Lecture 10 - Virus Assembly, Maturation and Exit

Question 1

Issue with virus assembly

Answer 1

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

Question 2

Virus whose structure is the minimum free-energy state

Answer 2

Tobacco mosaic virus

Question 3

How is discrimination of viral genomic material achieved?

Answer 3

Genetic ‘packaging’ sequences, often called ‘ψ’

Question 4

Packaging signal for DNA viruses

Answer 4

Often many repeated sequences on the enhancer

Question 5

Adenovirus packaging signal structure
1)
2)
3)

Answer 5

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

Question 6

RNA virus packaging signals

Answer 6

Genomes fold into 3D structures.

Question 7

HIV packaging signal
1)
2)
3)
4)

Answer 7

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

Question 8

Ways that viral structural units can assemble
1)
2)
3)
4)

Answer 8

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

Question 9

Example of proteins that assemble by association of individual, separately translated components

Answer 9

SV40 pentamer units
Adenovirus penton spikes
HBV capsid

Question 10

Examples of viruses that form large polyprotein subunits that are refolded after proteolytic processing

Answer 10

Polio

Retroviruses

Question 11

Example of a viral chaperone protein

Answer 11

Adenovirus L4 protein, that assists in the formation of correct hexamers

Question 12

How are SV40 pentamers formed?

Answer 12

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

Question 13

How are adenovirus penton spikes formed?
1)
2)

Answer 13

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

Question 14

Role of adenoviral L4

Answer 14

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

Question 15

Exceptions to the rule that DNA viruses that replicate and assemble genomes in the nucleus will assemble in the nucleus

Answer 15

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

Question 16

Common feature of NLS

Answer 16

High content of basic amino acids.

High charge also leads to strong nucleic acid binding

Question 17

What are concatemers?

Answer 17

Head-to-tail copies of a viral genome

Question 18

Example of a virus that forms concatemers

Answer 18

Herpesvirus

Question 19

How does herpesvirus incorporate its genome into capsids?
1)
2)
3)

Answer 19

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

Question 20

Example of a virus that uses scaffolding proteins

Answer 20

Herpesvirus

Question 21

How does herpesvirus use protein scaffold?

Answer 21

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

Question 22

Where does herpesvirus assemble its capsid?

Answer 22

Only in the nucleus

Question 23

Herpesvirus exit from the nucleus
1)
2)
3)

Answer 23

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

Question 24

Assembly of poliovirus from polyproteins
1)
2)
3)

Answer 24

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

Question 25

Maturation and exit of poliovirus 
1)
2)
3)
4)

Answer 25

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

Question 26

Where must a virus translate proteins if it has glycoproteins, or needs an envelope?

Answer 26

Golgi

Question 27

Implications of a virus using ribosomes on the rough ER

Answer 27

The virus forms glycoproteins, which must be formed in the RER

Question 28

How are proteins targeted to the ER?
1)
2)
3)

Answer 28

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.

Question 29

What does translation into the RER lumen allow?

Answer 29

1) Assistance with folding by chaperones

2) Post-translational modification (oligomerisation, glycosylation, proteolytic processing)

Question 30

What is myristilated?

Answer 30

Addition of a fatty acid to a protein

Question 31

What is prenylated?

Answer 31

Addition of a fatty acid to a protein

Question 32

Influenza assembly
1)
2)
3)
4)
5)
6)

Answer 32

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

Question 33

Different strategies that enveloped viruses can employ to attain an envelope
1)
2)
3)
4)

Answer 33

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)

Question 34

Determinants of how a protein moves through Golgi/ER

Answer 34

1) Thickness of transmembrane region determines which of the Golgi sacs a protein sits in
2) Golgi/ER localisation sequence

Question 35

Drug that can block formation of enveloped viruses

Answer 35

Brefeldin A

Question 36

How is movement of viral particles in a cell mediated?

Answer 36

Membrane traffic (secretory pathway)
Cytoskeletal (actin/microtubules)

Question 37

Where do HSV bud?

Answer 37

From nuclear membrane

Question 38

Where do coronaviruses bud?

Answer 38

Rough ER

Question 39

Where do Bunyaviruses bud?

Answer 39

Trans Golgi

Question 40

Where do HIV and influenza bud?

Answer 40

From plasma membrane

Question 41

Flavivirus maturation and exit
1)
2)
3)
4)
5)

Answer 41

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

Question 42

How does HIV assemble?

Answer 42

At the plasma membrane.

Accumulation of TM-SU (transmembrane/surface proteins), myr-MA-GAG (matrix and gag proteins) onto lipid rafts.

Question 43

How is HIV env processed in the host cell?

Answer 43

Extensively modified in ER/Golgi network.

Host enzyme furin releases TM (transmembrane) and SU (surface) receptors from env precursor.

Question 44

How is the HIV genome localised to the host-cell membrane?
1)
2)
3)
4)

Answer 44

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

Question 45

How does HIV mature into an infectious particle?

Answer 45

Viral polymerase cleaves gag polyprotein, leading to structural changes to capsid conformation

Question 46

Places to exit from host cell

Answer 46

1) Apical - to outside world
2) Basal - to deeper tissues
3) Lateral - to surrounding cells