Lecture 8 – Viruses – Microbiology PDF

BIOL371: Microbiology Lecture 8 – Viruses 1 Topics of today 1. 2. 3. 4. 5. Overview of viruses Structure of the virion Culturing, detecting, and counting viruses Replication cycle Examples of viruses causing human diseases Materials covered:  Chapters 5.1-5.3, 5.6, 11.8, 11.9, 11.11  Figures...

BIOL371: Microbiology Lecture 8 – Viruses 1 Topics of today 1. 2. 3. 4. 5. Overview of viruses Structure of the virion Culturing, detecting, and counting viruses Replication cycle Examples of viruses causing human diseases Materials covered:  Chapters 5.1-5.3, 5.6, 11.8, 11.9, 11.11  Figures 5.1-5.3, 5.5, 5.7-5.18, 5.20, 5.22, 11.22, 11.24, 11.27  Table 5.1 (human viruses) 2 Virus  Virus: genetic element that can multiply only in a living (host) cell  Not living organism; has its own nucleic acid genome  Obligate intracellular parasite: needs host cell for energy, metabolic intermediates, protein synthesis  Virion (virus particle) extracellular form of a virus, facilitates transmission 3 Viral components of naked and enveloped virus particles  Capsid: the protein shell that surrounds the genome of a virus  Naked viruses (e.g., most bacterial and plant viruses) have no other layers  Enveloped viruses (e.g., many animal viruses) have an outer layer consisting of a phospholipid bilayer (from host cell membrane) and viral proteins  Nucleocapsid: nucleic acid + protein in enveloped viruses  Virion surface proteins important for host cell attachment and may include enzymes involved in infection/replication 4 Viral activities: modes of infection  Virulent (lytic) infection: replicates and destroys host  Host cell metabolism redirected to support multiplication and virion assembly  Lysogenic infection: host cell genetically altered because viral genome becomes part of host genome 5 Viral diversity and classification  Viruses can be classified by nature of genetic material  Either DNA or RNA genomes  Either single-stranded or double-stranded  Classification based on the hosts they infect  Bacterial: bacteriophage or phage  Archaeal  Animal  Plant  Protozoan  Others 6 Not all viruses are bad  Disease-causing viruses are extensively studied  Some viruses exert beneficial effects  Arabidopsis infected with plus pox virus increases drought tolerance  Hepatitis G infection of HIV patients decreases HIV replication and infectivity – virus Watered plants + virus – virus + virus Drought conditions 7 Virion structure  Capsomere: individual protein molecules arranged in a precise and highly repetitive pattern around the nucleic acid making up the capsid  Some viruses only have one capsid protein because small size of viral genomes restricts number of proteins (e.g., tobacco mosaic virus)  Capsids can be put together through selfassembly (spontaneous) or may require host cell folding assistance 8 Basic virus symmetry  Two kinds of symmetry corresponding to two primary shapes: rod and spherical  Helical symmetry: rod-shaped; e.g., tobacco mosaic virus (previous slide)  Length determined by length of nucleic acid  Width determined by size and packaging of capsomeres  Icosahedral symmetry: spherical  20 triangular faces, 12 vertices; 5, 3, or 2 identical segments  most efficient arrangement of subunits in a closed shell  requires fewest capsomeres 9 Complex viral structure  Some viruses have complex structures  Virion contains several parts, each with their own shapes and symmetry  Most complex are head-plus tail bacteriophages; e.g., T4 phage 10 Enveloped viruses  Nucleocapsid surrounded by lipoprotein membrane  Most (e.g., Ebola) use outer surface proteins to attach and infect  Relatively few enveloped plant or bacterial viruses because of cell walls surrounding cell membrane  Entire virion enters animal cell during infection  Enveloped viruses exit more easily Reconstruction of Ebola virus (Panel b)  Red – spikes  Orange – lipid envelope  Green – membrane-associated proteins  Blue – nucleocapsid proteins Helical virion of Ebola Influenza