Animal Viruses, Prions, & Viroids PDF

Animal Viruses, Prions, & Viroids Chapter 11 Scanning electron micrograph of Ebola virus budding from the surface of a Vero cell Notice: This material is subject to the U.S. Copyright Law; furth An...

Animal Viruses, Prions, & Viroids Chapter 11 Scanning electron micrograph of Ebola virus budding from the surface of a Vero cell Notice: This material is subject to the U.S. Copyright Law; furth Animal and Plant Viruses ▪ Animal and plant viruses solve problems similar to those faced by bacteriophages: Host attachment, genome entry and gene expression, virion assembly, and virion release ▪ However, eukaryotic cells have a more complex structure than prokaryotic cells. Therefore, animal and plant viruses have greater complexity and diversity of viral replication cycles than we see in bacteriophages.  Animal Virus Replication ▪ Five-step infection cycle Understanding the infection cycle of viruses is important from a medical standpoint Knowledge of proteins involved in infection allows researchers to develop anti-viral medications © McGraw Hill, LLC Animal Viruses Show Tissue Tropism ▪ Animal viruses bind specific receptor proteins on their host cell. Receptors determine the viral tropism, or ability to infect a particular host, tissue, cell. For example, Ebola virus exhibits broad tropism by infecting many hosts and tissues within a host Speranza E, Connor JH. Host Transcriptional Response to Ebola Virus Infection. Vaccines. 2017; 5(3):30. https://doi.org/10.3390/vaccines5030030. Licensee MDPI, Basel, Switzerland. CC BY 4.0  Animal Virus Replication Five-step infection cycle 1. Attachment Viral spikes bind to receptors on host’s cell surface Usually glycoproteins on cytoplasmic membrane Often more than one required (ex: HIV binds to two) Function of host receptors unrelated to viral infection Specific host cell receptors required for attachment; limits tissue and host range of virus Some have narrow host range – influenza virus infects cells lining the respiratory tracts of humans Some have broad host range - rabies virus infects nerve cells of many mammal species Modification of image from Parker et al. 2022 OpenStax Microbiology CC BY 4.0.  Liu, J., Li, Y., Liu, Q. et al. SARS-CoV-2 cell tropism and multiorgan infection. Cell Discov 7, 17 (2021). https://doi.org/10.1038/s41 421-021-00249-2 CC BY SARS-CoV-2 tropism is correlated with ACE 2/TMPRSS2 distribution in the body Animal Virus Replication Five-step infection cycle 2-3. Entry and uncoating – fusion vs endocytosis Only enveloped viruses enter via fusion Lipid envelope of enveloped virus allows fusion Fuses with cytoplasmic membrane Leaving free nucleocapsid in cytoplasm © McGraw Hill, LLC Animal Virus Replication Five-step infection cycle 2-3. Entry and uncoating: Some enveloped and all non- enveloped virions enter by triggering endocytosis Take advantage of receptor- mediated endocytosis Binds to receptors that normally trigger this process If enveloped, viral envelope fuses with endosome membrane, leaving free nucleocapsid If non-enveloped, nucleic acid is released in cytoplasm once in endosome © McGraw Hill, LLC Animal Virus Replication Influenza Virus Five-step infection cycle Entry and uncoating: fusion or endocytosis In both fusion and endocytosis, entire virion enters cell (unlike phages) Uncoating then occurs – separation of nucleic acid from protein coat uncoating occurs through virus-specific, complex processes triggered by virus-host cell interactions Ex: Poliovirus and rhinovirus – attachment to surface receptors induces conformational changes in capsid, inducing uncoating EX: Influenza – low pH of endosome triggers uncoating Nucleic acid then can enter the nucleus via nuclear pores to replicate © McGraw Hill, LLC Animal Virus Replication Five-step infection cycle 4. Synthesis (production of new viral particles) requires two interrelated events I. Expression of viral genes to produce viral structural and catalytic proteins Ex: capsid proteins, enzymes required for replication II. Synthesis of multiple copies of the genome Three general replication strategies depending on viral genome type – DNA viruses – RNA viruses – Reverse-transcribing viruses Animal Virus Replication ▪ The primary factor that dictates the details of the synthesis stage of an animal virus is the form of its genome. ▪ DNA viruses Utilize some or all of the host replication machinery (DNA polymerase) ▪ RNA viruses Use an RNA-dependent RNA polymerase (replicase) to transcribe viral mRNA ▪ Retroviruses Use an RNA-dependent DNA polymerase (reverse transcriptase) to copy their genomic sequence into DNA for insertion in the host chromosome 11 Animal Virus Replication Five-step infection cycle Replication of DNA viruses Usually occurs in host cell’s nucleus Prefer to use host DNA polymerase But often encode their own DNA polymerase – allows replication even if host cell is not actively replicating its own chromosome dsDNA replication straightforward (follows Central Dogma of Molecular Biology) Illustration of the Replication of a Double-Stranded DNA Viral Genome and production of Viral mRNA.jpg by Gary E. Kaiser, Ph.D. https://cwoer.ccbcmd.edu/science/microbiology CC BY 4.0  Animal Virus Replication Five-step infection cycle Replication of DNA viruses ssDNA replication similar except complement first synthesized -> dsDNA intermediate ss(-)DNA viruses: (-) strand for transcribing mRNA; (+) strand for genome replication ss(+)DNA viruses: (-) strand used for both; still needs dsDNA intermediated Illustration of the Replication of a Single-Stranded DNA Viral Genome and production of Viral mRNA.jpg by Gary E. Kaiser, Ph.D. https://cwoer.ccbcmd.edu/science/microbiology CC BY 4.0 Animal Virus Replication Five-step infection cycle Replication of RNA viruses Majority are single-stranded; replicate in cytoplasm Uses virally encoded RNA polymerase – Replicase -> RNA-dependent RNA Poly Unique: normal RNA polymerase only can synthesize from DNA -> DNA-dependent RNA Poly Used to replicate genome and make mRNA for protein synthesis lustration of the Replication of a Single-Stranded Plus RNA Viral Genome and production of Viral mRNA.jpg by Gary E. Kaiser, Ph.D. https://cwoer.ccbcmd.edu/science/microbiology CC BY 4.0 Animal Virus Replication Five-step infection cycle Replication of RNA viruses ss(+)RNA functions as mRNA and immediately binds to host ribosomes First: Replicase translated from mRNA using host ribosome Then: Replicase replicates the viral genome The replication cycle of human picornaviruses (common cold) is representative of (+) sense single-stranded RNA viruses. © McGraw Hill, LLC ACE2 & TMPRSS2 receptors Majumder, J., Minko, T. Recent Developments on Therapeutic and Diagnostic Approaches for COVID- 19. AAPS J 23, 14 (2021). https://doi.org/10.1208/s12 248-020-00532-2 CCBY 4.0  Animal Virus Replication Five-step infection cycle Replication of RNA viruses ss(–)RNA (complement of mRNA) Carries replicase in nucleocapsid to synthesize (+) strand Host cell does not have a polymerase to transcribe ss- RNA to mRNA illustration of the Replication of a Single-Stranded Minus RNA Viral Genome and production of Viral mRNA.jpg by Gary E. Kaiser, Ph.D. https://cwoer.ccbcmd.edu/science/microbiology CC BY 4.0 Influenza virus Replicase (RdRp)  Animal Virus Replication Five-step infection cycle Replication of RNA viruses dsRNA viruses also carry replicase in nucleocapsid illustration of the Replication of a Double-Stranded RNA Viral Genome and production of Viral mRNA.jpg by Gary E. Kaiser, Ph.D. https://cwoer.ccbcmd.edu/science/microbiology CC BY 4.0  Animal Virus Replication Five-step infection cycle Replication of RNA viruses RNA viruses have high mutation rate because… Replicase lacks proofreading and generates mutations during replication Mutations may be “lethal” OR lead to genetic variation in surface proteins (which are recognized by the host’s immune system) The latter is called antigenic drift Ex: New variants of viruses over time Segmented RNA viruses have genes that are encoded by 2 or more nucleic acid strands Antigenic shift can occur if host cell is infected by 2 or more segmented viruses at the same time. New viral particles can be packaged with RNA segments from both Modification of images from Parker et al. 2022 OpenStax Microbiology CC BY 4.0. The Genome of Influenza A Modification of images from Parker et al. 2022 OpenStax Microbiology CC BY 4.0. By Ahmed Mostafa, Elsayed