3-4 Virus: Infectious cycle Flashcards
The infectious cycle
Steps
Summary
Do boundaries between steps exist?
- Attachment
- Entry
- Uncoating and Translation
- Replication
- Assembly
- Release
Summary
1. Attachment: Viral proteins on the capsid or phospholipid envelope interact with specific receptors on the host cellular surface. This specificity determines the host range (tropism) of a virus.
- Entry / Penetration: The process of attachment to a specific receptor can induce conformational changes in viral capsid proteins, or the lipid envelope, that results in the fusion of viral and cellular membranes. Some viruses can also enter the host cell through receptor-mediated endocytosis.
- Uncoating: The viral capsid is removed and degraded by viral enzymes or host enzymes releasing the viral genomic nucleic acid or nucleocapsid.
- Replication: After the viral genome has been uncoated, transcription or translation of the viral genome is initiated. It is this stage of viral replication that differs greatly between DNA and RNA viruses and viruses with opposite nucleic acid polarity. This process culminates in the de novo synthesis of viral proteins and genome.
- Assembly: After de novo synthesis of viral genome and proteins, which can be post- transrciptionally modified, viral proteins are packaged with newly replicated viral genome into new virions that are ready for release from the host cell. This process can also be referred to as maturation.
- Virion release: There are two methods of viral release: lysis or budding.
Steps to facilitate the study but not such artificial boundaries occur irl
Where do viral infections begin?
3 different ones
They begin at exposed epithelial surfaces
apical (top, presented to outside)
basal (bottom, prestented to inside)
lateral (side to side cell contacts)
What’s the difference between affinity and avidity?
What’s the characteristics of viral binding?
Affinity is the strength of an specific interaction between a single receptor and single virus binding protein, its the sum of attractive and repulsive forces between the 2 components
Avidity is a measure of the overall binding of receptor with ligand, involving multiple binding site, so its influenced by how many binding sites exist on receptor and ligand
Characteristics of viral binding
The interaction between receptor and viral ligand is low affinity reaction, but the combination if several receptors with viral ligand leads to high avidity reaction
Attachement
How is it mediated?
Which host cell molecules exist? (3)
What does it faciliate? (6)
What does entry receptors do? (3)
Attachment to host cell is mediated by virion proteins binding to specific host surface molecules:
- Glycoproteins
- Glycolipids
- Carbohydrates like sialic acid and heparan sulfate
Despite 1. low affinity, these receptors 2. facilitate adhesions and therefore 3. concentrate the virus near it’s entry 4. triggering signaling pathways. It can also 5. carry the virus to a specific organ. But the 6. ahesion alone does not trigger entry, that’s what entry receptors do
Entry receptors trigger endocytosis or membrane fusion, have a higher affinity, but they dont function as viral receptor, they are misused by the virus
Glycoprotein and different viruses
Rules and examples
Same glycoprotein can be receptor to multiple viruses, often ig-superfamily
CAR for CoxA and Adenovirus
DC-SIGN
Related viruses can bind different receptors
SARS (ACE2) and MERS (DPP4)
One virus might require multiple receptors
HCV
Viral glycoproteins can bind different receptors
HSV-1
HIV entry
Attachment via
Entry Mechanism (7)
Attachment via
Heparan Sulfate Proteoglycan
Entry
1. env gene codes for gp160
2. processed by host cell protease in gp 120 ans gp41
3. gp 120 binds to CD4
4. conformational change exposes co receptor binding site
5. binding of co receptors CCR5 and CXCR4
6. gp41 non covalently bound to gp120 is conformationally changed
7. Insertion of fusion peptide into host cell membrane, resulting in fusion of viral envelope with host cell membrane
Influenza
Attachment and involved (viral) proteins
Sialic acid 2 forms
Attachment
HA trimer binds sialic acid
NA cleaves sialic acid
M1 matrix
M2 ion channel
bound alpha 2,3 (avian) or alpha 2,6 (human) to Galactose
cleaved by Neuraminidase
Unspecific mechanism for uptake of macromolecules
2 examples and mechanism + how big?
example of viruses
Phagocytosis
particle broken down to simpler substances engulfed by plasma membrane
-> Pseudopodia (1-2µm)
Pinocytosis
ingested substances can be absorbed
-> Invagination (0,1-0,2µm)
example of viruses
Adenovirus
Specific uptake of macromolecules
2 examples and subtypes in general
Receptor mediated endocytosis
cell absorbs metaboilites, hormones, proteins, viruses by receptor specific inward budding of plasma membrane
Clathrin mediated
Caveolin mediated
others
Fusion with plasma membrane
enveloped viruses only
Fusion with plasma membrane
which viruses
Defintion
Mechanism (when active, which formation, mechanism)
only enveloped viruses
Defintion
Merging of 2 initially seperate lipid bilayers with result of mixing of 2 distict aqueous compartments
Mechanism
Viral fusion proteins need to hide and display the fusion peptide at the right moment to avoid fusion in wrong compartments
Active in trimieric conformation, some natively trimeric, some dimers on virion that are converted to trimers upon activation
- fusion peptide
- conformational change due to low pH induces insertion of fusion peptide into host cell membrane
- multiple event occur
- another conformational change leads to host cell membrane notches
- followed by hairpining and
- membrane fusion
Examples of different viruses and sequence
Dengue Virus, Mosquito borne viruses, Tick borne, Powassan
-> quite conserved sequence
Fusion protein
classes and example
classes, formation, facts and example
class 1:
alpha-helices
trimer
Influenza
class 2:
dimer
parallel to membrane, ß-sheet
Flaviviridae
class 3:
trimer
ß-sheet
Herpesviridae
Flaviviridae
examples
4 facts
example of fusion protein
examples
many mosquitp borne viruses like West-Nil or Dengue, Yellow Fewer Virus, tick born encephalitis
+ssRNA 10-11k, size 50nm, symetry, electron microscopy
fusion protein
class 2, dimer, tick born encephalitis E protein
Trafficking
Why necessary?
