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Assembly Lecture 11 Virology W3310/4310 Spring 2012 “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 • Hos 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 inacEvated 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 infecEon 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 oOen are concentrated so much that they are visible in the microscope (so-‐called ‘factories’ or ‘inclusions’ • Non-‐enveloped viruses oOen use internal membranes to concentrate proteins (poliovirus) 7 Viral proteins achieve high concentra3ons by several methods • Viral replicaEon and translaEon occur in a compartment (‘factory’ or localized site) • Localized producEon of viral proteins and protein-‐ protein interacEons enable formaEon of independent sub-‐assemblies • Local concentraEons of viral structural components can be boosted by lateral interac4ons 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, faWy acid modificaEons • Membrane proteins stay in the appropriate membranes -‐ RetenEon signals • Nuclear proteins go to the nucleus -‐ Nuclear localizaEon sequences (NLS) • Viral mRNA or ribonucleoprotein complexes move into the cytoplasm -‐ Nuclear export signals • Capsids and protein complexes exhibit directed moEon, not diffusion -‐ Microtubules and intermediate filaments are the tracks; dyneins, kinesins, myosins are the motors 10 414 CHAPTER 12 Localiza3on of viral proteins to the nucleus Plasma membrane Golgi apparatus Ribosome Rough endoplasmic reticulum Py(VP1)5 + VP2 /3 Ad hexon + 100 kDa Nuclear envelope: Outer nuclear membrane Inner nuclear membrane Nucleus Nuclear pore complex Mitochondrion Cytoskeleton: Influenza virus NP Intermediate filament Microtubule Actin filament bundle Extracellular matrix Figure 12.1 Localization of viral proteins to the nucleus. The nucleus and major membrane-bound compartments of the cytoplasm, as well as components of the cytoskeleton, are illustrated schematically and not to scale. Viral proteins destined for the nucleus are synthesized by cytoplasmic polyribosomes, as illustrated for the influenza virus NP protein. They engage with the cytoplasmic face of the nuclear pore 11 Figure 12.2 Localization of viral proteins to the plasma membrane. Viral envelope glycoproteins (red) are cotranslationally translocated into the ER lumen and folded and assembled within that compartment. They travel via transport vesicles to and through the Golgi apparatus and from the Golgi apparatus to the plasma membrane. The internal proteins of the particle (purple) and the genome (green) are also directed to plasma membrane sites of assembly. Localiza3on of viral proteins to plasma membrane Other viral proteins Cell surface viral protein Viral RNA Nascent viral protein Golgi Fusion Nucleus Transport vesicle Mitochondrion Microtubule ER Cytoskeleton Extracellular matrix 12 E X P E R I M E N T S Movement of vesicular stomatitis virus nucleocapsids within the cytoplasm requires microtubules stomatitis virus nucleocapsids be transported to the plasma e for assembly and budding of cles contain the (−) strand RNA nd several viral proteins, includrotein (see the text). To examine ar trafficking of nucleocapsids, e coding for a green fluorescent GFP) was inserted into that for region of the P protein. Coniments established that the Pon protein catalyzed both viral nthesis and genome replication, t exhibited somewhat reduced urthermore, mutant particles P-eGFP in place of the P proinfectious. cted cells, P-eGFP colocalized ly synthesized viral RNA, as h the N and L proteins, in cytoructures of the size predicted capsids. Time-lapse imaging of GFP-containing structures indit nucleocapsids move toward eriphery and, unexpectedly, do e association with mitochonignificance of this association is ear. However, as mitochondria n to move on microtubules, it kely that viral nucleocapsids o. Indeed, nucleocapsids were to be distributed throughout asm in close association with les (panel A below). Treatment d cells with drugs that disrupt les, such as nocodazole, dramatred this pattern: nucleocapsids became clustered in the absence of microtubules in large aggregates and did not reach the plasma membrane (panel B). Such drugs also reduced virus yield significantly, confirming the importance of microtubules in the transport of vesicular somatitis virus nucleocapsids to sites of assembly. Movement of VSV nucleocapsids in cytoplasm requires microtubules Das, S. C., D. Nayak, Y. Zhou, and A. K. Pattnaik. 2006. Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells. J. Virol. 80:6368–6377. Localization of P-eGFP-containing nucleocapsids (green) and microtubules (red) in cells infected by the mutant virus VSV-PeGFP and untreated (A) or treated with nocodazole prior to infection (B). Nuclei are in blue. Courtesy of Asit Pattnaik, University of Nebraska—Lincoln. 