M. Abdelwhab, Thomas C. Mettenleiter, and Stephan Pleschka - mdpi.com/1999-4915/10/9/497/htm, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=92987475 Influenza Receptors in Different Hosts Human influenza viruses (blue) typically bind to α2,6-linked receptors, while avian influenza viruses (red) often bind to α2,3- linked receptors. Avian influenza viruses typically cannot readily infect humans. Swine can act as a "mixing vessel" to facilitate the transmission of influenza viruses between avian and human populations due to their ability to express both types of receptors. Kerstetter LJ, Buckley S, Bliss CM and Coughlan L (2021) Adenoviral Vectors as Vaccines for Emerging Avian Influenza Viruses. Front. Immunol. 11:607333. doi: 10.3389/fimmu.2020.607333 CC BY  Reassortment between Human, Avian, and Swine Strains If two different strains of influenza virus infect a host simultaneously, their segments can reassort to generate a novel hybrid strain – antigenic shift. Because influenza genomes are capable of reassortment, they can rapidly generate a new strain that our immune system fails to recognize. For example, the pandemic H1N1 strain of 2009 was due to a quadruple reassortment of these strains The influenza viruses also continually acquire small mutations that can lead to new phenotypes with respect to drug resistance and host range –antigenic drift. Influenza is a major global public health challenge. © McGraw Hill, LLC Proofreading exonucleases in larger RNA viruses such as SARS-CoV (1 & 2). Smith EC, Denison MR (2013) Coronaviruses as DNA Wannabes: A New Model for the Regulation of RNA Virus Replication Fidelity. PLoS Pathog 9(12): e1003760. https://doi.org/10.1371/journal.ppat.1003760 CC BY “The discovery of 3′-to-5′ exoribonuclease (ExoN) activity within CoV nonstructural protein 14 (nsp14-ExoN), which is critical for CoV high-fidelity replication, has challenged the long-held paradigm that RNA viruses cannot proofread and raises the possibility of an entirely new model for how RNA viruses regulate replication fidelity.”  Animal Virus Replication Five-step infection cycle Replication of reverse-transcribing viruses aka Retroviruses, have ss (+) RNA genome (ex: HIV) Lack replicase gene, but carries reverse transcriptase in virion: makes ssDNA from RNA (RNA-dep DNA-poly) Integrates into host cell Complementary strand synthesized by chromosome host cell DNA polymerase (provirus) dsDNA integrates into host cell chromosome via integrase and forms provirus Can direct productive infection or remain latent Illustration of the Replication of a Single-Stranded Plus RNA Viral Genome and Production of Viral mRNA by way of Cannot be eliminated from the host Reverse Transcriptase.jpg by Gary E. Kaiser, Ph.D. https://cwoer.ccbcmd.edu/science/microbiology CC BY 4.0 cell Animal Virus Replication ▪ The replication cycle of human immunodeficiency virus (HIV) is representative of retroviruses. ▪ HIV is an RNA virus that uses reverse transcriptase to copy its RNA genome into double- stranded DNA. ▪ HIV causes the syndrome known as AIDS. © McGraw Hill, LLC HIV-1 Structure © McGraw Hill, LLC HIV Attachment and Host Cell Entry ▪ HIV binds the CD4 receptor of T lymphocytes together with the chemokine coreceptor CCR5. This triggers fusion of the viral envelope and host membrane, and the HIV core enters the cytoplasm. ▪ HIV capsid then dissolves and releases its contents. © McGraw Hill, LLC CCR5 and CXCR4 NIH Public Domain https://hivinfo.nih.gov/understanding-hiv/infographics/hiv-life-cycle Integrase immature HIV HIV RNA polyproteins Breaks up polyproteins into integrase and reverse transcriptase NIH Public Domain https://hivinfo.nih.gov/understanding-hiv/infographics/hiv-life-cycle Some research suggests that in certain cell types or under specific conditions HIV can enter via endocytosis © McGraw Hill, LLC Animal Virus Replication Five-step infection cycle Assembly Protein capsid forms; genome, enzymes packaged Occurs spontaneously, but in a step-wise manner Non-enveloped viruses are completely assembled in the cytoplasm Enveloped viruses are completed as they are released from the cell via budding  Animal Virus Replication Five-step infection cycle 5. Release Most enveloped viruses