Virus entry and Fusion / Virus Assembly and Release

Question 1

Fusion of viral membrane and cellular membrane
-> What are the problems that have to be overcome?

Answer 1

• Fusion should take place at target membrane, yet not on other membranes
  -> viral receptor-binding protein / receptor in target membrane partial proteolytic uncovering of fusion peptide by only locally expressed proteases, pH dependend fusion
• Membranes are biological barriers; energetic problem
  -> Conformational change in fusion protein draws target membranes into close proximity
  viral fusion peptide destabilize lipid bilayer
• newly formed intracellular virions have to be protected against fusion
  -> viral fusion peptide inaccessible due to either
  a, fusion part
  b, protein conformation
  c, helper protein

Question 2

Virus entry
-> First step of a viral infection

Answer 2

• Association with cell membrane / receptor binding
• Entry through target membrane
• Uncoating

Question 3

Principle of infection

Answer 3

1) Binding
2) Lateral diffusion
3) Signaling
4) Internalization
5) Vesicular transport
6) Membrane penetration
7) Intracytosolic transport
8) Nuclear import
9) Uncoating

Massive interaction between virus and cell!
e.g. more than 90 kinases are involved in the infection of HeLa cell with VSV
„Genome wide siRNA Screen“ „Kinaseom“

Question 4

Infection of the host cell by viruses
-> Different Viruses - different ways

Answer 4

Acrive process: Virus stimulates target cell for uptake - complex signaling cascades

Question 5

Virus entry strategies

Answer 5

• Barriers to infection -> Plasma membrane -> early endosome
• Viral entry pathways - receptor mediated endocytosis
• viral entry pathways - receptor mediated signaling

Question 6

Sites of virus particle fusion/penetration

Answer 6

PLASMAMEMBRANE
- Murine leukemia virus
- Epstein-Barr Virus
- Human immunodeficiency virus (?)

EARLY ENDOSOME
- Hepatitis C virus
- Semiliki Forest virus
- Vesicular stomatitis virus
- Human immunodeficiency virus (?)

LATE ENDOSOME/LYSOSOME
- Influenza
- Avian leukemia virus
- Human rhinovirus
- Dengue virus
- Reovirus
- SARS Coronavirus

ENDOPLASMIC RETICULUM
- SV40 polyomavirus

Question 7

Receptor-mediated endocytosis - Example HCV

Answer 7

RECEPTORS/CORECEPTORS:
- Low density lipoprotein (LDL) receptor
- Scavenger (SR-BI) Receptor
- CD81
- Claudin (CLDN1)
- Occludin (OCDN)

• attachment (reversible)
• stable receptor binding
• internalisation

Question 8

Receptors required for HCV infection
-> Pathogenesis of hepatitis C virus infection in Tupaia Belangeri

Answer 8

Northern tree shrews (Nördliches Spitzhörnchen)
- can be infected
- mild hepatitis
- intermittent viremia
- develop chronic infection

All identified receptor proteins on human cells required for HCV uptake are functionally conserved in Tupaias!

Question 9

Classic apoptotic mimicry: a virus acquires host cell phosphatidylserine and incorporates it into the viral membrane

Answer 9

Phosphatidylserine exposed on the viral surface binds to both direct phosphatidylserine receptors, such as T cell immunoglobulin and mucin receptor (TIM) proteins, and indirect phosphatidylserine receptors, such as AXL and tyrosine protein kinase receptor 3 (TYRO3), which require phosphatidylserine-bridging molecules.

Both EBOV and DENV have been shown to use both direct and indirect phosphatidylserine receptors, whereas VACV has only been shown to use the indirect receptor AXL. Whether EBOV and DENV can engage these various receptors simultaneously or whether VACV can use other phosphatidylserine receptors has not been determined. For some viruses, such as EBOV and VACV, engagement of phosphatidylserine receptors triggers their internalization by
macropinocytosis. For other viruses, including DENV, binding of phosphatidylserine to receptors
on the host cell surface induces clathrin-mediated uptake.

Question 10

Ways into the cell

Answer 10

F = Fusion with plasma membrane
V = Vesicular uptake

• Polyomavirus = V
• Newcastle disease Virus = Caveosom
• Influenza virus/Flavivirus = Endosome
• HIV = F
• Vaccinia Virus = F
• Adenovirus (without lipid envelope) = V
• Herpes Virus = F

Question 11

Endocytic mechanisms

Answer 11

Multiple mechanisms are defined as pinocytic, i.e., they involve the uptake of fluid, solutes, and small particles. These include clathrin-mediated, macropinocytosis, caveolar/raft-mediated mechanisms, as well as several novel mechanisms. Some of these pathways involve dynamin-2 as indicated by the beads around the neck of the endocytic indentations. Large particles are taken up by phagocytosis, a process restricted to a few cell types. In addition, there are pathways such as IL-2, the so-called GEEC pathway, and the flotillin- and ADP-ribosylation factor 6 (Arf6)-dependent pathways that carry specific cellular cargo but are not yet used by viruses. Abbreviations: Adeno 2/5, adenovirus 2/5; Adeno 3, adenovirus 3; CME, clathrin-mediated endocytosis; HPV-16, human papillomavirus 16; HSV-1, herpes simplex virus 1;
15 LCMV, lymphocytic choriomeningitis virus; mPy, mouse polyomavirus; SFV, Semliki Forest virus;
SV40, simian virus 40; VSV, vesicular stomatis virus

