Lecture 10 - Virus Assembly, Maturation and Exit Flashcards
Issue with virus assembly
1) Structural and functional compartmentalisation of eukaryotic cells results in different viral components in different cellular compartments
2) Viral genomes must be distinguished from host DNA and viral mRNA, and packaged into capsids
Virus whose structure is the minimum free-energy state
Tobacco mosaic virus
How is discrimination of viral genomic material achieved?
Genetic ‘packaging’ sequences, often called ‘ψ’
Packaging signal for DNA viruses
Often many repeated sequences on the enhancer
Adenovirus packaging signal structure
1)
2)
3)
1) Located near the LTR origin
2) Complex signal of repeated sequences, overlapping with enhancers that stimulate late transcription
3) Viral protein IV2a recognises sequence, is a transcription activator
RNA virus packaging signals
Genomes fold into 3D structures.
HIV packaging signal 1) 2) 3) 4)
1) Capsid only recognises diploid RNA. Stem loop complexes on genomic RNAs hybridise, forming kissing loops (diploid RNA)
2) HIV packaging signal is situated in spliced regions. Therefore, if genome is spliced (EG: if it is mRNA), it can’t be incorporated into the capsid
3) NC region of gag protein recognises ψ of HIV genome
4) NC only binds to region of genome that is revealed when stem loops hybridise to kissing loops
Ways that viral structural units can assemble 1) 2) 3) 4)
1) Association of individual monomeric proteins that are translated as separate components
2) Large polyprotein subunits that are refolded after proteolytic processing
3) Viral or cellular chaperone proteins that facilitate correct folding of protein
4) Using viral scaffold proteins, that aren’t included in the final virion, but are removed before maturity
Example of proteins that assemble by association of individual, separately translated components
SV40 pentamer units
Adenovirus penton spikes
HBV capsid
Examples of viruses that form large polyprotein subunits that are refolded after proteolytic processing
Polio
Retroviruses
Example of a viral chaperone protein
Adenovirus L4 protein, that assists in the formation of correct hexamers
How are SV40 pentamers formed?
SV40 spliced mRNAs translate individual monomeric proteins (VP1, VP2/3).
5 VP1 assemble around one VP2/3. This forms a structural component of SV40 capsid
How are adenovirus penton spikes formed?
1)
2)
1) Homomultimers of proteins that will make up membrane spike are formed. 5 protein III form penton base, 3 protein four make fibre.
2) Fibre and penton base form Ad2 penton
Role of adenoviral L4
Chaperone.
Ad2 hexon trimers are assembled with the assistance of L4.
Hexon proteins also lack a NLS. L4 has a NLS, so allows Ad2 hexon trimers to be imported into the nucleus
Exceptions to the rule that DNA viruses that replicate and assemble genomes in the nucleus will assemble in the nucleus
1) Hepadnaviruses form an RNA pre-genome, and assemble in the cytoplasm
2) Influenza and retroviruses have RNA genomes, but replicate in the nucleus, and assemble at the plasma membrane
Common feature of NLS
High content of basic amino acids.
High charge also leads to strong nucleic acid binding
What are concatemers?
Head-to-tail copies of a viral genome
Example of a virus that forms concatemers
Herpesvirus
How does herpesvirus incorporate its genome into capsids?
1)
2)
3)
1) pac1 and pac2 packaging signals lie within DR1.
2) UL15 recognises pac1 and pac2, UL6 brings genome into capsid.
3) Cleavage of concatemer genome when two pac1 and pac2 sequences are recognised
Example of a virus that uses scaffolding proteins
Herpesvirus
How does herpesvirus use protein scaffold?
Protein scaffold used to form procapsid, a transient intermediate structure.
VP24 protease breaks down scaffold when it is no longer needed, viral DNA enters capsid
Where does herpesvirus assemble its capsid?
Only in the nucleus
Herpesvirus exit from the nucleus
1)
2)
3)
1) Binds nuclear membrane, moves through ER. Envelopment/deenvelopment of nuclear membrane
2) Membrane-associated tegument proteins bind to glycoproteins that have been translated and translocated to the Golgi. This results in virus entering the Golgi.
3) This envelopes capsid. Exocytosis of enveloped virus
Assembly of poliovirus from polyproteins
1)
2)
3)
1) Polyprotein precursor folds into folded P1.
