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ZZPathology undergraduate year 3, first term. > Virology term - assembly, exit and entry. > Flashcards

Virology term - assembly, exit and entry. Flashcards

1
Q

Key points in viral structure essay.

A

Function of a virion

Restrictions on structure

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2
Q

Functions of a virion (4)

A

To package all genetic material + necessary proteins
To protect genetic material
To bind correct cell.
To deliver genetic material to correct compartment.

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3
Q

Delivery of genome and structure

A

Must be metastable structure: energy barrier prevents degradation, but can be overcome.

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4
Q

Limitations on structure

A

Limited coding capacity - use symmetry, either helical or platonic polyhedra.

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5
Q

How do you describe helical structures?

A

P = μ x ρ
where μ = number of structural units per turn,
where ρ = rise per structural unit,
where P = pitch of helix.

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6
Q

Helical nucleocapsid structures

A

Tobacco mosaic (just genome and capsid protein), paramyxo, rhabdo, orthomyxo.

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7
Q

Icosahedral structure

A

20 triangular faces, 12 vertices related by 2,3 and 5 fold symmetry. 60 identical subunits.

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8
Q

Quasiequivalence

A

Caspar and Klug.
T = triangulation number = number of structural units per face.
Non-covalent binding in different positions is similar but not identical. Pentamers maintained, but extra hexamers added.

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9
Q

Symmetry in helical capsids

A

Rotation and translation.

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10
Q

Example of small icosahedral virus.

A

Canine parvovirus.

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11
Q

Example of large icosahedral virus.

A

Tomato bushy stunt virus. T=3. Monomers have jelly-roll barrel formation.
Human rhinovirus.

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12
Q

Pseudo T=3

A

More than one structural protein, which are structurally similar but not identical.

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13
Q

Hiding binding sites

A

1) In canyon too narrow for antibodies e.g. ICAM-1 binding site in human rhinovirus. Pocket factor stabilises until binding.
2) Using glycosylation

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14
Q

Structure in treatment

A

Druggable binding pocket (rhinovirus).

Stabilising empty capsid with covalent bonds (FMDV).

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15
Q

Rhabdovirus nucleocapsid.

A

No specific interaction between N and RNA bases. 9 RNA molecules bind groove between N protein domains.

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16
Q

Rhabdovirus matrix protein

A

M protein required for condensation into tight helix. Forms another helix round N protien. Polymerisation links M layers to each other.

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17
Q

HSV capsid

A

VP5 is major protein for pentons and hexons. VP26 lies on top.
VP23 and VP19C form triplexes between hexons/pentons.

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18
Q

Unique herpesvirus structure

A

Portal formed by VP6

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19
Q

Dengue virus proteins

A

Capsid, membrane, envelope and 7 non-structural.

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20
Q

Dengue E protein.

A

With long prM protein on surface which needs pr peptide removed by furin in maturation.
Low pH induces large movement of domain two, switches from dimer to trimer.

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21
Q

Maturation of HIV-1

A

Cleavage of Gag by viral protease (dimer) –> conformational change. MA remains with lipid membrane. NC and RNA condenses. CA reorganises to form capsid. Forms fullerene core

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22
Q

Different structures of vaccinia virus

A

Immature virion - no envelope.
Intracellular mature virion - single envelope.
Intracellular enveloped virion - triple envelope.
Cell-associated enveloped virion - double envelope.
Extracellular enveloped virion, double envelope.

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23
Q

Basic virus assembly plan

A

Encapsidation of genome, selection of genome, localisation of virion components, acquisition of tegument, acquisition of envelope, escape from the cell, maturation events.

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24
Q

Encapsidation of genome mechanism.

A

Concerted assembly (either), empty shell (icosahedral). Requirement of chaperones or not.

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25
Q

Mechanism for selection of genome.

A

Specific signal, non-specific packaging, segmented viruses.

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26
Q

Localisation of viral components in assembly.

A

Virus factories, host export pathways, nuclear localisation.

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27
Q

Ways to acquire a membrane

A

At host membrane, in vesicular pathways.

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28
Q

Escaping the cell - details.

A

Downregulation of receptor, deal with tetherin.

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29
Q

Capsid assembly not requiring scaffold proteins.