Question 12

Endocytosis pathways used by viruses

Answer 12

A: Macropinocytosis: Adenoviruses
B: Cathrin-independent: Influenza, Arenaviruses
C: Clathrin-dependent: many viruses, Influenza too
D: Caveolar: SV40, Coxsackie B
E: Cholesterol dependent: SV40, Polyoma
F: See D but dynamin-2 dependent: Echovirus 1

VSV: fusion with multivesicular endosome: In infection and transport 90 cell. kinases are involved! Kinases have effect on cytoskeleton, membrane transport, cell growth, -cycle, endocytosis

Question 13

Entry of SV40 (non enveloped)

Answer 13

1. Endocytosis into Caveolae
2. Fusion with Caveosome, no pH-shift.
3. Long transport in vesicles;
   actin, Rho-GTPase and microtubuli-dependent into the ER
4. Structural rearrangement of the capsid in the reducing milieu of the ER, myristylated N-term. of VP2 exposed
5. Penetration into cytoplasm; ERAD pathway! (ER-associated protein degradation complex)
6. Import into nucleus by NPC and NLS in VP2/3

Question 14

Caveolar/raft pathways

Answer 14

Lipid and cholesterol uptake, transcytosis, proteins from lipid-rafts and with GPI-anchor

Question 15

Virus utilizes host cues in distinct combination to uncoat

Answer 15

(A) Receptor–Enzyme–Mechanical:
HIV-1 binding to its receptor structurally alters GP120, inducing membrane fusion (step i) and capsid release into the cytosol. Cytosolic peptidyl-isomerase conformationally alters the capsid (step ii), which is then trafficked to the nuclear pore by motor proteins to execute mechanical disassembly (step iii). (B) Receptor–Chemical–Mechanical: Herpes simplex virus-1 (HSV-1) engagement to its receptors alters the structural proteins (step i), which then induce endocytosis.
The low pH endocytic compartment further alters the structural proteins (step ii) to promote fusion
and capsid escape into the cytosol, where engagement with motor protein causes disassembly (step iii).
(C) Enzyme–Mechanical: SV40 binds to its glycolipid receptor and reaches the endoplasmic reticulum (ER) unaltered via endocytic route. In the ER, the protein disulfide isomerase (PDI)-family of isomerases/reductases rearrange the disulphide bonds (step i) to structurally alter the virus. The viral capsid is then engaged by cytosolic disaggregation machinery (step ii), which extracts and simultaneously disassembles the viral particle.
(D) Receptor–Chemical–Mechanical: Binding of human adenovirus-2 (HAdV2) to its receptors imposes mechanical strain due to drifting motion of the receptors (step i). The destabilized virus undergoes further structural distortion at low endosomal pH, which probably assists in capsid release into the cytosol (step ii). In the cytosol, the destabilized capsid engages the motor protein, which transports the capsid to the nuclear pore to
undergo mechanical disruption (step iii), leading to genome release. Note: small Roman numerals (i, ii, and iii)
represent virus coopting host cues. The background colors of the Roman numerals categorize them into receptor or enzyme (green), chemical (red), and mechanical (yellow).

Question 16

Dynamin: pinch-off of vesicles via GTP dep. motor proteins

Answer 16

• 96 kDA
• large GTPase

undecorated lipid tubes: Budding Clathrin-coated vesicle

Dynamin GTPgammaS on lipid tubes: “popase”

Dynamin GDP on lipid tubes: “Pinchase”, pinch off

Question 17

Membrane fusions between
- vesicles in animal cells
- Viruses/animal host cells

Answer 17

• HIV: Fusion from without the cell into the cytoplasm
• Influenza: Fusion from within the cellular endosome into the cytoplasm
  Bacteria/ animal host cells Yersinia

Question 18

Membrane fusions between Vesicles in animal cells

Answer 18

SNARES
Soluble NSF (N-ethylmaleimide-sensitive fusion protein) accessory protein (SNAP) receptor):
- contain a heptad repeat of 60–90 aa that participates in coiled-coil formation.
- family of SNARE proteins involved in intracellular fusion events and exocytosis.

Course of action in membrane fusion
- 3 tSNARES on target cell membrane and 1 vSNARE on vesicle membrane interact - formation of a supercoil with the coiled-coiled regions of the different SNARES corresponds to heterotetramer of 4 parallel a-helices
- driving force: hydrophobic and ionic interactions in the formation of helix bundles
- resulting conformational change (b) brings membranes into close proximity
- further factors for specificity of the membrane fusion: e.g. docking complexes at the 22 membrane, cytoskeletal changes and Rho-GTPases for directed vesicle transport.

Question 19

Membrane fusions between Viruses and animal cells

Answer 19

HIV -> Fusion from outside
Influenza -> Fusion from inside

1. Pinch-off of membrane vesicle into cell; virus in vesicle;
2. Fusion of virus and vesicle
3. Release into cytoplasm

Process of membrane fusion
- Binding to receptor leads to conformational change in fusion protein
- Hydrophobic fusion peptide becomes exposed and inserts itself into target membrane
- Fusion protein trimerizes; coiled-coil heptadrepeats of the different fusion proteins of the trimer form supercoil conformational change
- Membranes are brought in close proximity and fuse

Question 20

Membrane fusions between bacteria and animal cells

Answer 20

Salmonella/Yersinia
- Intracellular phase
- Uptake into vesicles
- Remaining in this vacuole
- Salmonella InvA
- Yersinia YadA

Process of membrane fusion
- Bacterial invasins mediate entry into host cell
- Invasins form homotrimer and are coiled – coil proteins
- Invasin monomers form supercoil Conformational change
- Membranes are brought in close proximity and fuse

Question 21

There are 3 classes of viral fusion proteins

Answer 21

Class I: e.g. Influenza Hemaglutinin
Class II: e.g. Flavivirus E
Class III: e.g. Rhabdovirus G
“The accepted model for enveloped virus entry posits that interactions with a target cell trigger an exothermic fusogenic conformational change of the fusion protein, which irreversibly transits from a metastable, activated prefusion form to its lowest-energy, postfusion conformation. The three classes adopt a common postfusion hairpin-like arrangement, juxtaposing the target membrane insertion element of the protein with its viral transmembrane anchor, suggesting that in spite of their altogether different structures, they display a similar mechanism for catalyzing the membrane fusion reaction.”

