Membrane remodeling by viruses

Question 1

Different viruses modify different intracellular membranes

Answer 1

ER-membranes:
- Poliovirus
- HCV
- Coronaviridae: Mouse hepatitis virus SARS virus
- Arterivirus: Equine arteritis virus
- Tobacco mosaic virus

ER/Golgi/Intermediate compartment:
- Kunjin virus

Lysosomes/and plasma membrane:
- Rubella virus
- Alphavirus: Semliki forest virus

Mitochondria:
- Flock house virus

Question 2

Generating membranous bending with proteins

Answer 2

Example:
- Clathrin on clathrin coated vesicles
- Coatamer proteins on COP I and COP II vesicles
- Common in viral proteins that induce vesicles

-> Amphipatic helices

Question 3

Double membrane vesicles in Nidovirales (Corona- and Arteriviruses)

Answer 3

The protrusion and detachment model: proposes that part of the endoplasmic reticulum discern starts to bend, pinches off and then seals to form a double-membrane vesicle. Interactions between the lumen domains of viral membrane proteins could mediate the tight apposition of the two bilayers and induce curvature. In the double-budding model, a single-membrane vesicle buds into the lumen of the ER and then buds out again, and the membrane proteins could mediate inward as well as outward budding.

Question 4

Cellulkar processes of membrane reshaping -> Autophagy

Answer 4

• Reaction to stress, starvation, infection
• Compassing of cytoplasmatic parts with sickle-shaped vacoules
• Fusion with lysosomes
• Content degradation in autophagosomes (double membrane)
• 27 Autophagy related genes (Atg)
• knock down of ATG 12 and LC3 does not inhibit replication of Poliovirus, but egress of new viruses (involvement of autophagy machinery?)

Question 5

Model: Oligomeric viral membrane proteins induce membrane concavity

Answer 5

Upon release of viral genomic RNA into the cytoplasm of the infected cell, the viral genome is translated into a poly protein that carries the structural and non-structural proteins. The viral non-structural protein NS4B induces the formation of membrane alterations, which serve as a scaffolds for the assembly of the viral replication complex. The RC consist of viral non-structural proteins, viral RNA and host cell factors. Within the induced vesicles, viral RNA is amplified via a negative-strand RNA intermediate.

Question 6

Remodeling of membranes in cells by (+)-strand RNA viruses

Answer 6

All (+)-strand RNA viruses modify intracellular membrane systems; generate vesicle-like structurs or vesicle-networks
RNA replication always at membranes

Functional properties:
- Shielding of ds RNA replicative intermediates from the innate immune system
- Resistance against RNases and proteases
- high locale concentration of viral components
- Contact with ER components of the cell?

Viral replicase proteins located at the outer side of the vesicles

Question 7

Architecture of the Chikungunya virus replication organelle

Answer 7

Alphavirus: Chikungunya virus (CHIKV)
- replicates its RNA genome in membrane spherules on the plasma membrane
- cryo-electron tomography imageas of CHIKV spherules in their cellular context
- viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud
- biochemical assays show that the viral helicase-protease nsP2, while itself not membrane bound, is recruited to membranes by nsP1
- full-sized spherules contain a single viral genome in double-stranded form
- the energy released by RNA polymerization is sufficient to remodel the membrane to the characteristic spherule shape.

A single copy of the genomic RNA determines the shape of the spherule membrane.

The force exerted by RNA polymerization is sufficient to drive spherule membrane remodeling

Question 8

Concepts for the release of progeny (+)-strand RNA genomes from “replication vesicles”

Answer 8

RNA synthesis takes place close to the pore structures for the release of this RNA into the cytoplasm.
Compare dsRNA viruses! RNA synthesis takes place in cores close to the exit channels!
Additional role for “crown“ in virion morphogenesis?

