands appearing during virus recovery lack virus. The light green areas contain large amounts of virus. The dark green islands and recovered tissue lack virus. The invaded leaf of the nontransgenic leaf has dark green islands that are resisting virus infection. The tip half of the transgenic leaf was invaded and the basal half exhibited recovery. Virus can not be detected in the dark green islands or in the recovered tissue. Recovery is based on gene silencing or RNAi. 1) GFP Expression from virus vectors is very useful for monitoring gene silencing during virus invasion. 2 - Systemic movement patterns of viruses & GFP post transcriptional gene (PTGS) silencing are similar. Silencing signals inactivating transgenic GFP follow the same exit patterns from veins as virus expressing GFP. GFP Engineered Plants GFP Virus Silencing 3 - Viruses fail to invade meristems & PTGS is not active in meristems. Some Tripartite RNA Viruses Bromoviruses: Brome Mosaic Virus (BMV) graminaceous, legume plants transmission: mechanical Family Bromoviridae Three Icosahedral Cucumoviruses: Cucumber mosaic virus (CMV) solanaceous, legume plants transmission: aphids Particles (26 to 35 nm) diameter Encapsidate four RNAs: gRNA1, gRNA2, gRNA3 & sgRNA4 Ilarviruses: Tobacco Streak Virus (TSV) woody plants seed and pollen transmitted Alfamoviruses: Alfalfa Mosaic Virus (AlMV) legume plants Four bacilliform particles transmission: aphids (30-57 X 18 nm) encapsidate: gRNAs 1, 2, & 3 plus sgRNA4. RNA 1 cap RNA 2 cap Replicase RNA 3 cap Replicase (sg)RNA 4 cap MP CP CP BROME MOSAIC VIRUS RNA1 RNA2 RNA3 RNA4 • BMV particles are Icosahedra consisting of 180 coat protein subunits. • Type member of the Bromovirus genus, family Bromoviridae. • Virions are nonenveloped icosahedral (T=3), 26 nm in diameter, contain 22% nucleic acid and 78% protein. • The BMV genome consists of three positive sense RNAs. RNA1 (3.2 kb) & RNA2 (2.9 kb), are encapsidated in separate particles. RNA3 (2.1 kb) & RNA4 (0.9 kb) are located in a third spherical particle. Divided RNA Genome of Brome Mosaic Virus Viruses with divided genomes can efficiently express genes needed early in infection & can regulate the timing and amounts of late genes by synthesis of sgRNAs. Brome mosaic virus is a tripartite RNA virus. Four proteins are expressed from three genomic RNAs RNA 1 encodes the helicase Brome Mosaic Virus RNAs subunit of the RDRP. tRNA-like RNA 2 encodes the polymerase Helicase Subunit subunit of the RDRP. 5’ m7G 3’ 3.2 kB RNA 3 is bicistronic and encodes Polymerase Subunit the movement protein and the 5’ m7G 3’ 2.9 kB coat protein. Ribosomes initiate at the 5’ m7G Movement Coat Cap of RNAs 1, 2 and 3 but can not 2.1 kB 5’ m7G 3’ initiate internally on RNA 3. P RNA 4 is a sg mRNA translated from an internal promoter on the minus strand of RNA 3. Coat 5’ m7G 3’ 0.9 kB Permits Genome Reassortment in hosts infected with two viruses. Molecular Genetic Analysis of Plus Strand RNA Viruses: 1) Transcription of synthetic RNAs from cDNA clones derived from viral genomes permit mutant analysis of specific biological traits and virus replication. 2) This strategy was first developed by Paul Ahlquist in 1984 with Brome Mosaic Virus. The method enabled analysis of Viral RNA for replication signals and promoters regulating mRNA transcription. In addition, application of foreign reporter genes provided numerous applications to assess replication. 3) Over the past 20 years Ahlquist’s strategy has been applied to most plus strand RNA viruses infecting plants, animals, and bacteria. Viral RNA Genome Phage T7 promoter Reverse transcribe RNA & Clone cDNA. Plasmid in bacteria to Amplify Cloned cDNA Mutate cDNA for specific changes & transcribe mutant Viral RNAs in vitro. Inoculate mutant RNAs to host cells or protoplasts. Evaluate Biological Effects. BMV REPLICATION: • Proteins 1a and 2a are localized in the endoplasmic reticulum, the site of BMV RNA synthesis. ER-derived membranes are the sites of RNA synthesis for all (+) sense RNA viruses • Promoter for the synthesis of (-) strand RNA is within the 134 nt tRNA-like sequence at the 3’ end of (+) sense genomic BMV RNAs • RNA dependent RNA polymerase binds to this promoter sequence and initiates synthesis of (-) strand RNAs by a primer-independent mechanism from a 5’-CCA- 3’sequence • A stem loop sequence within the tRNA-like domain and sequences upstream of the tRNA-like domain are required Bromovirus Multiplication mechanical RNA 1 1a cap mt hel tRNAtyr 109 kD tRNAtyr 94 kD (RdRp) cap RNA 2 cap 2a GDD Replication Replication RNA 3 Translation Translation 3a MP CP tRNAtyr 32 kD (MP) Transcription cap CP tRNAtyr sgRNA 4 Translation CP (20 kD) vRNA 3 RNA 1 vRNA 1 vRNA 2 RNA 2 RNA 3 + sgRNA 4 virus particles transmission ? [or vRNAs?] cell-to-cell movement From Lazarowitz (2001) in Fields' Virology 4th Ed. (Howley and Knipe, eds) Internal promoter for BMV subgenomic RNA synthesis Subgenomic RNA synthesis requires the interaction of replicase with the promoter sequence in minus strand RNA3 that is directly upstream of RNA4 initiation sequence A Yeast System to Study BMV RNA Replication 1a+2a directed RNA replication X = URA3 (select for growth without uracil, or against growth in 5-fluoroorotic acid) = CP, CAT, or GUS (assay for sgRNA synthesis and translation) 1a and 2a expressed from 2µ plasmids (ADH promoter driver) 5’-UTR and 3’-UTR sequences missing, so cannot replicate Replication is assayed by introducing RNA 3 either by introducing in vitro transcribed RNA 3 or by expressing RNA3 cDNA in vivo From Ishikawa et al. (1997) PNAS 94: 13810. Infection of yeast by Brome mosaic virus constructs In the presence of RNA 1 and 2, the RNA3 transcript, which has its 5’-UTR and 3’-UTR, is correctly replicated via its complementary strand and subgenomic RNA4 is transcribed. BMV REPLICATION IN YEAST • RNA3 is correctly replicated in the presence of 1a and 2a in yeast. • BMV 1a stabilizes RNA3 (+) sense RNA, increasing its half life from 5 min to 3 hours. • The stabilization of RNA3 blocks its translation, 1a interacts with RNA3 to recruit it away from translation and into RNA replication. • 1a interacts with the intercistronic 150 nt replication signal that includes a consensus box B-like element 5’GGUUCAAyyCC-3’ found in RNA pol III promoters and in invariant residues of tRNA loops. • Host proteins have been shown to be involved in stabilization of RNA3 through interactions with the 150nt element.