Assembly Lecture 11 Biology W3310/4310 Virology Spring 2013 Anatomy is des.ny. - - SIGMUND FREUD 1
All virions complete a common set of assembly reac3ons * common to all viruses common to many viruses 2
The structure of a virus par3cle determines How it is formed How it enters a new cell How it replicates 3
A tale of two picornaviruses Poliovirus virions survive passage through the stomach to replicate in the gut: acid resistant capsid Rhinovirus virions are inacovated in the gut and only replicate in the respiratory tract: acid sensi3ve capsid 4
Despite varia3ons in structure and biological proper3es, all infec3ous virions must be METASTABLE Stable enough to survive in the wild Unstable enough to come apart during infecoon The assembly pathway is irreversible in the cell that makes the virions, but reversible in the uninfected cell receiving the virion 5
Produc3on of virus par3cles depends on host cell machinery Cellular proteins catalyze or assist the folding of individual protein molecules Transport systems move viral proteins and nucleic acids to and from sites of assembly Secretory pathway processes and moves viral membrane proteins Nuclear import and export machinery moves viral nuclear proteins and nucleic acid in and out of the nucleus 6
Concentra3ng components for assembly: Nothing happens fast in dilute solu1ons Viral components owen are concentrated so much that they are visible in the microscope (so- called factories or inclusions Non- enveloped viruses owen use internal membranes to concentrate proteins (poliovirus) Negri bodies (rabies virus) 7
Viral proteins achieve high concentra3ons by several methods Viral replicaoon and translaoon occur in a compartment ( factory or localized site) Localized producoon of viral proteins and protein- protein interacoons enable formaoon of independent sub- assemblies Local concentraoons of viral structural components can be boosted by lateral interac.ons between membrane- associated proteins (e.g. membrane patches ) 8
GeFng things to the right place 9
Viral proteins have addresses built into their structure Membrane proteins go to the appropriate membranes - Signal sequences, fa]y acid modificaoons Membrane proteins stay in the appropriate membranes - RetenOon signals Nuclear proteins go to the nucleus - Nuclear localizaoon sequences (NLS) Viral mrna or ribonucleoprotein complexes move into the cytoplasm - Nuclear export signals Capsids and protein complexes exhibit directed mooon, not diffusion - Microtubules and intermediate filaments are the tracks; dyneins, kinesins, myosins are the motors 10
Localiza3on of viral proteins to the nucleus Plasma membrane Golgi apparatus Ribosome Rough endoplasmic reticulum Py(VP1) 5 + VP2/3 Ad hexon + 100 kda Nucleus Nuclear envelope: Outer nuclear membrane Inner nuclear membrane Nuclear pore complex Mitochondrion Influenza virus NP Cytoskeleton: Intermediate filament Microtubule Actin filament bundle Extracellular matrix 11
Localiza3on of viral proteins to plasma membrane Other viral proteins Viral RNA Nascent viral protein Cell surface viral protein Golgi Transport vesicle Fusion Nucleus Microtubule Mitochondrion ER Cytoskeleton Extracellular matrix 12
Movement of VSV nucleocapsids in cytoplasm requires microtubules uclei are in blue. Courtesy of Asit Pattnaik, University of Nebra A B Box 12.11 13
Three strategies for making sub- assemblies 14
viral chaperone 15
Sequen3al capsid assembly: Poliovirus 16
Assembly intermediates and the assembly- line concept Assembly- line mechanisms ensure orderly formaoon of viral parocles and virion subunits FormaOon of discrete intermediate structures Can t proceed unless previous structure is formed: quality control 17
{sequenoal} Viral scaffolding proteins establish transient intermediate structures viral proteases packaged in these intermediate structures become acovated to finalize structure 18
Sequen3al Assembly Adenovirus genome packaged into a preformed shell in the nucleus 19
Self- assembly versus assisted assembly reac3ons Self- assembly - HIV capsid proteins can form empty shells - Influenza HA glycoprotein can bud and form virus- like parocles - HBV surface anogen can assemble into virus- like parocles Assisted assembly - Can t assemble on their own - Proteins and nucleic acid genomes are required as scaffolds or chaperones to form structure 20
Concerted Assembly Influenza virus parocles form by budding 21
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Concerted Assembly Retrovirus parocles form by budding Mature a@er release 24
A Extracellular domain Signal Signal sequence peptidase Fusion Transmembrane Cytoplasmic domain ALV Signal peptidase HIV Variable regions B B S S TM SU Fusion peptide Viral membrane 25
AddiOon of lipid to viral proteins allows targeong to membranes independent of signal sequence Viral proteins are synthesized in the cytoplasm, and modified with lipids post- translaoonally Also added - geranylgeranol (C20) or palmitate 26
Changes at the myristoylaoon sequence prevent interacoon of Gag with the cytoplasmic face of the plasma membrane Virus assembly and budding are inhibited 27
Genome packaging Viral genomes must be disonguished from cellular DNA or RNA molecules where assembly takes place Requires discriminaoon among similar nucleic acid molecules Example: retrovirus genomes <1% cytoplasmic RNA, yet is the RNA packaged in majority of retrovirus parocles DiscriminaOon is the result of packaging signals in the viral genome - sequences necessary for incorporaoon of nucleic acid into virions; geneocally defined 28