virus Reconstruction of Ebola Vaccinia virus 11 Enzymes inside virions  Lysozyme: makes holes in bacterial cell wall to allow entry of nucleic acid  Lyses bacterial cells to release new virions  Neuraminidases (influenza virus)  Destroys glycoproteins and glycolipids  Allow liberation of viruses from cells  Nucleic acids polymerases  RNA replicases: RNA-dependent RNA polymerases  Reverse transcriptase: RNA-dependent DNA polymerase in retrovirus 12 Culturing, detecting and counting of bacteriophage  Plaque assay: plaques are clear zone of cell lysis that developed on lawns of host cells where successful viral infection occurs  Titer: number of infectious virions per volume of fluid  Calculate titer from the number of plaques 13 Animal cell culture and viral plaques  Similar to plaque assay of bacteriophage  Plating efficiency: estimates of viral titer by plaque assay  Number of plaque-forming units is always lower than direct count by electron microscopy  Efficiency of infection usually much lower than 100%  Defective virions or inappropriate conditions for infectivity 14 Steps in replication cycle of prokaryotic viruses  Five steps of phage replication in a permissive (supportive) host  Attachment (adsorption) of the virion)  Penetration (entry, injection) of the viral nucleic acid  Synthesis of viral nucleic acid and proteins by host cell as redirected by virus  Assembly of capsids and packaging of viral genomes into new virions  Release of new virions from host cell 15 Growth curve of viruses  One-step growth curve: virion numbers increase when cells burst  Eclipse phase: genome replicated and proteins synthesized  Maturation: packaging of nucleic acids in capsids  Latent period: eclipse + maturation  Release: cell lysis, budding, or excretion  Burst size: number of virions released 16 Receptors for bacteriophages  Attachment and entry of bacteriophage requires complementary receptors on the host cell surface – host specificity a major factor  Receptors on host cells carry out normal host functions for cell: e.g., proteins uptake, flagella, cell-cell interaction  Depending on the phage, receptors include proteins, carbohydrates, lipoproteins  For T4 phage, receptor is carbohydrates of lipopolysaccharide of the outer membrane 17 Attachment and penetration of bacteriophage T4  Virion attaches to cell via tail fibers that interact with polysaccharides on E. coli lipopolysaccharide layer  Tail fibers retract, and tail pins contact cell wall  T4 lysozyme forms a small hole in peptidoglycan  Tail sheath contracts, resulting in injection of viral DNA into cytoplasm  Capsid stays outside 18 Invasion of viral genome does not ensure infection  Prokaryotes possess mechanisms to diminish viral infections  Toxin-antitoxin molecules  Antiviral CRISPR (Clustered Regularly Short Palindromic Repeats)  Restriction endonucleases: enzymes that cleave foreign DNA at specific sites  Some viruses have modified genome that is unaffected by restriction enzymes 19 Virions synthesis  Within one minute of entry: host-specific protein synthesis ends and phage-specific protein synthesis starts  T4 genome encodes three major sets of proteins:  Early proteins: enzymes needed for DNA replication and proteins that modify host enzymes to transcribe viral genes  Middle and late proteins: head and tail proteins and enzymes required to liberate mature phage particles 20 Packaging and assembly  Packaging in three stages  Empty proheads (phage head precursors) assembled  Packaging motor assembled at prohead opening  Genome pumped into prohead using ATP  Motor discarded and capsid head sealed  After the head is filled, T4 tails, tail fibers, and other components are assembled  Late enzymes break membrane and peptidoglycan  Lysis occurs, 100+ virions released (b) Cryo-electron micrograph reconstruction of the T4 prohead and packaging motor 21 Temperate bacteriophages and lysogeny  Temperate: viruses establish long-term, stable relationship but are capable of virulence  Lysogen: host cell that