A

Poliovirus. Will self-assemble into capsids in cell-free translation system.

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30
Q

Capsid assembly requiring scaffold proteins.

A

Adenovirus needs them at 2 stages.
Herpesviridae family - removal of scaffolds requires protease and pH drop with DNA entry.
Polyomavirus VP1 can form aberrant capsid but needs hsp70 to form proper one.

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31
Q

Example of empty shell assembly.

A

Herpesviridae, entry via portal.

Adenovirus, mechanism not fully understood.

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32
Q

Concerted assembly.

A

Nucleation via binding of proteins to genome e.g. helical structures, HIV.
Can drive transcription-assembly transition if packaging region overlaps with promoters.

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33
Q

Virion assembly: selection of genome, SV40.

A

SV40 uses ses (italics) signal on genome. Cellular protein Sp1 recognises this, binds VP2/3, shuts down transcription and acts as nucleation point.

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34
Q

Virion assembly: selection of genome, herpes viridae.

A

Terminase subunit binds pac1/2 and docks at portal.
pUL15 has ATPase activity and pumps it into the capsid.
UL15 then cleaves DNA.

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35
Q

Model for genome segment selection in segmented dsRNA viruses.

A

Daisy chain or core filling models.

Not fully understood, but panhandle structures may be important.

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36
Q

Reoviridae - concerted assembly model.

A

RNA associated polymerase complexes associate. The core shell assembles round this.

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37
Q

Reoviridae - core filling model.

A

Polymerase complexes assemble with proteins to give complete shell. RNAs are inserted individually, concomitant synthesis of complementary strand.

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38
Q

Virion assembly - Localisation of proteins and genome.

A

In viral factory. Using host export machinery. Nuclear localisation machinery.

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39
Q

Virion assembly - nuclear localisation machinery - influenza virus and adenovirus

A

Influenza uses this. M1 imported in to asssociate with RNPs, then core genomic complex is exporeted.
Adenovirus: hexon trimerisation depends on L4-100K and cytosolic chaperones. Import depends on NLS on protein VI.

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40
Q

Virion assembly - localisation using host trafficking.

A

Common. E.g. HIV-1 uses this. Env synthesised in ER, trafficked to Golgi, cleaved by furin, trafficked via secretory pathway to plasma membrane. Interacts with Gag or is endocytosed.
MARV: nucleocapsid uses PT/SAP late domain to recruit Tsg101 for trafficking to membrane. ESCRT machinery.

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41
Q

Process of budding.

A

Accumulation of proteins at budding site.
Membrane deformation.
Membrane scission.

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42
Q

Accumulation of proteins at budding site, HIV.

A

Formation of budding site necessary because more cellular than viral proteins. Use protein interactions or lipid rafts. HIV depends on cholesterol and sphingolipid rich domains. Requires PIP2 as unsaturated fatty acid is displasced into Gag.

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43
Q

Membrane deformation.

A

Preformed capsids associating with membrane proteins.
Association of proteins causing membrane curvature - Gag, M1 polymerisation, VP40 rearrangement.
Possibly uses lipid rafts.

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44
Q

Membrane scission without ESCRT

A

Alphaviruses: precise stoichiometry. Semliki Forest virus.
Paramyxoviruses
Orthomyxoviruses
Poxvirus

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45
Q

ESCRT machinery.

A

ESCRT I binds membrane, recruits ESCRT II.
Bro1 and ESCRT I/II recruit ESCRT III, ubiquitination involved.
ESCRT III causes constriction of neck and scission.

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46
Q

Hijacking ESCRT I.

A

Recruited by HIV-1 tsg101 by p6 domain of Gag via PT/SAP, MARV VP40 via PPPY

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47
Q

ESCRT III action.

A

ESCRT III form tapering spirals or whorls that pull opposing membranes towards central fission spot. 3 models. Vps4A/B is an ATPase which drives scission. Spiralling CHMP4 subunits important. Vps4 may help form these by recycling CHMPs, or drive hemi-fission to completion.

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48
Q

Hijacking ESCRT III.

A

HIV recruits using ALIX. L domains of viruses often hijack this.

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49
Q

Exporting a virion from the nucleus - hepadnaviridae

A

Hepadnavirus; small enough to exit through the pores.

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50
Q

Herpesviridae exit from nucleus.

