Viruses Lecture Notes PDF

26 Viruses Classification is based on strandedness, type of nucleic acid and + or - envelop +ssRNA Can be translated on entry to make RNA-dependent RNA polymerase Trend is viral genome sequence and folding of capsid for phylogenies -ssRNA RNA-dependent RNA polymerase is part of the Virion Baltimore...

26 Viruses Classification is based on strandedness, type of nucleic acid and + or - envelop +ssRNA Can be translated on entry to make RNA-dependent RNA polymerase Trend is viral genome sequence and folding of capsid for phylogenies -ssRNA RNA-dependent RNA polymerase is part of the Virion Baltimore system emphasizes L/cycle, genome rep and mechanism for mRNA Retroviruses – Use reverse transcriptase Ds DNA viruses infect all cell types Reverse transcribing DNA Viruses ssDNA use dsDNA intermediate dsRNA use RNA Polymerase for genome rep and mRNA synthesis 1 27.1 Virus Taxonomy and Phylogeny 1. Distinguish between the Baltimore system of grouping viruses and the official taxonomy of viruses proposed by the International Committee on Taxonomy of Viruses 2. Determine if a virus has a positive- or negative-strand genome 3. Differentiate the Baltimore groups of viruses 2 Virus Taxonomy and Phylogeny Lack of information on origin and evolutionary history makes viral classification difficult A uniform classification system developed in 1971 by the International Committee for Taxonomy of Viruses (ICTV) – most current report ~2,000 viruses, 6 orders, 87 families, 19 subfamilies, and 349 genera 3 4 Virus Classification Classification based on numerous characteristics – nucleic acid type – presence or absence of envelope – capsid symmetry – dimensions of virion and capsid 5 Alternative Classification Scheme David Baltimore – focuses on viral genome and process used to synthesize viral mRNA – 7 life cycle groups based on double stranded (ds) DNA single stranded (ss) DNA dsRNA ssRNA (+ or – strand) retrovirus 6 7 Genomic Studies Retroviruses and reverse transcribing DNA viruses share a common evolutionary history as both have reverse transcriptase Nucleocytoplasmic large DNA (NCLD) viruses blur distinctions between cells and viruses 8 Double-stranded DNA Viruses Largest group of known viruses Most bacteriophages have dsDNA Important vertebrate viruses – herpesviruses, poxviruses Insect viruses Some rely on host’s DNA/RNA polymerases 9 Bacteriophage T4: A Virulent Bacteriophage Phage life cycle culminates with host cell bursting, releasing virions Steps – adsorption to receptor on E. coli outer membrane – tail sheath lysozyme/central tube pierce the cell wall – viral nucleic acid is injected into host cell through tube 10 Bacteriophage T4 Life Cycle 11 Bacteriophage T4 Life Cycle… Temporal transcription regulated by – alternative E. coli polymerase factors induced by virus – early viral gene products stimulate transcription of some late viral genes – genes with related functions are usually separated and clustered together early gene transcribed counterclockwise late genes transcribed clockwise 12 The T4 Genome A large proportion of the genome codes for replication-related products including – protein subunits of its replisome – enzymes needed for DNA synthesis synthesis of hydroxymethylcytosine (HMC), a modified nucleotide replacing cytosine in T4 DNA 13 T4 DNA HMC is then chemically modified by glucosylation – protects T4 phage DNA from E. coli restriction enzymes enzymes that cleave DNA at specific sequences – restriction is a bacterial defense mechanism used against bacteriophage infection 14 T4 DNA Is Terminally Redundant Base sequence repeated at both ends Allows for formation of concatamers – long strands of DNA consisting of several units linked together – structure allows for cleaving of genome for viral progeny packaging – genome is slightly longer than the T4 gene set – each genome unit begins with different gene 15 16 Assembly of T4 Phage Particles Complex self-assembly process – involves viral proteins and host cell factors for capsid assembly Set of proteins that package DNA – terminase complex generates double stranded ends – packasome moves DNA into phage head 17 Release of Phage Particles In T4 - E. coli system, ~150 viral particles are released – two proteins are involved in process T4 lysozyme attacks the E. coli