Viral strategies:
How do viruses enter their host cell?
Hans-Georg Kräusslich
Department of Virology
University Heidelberg
www.virology-heidelberg.de
History of virology
Pharao Ramses V
Ruma; 1.500 B.C.
Polio
Smallpox
History of virology, history of vaccination:
¾ 11th century: vaccination trials in China
¾ 1721 Lady Montague: Variolation in UK
¾ 1796 Edward Jenner: First Vaccination (cowpox; vacca=cow)
¾ 1885 Louis Pasteur: Attenuation of rabies virus
History of virology
Discovery of viruses:
¾ 1882 Adolf Mayer: Transmission of tobacco-mosaic-disease by plant
extract; Infectious agent could not be isolated
¾1892 Dimitri Ivanofsky: causative agent of tobacco mosaic disease is not
retained by filters and can not be cultivated in vitro
¾ 1898 Martinus Beijerinck: causative agent of tobacco mosaic disease can
be cultivated in live tissue
‚Contagium vivum fluidum‘; later: Virus (lat. poison)
Principles of Virology, 2004
What is a virus?
• DNA or RNA genome encased in a protective shell
• Dependent on host cell functions and metabolism
• Obligatory intracellular parasite with extracellular phase
envelope
(Lipid+Proteins)
Capsid
(protein shell)
Replication proteins,
Accessory proteins
Genome
(DNA or RNA)
Capsid and envelope:
• Protection of the genome in the extracellular environment
• Recognition of receptor molecules on cell surface, attachment and entry
Virus classification
RNA viruses
Plus strand
(= mRNA)
Minus strand
DNA viruses
Single strand
Double strand
Double strand
Genome segmented or non
segmented
Particle enveloped
or non enveloped
Classification of viral replication strategies
according to Baltimore
Hepatitis B
Virus
DNA
RNA
The viral replication cycle
attachment
entry
uncoating
genome
replication
gene
expression
assembly
release
How does a virus enter its host cell?
Viruses must access the cytoplasm
without destroying the host cell
Different entry mechanisms
depending on
• type of host:
animal, plant, fungi, bacteria
• type of virus:
enveloped, non enveloped
Flint et al., Principles of Virology
General mechanisms of virus entry
Adapted to the properties of the host cell
Animal cells
Lipid bilayer
Cortical actin
Bacterial cells
Bacterial cell wall
Plant cells
Plant cell wall
Bacteriophages
Plant viruses
Receptor
mediated
entry
Vector mediated (insects)
Mechanic damage to cell wall
Nucleic acid
+ protein shell
transferred
Nucleic acid
transferred
Nucleic acid
transferred
Enveloped
or non-enveloped
non-enveloped
non-enveloped
Animal viruses
Receptor
mediated
entry
Virus entry is mediated by many factors
Viral envelope or coat protein
Non-specific
attachment
factors
receptor (+co-receptors)
lipids
cytoskeleton
Uncoating factors
Restriction factors
Attachment:
• Independent of host cell metabolism
• Specific receptor determines host and
tissue tropism
Penetration:
• Delivery of virus genome to the cytoplasm
• Requires energy and host cell factors
Architecture of the plasma membrane
many possible attachment sites for viral proteins
Lateral movement of lipids
Lipid microdomains (rafts)
Flint et al., Principles of Virology
Non-proteinaceous virus receptors
Virus receptors do not have to be proteins
Sialic acid is the receptor for influenza virus
• First virus receptor identified
• Neuraminidase treatment of cells abolishes virus binding and infection
• Influenza virus uses its own neuraminidase to release virus progeny
from producer cells
Flint et al., Principles of Virology
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Examples for cellular molecules
(ab)used as virus receptors
Membrane penetration can occur at different sites
Fusion of viral envelope
and plasma membrane
Retroviruses: HIV, MLV
Measles Virus
Herpes Virus
Uptake, followed by
penetration of intracellular
compartment
All non-enveloped viruses
Influenza Virus
Vesicular Stomatitis Virus
Flaviviruses (HCV, TBE)
Cellular uptake of macromolecules
Flint et al., Principles of Virology
A variety of endocytic pathways
Marsh and Helenius, 2006
Steps in clathrin-mediated endocytosis
Example: internalization of LDL
EM eines “coated-pit”
Modell eines “coated-vesicle”
EM-Aufnahme gereinigter Triskelions
• Receptor binding
• Formation of coated pit
• Formation of clathrin coated vesicle
• Internalization
• Dissociation of clathrin coat
• Formation of early endosome
• Acidification
• pH iduced release of LDL
• Recycling of receptor
• Fusion with lysosome
• Degradation of lysosomal contents
Clatrin-coated-pits
LDL-Endocytose
Assembly-Intermediat
Molekularer Aufbau eines Triskelions
Semliki Forest virus enters via clathrin-coated pits
Helenius, Kartenbeck and Simons, 1980
Semliki Forest Virus
(S. Fuller, Oxford)
Ari Helenius
Kai Simons
Viruses enter cells using different pathways
plasma
membrane
fusion
Surfing
Clathrin mediated
endocytosis
Clathrin independent
endocytosis
Brandenburg and Zhuang,
2007
Some viruses use more than one pathway!