via budding Viral protein spikes insert into host cell membrane; matrix proteins accumulates; nucleocapsids extruded (buds) Some obtain envelope from organelles (ER) and then transported out of cell via a vesicle - exocytosis Non-enveloped viruses released when host cell dies – many trigger apoptosis (programmed cell death) Image cap tured at th e N IAID Integrated Res earch Facili ty (IRF) in Fort Det rick, © McGraw Hi ll, LLC Maryland. Credit : NIAID (CC BY 2.0) Structure of Influenza A 34 Influenza Virus Life Cycle Virion contains eight segments of RNA, each enclosed in a separate nucleocapsid. Enters host by receptor-mediated endocytosis. The low pH of the endosome causes conformational change. Facilitates contact with the endosome membrane, allowing fusion of endosome membrane and viral envelope to occur. Influenza components shuttle between the nucleus and cytoplasm using nuclear localization signals. Influenza Virus Is a segmented ss(-)RNA enveloped virus 1. Hemagglutinin (spike) attaches to sialic acid (host receptor) 2. Induces endocytosis 3. Viral envelope fuses with endosome, releasing nucleocapsid 4. Capsid uncoats and segmented ssRNA (-) is released, enters nucleus via pore and host transport machinery 5. Replicase makes ssRNA (+) 6. ssRNA (+) enters nucleus and is replicated via replicase and mRNA made 7. Mature mRNA leave nucleus and enter cytoplasm for translation 8. Viral proteins assemble around membrane and bud off 9. Neuraminidase (enzyme) on surface prevents attachment to same cell by cleaving sialic acid Influenza Virus Is a segmented ss(-)RNA enveloped virus 1. Hemagglutinin (spike) attaches to sialic acid (host receptor) 2. Induces endocytosis 3. Viral envelope fuses with endosome, releasing nucleocapsid 4. Capsid uncoats and segmented ssRNA (-) is released, enters nucleus via pore and host transport machinery 5. Replicase makes ssRNA (+) 6. ssRNA (+) enters nucleus and is replicated via replicase and mRNA made 7. Mature mRNA leave nucleus and enter cytoplasm for translation 8. Viral proteins assemble around membrane and bud off 9. Neuraminidase (enzyme) on surface prevents attachment to same cell by cleaving sialic acid  Why can’t they cram all the strains of influenza into a single dose? Vaccinating people against a disease they're never going to get is a risky proposition: We don't know how the body would respond to a barrage of flu vaccinations. The patient might also develop a strong immune response to an insignificant strain, while skimping on antibodies for a more prevalent one. National Institute of Allergy and Infectious Diseases Public Domain Animal Host Defenses ▪ Since viruses are ubiquitous, a wide range of defense mechanisms have evolved in animals and plants. ▪ Genetic resistance – Hosts continually experience mutations – alter receptor proteins ▪ Immune system – “Innate immunity”— interferons – “Adaptive immunity”— antibodies ▪ RNA interference (RNAi) – Alters gene expression and degrades viral RNA Antiviral Drugs Drug development has been slow because it is difficult to specifically target viral replication → Viruses use the metabolic machinery of their hosts, which limits many of the potential points of attack. Current drugs inhibit virus-specific enzymes and life cycle processes Mechanisms of Action of Antiviral Medications Mechanisms of Action of Antiviral Medications Prevent Viral Entry New group of chemicals prevent viral entry into host cell Maraviroc blocks HIV attachment by binding HIV spike for CCR5 Enfuvirtide binds to HIV spike that promotes fusion of viral envelope with cell membrane NIH Public Domain https://hivinfo.nih.gov/understanding-hiv/infographics/hiv-life-cycle  Mechanisms of Action of Antiviral Medications Interfere with Viral Uncoating Nucleic acid must separate from protein coat Amantadine Block influenza A viruses from uncoating by blocking M2-protein function Ion channel allows ions to enter nucleocapsid that aid in fusion and uncoating  Mechanisms of Action of Antiviral Medications Interfere with Nucleic Acid Synthesis Most antiviral agents work by inhibiting viral DNA synthesis. These drugs chemically resemble normal DNA nucleosides, but they lack the 3' OH group needed for chain elongation during DNA