cell wall holin creates holes in the E. coli plasma membrane 18 Bacteriophage Lambda: A Temperature Bacteriophage Phage lambda (λ) can enter either the lytic or lysogenic cycle upon infection of E. coli – lysogenic – dsDNA becomes prophage - integrated into host’s chromosome – upon induction, viral genome is excised and lytic cycle begins 19 Lambda Phage Linear ds DNA genome with cohesive ends – circularizes upon injection into host cytoplasm – 40 genes, genes clustered together by function – transcription from different promoters determine if lytic cycle or lysogeny occurs 20 21 Regulatory Proteins Determine Lysogeny or the Lytic Cycle Function as repressors, activators, or both – regulate transcription, termination, and antisense RNA molecules – cII activator plays pivotal role in determining if λ will establish lysogeny or the lytic cycle cII levels high early in infection – lysogeny cII levels not high early in infection – lytic cycle 22 λ Phage and High cII Levels Increases int gene transcription – integrase catalyzes integration of λ into host genome – lysogeny is established Increases transcription of cI gene (λ repressor) – λ repressor represses all transcription except – binds to PRM promoter and activates transcription of cI – therefore, lysogeny is maintained 23 λ Phage and Low cII Levels cII is quickly degraded by a host enzyme, HflB, unless it is protected by viral cIII If cII not protected, protein Cro increases – Cro is repressor inhibits transcription of cIII and cI genes this further decreases cII and λ repressor – Cro is activator increases transcription of itself, Cro increases transcription of regulatory protein, Q Q activates genes needed for the lytic cycle 24 If Lambda Repressor Wins Race with the Cro Protein… Lysogeny is established Lambda genome is integrated into the host genome in a reaction catalyzed by the enzyme, integrase 25 How Does Induction Reverse Lysogeny? Triggered by drop in λ repressor levels – due to UV light, mutagenic chemicals DNA damage alters host cell RecA protein interacting with λ repressor, causing repressor to cleave itself cI transcription decreased, λ levels reduced further Transcription increases – xis gene, excisionase increases and binds integrase reverse integration; λ phage freed from host chromosome – Cro protein levels increase synthesis of λ repressor blocked protein Q increases, lytic cycle proceeds 26 Other Temperate Phages Most exist as site specific integrated prophages Bacteriophage Mu – transposition allows random integration sites – repressor protein inhibits lytic growth E. coli phage P1 – lysogenic cycle occurs in absence of integration; P1 and E. coli replicate together 27 Eukaryotic Viruses Herpesviruses Nucleocytoplasmic Large DNA Viruses 28 Herpesviruses Herpesviridae subfamilies – alpha herpes simplex virus I and II varicella zoster virus – chicken pox, shingles – beta - cytomegalovirus – gamma Epstein-Barr virus - infectious mononucleosis some cancers – unclassified subfamilies 29 Herpesvirus Virons Icosahedral, 120–200 nm, pleomorphic, enveloped, surface spikes Envelope surrounds tegument (layer of proteins) which surrounds nucleocapsid Linear genomes, 50–100 genes Targets are epithelial or nerve cells 30 Herpesvirus Infections Productive (primary) infections – 50,000–200,000 virions produced/cell – cell dies due to degraded DNA Latent infections – occurs in neuronal cells – infectious virus not detected – can be reactivated in neurons – production infection recurs 31 Herpesvirus Productive Infection Receptor mediated attachment Virus envelope fuses with host cell membrane Linear dsDNA enters nucleus, circularizes – immediate early and early proteins made used for viral DNA replication – late gene transcription viral structural proteins 32 33 Herpesvirus Productive Infection… Nucleocapsid assembles and leaves nucleus Tegument proteins associate with nucleocapsid Virus envelope is generated by Golgi apparatus Mature enveloped virion leaves cell 34 Herpesvirus Latent Infection In epithelial cells, – viral protein, VP16 and host cell factor (HCF) may enter nucleus with the viral genome – both required for full expression of immediate early genes and lytic infection In neurons, – VP16 and HCF do not enter nucleus – immediate early gene expression decreased – small noncoding