Viruses also use clathrin-independent endocytosis:
SV40 entry (Pelkmans et al., Nat. Cell Biol. 2001)
SV40 enters through
caveosomes and accumulates
in a smooth ER compartment
Lucas Pelkmans
In caveolin negative cells, SV40 uses a different clathrin independent entry pathway
(Damm et al., 2005)
Cytoplasmic entry of a non-enveloped virus:
Poliovirus
Receptor binding induces conformational
changes in the capsid:
• loss of VP4
• hydrophobic N-termini of VP1 insert into the
cellular membrane
• RNA extrudes through the capsid opening
Flint et al., Principles of Virology
Virus-cell membrane fusion comprises multiple steps
•Bridging of both membranes by viral fusion
protein
•Conformational change > membranes
approach each other
•Fusion of both outer leaflets (Hemifusion)
•Early fusion pore
•Late fusion pore
•Complete fusion of both membranes
•Cytoplasmic entry of capsid
Membrane fusion mediated by viral fusion proteins
Class II
Class I
•Orthomyxoviruses (Influenza virus)
•Retroviruses (HIV, ASLV, MLV)
•Paramyxoviruses (Simian Virus 5)
•Filoviruses (Ebola virus)
•Coronaviruses (SARS-CoV)
•Flaviviruses (TBE virus, Dengue virus)
•Alphaviruses (SV40)
Intermediate: Vesicular stomatitis virus
Jardetzky and Lamb, Nature 2004
Pre- and post-fusion conformations of
viral fusion proteins
Conformational changes trigerred by: pH drop, receptor binding
Fusion peptide
Fusion peptide
Transmembrane
domain
Fusion peptide
Kielian und Rey, 2006
pH induced conformational change
during influenza virus fusion
Flint et al., Principles of Virology
Model for class II membrane fusion
Low pH:
• Conformational changes in
the envelope protein
• Exposure of the fusion
peptide
Kielian und Rey, 2006
Intracellular transport
• Following uncoating, the viral genome
is transported to the site of replication
• RNA genome replication can occur
exclusively in the cytoplasm, but
genomes of DNA viruses and some
RNA viruses have to enter the nucleus
for replication
• Mechanism for genome transport to
and passage through nuclear envelope
required
Marsh and Helenius, 2006
Nuclear import of viruses or viral replication complexes
• Uncoating at the plasma membrane or in the cytoplasm
> transport of viral nucleoprotein complex into the nucleus (Influenza virus, HIV)
• Docking of capsid to the nuclear pore
> (partial) diassembly
> release of viral genome through nuclear pore (Adenovirus, Herpes viruses)
• Entry of capsid through nuclear pore
> uncoating (HBV)
Influenza virus:
• release of 8 viral RNPs into the cytoplasm
by membrane fusion
• nuclear localization signal in viral NP
mediates import through importin β
Herpes simplex virus:
• capsid binds to the nuclear pore
• conformational changes in the capsid allow
viral genome to enter the nucleus
Smith and Helenius, 2004
Adenoviruses:
• capsid binds to the nuclear pore
• histone H1 mediates uncoating and
transport of viral genome
Experimental approaches
• Identification of attachment factors
and receptors
• Identification of entry pathway(s)
• Mechanism of cytoplasmic entry
• Intracellular transport pathway(s)
• Cell to cell transmission
• Inhibition of virus entry
Marsh and Helenius, 2006
How can a virus receptor be identified?
Identification:
• Monoclonal antibodies against cell surface factors
• Competition with defined soluble cellular components
• Biochemical approaches: Affinity chromatography
• Genetic approach: transfection of permissive, non-infectable
cells with cDNA from an infectable cell line
Experimental proof:
• Transfection of the cloned receptor gene into a non-infectable
cell line allows viral entry
• siRNA against putative receptor gene abolishes viral entry in
permissive cells
CD81 is necessary but not sufficient to mediate
Hepatitis C Virus entry. Which other factors are required?
