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Human Viral Disease; Virus Replication Cycle

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Human-Virus Interaction Virus extinction Clear virus, immunity Large number of deaths Small Population Favors persistent virus infection Virus infection with an immunological nave person i.e. herpes simplex virus, parent to newborn Large Population Many susceptible to infection Virus infected individuals available all the time Sporadic spread of virus i.e. common cold virus, school class room

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Asymptomatic infection no disease symptoms Acute infection disease symptoms Persistent infection long term Chronic: infectious virus Latent: no virus replication, virus reactivation Transformation alter cell regulation, tumor production, cancer No infectious virus Viral DNA, complete or partial Patterns of Virus Disease

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DNA Virus Infections

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RNA Virus Infections

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Acute Infection: Varicella- zooster virus (VZV) Herpesvirus one virus, two diseases Varicella virus Chickenpox; common childhood disease Primary acute mucosal/skin infection Resolves in 1-2 weeks Virus infect and latent in nerve cells

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Persistent Chronic Infection: VZV Zooster virus Shingles Latent in nerve tissue Presence of viral DNA, no infectious virus Virus held in check by host immune defense Later in life, reactivation of virus, replicates, descends down nerve tissue, replicates in skin cells

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One-Step Virus Replication In Cell Culture High level of virus infection (1-10 virus/cell) Synchronous virus replication in cells All events required for cell infection

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Virus Replication Cycle Attachment (Adsorption) Entry / Uncoating (Penetration) Gene Expression (Synthesis: Early, Genome, Late) Assembly (Maturation) Release (Lysis, Budding)

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Virus Attachment (Adsorption) Contact and interaction of virus to host cell Recognition of virus to host cell Virus molecule that binds to host cell called ligand (viral protein or glycoprotein)

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Attachment: Host Cell Virus binds to host cell molecule - receptor (i.e. cell protein, glycoprotein, lipid) Receptors are molecules that have a role in normal functioning of the cell

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Attachment: Cell Receptor Virus may bind up to three different cell receptors in succession: Low affinity receptor - in high abundance, virus contacts cell surface Primary receptor - in lower concentration Co-receptor follows binding of primary receptor

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Attachment: Specificity Host Range - the organism(s) that the virus is able to infect (narrow or wide) i.e. plant, animal, human Tissue Tropism- the cell type(s) a virus is able to infect i.e. skin, oral, GI, CNS

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Attachment: Binding 3-D fit between viral ligand and cell receptor Mainly weak electrostatic charges. Evidence for this is interaction may require: specific pH specific ionic strength presence of specific ions i.e. Ca ++, Mg ++

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Attachment: Nonenveloped Picornavirus Virus ligand - a deep cleft (canyon) in triangular face of capsid (viral proteins VP1, VP2, VP3) Binds to cell receptor ICAM 1 (intracellular adhesion molecule 1), normal function is to bind cells i.e. WBC

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Attachment: Nonenveloped Virus to Host Cell Membrane

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Attachment: Enveloped HIV Virus Host cell protein in virus envelope (cyclophilin A) initially binds HIV to low affinity receptor (heparin sulfate) of the cell Followed by binding of viral ligand (gp120) to primary receptor (CD4) on T helper cells, macrophages, and glial cells Binding of gp120 to CD4 results in conformational change of gp120, which then binds to chemokine co- receptor CXCR4 on T lymphocytes or CCR5 on macrophages

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Attachment: Enveloped Virus to the Host Cell Membrane

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Entry / Uncoating Entry is the mechanism used by the virus to penetrate into the host cell Uncoating is the separation of the nucleic acid from the capsid, and refers to changes that occur to make the viral nucleic acid ready for expression

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Entry: Nonenveloped Virus Receptor-mediated endocytosis Clathrin coated pits (seen by EM) Invagination, pinch off membrane Forms intracellular endosome, contains the virus Endosome becomes acidified

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Uncoating: Nonenveloped Virus Acid pH causes conformational changes in capsid protein Hydrophobic region interacts with membrane, forms a pore Viral nucleic acid released

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Entry / Uncoating: Nonenvelpoed Poliovirus

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Nonenveloped Virus: Endocytosis

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Entry / Uncoating: Enveloped Virus Receptor mediated fusion of virus envelope with cell plasma membrane Two modes of entry: Direct entry (pH independent) Receptor-mediated endocytosis (pH dependent; for uncoating)

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Direct Entry / Uncoating: Enveloped Sendai Virus At cell surface by a viral fusion protein (active upon cleavage) Viral capsid released into cytoplasm

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Fusion at the Cell Membrane: Enveloped Virus

