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Virus Assembly/Release Goal of virion structure: protect genome from environment facilitate disassembly upon infection (virions are metastable structures) Virus Assembly relies on host cell pathways Steps governed by structure enveloped or non-enveloped Steps influenced by cellular site of genome replication Overview of Assembly of Non enveloped viruses 1. Assembly of capsid structure 2. Insertion of genome into capsid 3. Release of virions from cell requires cell lysis (at least partially) 4. Assembly may occur in cytoplasm or nucleus depending upon virus family Nonenveloped virus Assembly Picornavirus Assembly--simplest Replication—cytoplasm and associated with membranes Other non-enveloped viruses (eg Adenovirus)) assembly is more complex (Capsomer--VP1, VP3, VP0) RNA 60 capsomers or 12 pentamers Packaging of RNA Linked to replication Only + RNA not –RNA Mechanism? Fields Virology Assembled Progeny Picornaviruses in Infected Cell Cytoplasm Release of Picornaviruses--cell lysis? Due to cell death or specific mechanism? Possible Role of Autophagic Vesicles in Polio Virus Release Overview of Assembly of Enveloped Viruses 1. Assembly of capsid or nucleocapsid with genome to form core 2. Assembly of envelope (modified patch of cell membrane 3. Association of core with membrane 4. Release from cells by budding from a membrane plasma membrane or Golgi membranes or RER membranes or Nuclear membranes 5. Does not require cell death Assembly and Release Enveloped Viruses Assembly Parts of virion budding Does not require Cell death Membrane scission Influenza Virus Assembly/Release Assembly of Influenza Proteins in Virion *HA *NA envelope M2 *M1 PB1, PB2, PA Core (RNP) *NP *major structural proteins A. Formation of the Envelope Envelope: HA, NA, M2 HA, NA, and M2 are transmembrane glycoproteins Use host secretory pathway for: membrane insertion pottranslational modifications transport to PM Types of viral glycoproteins HA= type 1 NA= type 2 Translation and Membrane Insertion of Glycoproteins Signal peptide of type 2=TM Post Translation Modification of Glycoproteins 1. Glycosylation (RER, Golgi) N linked (signal NXT/S) (O linked--Not flu) 2. Oligomerization (RER) HA trimer NA tetramer 3. Disulfide bond formation (RER) 4. Proteolytic cleavage (RER, Golgi, extracellular) 1. Glycosylation Addition of Core oligosaccharide to nascent chain Roles of Glycosylation folding protease protection mask from immune responses Modification of Core sugars-N linked Glycosylation NST NXS Endo H Mannose N acetyl glucosamine glucose various (peripheral sugars: N acetyl glucosamine, galactose, fucose, sialic acid) 2. Oligomerization—in the RER Structure of HA Trimer H1--blue H2--colors 3. Disulfide Bond Formation In RER Host cell disulfide isomerases (eg PDI) Most likely co-translational Intramolecular disufide bonds Sometimes intermolecular disulfide bonds stabilize oligomers 4. Proteolytic Cleavage Cleavage of HA required for virus entry (fusion) but not attachment Cleavage site TM HA H1, H2, H3 Single basic amino acids cleavage only in RT extra-cellular protease only in respiratory tract (RT) H5 Furin site = sequence RXR/KR cleavage in many tissues Golgi enzyme 5. Transport to cell surface via secretory pathway Problem: Acid pH--activates HA fusion activity M2 and passage of HA through Golgi during transport to the Plasma membrane for assembly H+ B. Assembly of Nucleocapsids 1. NP, P proteins, NS-2 transported to nucleus Each has an NLS 2. NP and P proteins assemble with vRNAs (-) (NP added as RNA is synthesized) 3. M1 transported to nucleus (has NLS) 4. NS2 (NEP) binds Crm-1-RanGTP 5. M1 binds to NP and M1 binds NEP 6. RNPs-M exit nucleus (LxxxLxxLxL) Exit of RNPs from nucleus From Fields Virology Further Issues in Assembly and Release from Plasma Membrane 1. Assembly and packaging of genome segments 2. Interactions Virion proteins BuddingMembrane curvature Membrane scission Specific mechanism to incorporate one copy of each segment--evidence EM--always 8 segments Serial sections through a single particle Assembly of Influenza From Noda, et al Nature 439: 490 Implications: virions asymmetric Electron Tomography of Budding virions Domains in vRNAs required for assembly into particles Mechanisms ??? What is role of specific packaging sequences?? Fields Virology Which segments Interact?? Approach Arrangements of Segments in Virions Arrangements By tomography data Tomography + in vitro Interaction data Interactions of Protein Virion Components— Proposed but not well established HA--M1 NA--M1 NP--M1 M2--M1 Central Role of M1— Connect membrane components with core Further Issues in Assembly and Release from Plasma Membrane 1. Assembly and packaging of genome segments 2. Interactions Virion proteins BuddingMembrane curvature Membrane scission