Question 9

Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly sites

Answer 9

• Electron microscopy (immune EM studies) - Electron tomography
• ER-derived membraneous network of convoluted membranes
• Vesicles represent invaginations of the ER membranes
• Dengue virus (DENV)-induced vesicles with pores
• Budding of particles on ER membranes opposed to pores into the ER - Virus particles are endoplasmic reticulum (ER)-derived

Question 10

“Membrane nests” of Dengue Virus

Answer 10

• sites of virus budding
• Budding of virus particles on ER membranes opposed to pores
• budding occurs into the ER

Question 11

Membrane rearrangements in HCV infected cells

Answer 11

• Membranes are also derived from the ER, like in the case of Dengue virus
• However, not by invagination; vesicles are protrusions of the ER membrane (in Dengue infected cells vesicles are invaginations, not protrusions of ER)
• HCV shows more similarities to Coronaviruses, Nidoviruses and Picornaviruses
• Main components of the membraneous web are single and double membrane vesicles (DMVs)
• DMVs are the predominant form; only there RNA replication?
• The vesicles are frequently connected to the ER membrane via a neck-like structure

Question 12

Hypothetical models for formation of double membrane vesicles

Answer 12

(A) By analogy to flaviviruses HCV proteins induce invaginations of the ER membrane. Extensive invagination leads to a local ‘shrinking’ of the ER lumen. This model assumes that enzymatically active HCV replicase (green dots) reside in the lumen of the invagination and remain active as long as the vesicle is linked to the cytosol. Upon closure of the DMV, the replicase would become inactive (grey dots). Alternatively, closed DMVs might be connected to the cytosol via proteinaceous channels. (B) HCV proteins might induce tubulation of ER membranes that undergo secondary invagination and thus double membrane wrapping. These DMVs could initially be open to the cytosol, but might close off as replication/infection progresses. The resulting DMV might stay connected to the ER via a stalk or be released as a ‘free’ DMV (left or right drawing, respectively). (C) Induction of DMVs follows the same pathway as described for panel B, but the viral replicase remains on their cytosolic surface as discussed e.g. for the poliovirus. (D) HCV RNA replication might occur on SMVs in close proximity of DMVs. In this case, DMVs might be an epiphenomenon
or serve some other purpose for the HCV replication cycle. For each model, structures identified in
the 3D reconstructions are shown next to or below the corresponding schematic drawing.

Question 13

Classification and morphologies of plus-strand RNA virus-induced membrane alterations

Answer 13

Invaginated vesicle (InV)/spherule type (A-C)
A: Flock-house virus (FHV);
B: Rubella virus (RUBV);
C: Dengue virus (DENV);

DMV type (D-F)
D: Poliovirus (PV);
E: Severe acute respiratory syndrome coronavirus (SARS-CoV);
F: Hepatitis C virus (HCV).

Question 14

Envelopment and cell egress for helper viruses

Answer 14

Two proteins (UL34 and UL31) enable the stride of the capsid through nuclear membr. into ER Thereby the 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

Question 15

Structural Basis of Vesicle Formation at the Inner Nuclear Membrane

Answer 15

A new alternative mechanism for nuclear export apart from nuclear pore transport

Question 16

Structural Basis of Vesicle Formation at the Inner Nuclear Membrane

Answer 16

UL31 + UL34 are located at inner nuclear membrane (INM) forming the nuclear eggress complex (NEC)
This NEC recruits the virus capsid to the INM

NEC recruits the virus capsid to the INM
pUL34 membrane anchoring organises the spatial geometry of the NECs in a way leading to membrane bending.
NEC coat architecture is an elegant solution for induction of membrane curvature based solely on the formation of a highly defined lattice of heterodimer interactions.
This is reminiscent to HIV budding from the plasma membrane.
Most cellular vesicle formation processes involve a dedicated cellular scission machinery and consume energy in form of e.g. ATP
The NEC appears capable of autoscission by continuing assembly of NEC units on the inside of the forming vesicle and requires at least under in vitro conditions no external energy input for both membrane budding and scission

Question 17

Cell-cell communication

Answer 17

(a) Neurological and immunological synapses transmit cell–cell signals through the extracellular space, relying on neurotransmitter release and receptor signaling through a tight cell–cell junction
(b) Cytonemes as thin filopodial bridges topologically identical to stretched out synaptic contacts.
(c) By contrast, cells communicating using gap junctions establish direct connectivity between two cytoplasms.
(d) Tunneling nanotubes represent thin cell–cell contacts with communicating cytoplasms.