Question 22

Fusion of viral and cellular membranes
-> Class I fusion proteins -> Examples

Answer 22

Influenza virus hemagglutinin
- HA0 is a trimer which displays in the virion an elongated conformation
- Proteolytic cleavage of HA0 by an extracellular protease (or in exceptions by Furin in the cell) generates HA1 and HA2; the fusion peptide is located at the N terminus of HA2; after cleavage HA2 is in a pH dependent metastable conformational state (ACTIVATION STEP!)
- Receptor binding leads to uptake into vesicle (virus is now in endosome)
- Acidification of the endosome (pH 5) triggers the exposure of the fusion peptide (cleavage into HA1 and HA2 is prerequisit) and conformational change (formation of supercoil) towards a hairpin structure (stable state); (TRIGGERING STEP!) this shortening of the HA structure leads to approach between the membranes
- The close proximity and the inserted fusion peptide induce membrane fusion
HIV (gp41), Coronavirus (spike)

Question 23

HIV (gp41), Coronavirus (spike)

Answer 23

• Central domain in the fusion protein forms trimer with helix bundles; approach of the membranes
• No pH change required, but second protease cleavage in endosome

January 2022: Delta vs. Omikron Delta: efficient TMPRSS2 cleavage; fusion pref. at plasma membrane
Omikron: inefficient TMPRSS2 cleavage; fusion pref. after uptake into endosome.
Consequence: different tissue tropism: lung vs. upper resp. tract

Question 24

Enhancing host cell infection by SARS-CoV-2

Answer 24

Proteolytic processing of SARS-CoV and SARS-CoV-2 S proteins facilitates virus entry. SARS- CoV and SARS-CoV-2 bind to ACE2 at a region on S1. Furin cleavage at the S1-S2 junction exposes the C-end rule peptide on SARS-CoV-2 S1 and allows binding to NRP1. Subsequent processing by cathepsins and TMPRSS2 allows S2 fusion peptide–mediated membrane insertion and merging of membranes. The absence of a furin cleavage site in SARS-CoV S1 and a SARS-
CoV-2 S1 mutant prevents binding to NRP1 and limits virus entry and infection.

Question 25

Mechanism of class I fusion proteins

Answer 25

1) Receptor binding or low pH
2) Extension, exposure of fusion peptide
3) inserted Fusion peptide
4) local enrichment of HAs
Conformational change in FP leads to mixing of the lipids of the double membranes
5) Transmembrane anchoring essential!
6) Approach of TM domain and fusion peptide
Hemifusion-intermediate: only 1 layer mixed
7) Fusion

Question 26

Alternative model for fusion

Answer 26

A) Target -> Attachment -> Deformation -> Hemifusion -> Pore
B) Target -> pH 5.5 -> Attachment -> Dimpling -> Scission -> Apposition -> Hemifusion -> Constrained Pore -> pH 5.0 -> pore

Question 27

HIV fusion

Answer 27

1. Attachment/receptorbindingviagp120
2. Coreceptorbinding
3. gp120release; fusion mediated by exposed gp41

Question 28

HIV fusion - Detailed knowledge allows development of new therapeutics

Answer 28

gp41 zipping:
- helical HR2 domain folds back upon itself and associates with a second helical structure, the HR1 domain
- increases intimacy between viral and cellular membranes
- membrane fusion

T-drugs interfere with this process
T-20, Enfuvirtide or Fuzeon
(Trimers Inc., Roche):
- e.g. 36-amino-acid peptides (different versions)
- bind to helical regions and interfere with zipping mechanism
- inhibit fusion

Approved drug!

Question 29

Neutralizing antibodies against a broad range of influenza viruses

Answer 29

• library of single chain abs (VH-VL)
• recombinant trimeric HA ectodomain expressed in insect cells
• selection of abs binding trimeric HA
• neutralize a broad range of influenza viruses

Mode of action: Inhibition of fusion!