A

Bud into INM. In HSV this uses gD and gH, but doesn’t in pseudorabies.
Nuclear envelopment complex includes pUL31 and pUL34 disrupts the nuclear lamina and associates with the membrane.

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51
Q

Nucleation site helical -ive ssRNA viruses.

A

Encapsidation occurs during synthesis (sometimes). Rdrp can act as nucleation point.

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52
Q

Nuclear localisation for assembly - polyomavirus

A

Polyomaviruses use this. VP1 most likely to import if with VP2/3, so probably import as pentamer. Localisation within nucleus to PML bodies depends on VP2/3 signals. Localisation of genome depends on Large T antigen.

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53
Q

Virion assembly selection of genome: adenovirus.

A

ψ, packaging signal, contains copies of A repeat.
Viral proteins IVa2 and L4-22K bind this.
IVa2 probably drives entry.

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54
Q

Accumulation of proteins at budding site, HCMV

A

Or target to specific budding compartment. E.g. HCMV formation of virion assembly compartment.

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55
Q

Segmented genome selection, influenza A.

A

5’ and 3’ ends important. Intersegment base-pairing creates packaged complex of 8 segments. Daisy-chain model = they each interact with one on each side. Master segment model = they all interact with central segment.

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56
Q

Herpesvirus full egress model.

A

Buds into INM, de-envelopment by unknown mechanism. Acquires tegument. Buds into golgi-derived vesicles and then is trafficked out by secretory pathways.

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57
Q

Herpesvirus proteins in budding

A

pUL31/pUL34.

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58
Q

Poxvirus assembly

A

Single lipid bilayer around virus core forms immature virion. Forms on scaffold made of D13.
IMV form s after proteolytic cleavage and core condensation.
Wrapping of IMV particles adds 2 lipid bilayers to form IEV.

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59
Q

Non-enveloped virus exit

A

Lysis, autophagy, exosome pathway.

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60
Q

Topics to consider in viral entry essay

A

Enveloped vs non-enveloped.
Mechanisms of entry (druggable?).
Receptor specificity.
Effects on host cell.

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61
Q

Similarities in entry between enveloped and non-enveloped.

A

Both are metastable entities. Both can be taken up by endocytosis.

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62
Q

Receptor binding env vs non-env.

A

Enveloped: spike proteins.

Non-enveloped: projections or indentations of the capsid.

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63
Q

Endocytosis of viruses

A

May require movement to an endocytic hotspot.
Often protects from host immune defences.
Can make conformational change pH dependent.

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64
Q

Mechanisms of viral endocytosis

A

Clathrin coated pit
Caveolar pathway
Clathrin and dynamin independent pathway.
Fluid phase uptake.

65
Q

Clathrin-dependent endocytosis.

A

Usually used for receptor internalisation; invaginations form that pinch off using dynamin.
Matures into early endosome, sheds protein pit, acidifies.
Late endosomes continue to acidify.
Viral fusion occurs at either EE or LE, may require proteolysis.

66
Q

Caveolar dependent endocytosis.

A

Dynamin and cholestrol dependent, slow, takes virus to pH neutral caveosomes.

67
Q

Clathrin and dynamin independent internalisation pathways.

A

Involves endocytosis of GPI-anchored proteins with fluid to give GEECs, which are very acidic.

68
Q

Fluid phase uptake

A

Macropinocytosis.
Stimulated by growth factor receptors.
Primarily actin driven, can be acidified, vaccinia taken up like this.

69
Q

Poliovirus uptake

A

Clathrin and caveolin independent uptake. Requires tyrosine kinase dependent pathway.

70
Q

Attachment factors

A

Concentrate virus on cell surface, do not cause change in viral anti-receptor.

71
Q

Rare alternative to attachment factor.

A

HPV. Wounding allows access to basment membrane. Leads to conformational change and L2 cleavage, which exposes L2 epitope, which allows transfer of virions to epithelial cell surface.

72
Q

Define receptor

A

An attachment factor or protein whose binding is necessary to trigger uptake or conformational change.

73
Q

Viruses requiring only one receptor

A

Influenza HA and sialic acid.

VSV G and phophatidylserine.

74
Q

Co-receptors example.

A

HIV gp120 - CD4 and CCR5 or CXCR4.