synthesis. DNA chain-terminating analogs are selectively toxic because viral polymerases are more prone to incorporate nucleotide analogs into their nucleic acid than are the more selective host cell polymerases. Acyclovir is an analog of thymine-> ceases DNA replication of HSV (makes its own viral DNA polymerase) These drugs do not work on RNA viruses like influenza. Mechanisms of Action of Antiviral Medications Nucleic Acid Synthesis Replicase Inhibitors Inhibits replicase Can bind to cellular polymerase = bad side effects. Reverse Transcriptase Inhibitors Inhibits reverse transcriptase Often used with nucleoside analogs to treat HIV infections  Mechanisms of Action of Antiviral Medications Prevent Genome Integration Raltegravir Inhibits HIV-encoded enzyme integrase Prevent Assembly and Release of Viral Particles Protease Inhibitors are virus specific In HIV, prevents cleavage of polyproteins During replication of HIV, integrase and reverse transcriptase translated as a polyprotein that must be cleaved by a protease. NIH Public Domain https://hivinfo.nih.gov/understanding-hiv/infographics/hiv-life-cycle Mechanisms of Action of Antiviral Medications Prevent Assembly and Release of Viral Particles Neuraminidase Inhibitors - Tamiflu Binds to and prevents function of neuraminidase (releases influenza from budding cell) Cultivating and Quantitating Animal Viruses ▪ To study, viruses must be grown in appropriate host Historically, done by inoculating live animals Cell culture or tissue culture now used Animal cells grown in liquid medium ▪ Quantitating Animal Viruses Plaque assays using monolayer of tissue culture cells Direct counts via Electron microscope Other Infectious Agents: Viroids and Prions Viroids are small single-stranded RNA molecules 246–375 nucleotides Forms closed ring Discovered in 1971 (potato tuber spindle disease) Thus far, only found in plants; enter through wound sites Many questions remain: How do they replicate? - Replicated by host RNA polymerases, but how? How do they cause disease? How did they originate? Do they have counterparts in animals? Pamela Roberts, University of Florida Institute of Food and Agricultural Sciences, USDA ARS)  Other Infectious Agents: Viroids and Prions Prions are proteinaceous infectious agents Composed solely of protein; no nucleic acids Linked to slow, fatal human diseases; animal diseases Mad cow disease – cattle Chronic wasting disease – deer and elk Creutzfeldt-Jakob disease – Humans Variant Creutzfeldt-Jakob disease - Humans (consuming MCD beef) Usually, transmissible only within species Mad Cow Disease is an exception, ~170 people died in England by eating infected tissue The risk of CWD transmission to humans is unknown Other Infectious Agents: Viroids and Prions Prions Once infected, prion proteins accumulate in neural cells Neurons die Tissues develop holes (spongiform lesions) Brain function deteriorates Characteristic appearance gives rise to general term for all prion diseases: transmissible spongiform encephalopathies “Figure 6.26 Creutzfeldt-Jakob disease (CJD) is a fatal disease that causes degeneration of neural tissue. (a) These brain scans compare a normal brain to one with CJD. (b) Compared to a normal brain, the brain tissue of a CJD patient is full of sponge -like lesions, which result from abnormal formations of prion protein. (credit a (right): modification of work by Dr. Laughlin Dawes; credit b (top): modification of work by Suzanne Wakim; credit b (bottom): modification of work by Centers for Disease Control and Prevention)” Modification of images from Parker et al. 2022 OpenStax Microbiology CC BY 4.0. Other Infectious Agents: Viroids and Prions Prions – how do they accumulate in tissue? Host cells normally produce cellular/endogenous prion form PrPC (prion protein, cellular) Proteases can readily destroy Infectious prion proteins PrPSC (prion protein, scrapie) Hypothesized that PrPSC induces PrPC misfolding to PrPSC Resistant to proteases; become insoluble, aggregate PrPsc prions in brain tissue can be the result of a spontaneous mutation in the PrPc protein gene, or it may originate from PrPsc prion consumed in food Modification of images from Parker et al. 2022 OpenStax Microbiology CC BY 4.0.