Question 18

Biogenesis of “Cytonemes” and “Nanotubes”

Answer 18

Transient filopodial contacts (a) can stabilize to form elongated stable cytonemes (b).
In the presence of additional adhesion proteins that favor strong cell–cell interfaces, cytonemes could mature into broad synaptic cell–cell interfaces (c). a-c is reversible!
At some synapses, cytoplasmic connectivity is established (d).
Downregulation of these cell–cell interfaces generates tunneling nanotubes with connected 41 cytoplasms (e,f).

Question 19

Abuse of filopodia by viruses

Answer 19

(a) Newly formed retroviruses accumulate on the cell surface, they surf on existing filopodia and are transported via retrograde transport on filamentous actin to the new target cell
(b) Long contact between target membrane and filopodia leads to their uptake; facilitates virus transmission followed by intracellular actin driven transport
(c) Vaccinia virus: induces from outside the formation of Actin-“comet‘s tail“; virus is thereby transported towards target cell; actin-flow in the target cell is used for further transport processes
(d) African swine fever virus; induces from inside the formation of filopodia

Question 20

Vaccinia virus: Time scale of spread is not in line with speed of replication

Answer 20

Time for spread from cell to cell is 75 min (live cell imaging) ?
Time required for complete replication cycle 5-6 h
Trick: Selective infection of uninfected cells
Findings:
Two viral proteins are expressed in a protein complex at the surface of infected cells.
These proteins mark within minutes the cell as infected and induce the repulsion of virions which otherwise would superinfect the cell
Two viral “early” proteins (A33 and A36) were required for this process
Cells expressing A33 and A36 repelled exogenous virions rapidly
Contact between this complex and an „infecting“ virus particle triggers actin tail formation, which effectively ‘bounces’ the new particle off and onto neighbouring cells
Infected cells serve as distributor of other virus particles

Question 21

Vaccinia virus spread

Answer 21

After completing the replication cycle new virions induce actin tails and membrane protrusions which facilitate transfer to noninfected cell
Infected cells express surface complexes including A33 and A36 which induce actin tail formation and repel of superinfecting viral particles

Consequence: Optimal efficiency for viral spread

Question 22

Interaction of viruses with actin

Answer 22

• Vaccinia virus with virus-induced actin-“comet’s tail”
• African swine fever virus induces filopodia

Question 23

Viral spread from cell to cell

Answer 23

Poxvirus -> VV -> Projection on actin tails
Retroviruses -> HIV-1 -> Virological synapses, Nanotubes
Retroviruses -> MLV -> Capture of filopodia
Asfarvirus -> ASFV -> Induction of filopodia
Herpesviruses -> CMV -> Cell-cell-fusion
Paramyxoviruses -> RSV -> Syncytium formation

Question 24

Reovirus encoded cell-cell fusion machinery Viral spread via formation of syncytial

Answer 24

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 step of fusion on surrogate adhesins.
• 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) membranous approach - viral FAST proteins mediate only step of fusion
• further approaching of membranes required: FAST protein-ectodomain too short
• fluctuation of membrane?
• actin-driven membrane concavity?
• adherens junction?
• gap junction?