Coreceptor tropism may change between strains or over time.

75
Q

HIV R5 and transmission

A

Traditionally thought to be macrophage trophic. Recent suggestion is that there are a series of inefficient barriers that are better overcome by R5 rather than 1 efficient one.
R5s are only ones commonly causing transmission.

76
Q

Non-enveloped entry

A

Tightly coupled with conformational change.
Pore formation.
Membrane disruption.
Penetration.

77
Q

Non-enveloped pore formation

A

Only nucleic acid enters. E.g. poliovirus.

78
Q

Poliovirus delivery of genome

A

Forms homo-multimeric size-selective membrane pore.
Pvr binds 160S. This expels pocket factor and causes conformational change.
VP1 N-terminus and myristoyl of VP4 externalise and form a pore. VP3 plug domain shifts and the RNA exits. Druggable target.

79
Q

Non-enveloped membrane disruption example.

A

Adenovirus. pH change leads to flipping of N-terminal domain of amphiphathic helix out of protein VI, inserts into membrane causing its lysis.

80
Q

Stages of membrane fusion

A

stalk, hemifusion and pore intermediates

81
Q

Alternative to producing membrane bound free viral particles.

A

cell to cell spread.

82
Q

Membrane fusion receptors: class 1

A

trimer of trimers

83
Q

Membrane fusion receptors class 1. Viral families.

A

Similar to proteins used in vesicular fusion. Retroviruses, influenza viruses, paramyxoviruses, filoviruses.

84
Q

Membrane fusion receptors class 1. Mechanism.

A

Fusion peptide liberated by proteolytic processing or pH change.
Trigger leads to formation of extended intermediate, which is embedded in both membranes. Collapse of the intermediate leads to hemifusion, and drives this to completion.

85
Q

Fusion peptides.

A
  • Usually type 1 glycoproteins with short hydrophobic stretch – the fusion peptide.
  • N-terminal fusion peptides
  • Internal fusion peptides
86
Q

N-terminal fusion peptides

A

orthomyxo,paramyxo, some retro

87
Q

Internal fusion peptides.

A

Rous sarcoma virus, VSV, Ebola, MHV.

88
Q

Further steps to influenza entry after membrane fusion

A

M2 channel allows acidification of interior, possibly leads to M1 conformational change. Result: RNPs released into cytosol.

89
Q

Membrane fusion receptors Class 2. Families

A

Flaviviruses, alphaviruses. Based on B-sheets not a-helices.
Classic example: Dengue.

90
Q

Membrane fusion receptors class 2.

A

Trimers from dimers.

91
Q

Membrane fusion receptors - Dengue.

A

E protein fusigenic, prM protein cleaved to give M.
PrM/E form trimers. Processing of prM leads to E forming homodimers. pH change leadst to E homotrimers concomitant with insertion of fusion loop.

92
Q

Membrane fusion receptors - class 3.

A

Non-spring loaded class; reversible as no proteolytic processing.

93
Q

Membrane fusion receptors - class 3: families.

A

Rhabdoviruses, herpesviruses.

94
Q

Membrane fusion receptors - class 3. Triggers.

A

Rhabdo: pH,
Herpes: conformational change in partner protein.

95
Q

Membrane fusion receptors - class 3. Mechanism.

A

 Example: VSV G fusion mechanism
Pre-fusion trimer with fusion loop held near viral membrane.
Hypothetical extended conformation leads to postfusion conformation.

96
Q

Herpes virus entry proteins.

A

gB/C for attachment.
gD has conformational change
gB and gHgL interact with the cellular membrane to cause fusion.

97
Q

Things to consider in receptor specificity.

A

Disease tropism.
Constraints on evolution.
Manipulation in vector delivery.
Druggable targets.

98
Q

HA and 2,3 vs 2,6-sialic acid.

A

Birds and horses have 2,3-sialic acid (linear) at sites of virus entry, humans 2,6 (more folded). Acts as one of the species barriers. Requires two substitutions and loss of glycosylation site to effectively overcome, although a single substitution improves 2,6-sialic acid binding. Alter binding, but also alter stability.

99
Q

Receptor specificity, disease tropism: examples

A

HA and sialic acid.