Evans et al., Nature 2007
Screen for cDNAs from Huh-7 cells that mediate HCV infectability of 293T cells
Claudin-1
Claudins: transmembrane proteins which
form the backbone of tight junctions
(24 known Claudin family members)
Confirm the function of claudin in HCV entry
Evans et al., Nature 2007
A. Expression of Claudin-1 confers HCV susceptibility to 293T cells
293T
293T+CLDN1
Huh7 cells
B. Knock-down of Claudin by siRNA inhibits HCV entry into Hep3B cells
VSV-Gpp
HCVpp
Extracellular movement:
Viruses surf along filopodia towards the cell body
Lehmann et al., J Cell Biol 2005
Walther Mothes
Murine leukemia virus (retrovirus)
attaches to filopodia of 293 cells
Surfing requires ATP andLehmann
the viral
receptor
et al.,
2005
How can you differentiate between entry
pathways?
• pH dependence?
• Co-localization or co-trafficking of virus with components of
cellular pathways and compartments:
Clathrin, caveolin, Rab proteins, endosomal markers, ER
markers, etc.
• (Inducible) expression of dominant negative variants of factors
involved in endocytosis
• siRNA mediated knock-down of factors involved in endocytosis
(e.g. dynamin, caveolin)
Differentiate between entry pathways:
pH dependent entry of ALV
Mothes et al., Cell 2000
Inhibitors of endosomal
acidification inhibit ALV
entry
NH4Cl blocks an early
stage in ALV infection
Bafilomycin sensitivity is mediated
by the ALV Env protein
Viruses use the cytoskeleton for intracellular trafficking
Experimental approaches:
• Effect of drugs interfering with the actin
cytoskeleton (latrunculin B, cytochalasin D,
jasplakinolide) or with microtubules (nocodazole,
colchizine, taxol)
• Analyze dynamic aspects of virus-cell interaction
using fluorescent viruses
Lynn Enquist
Radtke et al., 2006
Beate Sodeik
Adenovirus entry
Urs Greber
Dissection of HIV entry pathways using
double labelled particles
Outer shell: Matrix-mRFP
Inner core: eGFP-Vpr
Fusion
Endocytosis
Double-labelling strategy
designed to distinguish
fusion at the plasma
membrane from endocytic
uptake
Daecke et al., 2005; Müller et al., 2004; Lampe et al., 2007
Software aided analysis of fusion events
Transport of single
S
particles
within the cell
is observed in real time
(50 ms/frame)
Software follows the
track of each signal
over time
VSV-G pseudotypes, MA/Vpr
Software aided analysis of fusion events
12000
Fluorescence [AU]
10000
MA
8000
Vpr
6000
4000
2000
0
0
200
190
5
205
10
210
Pixel
nm / pixel]
15 [160 20
25
215
red fluorescence (background substracted)
P ix e l [ 1 6 0 n m / p ix e l]
195
time
220 225
Time [sec]
30
230
35
235
0.6 µm/sec
0.1 µm/sec
205
210
1.3 µm/sec
220
225
230
240
green fluorescence (background substracted)
200
215
40
0.02 µm/sec
235
VSV-G pseudotypes, MA/Vpr
Velocity consistent with microtubuli driven transport
Direct visualization of entry pathway
Assembly of endocytic machinery around individual influenza viruses during viral entry.
Rust et al., Nat Struct Mol Biol. 2004
Xiaowei Zhuang
• Virions can enter through both clathrin mediated and clathrin independent
pathways in parallel
• Virus-induced formation of clathrin coated pits
Brandenburg and Zhuang 2007
Inhibition of virus entry
HIV fusion at the plasma membrane
Doms, 2004
Inhibition of virus entry:
HIV co-receptor antagonists
HIV
Receptor
cell
X
Chemokine rezeptors
(CXCR4 or CCR5) function as
HIV co-receptors
Ko-Rezeptor
Co-Receptor antagonist
Maraviroc (Celsentri)
blocks virus binding to
CCR5
Inhibitors of virus entry:
HIV fusion inhibitor Enfuvirtide (T20, FuzeonTM)
HIV
Ko-Receptor
CD4
Zelle
Enfuvirtide
Inhibition of picornavirus entry by neutralizing antibodies
or compounds blocking conformational changes
Cell to cell transmission of viruses:
The virological synapse
Model for cell to cell spread of HIV
Jolly and Sattentau, 2004
Transmission from cell to cell
Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission.
Sherer et al., Nat Cell Biol. 2007
• MLV producing cells form stable filopodes
• Cytonemes are formed between infected cells and target cells
• Virus particles traffick along the cytonemes with an average speed of 1 µm /min