2) Folded P1 is cleaved by viral protease 3CDpro, into 5S structural unit
3) Virion becomes infectious once VP0 has been cleaved to VP4 and VP2
Maturation and exit of poliovirus 1) 2) 3) 4)
1) Genome replication occurs in the cytoplasm, in the smooth ER
2) Particles assemble in the cytoplasm from incompletely-cleaved polyprotein that retains VP0
3) A non-infectious virion forms.
4) Cleavage of VP0 into VP2 and VP4 results in an infectious virion
Where must a virus translate proteins if it has glycoproteins, or needs an envelope?
Golgi
Implications of a virus using ribosomes on the rough ER
The virus forms glycoproteins, which must be formed in the RER
How are proteins targeted to the ER?
1)
2)
3)
1) Ribosomes translating proteins that have an uninterrupted sequence of ~20 hydrophobic residues at the NH2 terminus attach to wall of ER
2) Hydrophobic signal sequence directs protein into RER lumen
3) Signal sequence cleaved off. Protein can be exported, secreted.
What does translation into the RER lumen allow?
1) Assistance with folding by chaperones
2) Post-translational modification (oligomerisation, glycosylation, proteolytic processing)
What is myristilated?
Addition of a fatty acid to a protein
What is prenylated?
Addition of a fatty acid to a protein
Influenza assembly 1) 2) 3) 4) 5) 6)
1) (-)ssRNA genomes are packed in nucleoprotein in the nucleus
2) M1 binds ribonucleoprotein and prevents further transcription. NEP binds M1.
3) NEP directs M1-RNP through nuclear pore to plasma membrane
4) HA, NA, M2 produced in ER/Golgi concentrate on plasma membrane, are bound by M1.
5) Binding of M1 to HA, NA, M2 triggers budding of virus.
6) NA cleaves sialic acid
Different strategies that enveloped viruses can employ to attain an envelope 1) 2) 3) 4)
1) Envelope glycoproteins and capsid are essential for budding alphaviruses
2) Retroviral internal matrix or capsid proteins drive budding
3) Coronavirus envelope proteins in the plasma membrane drive budding
4) Matrix proteins drive budding, membrane glycoproteins needed for accuracy of budding (rhabdoviruses)
Determinants of how a protein moves through Golgi/ER
1) Thickness of transmembrane region determines which of the Golgi sacs a protein sits in
2) Golgi/ER localisation sequence
Drug that can block formation of enveloped viruses
Brefeldin A
How is movement of viral particles in a cell mediated?
Membrane traffic (secretory pathway) Cytoskeletal (actin/microtubules)
Where do HSV bud?
From nuclear membrane
Where do coronaviruses bud?
Rough ER
Where do Bunyaviruses bud?
Trans Golgi
Where do HIV and influenza bud?
From plasma membrane
Flavivirus maturation and exit 1) 2) 3) 4) 5)
1) Assembles in the lumen of the rER (pH7, non-infectious)
2) Transported out of cell via secretory pathway (ER to Golgi to recycling endosome) (pH6 in Golgi)
3) pH and cellular proteins contribute to maturation of viral particle
4) Host enzyme furin cleaves PrM to M. M prevented protein E fusing with host membranes during cellular transport.
5) Pr dissociates from M and E once the virus has entered extracellular space
How does HIV assemble?
At the plasma membrane.
Accumulation of TM-SU (transmembrane/surface proteins), myr-MA-GAG (matrix and gag proteins) onto lipid rafts.
How is HIV env processed in the host cell?
Extensively modified in ER/Golgi network.
Host enzyme furin releases TM (transmembrane) and SU (surface) receptors from env precursor.
How is the HIV genome localised to the host-cell membrane? 1) 2) 3) 4)
1) Myristoylation and phosphorylation.
2) gag-NC protein has RNA binding sites, which bind to the viral genome. These are a basic region and two zinc-finger regions.
3) Myristoylation of gag promotes its binding to phosphatidylinositol-(4,5)-bisphosphate. This is only found on the plasma membrane
4) The effect of this is to localise both gag-NC and viral genome to the plasma membrane
How does HIV mature into an infectious particle?
Viral polymerase cleaves gag polyprotein, leading to structural changes to capsid conformation
Places to exit from host cell
1) Apical - to outside world
2) Basal - to deeper tissues
3) Lateral - to surrounding cells
