L7 - Virus Uncoating Flashcards
Ubiquitination, Microtubule, Nuclear Pore Complex, Endosomal Escape, Adenovirus Movement
Ubiquitin
Short peptide (76aa), free peptide which can be conjugated (covalently attached) to target proteins which mark for degradation by the proteosome.
Ubiquitin Ligase Pathway
E1 + ATP = activated ubiquitin -> E1 is ubiquitinated -> E2 receives ubiquitin from E1 -> E3 targets substrate to join with E2 and transfer ubiquitin from E2 to target protein > E3 is a ligase hence it’s role > ubiquitinated target is degraded by proteosome.
Microtubule
Primary pathway for movement of material within a cell. The positive end is dynamic and polymerises and depolymerises to structure the cell. The negative end is associated with the MTOC. Viruses can associate with the microtubules can move towards the negative end and be physically close to the nucleus.
MTOC
Microtubule organising centre, negative end of microtubule pathway from which they sprout. Viruses move towards the MTOC to be close to the nucleus.
Dynactin
Move material towards the MTOC. Viruses can use this to get closer to the nucleus during entry (-ve direction).
Kinesin
Move material away from the MTOC. Viruses can use this towards later replication cycle to get virus particles out of the cell (+ve direction).
GFP
Green Fluorescent Protein, emits green light under UV light. This has been used as tagging for tracking protein localisation and movement in living cells. They can be associated with <40KDa proteins to visualise entry through NPCs.
NPC
Nuclear Pore Complex, about 2000 per nucleus. Protein complex formed from many proteins that facilitate movement in and out of the nucleus.
NPC for <40KDa Cargo
Most cargo that is 40KDa or less will readily diffuse across the NPC. (Polarisation may affect this).
NPC for >40KDa Cargo
Cargo larger than 40KDa is too large to readily diffuse, instead will associate with nuclear import factors, on NPC filaments, which open the NPC for entry / associate with nuclear export factors which open NPC for exit. This requires ATP to function.
Adenovirus
Non-enveloped, linear dsDNA viruses. Replication and viral particle assembly both occur in the nucleus. Adenovirus type 2 and 5 are the relevant ones, they cause very mild common colds.
Adenovirus - Capsid
Hexon, Penton base and Fibre form a trimer which in turn forms the icosahedral shape. Fibre is attached to Penton base and protrudes from icosahedral, this fibre facilitates viral attachment.
Adenovirus - Cement proteins
IX, VIII, VI (endosomal escape) and IIIa associate with capsid trimers to bridge them together and provide structural integrity to virus particle.
Adenovirus - Core proteins
TP, Mu, pVII and V associate with vDNA by wrapping it up to condense DNA down.
AVP
Adeno-Viral Protease, once new viral particle is fully assembled, the cement proteins and core proteins are snipped at sequence specific points by AVP to go from long precursor proteins to a mature infectious particle structure. If AVP is not packaged into particle, new particle cannot escape endoscope in next host cell infection.
Adenovirus - Trafficking
Virus attaches to host cell receptor via fibre. Clathrin-mediated endocytosis takes particle across bilayer into endosome. Endosome is trafficked by microtubules and pH drops, loosening the virus particle. vProtein VI is released through the loosening of virus, which then lyses endosome. Escaped particle associates with microtubule and moves towards MTOC, further degrades particle at NPC to release DNA into the nucleus.
Adenovirus - Endosomal Escape
Viral protein VI is critical for inducing negative curvature of the endosomal wall causing disruption and escape. Acidification (drop in pH) assists this, but it is NOT absolutely necessary, VI is still released with no drop in pH. After endosome release, VI is ubiquitinated and is necessary for associating to microtubule for movement. - Side note: small amounts of VI are released at attachment stage to extracellular receptors which disrupt membrane to cause calcium influx, cell response is to aid movement of viral particle intracellular.
Adenovirus - Assisted Diffusion Model
After endosomal escape, the partially degraded particle will associate with both Kinesin (away from MTOC) and Dynactin (toward MTOC) randomly. This move the particle up and down microtubules until it finds a nuclear pore complex (NPC) to dock with. Relies on constant movement, the likelihood of reaching the NPC by one route of another will occur. It is not well understood, but Kinesin binds Penton base and Dynactin binds Hexon so moving between the two may be pulling apart the viral particle which might assist NPC association.
Adenovirus - NPC Association
Partially disassembled virus reaches NPC -> associates with Nup214 and Nup358 (protruding portions of NPC) -> recruitment of ubiquitin ligases and proteosome -> it is thought a host or viral factor is degraded here to allow Kinesins to further pull apart viral particle -> protein V ubiquitination by MIB1 to further disassemble virus and unravel VII and associated vDNA -> vDNA associated with protein VII (acting as histone) exposed -> VII sequence is recognised by nuclear import factor (transportin-1) -> importation of vDNA and VII. Side note: if protein V is not present, MIB1 does not associate and the viral particle does not disassemble sufficiently to allow VII interaction with transportin-1 for movement into nucleus, protein V holds the capsid and protein VII so it may be that no V or mutated V would mean VII is not brought close enough to NPC to interact and be pulled intranuclear.
Influenza
Enveloped, ss -ve RNA genome segmented into 8 portions, replication occurs in the nucleus, new particle assembly occurs in the cytoplasm. More frequently elongated filamentous particles, but can be round. Unlike adenoviruses, there are cellular proteins (ubiquitin, tubulin, actin and annexin. Structurally composed of HA (attachment), NA (cleaves sialic residues on viral exit), M1 (scaffold beneath envelope, for structure), M2 (ion channel, uncoating and maturation).
Influenza - Endosomal Escape
Clathrin-mediated endocytosis into early endosome -> acidification of endosome (including virus particle) -> change in HA exposes fusion motif -> fusion of viral membrane with endosome -> pH drop also triggers dissociation of M1 from viral genome segments -> free ubiquitin within virus particle recruits host HDAC6 -> HDAC6 will also associate with Dynactin and Kinesein and cellular actin which causes a physical shearing effect from two different pathways (microtubules and actin pathway) -> this pulls apart virus particle -> HDAC6 recruits aggresome components (MTOC associated complex, serves as proteasome when cell is overloaded), host E3 ubiquitinates M1 causing HDAC6 to associate -> M1 releases vRNPs which associate with host importins (alpha and beta) which facilitate import through NPC -> viral RNA inserted into nucleus.