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 formaEon of viral parEcles and virion subunits • FormaEon of discrete intermediate structures • Can’t proceed unless previous structure is formed: quality control 17 {sequenEal} Viral scaffolding proteins • establish transient intermediate structures • viral proteases packaged in these intermediate structures become acEvated to finalize structure 18 Sequen4al 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 parEcles -‐ HBV surface anEgen can assemble into virus-‐like parEcles • 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 parCcles form by budding 21 22 23 Concerted Assembly Retrovirus parCcles form by budding Mature a6er release 24 434 434 CHAPTER 12 A A CHAPTER 12 Signal Signal sequence peptidase Signal Signal sequence peptidase ALV Fusion Extracellular domain Fusi Cytoplasmic domain Transmembrane Signal peptidase ALV Signal peptidase HIV HIV Variable regions Variable regions B S S TM B Fusion peptide SU SU Viral membrane Table 12.4 Figure 12.13 Modification and processing of retroviral Env polyproteins. (A) Sequence features and modifications of the Env proteins of avian leukosis virus (ALV) and human immunodeficiency virus type 1 (HIV) are depicted as in Fig. 12.3. The variable Env TM regions of human immunodeficiency virus type 112.13 Modific differ greatlySin Ssequence among viral isolates. The translocation Figure products shown here are cleaved by the ER signal peptidase (red arrows) and (A) by furin family Sequence features proteases in the trans-Golgi network (orange arrowheads). The latter liberates (ALV) and hum Fusion peptidefrom thevirus the transmembrane (TM) and surface unit (SU) subunits Env precursor. in maintain Fig. 12.3. (B) Both noncovalent interactions and a disulfide bond (-S-S-) can the The variab association of the SU and TM proteins. differ greatly in sequen Examples of acylated or isoprenylated viral proteins Virus Protein Lipid Viral membrane Probable function here are cleaved by th proteases in the transthe transmembrane (T (B) Both noncovalent association of the SU a Envelope proteins Alphavirus Sindbis virus Coronavirus E2 Palmitate Required for efficient budding of virus particles 25 • AddiEon of lipid to viral proteins allows targeEng to membranes independent of signal sequence • Viral proteins are synthesized in the cytoplasm, and modified with lipids post-‐ translaEonally • Also added -‐ geranylgeranol (C20) or palmitate 26 • Changes at the myristoylaEon sequence prevent interacEon of Gag with the cytoplasmic face of the plasma membrane • Virus assembly and budding are inhibited 27 Genome packaging • Viral genomes must be disEnguished from cellular DNA or RNA molecules where assembly takes place • Requires discriminaEon among similar nucleic acid molecules • Example: retrovirus genomes <1% cytoplasmic RNA, yet is the RNA packaged in majority of retrovirus parEcles • DiscriminaEon is the result of packaging signals in the viral genome -‐ sequences necessary for incorporaEon of nucleic acid into virions; geneEcally defined 28 Packaging signals -‐ DNA genomes Adenovirus • Packaging signal near leO inverted repeat and origin • Signal is complex: a set of repeated sequences; overlapping with enhancers that sEmulate late transcripEon • Structure is recognized by viral protein IV2a (also a transcripEon acEvator) 29 •Herpesvirus genome replicaEon produces concatemers with head-‐to-‐tail copies of viral genome •HSV-‐1 packaging signals pac1 and pac2 needed for recogniEon 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 selecEve encapsidaEon 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 encapsidaEon of viral nucleic acid with its synthesis may contribute to specificity, e.g. poliovirus RNA replicaEon on membrane vesicles; no packaging signal idenEfied in genome 33 Packaging of segmented genomes • How to ensure that virions receive one copy of each segment? Random or selec4ve mechanisms for influenza virus (8 segments) cannot be disEnguished • Random mechanism would yield 1 infecEous parEcle per 400 assembled -‐ within the known parEcle to pfu raEo • If 12 segments are packaged, 10% of parEcles would contain complete viral genome • New evidence for specific packaging sequence on each RNA segment 34 Influenza virus RNA packaging hWp://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 ParEcle:pfu raEo ~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 essenEal for budding -‐ alphaviruses Internal matrix or capsid proteins drive budding -‐ retroviruses Envelope proteins drive budding -‐ coronavirus Matrix proteins drive budding, but addiEonal components (glycoproteins, RNP) needed for efficiency or accuracy 38 Internal structure assembly and budding spaEally & temporally separated 39 40 When synthesized alone, Gag directs budding of virus-‐like parEcles Internal structure assembly and budding are coincident in space & Eme 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 45 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, oVen 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) • DirecEon and mode of egress influence pathogenesis 49 Polarized spread of infec4on by an alpha herpesvirus: Release from axon terminals and infec4on of epithelial cells in vitro Single Axon Purple: nuclei of epithelial cells Green: GFP expressing virus 50 Why do infected cells lyse? • InhibiEon of cell macromolecular processes and transport • ApoptoEc 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