RNAs (microRNAs) produced by virus also reduce immediate early genes 35 Nucleocytoplasmic Large DNA (NCLD) Viruses NCLD group thought to have arisen from common ancestor Similar life cycle, most in cytoplasm – May carry either proteins or genes to make proteins for DNA replication in cytoplasm Enveloped, icosahedral capsids Virion and DNA are large 36 NCLD Virus Members Virus members – Poxviridae – infect mammals, e.g., smallpox – Iridoviridae – infect fish and amphibian – Phycodnaviridae – infect algae – Mimiviridae - mimivirus only member largest NCLD virus, infects Acanthamoeba as large as some bacteria genomes Most encode all proteins needed for DNA replication Encode recombination enzymes, RNA polymerases, transcription factors, and chaperones – caused rethinking of definition of cell 37 27.3 Single-Stranded DNA Viruses 1. Describe in general terms the strategy used by singlestranded (ss) DNA viruses to synthesize their nucleic acids and proteins 2. Choose one specific bacterial and eukaryal ssDNA virus and illustrate the major events in their life cycles, noting, when possible, the specific mechanisms used to accomplish each step 38 Bacteriophage φX174 ss circular plus-stranded DNA injected into E. coli – phage converted to replicative form (RF) directs synthesis of more RF copies and plus strand DNA by rolling circle method virion assembled with viral enzyme E – blocks peptidoglycan synthesis – cell wall weakens and lyses 39 Filamentous Bacteriophage fd Circular, positive-strand DNA genome DNA virus enters F+, Hfr, F’ E. coli cells – RF produced, template for mRNA synthesis – rolling circle replication – phage and host have symbiotic relationship – new virions are continually released by a secretory process 40 27.4 RNA Viruses: Unity Amidst Diversity 1. Identify which RNA viruses use RNA-dependent RNA polymerases and which use DNA-dependent RNA polymerases to complete their life cycles 2. Describe the different approaches used by RNA viruses to synthesize the proteins they need to complete their life cycles 3. Propose how a virus with a single RNA molecule as its genome might generate multiple proteins from that molecule 41 How do RNA viruses replicate their genomes? Methods vary from group to group – dsRNA, + sense, and - sense ssRNA viruses use an RNA-dependent RNA polymerase to create a template intermediate of their genome This template can then be used by the same enzyme to create more genome copies – Retroviruses use reverse transcriptase enzymes to convert their RNA genome into a DNA intermediate This intermediate is transcribed into more genome copies by a DNA-dependent RNA polymerase (often cellular in nature) 42 How do viruses use the least genetic material to create their proteins? Methods vary – RNA levels polycistronic mRNA shorter length subgenomic RNA molecules alternative splicing of RNA molecules – Translational levels overlapping coding regions with independent start/stop signals different reading frames production of large polyproteins, cleaved by proteases ‘read-through,’ not stopping at a stop codon 43 27.5 Double-Stranded RNA Viruses 1. Describe in general terms the strategy used by dsRNA viruses to synthesize their nucleic acids and proteins 2. Describe the major events in the life cycles of φ6 and rotaviruses, noting, when possible, the specific mechanisms used to accomplish each step 44 Reproduction of RNA Phages RNA genomes cannot rely on host cell enzymes for genome replication or mRNA synthesis RNA-dependent RNA polymerase completes life cycles – replicase and transcriptase activities 45 Rotavirus Human rotavirus kills >600,000 children worldwide each year – transmitted by fecal material – virus stable in environment Virion – wheel-like appearance, non-enveloped, segmented genome, dsRNA, three concentric layers of proteins – virus loses outer layer of protein when it enters host cell – double layered particle (DLP) mRNA transcription, translation proteins form inclusion called viroplasm RNA genome replication occurs here third layer added in ER 46 Rotavirus… 47 Question 1 – Attendance 18 Glucosylation of hydroxymethylguanine residues protects phage T4 DNA from cleavage by bacterial restriction enzymes. True False 48 27.6 Plus-Strand RNA Viruses 1. Describe in general terms the strategy used by plusstrand