Question 30

Fusion of viral and cellular membranes
-> Class II fusion proteins e.g. Flavivirus E protein (also Alphaviruses)

Answer 30

• Co-translational association with helper protein (e.g. prM in Flavi-, E2 for Alphaviruses)
• Helper protein blocks fusion activity; protects virus from premature fusion
• Proteolytic cleavage of helper protein is a requirement for fusion activity of the fusion protein
• Due to a low pH in the secretory pathway, pr peptide remains bound to E even after proteolysis and still protects virus from premature fusion with the host cell membrane
• After secretion into the extracellular milieu (neutral pH), pr peptide is released and the fusion loop in E (internal loop not terminal like class I fusion peptide) becomes fusion active
• At neutral pH E is a dimer lying flat on the virion; at low pH in the endosome E is converted into a conformation protruding from the virion surface which allows insertion of the fusion loop into the endosomal membrane
• This triggers trimerisation of E which triggers the conformational change leading to the approach of membranes and their fusion

Question 31

Mechanism of class II fusion proteins

Answer 31

Flavivirus E dimer (shielded fusion peptide) -> low pH -> accessible fusion peptide -> lateral rearrangement into trimers -> inserted fusion peptide
Conformational changes in and between E-domains leads to membrane approach and fusion
-> Approach of TM-domain and fusion peptide, hemifusion -> Fusion

Question 32

Capturing a Flavivirus Pre-Fusion Intermediate

-> CryoEM image reconstruction of WNV in complex with E16 antibody fragments at pH 8

Answer 32

Trick:
Antibody blocks formation of fusion active E trimers = freezing of intermediate

Allows to capture a structural intermediate that likely occurs during cell entry when flaviviruses transit through the acidic pH environment of early endosomes.

Question 33

Equatorial slices of cryoEM image reconstruction of WNV in complex with E16 antibody fragments in different pH environments

Answer 33

A) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 8, rendered at 23 Å resolution. Red arcs (1 through 5) specify the outer radii of the nucleocapsid core (154 Å),
lipid bilayer (205 Å), E glycoprotein shell (247.5 Å), scFv molecules (278 Å),
and Fab fragments (318.5 Å) from the viral center. The positions of the icosahedral two-,
three- and fivefold axes are indicated with black arrows and numbers.
(B) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 6, rendered to 25 Å resolution. The red arcs (6 and 7) specify the outer radius of the expanded E/scFv (317.5 Å) and E/Fab (347 Å) protein layer, respectively. The low pH triggered radial expansion of the E/scFv or E/Fab protein shell resulted in a ~60 Å wide shell of low density between the lipid bilayer and the expanded outer protein layer, as indicated by the green arrow. The scale bars represent 100 Å.

E16 antigen binding fragments (Fab)
single-chain antibody derivative (scFv)

Question 34

pH dependent widening of the virion radius

Answer 34

Domains DI, DII, and DIII of E are colored red, yellow, and blue, respectively. The partially alpha-helical stem region is shown in magenta and the transmembrane helices in grey. The fusion loop at the distal end of DII is indicated in green.
Low endosomal pH weakens inter- and intramolecular E contacts and induces the outward extension of the whole stem region or its N-terminal portion including helix H1, followed by the dissociation of the E dimers. Further structural repositioning of the E monomers allows the interaction with the endosomal target membrane via the fusion loop. E16 was shown to inhibit the formation of fusion-active E trimers, most probably by interfering with the rearrangement of the E domains as a consequence of adding mass to DIII and sterically clashing with neighboring
E and Fab molecules. However, E16 may also sterically prohibit the contact of E with the target membranes.
Domains DI, DII, and DIII of E are colored red, yellow, and blue, respectively. The partially alpha-helical stem region is shown in magenta and the transmembrane helices in grey. The fusion loop at the distal end of DII is indicated in green.
B: pre-fusion intermediate with the E glycoprotein/Fab layer expanded radially outwards leaving an ~60 Å-wide gap between the lipid bilayer and the outer protein shell. These structural data suggest that the low pH-triggered formation of fusion-active E homotrimers on the viral surface is preceded by the outward extension of the E stem region.

Question 35

Structural protein region of flaviviruses

Answer 35

prM: protection from fusion during budding; Furin cleaves prM (blue) into pr and M (in TGN)

Question 36

Maturation of Flavivirus (genus!) particles

Answer 36

Secretory pathway (do not interchange with fusion in endosome!!!)

pr release is pH dependent!

pH 7.0 = Dimer
pH 6.0 = Dimer -> Furin!
pH 7.2 = Trimer

Important: Protection of visions from premature fusion while budding

Fusions-loop is shielded by prM

Question 37

Change of the extent of oligomerisation of E during the maturation of the vision

Answer 37

PREMATURE PARTICLE I
Trimer of prM/E heterodoxer

PREMATURE PARTICLE II
Dimer of prM/E heterodimer

PREMATURE PARTICLE III
Dimer of pr+M/E heterodimers

MATURE PARTICLE
Dimer of E homodimers

1) Immature virus / pH 7.0 / Non-infectious (prM inaccessible for Furin)
2) Immature virus / pH 6.0 / Non-infectious (prM is accessible for Furin, due to a pH dependent conformational change)
3) Immature virus / pH 6.0 / Non-infectious ?
4) Mature virus / pH 7.0 / Infectious (pr release is pH dependent!)

Dengue virus: Furin treatment leads to a 1000 fold increase in infectivity of virions

Question 38

Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers

Answer 38

DENV serotype 2 (DENV2)–specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody- enhanced severe dengue disease.
We determined the cryo–electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 “locks” two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency.

Important:
The recombinant antibody used is unable to bind to Fc receptors due to two mutations in the heavy chain: no ADE induction!

Question 39

A potent neutralizing antibody with therapeutic potential against all four serotypes of dengue virus

Answer 39

We describe the neutralizing and protective capacity of a dengue serotype-cross- reactive antibody isolated from the plasmablasts of a patient. Antibody SIgN-3C neutralized all four dengue virus serotypes at nano to picomolar concentrations and significantly decreased viremia of all serotypes in adult mice when given 2 days after infection. Moreover, mice were protected from pathology and death from a lethal dengue virus-2 infection. To avoid potential Fc-mediated uptake of immune complexes and ensuing enhanced infection, we introduced a LALA mutation in the Fc part. SIgN-3C-LALA was as efficient as the non-modified antibody in neutralizing dengue virus and in protecting mice while antibody-dependent enhancement was completely abrogated. The epitope of the antibody includes conserved amino acids in all three domains of the glycoprotein, which can explain its cross-reactivity. SIgN-3C- LALA neutralizes dengue virus both pre and post-attachment to host cells. These attributes likely contribute to the remarkable protective capacity of SIgN-3C.