Receptor based resistance - CCR5

100
Q

Receptor based resistance - CCR5

A

CCR5 deletion homozygotes protective against acquisition of HIV, delays death in heterozygotes.

101
Q

Entry: beyond the plasma membrane. Topics.

A

Effects of binding.
Delivery of effector proteins.
Delivery to the correct compartment.

102
Q

Effects of binding

A

Altered uptake
Modulation of signalling cascades.
Delivery of PAMPs.
Pathogenesis

103
Q

Effects of binding: altered uptake.

A

CD4+ binding triggers signaling turning on actin co-regulator cofilin, disrupting cortical actin, aiding viral entry.

104
Q

Effects of binding: signalling. Examples

A

Coxsackie virus B
HIV gp120
Binding integrins.
HCMV

105
Q

Effects of binding: signalling. Coxsackie virus B.

A

Binding leads to DAF clustering.
Abl activation, Rac activation, actin reorganization and movement to the tight junction and binding of CAR, necessary for particle conversion
Fyn activation, caveolin phosphorylation and virus entry at the tight junction.

106
Q

Effects of binding: signalling. HIV gp120.

A

Binding CD4 leads to either activation or anergy.

Chemokine receptors leads to cytoskeletal rearrangements, alterations in host transcription and chemotaxis.

107
Q

Binding of integrins

A

Stimulates endocytosis, conformational change and other.

108
Q

Effects of binding: signalling. HCMV.

A

Binds growth factor receptor, stimulates host cell metabolic activity.

109
Q

Effects of binding: pathogenesis. Example

A

 Influenza and some other respiratory viruses bind epithelial amiloride-sensitive sodium channel resulting in fluid accumulation, coughing and sneezing. Helps transmission, causes symptoms.

110
Q

Delivery of proteins altering cellular milieu - example

A

Lateral bodies in vaccinia – VH1 is a phosphatase which dephosphorylates STAT1 to prevent interferon-y-mediated viral responses.

111
Q

Delivery to the correct compartment.

A

Use endocytosis.
Exposure of sequences due to capsid destabilisation.
Nuclear entry.

112
Q

Nuclear entry mechanisms

A

Enter during mitosis (some retroviruses)
Small: through via nuclear pore (HBV)
Long and thin: through nuclear pore (flu)
Uncoating at nuclear membrane and delivery of genome through pore. Ejection of DNA due to conformation change. Viruses with portals: this may be significant here.

113
Q

Ways to identify receptors.

A

Viral anti-receptor as affinity hook.
Functional cloning
Inhibitory antibody based assay.

114
Q

Identifying HIV co-receptors

A

 Use HeLa cells. In some express viral gp120, in others express HeLa cDNA library.
 Fusion of cell detected
 cDNA library subfractionation used to home in on co-receptors

115
Q

Caveats to lab work in identifying receptors.

A

 Viral concentrations might be different
 Virus may become tissue-culture adapted
 Cultured cells may be different from cells in natural tissues.

116
Q

HIV co-receptors

A

mannose binding protein, DC-SIGN. Possibly tether to allow interactions with suboptimal levels of CD4+?

117
Q

HIV uncoating

A

Occur somewhere between PM and nuclear pore.
MA phosphorylation by MAP kinase may have a role of some sort.
Cyclophilin A is a cellular factor packaged in virions which may have a role.

118
Q

DNA flap

A

Involved in nuclear entry of PIC.

119
Q

Pre-integration complex

A

dsDNA, IN, MA, Vpr, RT

120
Q

Pox virus entry

A

Uncoating in 2 stages. First outer membrane removed during entry into cell, then the next membrane is uncoated in the cytoplasm.

121
Q

Virus budding essay

A 
Intro
Virus budding basics
ESCRT mechanism
Recruitment of ESCRT
Other techniques
122
Q

Intro to virus budding essay

A

Compare with fusion: most encode own fusion proteins, but use host budding proteins.
Virus budding sheds light on ESCRT mechanisms.

123
Q

Places viruses bud

A

Plasma membrane - HIV, influenza.
Acquired in ER or Golgi: flavi, herpes.
Acquire in cytoplasmic viroplasm: pox.

124
Q

Energy for membrane deformation.

A

Protein-protein interactions.

125
Q

Why use ESCRT?

