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Bio 260 Viruses!!! Chapter 13 Viruses, and a half a second about viroids and prions Viral topics today • • • • • • Characteristics Structure Classification Culturing Replication Things that aren’t viruses Some questions first • • • • How big is a virus? What is its structure? How does it get into cells? Can you tell viral relatedness based on symptoms? How big? 5 um General Characteristics of Viruses • • • • • • • Obligatory intracellular parasites DNA or RNA Protein coat Some enclosed by envelope Some have spikes Most infect only specific types of cells in one host Host range determined by specific host attachment sites and cellular factors Virus Sizes Figure 13.1 Filtration – will it remove viruses? • Membrane filtration removes microbes >0.22 µm Other methods: Autoclaving (steam heat) Chemicals Dry heat (longer time) Figure 7.4 Virion Structure • What’s a virus made of? • Chemical composition? • Structural components? Figure 13.2a Virion Structure • Nucleic acid – DNA or RNA • Capsid (protein) – Capsomeres • Attachment proteins – spikes • Optional extras – Envelope (lipid) Figure 13.2a Virus – naked or enveloped • Naked viruses: outer coat is protein capsid made of capsomeres (subunits) (eg.phages and some animal) • WHY repeating subunits? • Enveloped viruses: lipid bilayer covers protein capsid (some animal) • WHERE did envelope come from? • Attachment proteins (spikes) either in capsid or envelope • WHAT determines host specificity? Virus Morphologies • polyhedral: composed of flat equilateral triangles – Icosahedral: 20 faces • Helical: filamentous or rod-like appearance • Enveloped: irregular shape • Complex: Isometric head and helical sheath or tail eg. phages Morphology of a Polyhedral Virus • Nucleic acid (DNA or RNA) • Capsid (triangular faces) made of capsomeres • Spikes (for what?) Adenovirus – common cold Check the size Morphology of a Helical Virus What’s this subunit? Morphology of a Helical Virus What’s this subunit? Morphology of an Enveloped Virus Morphology of a Complex Virus If you were to guess, which viruses would be the most complex? • Bacteriophages • Animal viruses • Plant viruses Figure 13.5 Morphology of a Complex Virus Figure 13.5 Viral Genome • Usually very small, few genes • RNA or DNA, ss or ds (matters for replication and transcription) • What GENES will the virus need? (what proteins…) Viral Genome • Usually very small, few genes • RNA or DNA, ss or ds (matters for replication and transcription) • Genome must code for three things: – 1) Viral protein coat – 2) Replication of viral nucleic acids – 3) Movement into and out of host cells Genome structure matters • Genome structure: ss or ds DNA or RNA – Only (+) mRNA is translated into protein – Other genomes need to be transcribed to (+) mRNA • RNA replication unique to viruses; usually in cytoplasm • DNA replication usually in nucleus Different strategies to achieve two goals • Make new genome (how??) • Make new capsid and other proteins (process??) Different strategies to achieve two goals • Make new genome – DNA or RNA polymerase • Make new capsid and other proteins – transcription and translation aka expression • Viruses are all about genetics! • Terminology note: “viral replication” means all of the above (genome replication plus gene expression plus capsid assembly) Preview of viral replication and gene expression But don’t worry… we’ll talk more about that later. Meanwhile… • • • • • • Characteristics Structure Classification Culturing Replication Things that aren’t viruses Classification of Viruses based on structure • Virus particle structure – Isometric, helical, pleomorphic, complex (bacteriophages) • Naked vs. enveloped • Genome structure – DNA or RNA; ss or ds; one molecule or segmented Classification based on transmission • Enteric viruses: fecal-oral route YAY! – (e.g., poliovirus, norovirus) • Respiratory viruses: inhaled droplets – (e.g., influenza virus, rhinovirus) • Sexually transmitted viruses – (e.g., herpes, papillomaviruses, HIV) • Zoonotic viruses: animal or arthropod vector – transmission can occur to humans or other animals – (e.g., rabies, West Nile virus, cowpox, dengue) Growing Viruses - phage • Obligate intracellular parasite… meaning? • Viruses must be grown in living cells – Bacteriophages