Question 40

Fusion of viral and cellular membranes
-> Class III fusion proteins e.g. VSV, HSV-1 gB, EBV gB and baculovirus gp64

Answer 40

• trimeric in pre and post fusion state
• no cleavage event needed at all for conformational changes required for fusion
• no terminal fusion peptide but fusion loop (comp. class II)
• reversible, pH dependent conformational change due to reversible protonation of His residues

Question 41

Class III viral membrane fusion proteins
-> Conformational change in VSV G

Answer 41

The ectodomain of G has been crystallized in its pre-fusion (panel A) and post-fusion (low-pH) (panel B) state. The conformational change results in flipping of domain I, carrying the fusion loops, and the C-terminus, to the opposite side of the molecule, relative to domain IV and helix F2 of domain III, which can be viewed as a rigid body and are shown in the same orientation in panels A and B. During the structural rearrangement, domains I, II and IV retain their folds, while domain III (yellow) undergoes significant refolding (central helix F2 is prolonged into the longer helix F in post-fusion form, through recruitment of helix F1, as indicated by the yellow arrow in panel A). The linker or hinge regions, which suspend domain I off the rest of the molecule (residues 47-52 and 173-180), are shown in violet. These regions undergo structural changes important for the initial stage of the conformational change, during which domain I separates from the C-terminus, swings out and rotates 94° relative to domain II (the direction of movement is indicated by the blue arrow in panel A). This is followed by repositioning of domain IV on top of domain III, and results in the more extended post-fusion conformation.

Question 42

After receptor binding

Answer 42

• Conformational change
• Exposure of hydrophobic surfaces
• In some cases release of lytic factors
• Membrane destabilisation
• penetration

Question 43

Membrane penetration of non enveloped viruses

Answer 43

ENVELOPED VIRUS
Fusion of viral and cellular membranes -> Membrane penetration

NONENVELOPED VIRUS
Binding and/or disruption of cellular membrane -> Membrane penetration

Question 44

After membrane fusion viruses end up in different cellular compartments

Answer 44

PLASMAMEMBRANE: Poliovirus
ENDOSOME: Parvovirus, Reovirus, Adenovirus
GOLGI: Papillomavirus
ENDOPLASMATIC RETICULUM: Polyomavirus SV40

Question 45

Poliovirus uncoating
-> Release of genomic RNA for Poliovirus

Answer 45

• Binding to PV-receptor (CD 155) displaces „pocket factor“ from capsid structure and allows deeper binding to CD155; results in a conformational change in the particle:
• VP4 is released and inserts into endosomal membrane
• Hydrophobic N-terminus of VP1 is relocated from the inner side of the capsid to the outside and also inserts into the endosomal membrane
• RNA genome migrates through membrane pore into the cytoplasm
• Pocket factor: Fatty acid molecule

Question 46

Picornavirus unpacking

Answer 46

PLA2G16 facilitates genome dislocation from LGALS8 clusters and enables viral genome translation

PLA2G16:
Phospholipase; allows the release the viral RNA from the vesicle; if gene is deleted, degradation of RNA
LGALS8:
Galectin 8, binds to glycans of intracellular bacteria, leading to their autophogosomal destruction; if gene is deleted together with PLA2G16 no degradation of the genome

Question 47

Fusion of reovirus ISVP with endosomal membrane

Answer 47

• μ1N peptide and f peptide, both proteolytic cleavage products of μ1 protein, integrate into the endosomal membrane and contribute to pore formation
• μ1N peptide alone is sufficient to form a pore in the target membrane
• ISVP are recruited to the pores, dock, and finally passage through membrane

µ1 outer capsid protein: is processed to allow fusion

Question 48

Virus -> Trigger -> Membrane interaction

Answer 48

a) Poliovirus -> cell receptor -> myristylated VP4 peptide
b) Reovirus -> cell protease -> Autolysis? -> Pore formation
c) Rotavirus -> cell. protease -> pore formation
d) Adenovirus -> in endosome -> pore formation
e) Polyomavirus -> cell chaperone

Similar: Infectious bursitis virus (Birnaviridae), pep46, Flock house virus (insect virus), gamma-peptide
Cellular interacting partner/ or cellular surroundings
- mediates conformational change or proteolysis in viral capsid
- allows membrane interaction of free peptides or as a part of the virion

Peptides which can insert into target membranes play an essential role in the membrane penetration of nonenveloped viruses

Question 49

Escape from the endoscope

Answer 49

Since parvoviruses are non-enveloped, they cannot escape from the endosomal system by membrane fusion, as enveloped viruses do. Instead, parvoviruses deploy a phospholipase A2 (PLA2) domain located at the N-terminus of VP1.
Studies on the prototypic minute virus of the mouse, MVM, revealed that the PLA2 domain, which is hidden in the interior of the capsid, becomes exposed in the endosome when the N-terminus of VP1 is extruded, but remains capsid-tethered. PLA2 activity was found to be required for virus escape from the endosome into the cytoplasm.