A

Only known machinery to perform membrane scission with reverse topology to endocytosis.

126
Q

ESCRT model system

A

Genetic analysis in yeast.

Used for formation of multivesicular bodies, among others.

127
Q

Late domains used to recruit ESCRTs

A

At least 5 different classes - discovery of new classes often leads to discovery of new ESCRT proteins.
Retrovirus gags e.g HIV p6 : PTAP
Rhabdovirus M protein : PSAP, PPxY
Filovirus : VP40 : PTAP, YPXL, PPXY

128
Q

PTAP recruits

A

Tsg101

129
Q

YPxL recruits

A

Alix, a Bro-1 protein

130
Q

PPxY recruits

A

Nedd4-like ubiquitin ligases.

131
Q

Models for ESCRT III.

A

ESCRT-III filaments could form tapering spirals or whorls that pull the opposing membranes toward a central fission point
the “dome”, “whorl”, and “hourglass” models
and/or spiraling filaments constrict membranes by sliding past themselves, assistance of VPS4 (e.g., the “break and slide” and “purse string” models).

132
Q

Ubiquitin dependent recruitment of ESCRT III>

A

Retroviral virions concentrate ubiquitin and ubiquitin depletion inhibits virus budding.
Covalent ubiquitin can sometimes function as a late assembly domain when fused directly to retroviral Gag proteins.
The known early-acting mammalian ESCRT factors ALIX, ESCRT-I and ESCRT-II, all contain ubiquitin-binding domains (UBDs);
genetic analyses indicate that ALIX binding to K63-linked ubiquitin chains enhances EIAV and HIV-1 budding

133
Q

Some new potential late domains.

A

FPIV
AMOTL1
a-taxilin
IQGAP

134
Q

FPIV

A

New potential late domain. “FPIV” within the M proteins of the paramyxoviruses human Parainfluenzavirus Type 5 (hPIV-5) and Mumps

135
Q

AMOTL1

A

New potential late domain. AMOTL1 can also bind hPIV-5 M and facilitate virus release

136
Q

NEDD4 family members.

A

New potential late domains?
in several cases, NEDD4 family members can stimulate release of retroviral Gag proteins that lack PPXY late assembly domains, implying novel interaction modes
HIV-1 overexpression of NEDD4L can “rescue” the release and infectivity of viral constructs that lack TSG101 and ALIX binding sites

137
Q

IQGAP

A

The actin remodeling protein IQGAP is required for efficient release of Ebola virus-like particles. IQGAP also can bind the structural proteins of Ebola and MLV and can bind TSG101

138
Q

HIV budding - example for ESCRT.

A

Driven by gag
Recruited to membrane by myristate
Final step requires ESCRT
Otherwise you get particle on a stalk

139
Q

Herpesvirus egress and budding - ESCRT

A

ESCRTIII but not I and II and essential for herpesvirus egress
siRNA showed hsv1 budding with Alix, ESCRT1 and 2 knockdown
Vps4 negative mutants no budding
Multiple redundant pathways for viral recruitment
But ESCRT machinery essential for scission
Not required for nuclear egress which is driven by the interaction between pUL31 and pUL34

140
Q

Alphavirus budding

A

Precise stoichiometry provides energy for membrane scission.
Semiliki forest viruse : COCKBURN 2004
E1/E2 trimers of spike protein
E2 tail interacts with capsid protein
Precise interaction of 80 spike: 240 capsid drives membrane scission.

141
Q

Paramyxovirus budding

A

RSV - Require a functional Rab11 pathway
Unclear whether this is due to budding
Rab11 typically intracellular vesicle trafficking
Sendai virus uses requires actin.

142
Q

Orthomyxovirus budding

A 
Use viral proteins. 
Vps28 and Vps4 independent 
M1 polymerisation and M2 scission 
M2 ampipathic helix sufficient for membrane fission in vitro 
Antiviral target? 
Filamentous forms
143
Q

Pox virus acquiring membrane

A

Denovo lipid membrane synthesis

Triple layered particle

144
Q

Non-enveloped viruses using ESCRT

A

Blue tongue: Reovirus
Picornavirus : Hep A
Hep C within exosomes

145
Q

Herpes egress overview

A 
Capsid and DNA encapsidation.
Perinuclear virions.
Fusion with ONM.
Acquisition of tegument proteins.
Budding into trans-Golgi network.
146
Q

Herpes egress - nuclear exit.

