MCB3024S
Mammalian immunity, immunity to viruses and vaccines
Viruses and immunity
What are viruses
- viruses may be defined as acellular organisms whose genomes consist of nucleic acid, and which obligately replicate
inside host cells using host metabolic machinery and ribosomes to form a pool of components which assemble into particles
called virions, which serve to protect the genome and to transfer it to other cells
- they are distinct from other virus-like agents such as viriods and plasmids and prions
- the “virocell” is how viruses are alive, when a virus enters a cell, they express themselves and the cell becomes a vehicle
to make more virions
- plasma membranes with membrane proteins protruding on the outside are potentially membrane receptors for viruses
Animal cells
- animal cells do not have cell walls, only a lipid bilayer cell membrane (plasma membrane) with embedded proteins
- they have a highly developed internal membrane complex which consists of:
a) a nuclear (double) membrane
b) rough and smooth endoplasmic reticulum
c) Golgi apparatus
d) specialized protein-containing vesicles such as lysosomes and endosomes, involved in intracellular digestion of
macromolecules
e) peroxisomes for breakdown of very long chain fatty acids
Vesicle transport
- eukaryote cells have an intricate system of vesicle transport, centered on the Golgi
- involves: export of proteins and vesicles from the ER to the Golgi
- production of export vesicles containing proteins from this for fusion with the cell membrane in a process called
exocytosis (SARS-CoV2 uses this mechanism to get whole virus particles out of a cell)
- production of lysosomes to fuse with endosomes
- cytoskeleton-directed vesicle trafficking mediated via molecular motors such as myosin, kinesin and dynein which
involves targeting of vesicles back to the Golgi and to the nuclear membrane (cytoskeleton is a network of fibrils that span
the whole cell and give it structure)
- endosomes result from and digest material internalized by receptor-mediated or non-specific endocytosis (phagocytosis for
particulates; pinocytosis for liquid)
Clathrin-coated pit with receptors binding target molecules, cross-linking and invagination
- clathrin is a structural protein found in the cytoplasm and is also associated with the inside of the cell membrane and
vesicles derived from the membrane
- a pit forms on the surface if a cell, this can be caused by cross-linking of receptor molecules in a region of the cell
membrane that cause an accumulation of them, they become immobilized in the cell membrane which induces some
curvature to the membrane and formation of a clathrin-pit is then energetically favored
- end result is that clathrin curves up and forces the membrane to curve up and around enclosing the receptors and
molecules bound to the receptors until the cage is complete and the clathrin-coated vesicle is fully enclosing the target
molecule-receptor complex and the vesicle can bud off from the cell
- this is a process that many viruses have co-opted to get inside of cells
- disassembly of clathrin cage by heat-shock protein (Hsc70) and the J-domain-containing protein, auxilin in an ATP-
dependent process
Receptor-mediated endocytosis
- results from cross-linked membrane receptors collecting in clathrin coated pts which develop into vesicles due to
assembling clathrin which promotes curvature of the membrane
- clathrin-coated pits that do not stabilize by cargo binding to receptors do not proceed to a complete clathrin-coated vesicle
but dissemble rapidly (i.e. are transient)
- clathrin is recycled back to the cell membrane after endocytosis, to allow fusion of the vesicle with an endosome, which
increases its size (and floods it with enzymes and cytoplasm at a low pH)
- clathrin-coated cage has a triskelion subunit structure
- about 2500 clathrin-coated vesicles leave the plasma membrane of a cultured fibroblast every minute, and within seconds
of being formed, they shed their coat and are able to fuse with early endosomes
Caveolin-dependent endocytosis
- are three mammalian caveolins (21 kDa; cytoplasmic N- and C-termini, transmembrane hairpin between)
, - are transported to the Golgi as monomers and then associate with lipid rafts to form oligomeric structures (14-16 mol)
- oligomers form caveolae: 50-100 nanometer invaginations of the plasma membrane in many vertebrate cell types
- caveolae protect cells from mechanical stress (by providing a spare membrane that is in the form of dimples), have a role
in cell signaling and in lipid regulation and as mechanosensors
- they can also be used for entry to the cell by some pathogens which avoids degradation in lysosomes
How viruses get into animal cells
- all entry of virus genomes into animal cells is mediated by specific recognition of cellular receptor molecules by virus
“attachment proteins”
- the cellular receptor molecules are generally glycoproteins and are a wide variant of integral cell membrane proteins: they
are normal cell proteins that have been co-opted by viruses
- attachment proteins are either integral virion membrane proteins or proteins on the surface of non-enveloped virions (such
as papillomaviruses)
- binding of individual cellular receptor molecules by virus attachment proteins can be followed by consolidation of binding
as more receptor proteins migrate in the fluid cell membrane to bind virus proteins
- consolidation of binding: virion could attach itself to cell surface, it could by one or many receptors, it could remain
attached in which case the tendency would be for more receptors to migrate to the area and they could bind the surface of
the virion as well causing a curving on the membrane ; or it could just dis-attach (transient binding)
- non-transient binding is accompanied by changes in the cell membrane (accumulation of receptor proteins in a pit)
- binding of many receptors in one region of cell membrane can trigger a number of processes, including internalization of
virions in vesicles or direct virion envelope to cell membrane fusion (pox, herpes and paramyxovirus) which would then
liberate anything that is inside the membrane of the virus unto the cytoplasm of the cell
Fusion proteins of enveloped viruses
- the fusion proteins are derived from host to virus or virus to virus acquisition
- they are common to all enveloped viruses and there are 4 types
1) class I fusion proteins
- form virion spikes that are trimers of 3 identical subunits, largely alpha-helical, with 2 subunits generated by proteolytic
cleavage from a precursor, the original C-terminus is anchored to the viral membrane; the new N-terminus has a stretch of
about 20 hydrophobic amino acids: this is the fusion peptide
- the class I proteins all have a trimeric helical coiled-coil rod adjacent to the fusion peptide: this may act as a template for
the refolding of protein segments during fusion when the “six helix bundle” forms
- they are found in retroviruses (HIVs, HTLVs), orthomyxoviruses (influence), coronaviruses and paramyxoviruses Mumps
and measles) and filoviruses (Ebola), among others
Influenza virus cell entry
- the virion attaches, a clathrin cage forms, (is a class I fusion protein so see trimers clustered), the vesicle is internalized,
the clathrin falls off , fuses with an early endosome then lysosome and the interior is acidified, the fusion protein interacts
with the cell membrane which results in the fusion of the virus membrane with the endosome membrane
- the fusion peptide is hydrophobic and it binds into the insdide membrane of the endosome (now have protein briding from
the virus membrane to the cell membrane)
- the cyan parts associate with eachother and they bend “single extended intermediate”
- only HA2 shown, alrhough HA1 remains tetheredtoit through a disulphidebond
- fusion peptides engage the target membrane
- arrows show direction of folding back of the coiled-coil to get a structure, forcing the 2 lipid bilaers into 1
- there is nowa pore betweent the viral membrane and inside the endosomal membrane which opens into the cytoplasm
- final refolding steps stabilize nascent fusion pore, with formation of the “siz helix bundle”
Influenza or other Group I fusion protein summary