Packaging signals - DNA genomes Adenovirus Packaging signal near lew inverted repeat and origin Signal is complex: a set of repeated sequences; overlapping with enhancers that somulate late transcripoon Structure is recognized by viral protein IV2a (also a transcripoon acovator) 29
Herpesvirus genome replicaoon produces concatemers with head- to- tail copies of viral genome HSV- 1 packaging signals pac1 and pac2 needed for recognioon of viral DNA and cleavage within DR1 30
Packaging signals - RNA genomes 350 nt; necessary and sufficient env mrnas not packaged Necessary but not sufficient for HIV- 1 genome packaging 31
Packaging signals - RNA genomes NC of Gag mediates selecove encapsidaoon of genomic RNA during assembly Central region binds RNAs with Ψ sequences Structure of HIV- 1 NC bound to Ψ SL3 shows protein contacts with RNA 32
Packaging signals - RNA genomes Packaging limits - upper limit on size of viral nucleic acid that can be accommodated in icosahedral capsid or nucleocapsid; ~5-10% larger than viral genome Coupling of encapsidaoon of viral nucleic acid with its synthesis may contribute to specificity, e.g. poliovirus RNA replicaoon on membrane vesicles; no packaging signal idenofied in genome 33
Packaging of segmented genomes How to ensure that virions receive one copy of each segment? Random or selec.ve mechanisms for influenza virus (8 segments) cannot be disonguished Random mechanism would yield 1 infecoous parocle per 400 assembled - within the known parocle to pfu raoo If 12 segments are packaged, 10% of parocles would contain complete viral genome New evidence for specific packaging sequence on each RNA segment 34
Influenza virus RNA packaging h]p://www.virology.ws/2009/06/26/packaging- of- the- segmented- influenza- rna- genome/ 35
Selec3ve packaging Bacteriophage ϕ6-3 dsrna segments S, M, L S segment enters alone; entry of M depends on presence of S; entry of L depends on presence of S + L Serial dependence of packaging ParOcle:pfu raoo ~1 36
Acquisi3on of an envelope May follow assembly of internal structures (most enveloped viruses) May be simultaneous with assembly of internal structures (retroviruses) 37
Four budding strategies Envelope glycoproteins and capsid are essenoal for budding - alphaviruses Internal matrix or capsid proteins drive budding - retroviruses Envelope proteins drive budding - coronavirus Matrix proteins drive budding, but addioonal components (glycoproteins, RNP) needed for efficiency or accuracy 38
Internal structure assembly and budding spaoally & temporally separated 39
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When synthesized alone, Gag directs budding of virus- like parocles Internal structure assembly and budding are coincident in space & Ome 41
Amino acid change in Gag cause arrest of budding at a late stage (late or L domains) Three different classes of L domains, found in + and - strand enveloped viruses L domains bind cell proteins, involved in vesicle trafficking, needed for virus release 42
L domain mo3fs 43
Involvement of the ESCRT machinery in three topologically equivalent types of membrane abscission 44
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Exocytosis: reverse of endocytosis; used by viruses that assemble within vesicular compartments of the ER or Golgi Pathways of herpesvirus assembly and egress 46
The majority of viruses leave an infected cell by one of two general mechanisms Release from the cell by budding or lysis Movement from cell to cell 47
HIV- 1 infected T- cell Polarized release Of HIV from Infected T- cell Epithelial cell 48
Egress (exit) in vivo is a controlled process, ozen polarized Via apical surface places virus in the outside world (sneezing) Via basal surface of epithelial cells places virus in contact with blood, lymph, nerves (systemic spread, bad news) Can occur at sites of cell contact (lungs, gut, synapses) DirecOon and mode of egress influence pathogenesis 49
Polarized spread of infec3on by an alpha herpesvirus: Release from axon terminals and infec3on of epithelial cells in vitro Single Axon Purple: nuclei of epithelial cells Green: GFP expressing virus 50
Why do infected cells lyse? InhibiOon of cell macromolecular processes and transport ApoptoOc lysis Specific mechanisms - Adenovirus L3 protease cleaves intermediate filament proteins - Damages structural integrity of cell, facilitates virus release - Adenovirus glycoprotein required for cell lysis, mechanism not known 51
439 Intracellular Trafficking B Inhibition of secretion Induction of autophagy Endoplasmic reticulum Model for nonly3c release of poliovirus par3cles Transport vesicle Cytosolic contents Intermediate compartment Protein 3A Endosomal fusion cis Medial trans Golgi Lysosomal fusion 2BC 3A Lc3-pe Poliovirus polymerase Lamp-1/2 llular secretory pathway in poliovirus-infected cells. (A) Electron lls (left) and HeLa cells 5 h after poliovirus infection (right) preserved fected cell-specific vesicles can been seen in the infected cells. G, Golgi virus particles. The bars indicate 1 µm. Adapted from A. Schlegel with permission. Courtesy of Karla Kirkegaard, University of Colorado, 52
Extracellular virion matura3on Acidianus convivator virus, from acidic hot spring (ph 1.5, 85-93 C) 53