Question 25

Viral spread from cell to cell -> specific examples

Answer 25

a Herpes-, paramyxo-, retroviruses: membrane fusion, syncytia formation
b Herpesvirus: basolateral budding between tight junctions
c Herpes-, paramyxo-, rhabdoviruses: budding into synaptic cleft
d Virus-induced actin-containing membrane structures carry virions into the adjoining cell
e: murine leukemia virus (retrovirus) uses existing actin-containing membrane structures which carry the virions into adjoining cells
f: HIV uses actin-containing nanotubes for transport into adjoining cells
g: „Virological synapse“: Immunological synapse is abused; induction of polarity in the secretory apparatus

HTLV-1:
ICAM1 induces polarisation of the secretory apparatus. Viral protein Tax accumulates at the MTOC

HIV: assembly and budding happens in a polarized manner at the synapse

Question 26

Viral spread from cell to cell: Herpesvirus

Answer 26

• between neurons
• between neurons and adjoining cells

Question 27

Retroviruses induce membrane bridges between cells

Answer 27

Contact between the viral coating proteins in the filopodia membrane and its cellular receptor in the target cell membrane is essential for the stabilisation of the membrane bridge; mAbs against one of the two partners are sufficient to prevent stabilisation

Question 28

MLV Vision assembly randomly distributed?

Answer 28

Result
- Efficient assembly/budding of MLV especially at sites of cell/cell contact
- 10x more efficient than at other regions of the plasma membrane
- Polarized assembly depends on the presence of the cytoplasmic tail of Env
- Highly coordinated process

Question 29

HIV assembly observed in real-time

Answer 29

Gag molecules are recruited from the inside of the cell and travel to the cell’s surface. When enough Gag molecules get close and start bumping into each other, the cell’s outer membrane starts to bulge outward into a budding virion and then pinches off to form an individual, infectious particle.

Techniques critical for progress:
- fluorescently tagged derivatives of Gag (Gag-GFP and Gag-mCherry)
- fluorescence resonance energy transfer (FRET)
- total internal reflection fluorescent microscopy (TIR-FM) in living cells
-> only very small volume between glass plate and cell surface is illuminated

Question 30

How to detect assembly of protein complexes in vivo?

Answer 30

• Apply FRET
• Differentially labeled Gag fusion proteins
• When they hetero-oligomerize FRET can be observed

Question 31

Fluorescence imaging of single virus particles

Answer 31

Spinning disc confocal microscope
- pinhole disc
- lense disc
subdivision of exciting light beam wide field image

TIRF: total internal reflection fluorescence

Question 32

Retroviruses use CD169-mediated trans-infection of permissive lymphocytes to establish infection

Answer 32

Dendritic cells can capture and transfer retroviruses in vitro across synaptic cell-cell contacts to uninfected cells, a process called trans-infection. Whether trans-infection contributes to retroviral spread in vivo remains unknown. Here, we visualize how retroviruses disseminate in secondary lymphoid tissues of living mice. We demonstrate that murine leukemia virus (MLV) and human immunodeficiency virus (HIV) are first captured by sinus-lining macrophages (sLM). CD169/Siglec-1, an I-type lectin that recognizes gangliosides, captures the virus. MLV- laden macrophages then form long-lived synaptic contacts to trans-infect B-1 cells. Infected B- 1 cells subsequently migrate into the lymph node to spread the infection through virological synapses. Robust infection in lymph nodes and spleen requires CD169, suggesting that a combination of fluid-based movement followed by CD169-dependent trans-infection can contribute to viral spread.
“The direct study of viral pathogenesis within living animals should reveal more surprises in the future,” Mothes said.

Our data support a model in which trans-infection and virological synapses can both contribute to the spread of viral infections in vivo.
The data are also consistent with a role for cell-free virus in spreading, as an alternative mode to virus spread via migration of infected cells.
CD169+ macrophages are located at the interface between fluid phases such as the lymph, blood, and lymphoid tissue. They can concentrate cell-free viruses from the fluid phase to deliver them efficiently to permissive lymphocytes for infection.

Virus collected by Macrophage -> Trans-infection of B1 cell via long-lived synaptic contacts -> B1 cells spread infection through virological synapses in lymph node

Question 33

Filopodia and Viruses: An Analysis of Membrane Processes in Entry Mechanisms

Answer 33

Heparane sulfate on Filopodia is an important binding molecule for viruses