most common type and mechanism
Which viruses mostly use it?
In the cell, simple diffusion is not sufficient because the cytoplasmic enviroment is highly packed
strcutures above 20nm like viruses require an energy dependent motility to travel, therefore most viruses utilize molecular motors like microtubules (most) or actin skeleton (rare)
Microtubuli
Dynein motors on microtubule move towards minus end (nucleus)
Kinesin motors on microtubules move towards positive end (periphery)
Receptor for motor molecule required for e.g. cargo proteins
Which viruses?
Mostly used by viruses that target their genomes to nucleus
Also neutrotropic viruses to enter nearby nerve endings and their viral genome must be moved to cell bodies by aonomal transport (retrograde = towards center)
Nuclear transport
mechanism
via nuclear pore complexes (4000 present per cell, highly efficient)
Protein with NLS is recognized and transported to complex by importin
Then protein + alpha subunit of Importin translocated through nuclear pore complex in energy requiring step
Viral capsid
function
metastability
Mechanism
protects genome
delivers/releases the genome (metastable)
optional
binds to host cell receptors
involved in mebrane fusion
trafficking inside cell
Metastability
Have not attained minimum free energy conformation, this conformatio is only gained when unfavorable energy barrier is reached
Followed by irreversible conformational changes during attachment and entry
Endosomal escape and uncoating
3 ways, mechanism and example
Fusion with endosomal membrane
enveloped without capsid like Influenza
Viral fusion proteins undergo conformational change, often due to low pH of late endosome, then fusion of endosome mebrane and virus for release
Binding to sialic acid, cleavage of host enzyme and conformational change leads to membrane fusion
Permeaboilization of endosome
Polio via capsid portal
some non enveloped viruses induce local permeabilization through interaction between host membrane and membrane penetration protein right after fusion with cell membrane (capsid)
Lysis of endosome
without envelope like Adeno-, Rhino or Rotavirus
rupture of endosomal membrane after confornational change of capsid protein tiggered by low pH or receptor binding
results in genome release into cytoplasm (Rhinovirus) or particle release from endosome (Adenovirus 2)
Whats special about Reovirus?
Genome stays in capsid, Infectious subviral particle penetrates lysosome to enter cytoplasm, then mRNA synthesis happens with cores that makes it possible to enter the infectious subviral particle
Formation of individual structure units
different steps and example
example
Polio
Protomer
subunit: single folded polypeptide chain
Structural unit: different subunit as protomer
Capsomer
subunit of capsod, aggregated protomer
structure: penton or hexon
Capsid
protein shell surrounding genome
Nucelocapsid: nucleic acid enclosed by protein coat/capsid
Envelope and other virion components
Envelope
host derived membrane
Enzymes
Polymerases, Integrases (e.g. HIV) and associated proteins
Proteases
Poly(A) Polymerase for -ssRNA
Capping enzymes
Topoisomerase
Activators
mRNA degradation, required for eeficient infection, mRNAs
Cellular components
Histones, tRNAs, myristate, lipid, cyclophillin A, …
Virus assembly
Reactions (6)
Structure vs Function
Virus assembly reactions
1. Formation of individual structural units of protein shell from 1 or several viral proteins
2. Assembly of protein shell by appopriate, interactions among strucutural units
3. Selective packaging of nucleic acid genome and other essential virion components
(4. Acquisition of envelope)
5. Release from host cell
(6. Virion maturation)
Structure created by symetrical arrangement of identical capsomers, protomers provides max contact and non covalent bonding
Function is genome delivery because the strcuture is not permanently and can be taken apart to release or expose genome
Capsid symetry
What did Watson and Crick observe and what was the conclusion?
Observation
particles made of few proteins
regular and repetititve interactions
limited volume of capsid
Conclusion
symetric arrangement
based on EM analyis helical and isosahedral
Symmetry rules
and Euclid rule
Rule 1:
Each subunit same bonding contact with neighbours, this repeated ineraction leads to symetric arrangement
Rule 2:
Non covalent bonds lead to reversible formation
error free assembly and minimization of free energy
Euclid
only 5 regular convex polyhedra
Tetra (Pyramide)
Hexa (Würfel)
Octa (2 Pyramiden)
Icosa (20 Dreiecke)
Dodeca (20 Fünfecke, Fußball)
Helical symmetry
example virus
formation
Examples
enveloped Rabies or VSV
non enveloped TMV
Formation
simpliest genome packing arrangement forming a long rod like helix leading to a protective shell, any genome size is possible
Direct contact between viral nucleic acid and proteins
Capsid proteins engage in identical equivalent intercation with themselves and viral genome
Icosahedral structures
Subunit
Multimerization
How many identical units at least and why?
