Citrus Virology- Introduction Amit Levy [email protected] 863-956-8704 What is a Virus? • Viruses are very small particles that can infect animals and plants and sometimes make them sick. • Viruses are made up of genetic materials like DNA or RNA and are protected by a coating of protein (CP). • Are all sub-microscopic • Viruses hijack the cells of living organisms. They then use the cell to replicate and take over more cells. Only reproduce in living organisms. • Are viruses alive? Most scientists will say they are non-living because they cannot reproduce without the aid of a host. Viruses also do not metabolize food into energy or have organized cells, which are usually characteristics of living things. Also do not divide like bacteria. Outline: • How are viruses packed? • How do viruses multiply? • How do viruses spread? Name Genus Genome Size particle Citrus leaf blotch Citrivirus Linear 8.7 kb helical virus ssRNA(+) 960 nm long genome Citrus chlorotic Geminiviridae circular, 3.64 kb capsid dwarf (family) ssDNA genome 38 nm length Citrus psorosis virus Ophiovirus Segmented RNA1- 7.8kb nucleocapsids negative- RNA2- 1.7kb stranded RNA RNA3- 1.5kb RNA4- 1.4kb Citrus tristeza virus Closterovirus Linear 19.3 kb helical ssRNA(+) 2000 nm long genome Citrus leprosis Cilevirus / Bipartite/ tripartite 8.7, 5 kb Higrevirus ssRNA(+) 8.4, 3.2, 3.1 kb genome 150 nm length Outline: • How are viruses packed? • How do viruses multiply? • How do viruses spread? Name Genus Genome Size particle Citrus leaf blotch Citrivirus Linear 8.7 kb helical virus ssRNA(+) 960 nm long genome Citrus chlorotic Geminiviridae circular, 3.64 kb capsid dwarf (family) ssDNA genome 38 nm length Citrus psorosis virus Ophiovirus Segmented RNA1- 7.8kb nucleocapsids negative- RNA2- 1.7kb stranded RNA RNA3- 1.5kb RNA4- 1.4kb Citrus tristeza virus Closterovirus Linear 19.3 kb helical ssRNA(+) 2000 nm long genome Citrus leprosis Cilevirus / Bipartite/ tripartite 8.7, 5 kb Higrevirus ssRNA(+) 8.4, 3.2, 3.1 kb genome 150 nm length How are viruses packed? Virion • Virion is the infectious particle • composed of : • nucleic acid • protein capsid • (+/- envelope) Viral Capsids Icosahedral Helical Wendell M. Stanley 1946 Nobel Prize in Chemistry Crystallized Tobacco Mosaic Virus and Investigated its Biochemistry Helical- protein forming a ‘tube shape’ Tobacco mosaic virus is a ssRNA virus composed of 6000 nucleotides. The capsid is made of 2100 copies of a single protein subunit that contain 158 amino acids. Icosahedral- triangular structures laid side by side T=triangulation number Icosahedral Helical Epcot center Outline: • How are viruses packed? • How do viruses multiply? • How do viruses spread? How do viruses replicate? Virus genome can be: DNA OR RNA Shape Circular Linear Number One Or more Strands ss ds + or - if RNA Name Genus Genome Size particle Citrus leaf blotch Citrivirus Linear 8.7 kb helical virus ssRNA(+) 960 nm long genome Citrus chlorotic Geminiviridae circular, 3.64 kb capsid dwarf (family) ssDNA genome 38 nm length Citrus psorosis virus Ophiovirus Segmented RNA1- 7.8kb nucleocapsids negative- RNA2- 1.7kb stranded RNA RNA3- 1.5kb RNA4- 1.4kb Citrus tristeza virus Closterovirus Linear 19.3 kb helical ssRNA(+) 2000 nm long genome Citrus leprosis Cilevirus / Bipartite/ tripartite 8.7, 5 kb Higrevirus ssRNA(+) 8.4, 3.2, 3.1 kb genome 150 nm length Citrus leaf blotch virus + strand RNA virus Replicase: RNA Dependent RNA Polymerase MP- Movement Protein CP- coat protein Citrus leaf blotch virus + strand RNA virus Translation by cellular + sense genome ribosomes Protein production (RDRP- RNA dependent RNA polymerase replicase) Transcription by RDRP Translation by cellular (-) sense genome) sgRNAs ribosomes Internal promoters Proteins: structural- Movement Protein, Replication by Coat protein- assembly RDRP and exit Citrus Psorosis virus: (-) sense RNA virus (-) sense genome) Transcription by RDRP (-) sense genome) + sense genome Protein Products Citrus chlorotic dwarf ssDNA virus 1. Synthesize proteins: Cellular DNA Single strand DNA genome replication protein Double stranded DNA genome Transcription by host cell RNA polymerase mRNA Proteins Citrus chlorotic dwarf ssDNA virus 2. Replication Cellular DNA Single strand DNA genome replication protein Double stranded DNA genome Cellular DNA replication proteins Single strand DNA genome Outline: • How are viruses packed? • How do viruses multiply? • How do viruses spread? Tobacco Mosaic Virus 1.Inoculated leaf- local lesions 2.Systemic movement to sink leaves in phloem TMV-GFP Viruses use Movement Proteins to move Size of plasmodesmata ~50 nm With available space ~5-10 nm CTV is 2000 nm! Movement Proteins: • Alter the formation or function of PD • Increase the available space inside PD • Coordinating replication of viral gnome with transport to and across wall • Associate with membranes • Function is highly regulated MP:GFP in PD RdRP PD (PDLP1-GFP) 5 MT/HEL 30 K RFP 3 cell1 cell2 Merge 2. Systemic movement (Red arrows in the figure) Hipper et al. 2013 • Plant viruses spread directly cell-to-cell within a leaf without an extracellular phase (local movement) • For systemic long distance infection an extracellular form is transported leaf- to-leaf through the phloem • For most plant viruses the extracellular form is the virus particles • Some viruses move only systemically (no local movement) and some will move only locally (no systemic movement) • Plant-to-plant movement it tied to systemic movement strategy Name Genus Genome Size particle Citrus leaf blotch Citrivirus Linear 8.7 kb helical virus ssRNA(+) 960 nm long genome Citrus chlorotic Geminiviridae circular, 3.64 kb capsid dwarf (family) ssDNA genome 38 nm length Citrus psorosis virus Ophiovirus Segmented RNA1- 7.8kb nucleocapsids negative- RNA2- 1.7kb stranded RNA RNA3- 1.5kb RNA4- 1.4kb Citrus tristeza virus Closterovirus Linear 19.3 kb helical ssRNA(+) 2000 nm long genome Citrus leprosis Cilevirus / Bipartite/ tripartite 8.7, 5 kb Higrevirus ssRNA(+) 8.4, 3.2, 3.1 kb genome 150 nm length 1. Local AND systemic movement Citrus leaf blotch virus Aguero Can be transferred et al. mechanically between plants 2013 2. Local movement Only Citrus leprosis- Transmitted between plants by insects feeding on leaf cells 3. Systemic movement only- Citrus tristeza virus- PDS silencing, Hajeri et al. 2014 Transmitted by phloem sucking insects Citrus viroids Viroids are “sub-viruses” composed exclusively of a single circular strand of nucleic acid (RNA) No coding capacity -do not program their own polymerase, no coat protein Use host-encoded polymerase for replication. Viroids: potato spindle tuber viroid (Pospiviroidae)-rod like structurre peach latent mosaic viroid (Avsunviroidae)- complex structure Replication: Pospiviroidae Use HOST proteins RNA dependent RNA polymerase RNA cleavage RNA ligase Ding et al. 1999.