Question 50

Rotavirus entry and cry-EM structures of the penetration protein in upright and reversed conformations

Answer 50

Infection requires the cleavage of VP4 into an N-terminal fragment, VP8, and a C-terminal fragment, VP5. VP8* attaches the virion to its cellular receptor (in many cases, a glycolipid). VP5* perforates the lipid bilayer of the endosomal membrane.

The function of VP7, a Ca2+-stabilized trimer, is to anchor VP4 onto the virion surface and to respond to loss of Ca2+ at an early stage of infection by dissociating and releasing VP8* and VP5* from the double layer particle (DLP). On a virion, the ‘spike’ protein VP4 is a trimer with an unusual, asymmetric conformation, both before and after its activation by cleavage.

• rearrangement leading to insertion of VP5* into membrane

Question 51

Reovirus-coded cell-cell fusion machinery / Viral spreading via syncytial formation

Answer 51

reovirus fusion-associated small transmembrane (FAST) proteins: NSPs (not in virion)!

• spatial proximity of the membranes due to cellular adhesion
• viral FAST proteins mediate only the fusion step
• active cellular actin remodelling required for max. efficience
• FAST proteins interact only with the membrane but not with adhesins
• Cellular adhesins generate (without interaction with FAST proteins) approach between membranes
• viral FAST proteins mediate only step of fusion
• further approach of membranes required: FAST proteins-ectodomain is too short
• fluctuation of membrane?
• actin-driven membrane concavity? - adherens junction?
• gap junction?

Viral component for cell-cell fusion:
- nectin
- cadherin
- HA
- FAST protein
- F-actin
- actn monomers
- adaptor proteins
- connexin

Question 52

Release of viral genomes

Answer 52

PARAMYXOVIRIDAE
Cytoplasm -> Uncoating
HERPESVIRUS
Cytoplasm -> Dynein Microtubuli -> Docking onto nuclear membrane
FLAVIVIRIDAE (UNCOATING WITHIN ENDOSOMES)
Cytoplasm -> Endosome -> Microtubuli -> Uncoating
ADENOVIRUS (UNCOATING AT THE NUCLEAR MEMBRANE)
Cytoplasm -> Endosome -> Dynein -> Microtubuli -> Docking onto nuclear membrane

Question 53

Nuclear transport: Herpesviridae, Adenoviridae, Orthomyxo- viridae, lentiviruses (which are part of Retroviridae), Hepadnaviridae, Parvo- viridae and Polyomaviridae

Answer 53

Most viruses use the canonical nuclear pore complex (NPC) in order to get their genome into the nucleus. Viral capsids that are larger than the nuclear pore disassemble before or during passing through the NPC, thus allowing genome nuclear entry. Surprisingly, increasing evidence suggest that parvo- viruses and polyomaviruses may bypass the nuclear pore by trafficking directly through the nuclear membrane.
Parvo: Caspase dependent pathway? See below (slide 78)
Polyoma: pore formation in ER and nuclear membrane by VP2 and/or VP3?
Papilloma: Nuclear membrane breakdown in mitosis essential for entry of viral DNA genome into nucleus

Question 54

Adenovirus DNA genome release into the nucleus

Answer 54

-Fiberprotein binds to receptor
-Interaction of penton base protein with integrin receptor on cell leads to endocytosis
-Acid-dependent partial disassembly
-Capsid release and transport via microtubuli to nuclear pore complex
-Binding to CAN/Nup124
-Bindung of cellular histone H1 to capsid leads by binding of importin-7 and –b to disassembly
-Import of viral DNA into nucleus

Question 55

Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis

Answer 55

• parvoviruses induce nuclear membrane breakdown to get into the nucleus
• direct binding of parvoviruses to distinct proteins of the nuclear pore causes structural rearrangement of the parvovirus capsid
• exposure of domains comprising amphipathic helices is required for nuclear envelope disintegration (disruption of inner and outer nuclear membrane)
• Ca++ essential, activates PKC, which activates cdk-2 (further activation by caspase 3)
• Parvovirus abuse and uncouple this process from the initial phases of mitosis

Question 56

Feline Calcivirus Cell entry
-> Model of FCV entry and endosome escape

Answer 56

• FCV binding to fJAM-A (cellular receptor) at the cell surface leads to endocytosis
• the enwrapping of the virion within the endosome leads to further binding of fJAM-A molecules triggering conformational changes in the capsid
• results in formation of the portal-like assembly at a unique vertex
• the hydrophobic N termini of VP2 insert into the endosomal membrane, and form a channel through which the genome may be released
• in our in vitro system, viral genomic RNA remained associated with the capsid after receptor engagement; it was released only at low pH, as a consequence of virion disassembly. Further structural changes—and possibly the action of an unknown cofactor—may therefore be required to trigger release of the viral genome through the portal vertex in vivo.