harbours temperature virus  In lysogeny, viral genome is integrated into bacterial genome forming prophage (viral DNA)  Lysogeny maintained by phage-encoded repressor protein  Inactivation of repressor protein induces lytic stage (induction)  Viral DNA excised and starts lytic cycle  Cell stress (e.g., DNA damage) induces lytic pathway 22 Animal viruses  Processes universal to all viral infections  Capsids and DNA/RNA genome  Infection and takeover of host  Assembly and release  Differences between animal and bacterial viruses  entire virion enters the animal cell  Eukaryotic cells contain a nucleus, the site of replication for many animal viruses  Viroplasms (membrane-bound viral factories) form in some eukaryotic cells to increase virion assembly rate and protect from host defense 23 Representative viral diseases of human GenomeDNA or RNA a Sizeb Disease or host Virus Cold sores/genital herpes Herpes simplex dsDNA 152,000 Smallpox Variola major dsDNA 190,000 Influenza Influenza A virus ssRNA 13,600 Ebola hemorrhagic fever Ebola virus ssRNA 19,000 Severe acute respiratory syndrome (SARS) SARS virus ssRNA 29,800 Infant diarrhea Rotavirus dsRNA 18,600 Acquired immunodeficiency syndrome (AIDS) Human immunodeficiency virus (HIV) ssRNA (retrovirus) 9,700 24 Viral infection of animal cells  Bind specific host cell receptors, typically used for cell-cell contact or immune function  Often infect only certain tissue because different cell surface proteins expressed by different tissues/organs  Host cell entry by fusion with cytoplasmic membrane or by endocytosis  Uncoating occurs at cytoplasm or cytoplasmic membrane  Viral DNA genomes enter nucleus, most viral RNA genomes are replicated or converted to DNA within nucleocapsid  After genome packaging, many animal viruses are enveloped, during lysis or budding 25 Outcomes of virulent infection     Virulent infection: lysis of host cell, most common Latent infection: Viral DNA exists in host genome as provirus (similar to lysogeny) and virions are not produced; host cell is unharmed unless/until virulent pathway is triggered. Persistent infections: slow release of virions from host cell by budding does not result in cell lysis.  Infected cell remains alive and continues to produce virus Transformation: conversion of normal cell into tumor cell – rare event 26 Retrovirus – integration into the host genome  Includes HIV-1 (human immunodeficiency virus-1), the causative agent of AIDS (acquired immunodeficiency syndrome) in humans  Enveloped virion contains two copies of the ss(+)RNA genome  Retroviral genome is not used as mRNA  Virion contains several enzymes and specific viral tRNA  Reverse transcription: synthesize DNA from RNA template  Ribonuclease activity degrades RNA strand of RNA:DNA hybrid  DNA polymerase to make dsDNA from ssDNA using viral tRNA as primer  dsDNA integration into genome by integrase 27 Influenza virus  Segmented genome – influenza A genome has eight linear RNA molecules  Surface proteins interact with host cell surface  Hemagglutinin highly immunogenic (stimulates immune system); anti-hemagglutinin antibodies prevent infection (immunization)  Neuraminidase breaks down sialic acid component of host cytoplasmic membrane, functions in virus assembly  Nucleocapsid goes to nucleus for transcription  Enveloped virion forms by budding  Antigenic shift: RNA genomes from two different strains of the virus infecting the same cell, leading to re-assorted genome  Surface antigen of hybrid influenza virus not recognized by the immune system  Cause of major outbreaks of influenza 28 Coronaviruses  Enveloped virus with glycoprotein spikes on surfaces giving “crown” (corona) appearance  Cause respiratory infections in humans and other animals  Include Severe Acute Respiratory Syndrome Coronavirus-2 virus (SARS-CoV-2), the causative agent of COVID-19  Genomic RNA used as template to produce –strand from which mRNA is produced and translated  Virions assembled in Golgi complex  Released from cell surface 29