C. Budding and Release Membrane curvature? Membrane scission? Model: General mechanism of enveloped virus budding and release Observations in HIV system mutation in gag--particles assemble but fail to release from cell membrane Mutation called late domain mutation At least 5 types of late domains PTAP PPXY YPXL ΦYX0/2(PΦ) FPIV Late domains bind host proteins For example: PTAP binds Tsg 101 Tsg 101 member of VPS system VPS=vacuolar protein sorting system VPS involved in sorting proteins to Multivesicular Body--compartment destined for lysozome What is the VPS System? VPS system for handling proteins for degradation VPS and virus budding Viral budding And release Host pathway ESCRT mediated budding Virology 372: 221 (2008) Mechanisms for HIV release Inhibition VPS4 blocks release Some viral late domains Phenotype of Late domain mutations HIV MLV Late domains in RNA viruses usually in M proteins Expression of M alone=particle release From Bieniasz, Virology 344: 55 Also see Votteler and Sundquist Cell Host and Microbe 14: 232 Problem with Influenza Budding and the VPS No late domains in M1 M1 expressed alone—no particles VPS 4 not required for virus release What drives influenza budding? M1 protein alone—no budding unless targeted to membranes NA or M2 expressed alone will release particles (inefficiently) HA alone will drive budding HA+NA+M+M2 best for budding Mutation of helix in CT domain of M2 M2 expressed alone forms particles Cell 142: 902 (2010) M2 is localized at base of budding virus Cell 142: 902 (2010) Model of Influenza Budding Virology 411: 229 (2011) Predicts Asymmetry of Virions 1. Segment organization 2. Localization of M2 Implications for: Envelope-Core Interactions Glycoprotein-M1 interactions Organization of Glycoproteins Asymetry of Virions Lab strains are basically spherical Virus from infected lungs are filamentous Growth of a human isolate in eggs selects spherical virions Questions—structure of filamentous virus implications for assembly (and mechanisms of entry) Issues in Assembly Filamentous virions RNP More spherical virions RNPs contact membrane only at top NA tends to cluster at one end (opposite from RNP) HA X-31 Udorn NA M2 PNAS 107: 10685 What drives budding?? RNP envelope (HA-RNP?, M1-RNP?) interactions?? What drives membrane scission? M2 likely but mechanism unclear budding Membrane scission Model of Influenza Budding Virology 411: 229 (2011) D. Assembly and Reassortment Infection of a cell with two different influenza viruses results in reassortment of the gene segments generating new viruses Likely origin of new pandemic viruses/Antigenic Shift Classification of Influenza A Viruses Divided into subtypes or species Based on HA and NA In nature 15 different HA genes (H1--H15) 9 different NA genes (N1-N9) Subtypes designated by which HA and which NA are present eg. H1N1 or H3N2 All exist in wild birds Humans--usually one or two Influenza A strains Influenza A and Pandemics 1890 H2N2 1900 H3N8 1918 H1N1 1957 H2N2 1968 H3N2 (1977 H1N1) 2009 H1N1 Antigenic Shift Responsible for periodic Appearance of new virus Mechanism of Antigenic Shift Humans Birds H5N8 Pig H1N1 supports replication of both human and avian viruses Progeny Virus H5N8, H1N1, H5N1, H1N8 And different combinations of other segments 1957--HA,NA,PB1 avian 1968--HA, PB1 avian Pandemic due to new virus H5N1 or H1N8 Internal genes also reassort with consequences Requirements for a pandemic virus 1. New HA subtype 2. HA bind to human respiratory epithelium 3. Human to human transmission 4. Replication efficient in human cells (PB2 implicated) Pathogenicity varies with particular combination of genes (varies with pandemic virus—1918 vs others) Determinants of pathogenicity incompletely understood (HA and PB2 definitely implicated) Originof ofSegments Segments ofinNew Pandemic H1N1 virus*** Origin 2009 Pandemic Virus H1N1 From Garten, et al Science 325: 197 (2009) Remaining Questions in Influenza Assembly Role of segment interactions and generation of reassortants (implications for potential types of antigenic shift viruses that are possible) Specifics of interactions between components of virions (glycoproteins with M1, with RNP structures) (M1 with RNP structures) Mechanisms involved in budding Mechanisms of membrane scission Role of NA in release NA releases reattached virus (Tamiflu Relenza--sialic Acid analogues) Consequences of NA defective influenza: Aggregation and failure to release from Infected cells Note location of NA in virions Budding from specific cell domains--polarized budding implications for pathogenesis flu Mechanism? Reading: Fields Virology Chapter 6 (General discussion of assembly) Relevant sections of Chapters 24, 47 Also: Polio assembly PlosPathogens 8: e1003046 Influenza: PlosPathogens 10:e1003971 Nucleic Acids Research 40: 2197 PNAS 110:e3840