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Entry By Receptor- Mediated Endocytosis: Enveloped Influenza Virus Lower pH in endosome Conformational change in HA of influenza exposes a fusion peptide Fusion of viral envelope with endosomal envelope Release capsid into cytoplasm

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Influenza Virus Envelope : Cell Membrane

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Endocytosis: Enveloped Virus

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Receptor-Mediated Endocytosis: Enveloped Virus

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Synthesis: Early Gene Expression Release of viral genome into cell (cytoplasm or nucleus) Virus regulates host cell metabolic machinery Only some viral genes expressed (early transcription & translation) Viral regulatory proteins and enzymes for initial synthetic events

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Synthesis: Genome Replication Replication of viral nucleic acid Cellular or viral polymerase New genome synthesis signals for additional viral synthetic events

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Synthesis: Late Gene Expression Further expression of viral genome late transcription and translation Some regulatory proteins Mainly structural (capsid, envelope) proteins for progeny virus

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Assembly (Maturation) This phase of viral replication is FUNDAMENTALLY DIFFERENT from organisms Viruses assembled from component parts, not from division of a pre-existing virus i.e. not exponential growth kinetics, but burst of new virions

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Self Assembly Concentration of viral structural proteins and genomes (reactants) adequate Self forming process (recognition between viral components) Assembly follows basic laws of thermodynamics

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Virion Assembly Assembly requires protein-protein interactions and protein-nucleic acid interactions The order of assembly occurs two ways: The genome serves as a focus for assembly of the capsid surrounding it (helical viruses) A hollow capsid formed and then filled with the genome (icosahedral virus)

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Assembly Helical Virus: TMV Rigid helical virus Composed of RNA plus identical capsomers arranged in a helix TMV capsid proteins only recognize TMV RNA This means that the protein-nucleic acid interactions are very specific

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TMV Assembly: Proteins First, 34 capsid proteins assemble into a pair of disks The outer portions interact to hold the two disks together, while the inner portion has a gap where RNA binds When the RNA enters, the gap is closed to hold the RNA in place

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TMV Assembly: Genome RNA interacts with the disks beginning at the pac (packaging signal) site, which is about 1000 bases from the 3 end of the genome The pac site consists of ~ 500 bases that can form a series of hairpin loops

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Summary: TMV Assembly Capsomers Disc Multiple helical disc RNA binds to disc Helix elongation of RNA through central hole

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Assembly: Icosahedral Virus Has 20 triangular faces and each face is composed of 3 subunits (or multiples of 3). The subunits may be identical or different

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Assembly: Poliovirus Protomer is made with Vp0, VP1, and VP3 Five protomers combine to form a pentamer Twelve pentamers combine to form an empty procapsid (60 protomers) RNA enters the procapsid A maturation cleavage converts VP0 into VP2 and VP4 to form intact virion

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Cell Lysis Virus lytic infections cause distinct changes of infected cell Changes called cytopathic effect (CPE) and include: Inclusion body Nuclear pyknosis (shrinking) Vacuole Apoptosis Syncytia (multinucleated cells)

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Inclusion (Negri) Body - Rabies Virus

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Syncytia (giant cell) Formation - Herpesvirus

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Virus Release: Cell Lysis CPE usually secondary result of changes in host cell metabolism by viral replication Virus may halt or alter host cell DNA synthesis, transcription, and/or protein synthesis (translation) Results in disintegration of infected cell and release of progeny virus

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Virus Release: Budding (Exocytosis) Synthesis and insertion of viral glycoproteins in host cell membrane (nuclear, ER, Golgi, plasma membrane) Assembly of viral nucleocapsid Nucleocapsid and virus modified membrane brought together (capsid protein may interact directly with viral glycoprotein or via a viral matrix protein) Exocytosis, or budding - may or may not kill the cell

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Virus Budding Through Cell Plasma Membrane

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Polarized Cell Plasma Membrane Exit Viral envelope proteins contain apical or basolateral plasma membrane transport signals Virus that bud apically tend to cause localized infections (release via surface) Virus that bud basolaterally tend to cause systemic infections (release via interior)

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Reading & Questions Chapter 4: Patterns of Some Viral Diseases of Humans Chapter 6: The Beginning and End of the Virus Replication Cycle (omit Questions 3, 4)

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QUESTIONS???

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Class Discussion Lecture 3 1. How does an acute virus infection differ from a persistent (chronic, latent) infection? 2. Is virus attachment/entry similar to a normal cell process? 3. How is the capsid of a helical virus (TMV) assembled? 4. How is the capsid of a spherical virus (poliovirus) assembled? 5. Non-enveloped virus are able to self- assemble in vitro, but not enveloped viruses. Why?