form plaques on a lawn of bacteria Figure 13.6 Growing Viruses - animal • Animal viruses – may be grown in living animals OR – embryonated eggs Figure 13.7 Growing Viruses - common • Animal and plant viruses can grow in cell culture – Continuous cell lines maintained indefinitely Figure 13.8 Virus Identification • Cytopathic effects • Serological tests • DNA or RNA sequence Virus Identification –cytopathic effect Any detectable change after infection – range from shape change to death Which is the infected side? A or B Figure 13.9 Virus Identification –cytopathic effect Any detectable change after infection – range from shape change to death Uninfected – monolayer VSV infection: rounded Figure 13.9 Serological tests • Detect antibodies against viruses in a patient • Use antibodies to identify viruses in neutralization tests, viral hemagglutination, and Western blot How do viruses replicate? Virus life cycle in brief • • • • Get in Make more Get out Some variations on this theme Figure 13.9 A Viral One-Step Growth Curve Figure 13.10 Bacteriophage replication • DNA genomes • Lytic cycle • Lysogenic cycle Lytic cycle • Attachment and penetration • Biosynthesis and maturation • Lysis and release get in make more get out The Lytic Cycle (bacteriophage) • Attachment: Phage attaches by tail fibers to host cell • Penetration: Phage lysozyme opens cell wall; tail sheath contracts to force tail core and DNA into cell • Biosynthesis: Production of phage DNA and proteins • Maturation: Assembly of phage particles • Release: Phage lysozyme breaks cell wall Lytic Cycle of a T-Even Bacteriophage 1 attachment 2 penetration 3 biosynthesis Figure 13.11 Lytic Cycle of a T-Even Bacteriophage maturation 4 release Figure 13.11 Viral infection of bacteria • Lytic cycle – Phage causes lysis and death of host cell • Lysogenic cycle – Prophage DNA incorporated in host DNA The Lysogenic Cycle “prophage” – when the phage DNA is in the host bacterial chromosome Figure 13.12 The Lysogenic Cycle • phage DNA incorporated in host DNA ________ • A form of horizontal gene transfer ___________ Figure 13.12 The Lysogenic Cycle • phage DNA incorporated in host DNA Prophage • A form of horizontal gene transfer Transduction Figure 13.12 Why should we care about lysogeny (or transduction) • This exchange mediated by bacterial viruses is responsible for pathogenicity of many bacteria – – – – – Botulism toxin of C. botulinum originally from a virus C. diphtheriae, also originally a viral (phage) source Shiga toxin of E. coli, from Shigella toxic shock syndrome of Streptococcus I could go on… Multiplication of Animal Viruses • Attachment: Viruses attach to cell membrane (spikes not tail fibers) • Penetration by endocytosis or fusion • Uncoating by viral or host enzymes • Biosynthesis: Production of nucleic acid and proteins • Maturation: Nucleic acid and capsid proteins assemble • Release by budding (enveloped viruses) or rupture Getting in… • Animal virus options If you’re naked… • Attachment (how?) • Penetration by endocytosis • Uncoating (what’s coming off?) Figure 13.14a Attachment, Penetration, Uncoating • By fusion (enveloped) Figure 13.14b A question about entry • Do both naked and enveloped viruses need to remove their coats?? A for yes, B for no Getting OUT • • • • What do you think? Animal virus 2 options – depend on structure LYSIS BUDDING The dramatic way… lysis Budding of an Enveloped Virus How is viral structure related to entry/exit? ENTER BY NAKED VIRUS ENVELOPED VIRUS EXIT BY How is viral structure related to entry/exit? NAKED VIRUS ENVELOPED VIRUS ENTER BY EXIT BY ENDOCYTOSIS LYSIS FUSION BUDDING What happens in between • Biosynthesis, maturation – what’s the point? • Make new virus particles!!! Multiplication of DNA Virus release uncoat maturation “early” proteins (viral replication) “late” proteins (capsid) Get into nucleus Figure 13.15 If you’re a DNA virus • • • • • What is copying your genome? What’s making your mRNA? DNA DNA by host DNA polymerase DNA m RNA by host RNA polymerase mRNA protein by host ribosomes If you’re an RNA virus • What is copying your genome? • What’s making your mRNA? • Are these obvious or simple questions to answer? Some RNA virus terms • Single stranded “+” RNA • Single stranded “-” RNA • Double stranded RNA Types of RNA genome Multiplication