How is it possible to get more space for bigger genomes?
Subunit
one protein, homo-multimer or hetero-multimer
Multimerization
genetic economy
How many identical units at least and why?
At least 60, because we can divide it into symmetry related units based on their axis
2-fold axis: 30x 2 units
3-fold axis: 20x 3 units
5-fold axis: 12x 5 units = 60 proteins at least
How is it possible to get more space for bigger genomes?
Pseudo 6 fold symmetry, when Triangulation number >1, only certain triangulation numbers like 1,3,4,7,… occur to form a bigger space for genome
even T=1179 is possible
Triangulation number
who discovered it and when?
Rules and formula
Caspar and Klug 1962
Capsid subunit arranged as hexamers and pentamers, number of subunit follwed T value x 60
formula
t = h^2 + kk + k^2
h= number of units in straight line toward next pentagon
k= number of unit shifted to reach next pentagon
What’s special about Reovirus symmetry
Complex capsid with 2 icosahedral protein layers, T=13 and T=2
**Protein shell assembly **
2 major strategies
Assembly line concept and intermediates
2 major strategies
sequential (protein shell formed, then nucleic acid loaded in)
concerted (protein shells formed directed by nucleic acids)
Assembly line concept
ensures orderly formation of viral particles and virion subunits
Assembly intermediates
Formation of intermediate strcutures that can’t proceed unless previous structure is formed (quality control)
Sequential assembly
3 types
Assembly from individual protein molecule
Adenovirus, SV40: different proteins trannslated, then binding
and building the protomer
Assembly from polyprotein precursor
Poliovirus: Polyprotein being cleaved to form structural subunit, Capsid assembly and loading of genome to form Provirion, then cleavage of protein subunit for infectious virion
chaperone assisted assembly
Adenovirus: proteins that assist folding/unfolding, subassembly fibers, pentons, hexons
Concerted assembly
Example of viruses
Example
-ss Influenza (M1) or HIV (Gag)
things happen together, often membrane and nucleic acid guided
via secondary structures in genome leading to packaging signals
Sites of viral assembly
4 different sites
Cellular chaperons
Transport systems
Secretory pathways
Nucelar import and export machinery
Transport of virions components for assembly
Wha transport necessary?
Negri bodies
Adresses
Nucleic acid and proteins synthesized seperately and must be brought together
Therefore transport is important, non enveloped viruses often use internal membranes to concentrate proteins
Negri bodies:
inclusion bodies found in cytoplsam of nerve cells containing rabies replication istes
Adresses
membrane proteins to membrane: N terminal signal peptides
Membrane proteins stay put: retention signals
Nuclear proteins: NLS
viral mRNA or nibonucleoprotein complexes: NES
Binding to cellular motor proteins: capsid proteins
Genome packaging
Packiging singnals
No packiging signals
Diploid genomes
Segmented genomes
other things packed
Packiging singnals
Adenovirus:
Viral genome needs to be distinguished from host because packaging limit is 10% more than genome size
No packiging signals
Poliovirus: RNA synthesis and assembly coupled
Diploid genomes
HIV-2: kissing complex with 2 intermolecular kissing loop interactions initiates dimerization and packaging
Segmented genomes
complex
Influenza: 8 segments: at least one each
selective
RNA protein intercations and RNA,RNA interactions
other things
pack enzymes to initiate translocation and genome replication like RNA viruses (RdRp) and Retroviruses (RT, tRNA primers)
Enveloped viruses
Mechanism of gaining envelope
Viral membrane glycoproteins inserted intonplasma membrane
Then assembly of the virion and in the end budding
Release from cell by lysis
Release from cell by budding/exocytosis
direction
Lateral cell to cell spread
Release from cell by lysis
Inhibition of cellular processes
Apoptosis
Virus specific filament cleavage, structural integrity and protein accumulation
Release from cell by budding/exocytosis
Reverse of endocytosis, used by viruses that assemble within vesicular compartmnents of ER/Golgi like Herpesvirus
apical (transmission, sneezing e.g.)
Basal (blood, nerves, etc., mostly bad news)
at sites of cell contact
influences pathogenesis
Lateral cell to cell spread
Syncytia: multinucleated cells (RSV)
Maturation
defintion
examples and mechanism
proteolytic processing by virus-encoded enzymes to from mature infectious virions
HIV mechanism
HIV-1: gag cleaved 5 sites triggering major changes in the virion leading to its infectivity
Influenza mechanism
HA protein processed by host protease for virus to bind the sialic acid residue and infect next cell
Host protease restricted to RT, therefore limited to RT
Avian influenza processed by ubiquitinous protease, there leading to systemic infections