RNA viruses to synthesize their nucleic acids and proteins 2. Outline the major events in the life cycles of poliovirus and tobacco mosaic virus, noting, when possible, the specific mechanisms used to accomplish each step 49 Plus-Strand RNA Viruses Nonsegmented plus-strand RNA genomes Replicate in cytoplasm and synthesize RNAdependent RNA polymerase – synthesizes negative strand RNA Replication complex for assembly – derived from different cell organelles 50 Poliovirus Causative agent of poliomyelitis – transmitted by ingestion – may cripple and paralyze – vaccine is eradicating the disease Virion – Nonenveloped – Read up life cycle 51 Tobacco Mosaic Virus (TMV) Most plant viruses are plus stranded RNA, enter host through abrasion, wound TMV – filamentous, helical virion – TMV genome translated into 2 proteins, one with replicase and transcriptase activities – synthesis of coat protein and genome – self-assembly highly organized process 52 Plant Viruses Movement depends on virus’s ability to spread throughout plant – travel in phloem (vasculature) – move cell to cell through plasmodesmata – requires viral “movement proteins” coats RNA to linear conformation squeezes through plasmodesmata 53 Minus-Sense RNA Viruses More recent evolutionary development Enveloped virions, pleomorphic shape Segmented and nonsegmented genomes – segmented may have evolved from nonsegmented genomes by reduction of redundant genetic regions – nonsegmented arranged in highly conserved order, tandemly linked separated by nontranscribed intergenic sequences 54 Minus-sense RNA Virus Families and Examples Rhabdoviridae – rabies virus Filoviridae – Ebola and Marburg viruses Paramyxoviridae – measles virus Bunyaviridae – segmented, hantaviruses Orthomyxoviridae – segmented, influenza virus 55 Negative-Strand Viruses Cannot serve as mRNA to form viral proteins Must bring into cell preformed RNA-dependent RNA polymerase – new plus strand intermediates synthesized – the newly synthesized plus strand serves as template for genome synthesis and mRNA as well 56 Influenza Virus Causative agent of the flu – transmitted by inhalation or ingestion Three types of viruses – A, B, and C Seven to eight segments of linear RNA – hemagglutinin binds host receptors – neuraminidase hydrolyzes mucus, cleaves virus from host receptor 57 Influenza Virus Life Cycle Enters in endosome – low pH causes conformational change in hemagglutinin protein hydrophobic ends swing outward and fusion of membranes nucleocapsid released Genome template for genome synthesis and mRNA synthesis Virus buds from host cell acquiring envelope 58 59 Retroviruses Convert ssRNA into dsDNA using reverse transcriptase dsDNA integrates into host cell genome and serves as template for mRNA synthesis and genome synthesis 60 Retroviruses - HIV Human immunodeficiency virus (HIV) – cause of acquired immunodeficiency syndrome (AIDS) – globally important pandemic Member of genus Lentivirus – HIV-1 (most common cause of AIDS in US), HIV-2 (common in Africa) HIV-1 – enveloped virus two copies of RNA genome reverse transcriptase and integrase 61 HIV… gp120 binds CD4+ T cells, macrophages, dendritic cells, and monocytes – coreceptor (which can vary) also required to gain entry into cell – virus enters by receptor-mediated endocytosis Reverse transcriptase – RNA dependent DNA polymerase – DNA dependent DNA polymerase – ribonuclease RNase H – error prone, has no proofreading capability 62 Hepadnaviruses e.g., hepatitis B virus – genome is 3.2 kb, four partially overlapping reading frame – Production of new virions takes place largely inside of liver cells (hepatocytes) 63 Hepatitis B Virus Circular, dsDNA genome – one complete, nicked strand – complementary strand has large gap (incomplete) Viral infection – gapped DNA released into the nucleus – host repair enzymes repair gap 64 Hepatitis B Virus Genome transcribed by host RNA polymerase – generates several mRNA molecules one is large RNA (pregenome) others encode polymerase with reverse transcriptase activity Pregenome converted to dsDNA by virus polymerase 65 Question 2 Retroviruses have a +ssRNA as their genome. In order to complete an infection, they must first make a __________ molecule, which they use to direct the synthesis of mRNA. A. dsRNA B. dsDNA C. DNA:RNA hybrid D. -ssRNA 66