of RNA-Containing Viruses Only (+) mRNA is translated into protein Other genomes need to be transcribed to (+) mRNA Genome + mRNA translate to protein and transcribe to –RNA -RNA + RNA for mRNA and genome RNA to RNA HOW? Viral RNA-dependent RNA polymerase (in the genome) Figure 13.17 “+” stranded RNA genetics Multiplication of RNA-Containing Viruses Only (+) mRNA is translated into protein Other genomes need to be transcribed to (+) mRNA Genome - RNA transcribe to +RNA +RNA translate to protein, transcribe to –RNA genome RNA to RNA HOW? Viral RNA-dep RNA pol IN THE CAPSID Figure 13.17 “-” stranded RNA genetics Multiplication of RNA-Containing Viruses Only (+) mRNA is translated into protein Other genomes need to be transcribed to (+) mRNA Genome dsRNA use +RNA to make protein RNA polymerase transcribes both strands to make new ds genome RNA to RNA HOW? RNA-dep RNA pol in the genome (+ side of ds RNA can be mRNA) Figure 13.17 “ds” stranded RNA genetics How is viral genome related to replication? GENOME dsDNA FUNCTION REPLICATION requires DNA polymerase “+” RNA or dsRNA Genome is an mRNA Gene for RDRP (RNA dependent RNA pol) “-” stranded RNA RDRP protein loaded into the capsid Anything ELSE??? Of COURSE there is. :D Multiplication of a Retrovirus HIV, leukemia, animal tumors Single-stranded, enveloped, RNA viruses reverse transcriptase: ss RNA ds DNA NO PROOFREADING, mistakes made, capsid proteins highly variable ds DNA integrated into host chromosome = provirus Figure 13.19 Multiplication of a Retrovirus Release by budding Viral proteins to cell surface Entry by fusion Uncoating releases genome enzymes like RT, integrase RT ssDNAdsDNA transcription viral genome and proteins Integration of provirus Figure 13.19 Retrovirus replication How animal viruses get mRNA and genomes • DNA viruses – DNA DNA – DNA mRNA host DNA polymerase sometimes also viral DNA polymerase host RNA polymerase • “+” RNA or ds RNA – their genome is already an mRNA – Genome is an mRNA used directly by ribosome – Encodes a Viral RNA RNA pol; makes more genomes • “-” RNA – their genome is NOT an mRNA – In capsid: viral RNA RNA pol makes mRNA – This RNA RNA polymerase also makes more genomes • Retroviruses – not “+” or “-” because of life cycle – In capsid: RNA DNA reverse transcriptase – Enters nucleus, incorporates into chromosome as provirus – Now treated as a gene so mRNA is made by host RNA pol Cancer • oncogenes – transform normal cells into cancer cells – normal cell protein that is overactive or has extra functions due to mutation or misregulation – oncogenes are the result of mutation – radiation, chemicals, and 10% of the time gene transfer by viruses • Transformed cells how can you tell – Change shape: increased growth, loss of contact inhibition – Cell surface: tumor-specific transplant antigens – Genetic: oncogenic virus’ DNA integrated into host DNA Do plants get viruses? • Yes! • Is their life cycle the same? Figure 13.23 Plant Viruses -Very common - instead of attaching to receptors on cells enter through cell wall wounds - Many extremely hardy - Human, insect, worm, fungal vectors Things that aren’t cells or viruses • But still cause disease • And they are REALLY REALLY small Viroids are infectious RNAs in plants • It’s not PTSD, it’s PSTD: potato spindle tuber disease Figure 13.23 Viroids Circular ssRNA ~300 to 400 nucleotides Replicate autonomously within susceptible cells using host RNA polymerase Single viroid capable of infecting cell Resistant to digestion by nucleases So far only host is plants Sequences similar to introns - Many questions: how do they replicate, how do they cause disease, how did they originate (“RNA world”), are they restricted to plants? Prions • Proteinaceous Infectious particle • No nucleic acids! • Inherited and transmissible by ingestion, transplant, and surgical instruments – Spongiform encephalopathies: Sheep scrapie, Creutzfeldt-Jakob disease, Gerstmann-SträusslerScheinker syndrome, fatal familial insomnia, mad cow disease PrPC: Normal cellular prion protein, on cell surface PrPSc: Scrapie protein; accumulates in brain cells, forming plaques Sphongiform encephalopathies PrPC: Normal cellular prion protein, on cell surface PrPSc: Scrapie protein; accumulates in brain cells, forming plaques How a Protein Can Be Infectious Figure 13.22