A

Unlikely to be through pores, since too large, and pores intact til late in infetion.
Fusion of perinuclear particle clearly seen: budding into INM then fusion with ONM widely accepted.
Breakdown of nuclear envelope rare.

147
Q

Herpes egress. Fusion with ONM.

A

Mechanism unknown.

148
Q

Herpes egress - proteins in nuclear exit

A

Some evidence for a role of entry (fusion) proteins gB or gH in HSV-1 nuclear egress
Good evidence for NO role of gB, gD, gH or gL in pseudorabies virus (swine alphaherpesvirus) nuclear egress
Viral kinase US3 is important for efficiency of perinuclear particle fusion (deletion viruses have increased numbers of perinuclear virions)
Possible cellular proteins involved

149
Q

Tegumentation of herpesvirus.

A

Two routes: Recruitment to free capsids
Recruitment to the cytoplasmic domains of viral envelope proteins
Tegument can be divided into two layers, an inner tegument layer (capsid bound) and an outer tegument layer (envelope associated)

150
Q

Capsid - inner tegument interaction.

A

The ‘most’ inner tegument protein is thought to be VP1/2 (UL36) a very large protein of >3000 aa that can interact with UL37, UL25 and VP16.

151
Q

Herpesvirus portal - tegument around it.

A

Extra tegument density specifically around portal complex has been resolved by cryo-EM
Currently unknown what protein(s) contribute to this density
Could be VP1/2 in a different or more stable structure
This would fit with a likely role of VP1/2 during genome release at nuclear pores after entry
Could be terminase subunit(s) that remain attached after genome encapsidation (unlikely)
Or any other tegument protein

152
Q

Single-Molecule Localisation Microscopy: dSTORM

A

Very precise localised imaging technique using reconstruction.

153
Q

Localisation of tegumentation.

A

1) VP1/2 contains an NLS and can directly interact with the capsid/CCSC protein UL25 and so could be acquired in the nucleus
2) Some reports have shown the presence of VP1/2 on capsids within the nucleus but not likely since no evidence of this if nuclear egress is inhibited.
3) Others reports showing VP1/2 is only present in the cytoplasm
4) Some tegument proteins have been observed in perinuclear virions (US3 and VP16) – evidence that some tegument can be acquired before nuclear egress.
5) The vast majority of the tegument proteins are certainly acquired in the cytoplasm

154
Q

Membrane protein localisation

A

During their synthesis, membrane proteins are co-translationally inserted into the ER membrane.
Secretory pathway to get to plasma membrane.
Endocytic pathway to prevent sustained expression.

155
Q

Herpesvirus secretory pathway from ER.

A

After correct folding, glycosylation, disulphide bond formation etc., the default pathway for membrane proteins is to exit the ER in transport vesicles and travel sequentially through the Golgi stack, the trans-Golgi network (TGN) and then reach the plasma membrane

156
Q

Herpesvirus endocytic pathway to ER.

A

Upon reaching the cell surface, some membrane proteins can be internalised via endocytosis and targeted to a variety of endocytic organelles: early endosomes, sorting endosomes, late endosomes, lysosomes or the TGN

157
Q

Herpes glycoprotein M

A

When expressed on their own, both gD and gH/L localise to the plasma membrane.
When these glycoproteins are co-expressed with gM, they localise to intracellular membranes.
gM one mechanism by which herpesviruses can localise membrane proteins to the correct compartment for final envelopment.
gM also drags them back to endosomes, from the surface.

158
Q

HSV envelope protein localisation mechanisms.

A

gM
Other HSV-1 proteins also involved in localising gD and gH/L
Glycoprotein K (+UL20) also required for gH/L internalisation
Redundancy between gM and gK/UL20 for gD internalisation

159
Q

Transport of enveloped herpes virion to surface.

A 
Transport of virion containing vesicles is achieved using microtubles and kinesins (+ end directed motors)
 Little understanding of mechanism – viral membrane proteins gE and US9 may bind kinesin
 Cellular pathways (SNAREs, Rabs, tethers) most likely involved

ZZPathology undergraduate year 3, first term. (15 decks)

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