Question 57

Release of the genome from capsid

Answer 57

• Disassembly of the capsid (easyJ)
• But how to get a large genome out of a stable capsid?
• Negative charge of the densly packed genome induces in the capsid high osmotic pressure
• For herpesviruses (HSV-1) 20 atmosphers
• Required for injection of genome from the capsid into the nucleus
• This high internal capsid pressure is generated by an ATP-driven packaging motor located at a unique capsid vertex
  (strongest molecular motor known!)
• Trigger is the bindig to the NPC
• Can be turned off by exposing the capsid to an external osmolyte (e.g. PEG); Mechanism: capsids are permeable for water and small ions; addition of PEG to isolated capsids will induce the egress of water from capsid and thereby reduce osmotic pressure

Question 58

Virus assembly and egress

Answer 58

• Alphaviruses (e.g. SFV): Assembly and budding at PM
• Retroviruses (e.g. HIV): Assembly and budding at PM
• Rhabdoviruses, Orthomyxoviruses, Paramyxoviruses: Assembly and budding at PM
• Coronaviruses (e.g. SARS V): Assembly and budding at intermediate compartment
• Hepadnaviruses (HBV): Assembly and budding at ER
• Genus Flavivirus (e.g. YF Virus): Assembly at ER/Golgi

Coronaviruses, Hepadnaviruses, Genus Flavivirus: Vesicular transport to the PM, Release via exocytosis

Question 59

Virus assembly and egress
-> The viral elements essential for budding

Answer 59

TYPE I
- Spike (red)- and NC (blue)-dependent budding of alphaviruses

TYPE II
- Gag protein (blue)-driven budding of a C type retrovirus assembly at membrane
- D type retrovirus preassembled “Gag” particles

TYPE III
M (red, unfilled) and E (red, filled) membrane protein-driven budding of coronaviruses

TYPE IV
RHABDOVIRUS
- efficient with spike (red) and M (brown)
ORTHO- AND PARAMYXOVIRUSES
- less efficient with M-only
- M and cyto- plasmatic tails of spike protein(s)

Question 60

Virus assembly and egress
-> The viral elements essential for budding

Answer 60

HA and M2 are required for budding release
- amphipathic helix of M2 is sufficient to induce vesicle budding and release
- localizes to the point of membrane scission (neck) (immuno-gold labeling)
- virus mutant in M2 helix is impaired in membrane scission -> virus arrays: like beads on a string
- M2 is essential for pinch off

Question 61

Herpes simplex virus (HSV): assembly

Answer 61

• Translation of viral proteins in the cytoplasm
• Import via nuclear pores
• Assembly of B-capsid with scaffold proteins
• Viral protease cleaves scaffold and thus allows DNA-entry

Question 62

Genom packaging for herpes simplex virus

Answer 62

• Portal complex at the nuclei-capsid binds terminus complex, which assembles at the recognition sequence of the concatameric genome
• DNA is threaded by ATP-dependend helicase; head full; nuclease cuts at the recognition sequence

Question 63

Envelopment and cell egress for herpes viruses

Answer 63

• Nuclear lamina stabilizes and maintains structure of nuclear envelope
• lamins are connected via membrane bound proteins with nuclear membrane
• the nuclear lamina has to be disassembled during mitosis and reassembled afterwards
• this process is regulated by kinases phosphorylating the lamins
• Cdc2/cyclin-dependent kinase (CDK) 1 phosphorylates lamins which interfere with lamin/lamin interaction

Question 64

Envelopment and cell egress for herpes viruses
-> How does the virus get out?

Answer 64

Two highly conserved herpes virus proteins - one a nuclear membrane protein (UL50) and the other a nucleoplasmic protein (UL53) - form what is termed the nuclear egress complex (NEC); required for binding to and bending of nuclear membrane

Nuclear lamina as barrier
HCMV (human cytomegalovirus) tegument protein UL97 has kinase activity and phosphorylates lamins at the sites where CDK1 would phosphorylate during mitosis: disassembly of lamins

Molecular mimicry of cellular kinase

Question 65

Envelopment and cell egress for HCMV

Answer 65

HCMV UL50 / UL53 together with UL97 (kinase) enable the capsid to pass through the nuclear membr. into ER;
Nuclear membrane is taken along as an envelope, but is lost during the egress out of the ER Entry of capsid into golgi leads to envelopment with golgi membrane (this one remains!) Egress out of Golgi via „secretory vesicles“ leads to a second lipid envelope; this one is lost during the egress at the PM

a | Capsids in the nucleus.
b | Primary envelopment, showing the close apposition of the capsid and the inner nuclear membrane (INM).
c | Enveloped capsids present within the perinuclear space. The membrane surrounding the capsid is derived from the INM.
d| Initial steps of secondary envelop- ment. The unenveloped capsids in the cytoplasm interact with trans-Golgi network (TGN) membranes and become wrapped in these membranes (white arrow).
e| Final steps of secondary envelop- ment. Enveloped particles (white arrow) are present within the lumen of TGN- derived membranes.
f | Release of virions. Enveloped virions are transported to cell surfaces and released or remain bound to the plasma membrane.
ONM, outer nuclear membrane.

Question 66

Structural Basis of Vesicle Formation at the Inner Nuclear Membrane
-> A new alternative mechanism for nuclear export apart from nuclear pore transport

Answer 66

• UL31 + UL34 are located at inner nuclear membrane (INM) forming the nuclear eggress complex (NEC)
• This NEC recruits the virus capsid to the INM
• pUL34 membrane anchoring organises the spatial geometry of the NECs leading to membrane bending

Question 67

Abuse of the „Vacuolar Protein Sorting Pathways“ (VPS) by viruses

Answer 67

Problem for viruses: membrane is a barrier when leaving the cell
Solution: Abuse of the cellular apparatus for this problem

VPS: cellular budding process in the lysosomal degradation pathway for membrane proteins
- small vesicles with membrane proteins are budding with help of ESCRT- protein complex (endosomal sorting complex required for transport) in „multivesicular bodies“
That means, a machinery for „vesicle budding“ already exists in the cell
Viruses mediate a „conversion“ to a new function at the cell membrane

