Lecture 5 - Viral exit from living cells Flashcards
What is the life cycle of a bacteriophage (lytic cycle)?
- Phage infects a bacterial cell
- The phage DNA circularises remaining separate from the host DNA
- Phage DNA replicates and phage proteins are made. New phage particles are made
- The cell lyses, releasing the phage
What is the life cycle of a bacteriophage (lysogenic cycle)?
- Phage infects a cell
- Phage DNA becomes incorperated into the host genome
- The cell divides and the prophage DNA is passed onto daughter cells
- Under stressful conditions, the phage DNA is excised from the bacterial chromosome and enters the lytic cycle
- Phage DNA replicates and phage proteins are made. New phage particles are assembled.
- The cell lyses, releasing the phage
How does assembly occur in bacteriophages?
- Each element is assembled separately.
- Tail is key for entry next time.
- Assembly happens in the cytoplasm.
Tail assembly
- Tail core added to the baseplate
- Sheath is added around the core
Head assembly
- DNA packaged inside head
Head, tail and tail fibres come together to form a mature phage
How does bacteriophage T4 lead to lysis of the cell?
Endolysin E:
- early gene promoter
- accumulates fully folded in the cytoplasm
- Does not have a secretory signal sequence so cannot pass the inner membrane
- Relies on the timing program of Holin T
Holin T: -
- late gene promoter - only under stress conditions
- control lysis timing
- accumulates in the membrane until a programmed time
- Upon activation Holins aggragate in multimers that can permealise the inner membrane
- Required to form a channel in the membrane
Endolysin can then degrade the cell wall (peptidoglycan - glycosyl action)
Also other lysis proteins: Spanins : -
- inserted in both the inner and outer membranes to open up membrane
- can diffuse freely in space after endolysin degradation
- aggregate and lead to full lysis by opening the inner and outer membrane leading to fusion
In the lysogenic cycle, what protein is expressed to maintain the lysogenic cycle?
Antiholin is expressed to prevent Holin activity and therefore inhibit bacterial lysis
What is the process of formation and exit of the bacteria enveloped phage phi 6?
- Viral envelope and the viral core assembly are independent processes
- Protein P8 attaches to the prohead to form the nucleocapsid
- Envelope lipids are derived from the host cytoplasmic mebrane
- P9 and P12 induce vesicularisation of the membrane to associate with the nucleocapsid
- the spike protein P3 is assembled onto the phage membrane via protein P6
- The lysin P5 and the membrane protein P10 participate in lysis of the host cell to release new virions
What are the features of the phage M13?
- Filamentous phage
- Nucleoprotein particle is long (900nm) and thin (6.5nm)
- Genome is a atwisted closed circular single-stranded DNA
- The major protein coat gp8 is capped at the ends with minor coat proteins, g3, g6, g7, g9
- M13 genome codes for other gps involved in the release of new phages from the bacterium
How does the phage M13 acheive extrusion but not lysis?
- gp5 coated DNA gets to the release site
- gp1 inserted into the membrane to determine assembly site
- all other coat particles accumulate in the inner membrane
- gp5 coated DNA associates with gp1 which triggers the formation and opening of the extrusion channel
- Elongation of the phage before extrusion (replacement of gp5 by gp8 coat of DNA and the addition of gp3, gp6 to close filament)
Why does the phage M13 need to change it coat during viral extrusion from the cell?
gp5 not adapted to maintain a fit bacteriophage when islated
needs to change coat to survive and gain entry into next cell
By what is cell lysis of animal cells actively induced by viruses?
Viroporins: some eukaryotic lytic viruses like the adenoviridae and picornaviridae encode viroporins in the late phase of infection to disrupt the cell membrane
Describe the process of viral budding in terms of ‘pull’ and ‘push’ forces
- Viral membrane glycoproteins assemble on the cellular membrane and create a pulling force driving membrane curvature. The more glycoproteins the more the tension and larger pull force
- Inner viral structural proteins or pre-assembled viral nucleocapsids attach to the cytoplasmic side creating a pushing force that also drives membrane curvature
- Pushing and pulling forces act in conjunction to facilitate budding
What is the process of budding of influenza?
- Clustering of HA and NA in lipid raft domains denoted by the presence of the glycoproteins. M1 binds to the cytoplasmic tails of HA and NA and serves as a docking site for the vRNPs
- Elongation of the budding virion is caused by a polymerisation of the M1 protein which polarises the localisation of the vRNPs. M2 is recruited to the periphery of the budding virus through interactions with M1.
- Membrane scission is caused by insertion of the M2 ampiphatic helix at the lipid phase boundary. This alters membrane curvature at the neck of the budding virus and leads to a release of the budding virus.
What is the structure of the influenza vRNP (viral ribonucleoprotein)?
Each - sense RNA segment is associated with the virus nucleoprotein and three polymerase subunits (PA, PB1 and PB2)
How is influenza M2 (viroporin 80) targeted to the viral budding site?
M2 binding to cholesterol is thought to target M2 to lipid rafts, the viral budding site
How are vasoporins thought to be linked to membrane curvature?
- The action of vasoporin depolarises the membrane
- Thought to be acting as a proton channel
- Reduces the electrical or contact repulsion between opposing monolayers of the membrane at the neck of the bud, which could provide the stimulus and even the energy to promotw budding and release
How often do animal viruses undergo lysis?
Only go through occasional lysis
They can inducce lysis under special circmstances
What is the occlusion body and when does it form?
- a crystalline protein matrix where the virus becomes occluded in the protein polyhedrin and the polyhedral envelope (calyx) is produced
- used for horizontal transmission - viruses trapped in the occlusion body are often involved in a host-to-host infection (e.g. by baculoviridae)
- Produced during the occlusion phase
- formed in the late phase of infection
What occurs in the occlusion phase?
The virus becomes occluded in the protein polyhedrin and the polyhedral envelope (calyx) is produced. Lysis of the cell relases the occluded virus
Why does the bacculovirus use the occlusion body strategy?
Buculovirus hosts live in water, and crustaceans are hard to infect (shell), best to infect through injection. Viruses are released when in the gut, can pass through mucus layer and then live as a classic virus
How do animal enveloped viruses exit?
Using budding
- meed to make membrane curve to its own shape
- virla glycoproteins choose the site -> curvature -> budding
Describe how the HCV entry and exit rely on lipoproteins
- HCV lipoviroparticles enter target heptocytes via receptor-mediated endocytosis
- Released viral RNA is translated at the ER producing a single polyprotein preursor that is cleaved by host and viral proteases
- The viral NS proteins (NS2 and NS6B) form RNA replication complexes in detergent-resistance lipid rafts, where + strand RNA is replicated by the viral RNA-dependent RNA polymerases NS5B via a negative strand intermediate
- Newly synthesised positive strand RNA is encapidated by the viral nucleocapsid core in close proximity to the LDs, and envelope glycoproteins are aquired through budding into the ER lumen
- Lipoviroparticles mature in the ER through interactions with lipoproteins
- Exit the cell via the cellular golgi apparatus