Two steps:
I) Formation of a budding pore
II) Membrane fusion

Question 68

Host factors in virion budding
-> Ubiligase: transfer of Ubi to viral glycoprotein

Answer 68

The process of retroviral budding requires the endosomal sorting complex required for transport (ESCRT) machinery, which is a collection of approximately 20 proteins that form four complexes. ESCRTs are usually responsible for sorting cargo proteins for delivery to the late end-some compartment or multivesivular body. The ESCRT machinery includes: Nedd4-like proteins, E3 ligases for ubiquitin transfer, Hrs, a ubiquitin binding protein associated with STAM1 and 2; ESCRT-I components, including Tsg101, Vps28 and Vps37; the ESCRT-II complex; ESCRT III components, named CHMPs, in association with LIP5; Vsp4, an AAA+ ATPase family member involved in recycling the machinery; and AIP-1/Alix, a bridge between ESCRT-I and -III. Endophilins associate with the ESCRT components and induce membrane curvature, which facilitates virus budding and exit from cells.

Gag = group-specific-antigen-protein
Ub = Ubiquitin

Question 69

Abuse of the “Vacuolar Protein Sorting Pathways” (VPS) by viruses

Answer 69

• Gag recruits ESCRT complex from the endosome to the plasma membrane - Budding out of the cell and not into MVB
• Interaction of Gag with many ESCRT factors
• Recognition signals in viral proteins: „late domains“, AS motifs
  PPPY Interaction with NEDD4, PTAP Interaction with Tsg101, YPxL Interaction with AIP-1/Alix
• HIV Gag is sufficient for budding; association with Env 105
• In other cases, e.g. Foamy virus, glycoprotein despite to L-domain not sufficient

Interaction of virion components with ESCRT components via late domain:
- internal building blocks of virions (e.g. Gag) or viral glycoprotein (Bluetounge NSP3)

Question 70

Retrovirus assembly and egress

Answer 70

Gag trafficking during vision assembly and release
Gag is either transported to the plasma membrane where visions are assembled and budded from the cell or trafficked by vesicles, or it buds into exosomes. In some cell types, Gag is transported by the centrosome, or might even detour through the nucleus, en route out of the cell.

Essential role of ESCRT complex for pinch-off during cell division. Do viruses use this pathway?
- Scission by ESCRT-III protein Snf7

Question 71

Hepatitis A virus (HAV) and hepatitis E virus (HEV), both considered by virologists for many years to be non-enveloped viruses but recently recognized to have a more complicated modus operandi

Answer 71

Historically, animal viruses have been classified on the basis of the presence or absence of an envelope – an external lipid bilayer membrane typically carrying one or more viral glycoproteins. However, growing evidence indicates that some ‘non-enveloped’ viruses circulate in the blood of infected individuals enveloped in host-derived membranes that provide protection from neutralizing antibodies. In this opinion article, we discuss this novel strategy for virus survival and consider how it contributes to the pathogenesis of acute viral hepatitis.
The acquisition of an envelope by non-enveloped viruses profoundly influences their interaction with the host at both the cellular and system level and challenges how we think about vaccine protection against these infections.
HAV and HEV are phylogenetically unrelated, small RNA viruses, each with a single-stranded, positive-sense genome. Both infect the liver and cause acute inflammatory hepatitis. Remarkably, they circulate in the blood during acute infection in a membrane-enveloped form, but are shed in feces as non-enveloped viruses.

Transient envelopment of HAV and HEV: both are infectious also without envelope!

Question 72

Cellular entry and uncaring of naked and quasi-enveloped human hepatoviruses

Answer 72

Many ‘non-enveloped’ viruses, including hepatitis A virus (HAV), are released non-lytically from infected cells as infectious, quasi-enveloped virions cloaked in host membranes. Quasi-enveloped HAV (eHAV) mediates stealthy cell-to-cell spread within the liver, whereas stable naked virions shed in feces are optimized for environmental transmission. eHAV lacks virus-encoded surface proteins, and how it enters cells is unknown. We show both virion types enter by clathrin- and dynamin-dependent endocytosis, facilitated by integrin β1, and traffic through early and late endosomes. Uncoating of naked virions occurs in late endosomes, whereas eHAV undergoes ALIX-dependent trafficking to lysosomes where the quasi-envelope is enzymatically degraded and uncoating ensues coincident with breaching of endolysosomal membranes. Neither virion requires PLA2G16, a phospholipase essential for entry of other picornaviruses. Thus naked and quasi-enveloped virions enter via similar endocytic pathways, but uncoat in different compartments and release their genomes to the cytosol in a manner mechanistically distinct from other Picornaviridae.

Question 73

Virus assembly and egress -> Alphaviruses

Answer 73

• budding without involvement of the VPS pathway
• integral membrane proteins are driving force

Question 74

Virus assembly and egress -> Influenza viruses

Answer 74

• fatty acid (myristylation) at the cytoplasmatic part of HA localizes HA in „lipid rafts“, lipid microdomains
• high local concentration promotes budding
• hydrophilic interactions of proteins with lipid surroundings can induce membrane bending and vesicle formation
• pinch off induced by M2 protein (functional analog of Snf7?)

Question 75

Virus assembly and egress -> VSV

Answer 75

• M protein can induce vesicle formation in liposomes
• but: pinch-off remains to be mediated, host factors?

Question 76

Virus assembly and egress -> Different viruses - Different ways

Answer 76

PULLING FORCES: Membrane-microdomains / viral proteins
PUSHING FORCES: Host cell proteins / machinery