Attachment and Entry of Viruses

Contact:
Dr. Eike Steinmann
Centre for Experimental and Clinical Infection Research
Department of Experimental Virology, Twincore
Feodor-Lynen-Str. 7, 30625 Hannover
Tel.: 0511220027133, eike.steinmann@twincore.de
www.twincore.de
 Lecture Overview

• Introduction virus properties and viral life cycle

• The architecture of cell surfaces

• Interaction of viruses with cellular receptors

• Mechanisms of viral entry

• Examples of virus entry strategies

Virus diversity
 Introduction viruses
1. Viruses produce (almost) identical offspring
They contain the information for reproduction - not the machinery to do so.

2. All viruses are smaller than a cell

DNA-Helix 3,4 nm, smallest spherical viruses 25 nm, length TMV 300 nm, length of E.coli 2000 nm

3. Viruses do not have an own metabolism (neither catabolic nor anabolic)

No energy and cannot perform de novo biosynthesis using metabolised nutrients => depend on host

4. Viruses do not replicate by growing or division

Assembly of components
 Introduction viruses II

5. Viruses have only one sort of nucleid acid

either RNA or DNA

6. Viruses have an extra-cellular phase (difference to plasmides)

Creation of progeny viruses depends on genetic information
instability of nucleic acids (pH, temperature, nucleases) = formation of capsids

7. Viruses and host co-evolve

Balance/„Co-evolution"/Predator-Prey relationship

8. Classification:
Pathogenic effect
Host specificity, morphology, genom-structure
Replication mechanism/m-RNA synthesis strategy
…
The viral life cycle
 The viral life cycle

1. Attachment or adsorption
Viral receptor: specific recognition event at plasma membrane

2. Penetration
Virion or subviral particle enter cytosol, some via endosomes „first uncoating“

3. „Second uncoating“
At the site where transcription and replication take place;
Release of the viral genome into nucleoplasm or cytosol

4. Synthesis of viral „early proteins“

Begins actual replication of virus; viral proteins and
enzymes synthesized at this time usually aid in mobilizing
host resources for viral synthesis
 The viral life cycle

5. Genome replication
Synthesis of viral nucleic acid

6. Synthesis of viral „late“ structural proteins

May occur concomittingly with genome replication; usually lags behind, viral genomes and
structural proteins accumulate in infected cells

7. Assembly
Genomes and structural proteins are added together; often via intermediates e.g. capsid
assembly, incorporation of genome, aquisition of viral envelope

8. Maturation
Proteolytic cleavages, phosphorylation, structural
reorganization

9. Egress of progeny virus

Either by cell death (CPE) or via specific secretion or
export mechanims
 Overview of viral entry with Hepatitis C Virus (HCV) as example

Movie HCV entry: http://spiral.univ-lyon1.fr/files m/M5502/WEB/index.html

 Attachment and entry

- Binding to a specific receptor (cell surface molecule) and sometimes also co-receptor

- Knowledge increased in 1980s with cellular, molecular and structural techniques (mAbs, recomb. DNA)

- Understanding viral-receptor interaction at molecular level and process of viral uncoating

- Entry as antiviral target

- Entry no passive process; viral usurpation of normal processes like endocytosis, membrane fusion,
vesicular trafficking, transport to the nucleus, genome uncoating
 Viral attachment and entry definitions

- Virus receptor: cellular structure which mediates a productive infection in

permissive cells

- Permissiveness: permissive cells support replication of viral genomes

(intracellular components)

- Susceptibility: capability of a cell to bind and mediate entry of a virus via

receptors
The architecture of cell surfaces

virus entry virus exit

Influenza
Influenza
SV40 apical SV40

tight
junction

Influenza HIV
VSV basolateral
VSV
Extracellular matrix
Plasma membrane - possible binding sites for viral surface proteins

-phospolipid/glycolipid bilayer
-specific microdomains e.g. lipid rafts
Proteins of the plasma membrane
Experimental strategies for the identification and isolation of
cellular genes encoding viral receptors
 PVR
CD4
ICAM-1

VLA-2
LDLR

Am
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Examples of virus receptors

Ca
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 Sialic acids as virus receptors

- Viral receptors are not necessarily protein structures

- Example: Sialic acid = N-Acetylneuraminic acid for influenza A and B virus

- First identified virus receptor (until 1985 the only one)

- Treatment of susceptible cells with neuraminidase lead to abrogation of

both, virus binding and infection
Uptake of macromolecules from extracellular fluid
Receptor-mediated endocytosis
 Virus entry and uncoating

Paramyxoviridae

HSV1

HCV

Adenovirus
 Cell membrane fusion
- fusion with plasmamembrane

- fusion with endosomal membrane

viral membrane hemifusion small pore large pore

cell membrane
Transport of the viral genome into the nucleus
Transport of the viral genome into the nucleus II
 Entry pathway used by viruses

(A) Macropinocytosis: e.g. adenoviruses

(B) Clathrin-independent pathway: influenza virus and arenaviruses
(C) Clathrin-mediated pathway: commonly used. Transported via early and late endosomes
proceeds evtl. to lysosomes.
(D) Caveolar pathway: cholesterol-dependent: SV40, coxsackie B, mouse polyoma, and Echo
(E) Cholesterol-dependent endocytic pathway devoid of clathrin and caveolin-1: polyomavirus
and SV40
(F) A pathway similar to (D) except dependent on dynamin-2. It is used by Echo virus 1.
(from Marsh and Helenius, Cell, 2006)
 Semliki Forest Virus entry

- Entry by clathrin dependent receptor-mediated

endocytosis

- Fusion catalyzed by acidification of endosomes

- Fusion results in exposure of the viral nucleo-

capsid to the cytoplasma

- Cellular ribosomes then bind capsid,

disassembling it and distributing the capsid protein

- Viral RNA accessible to ribosomes, which initiates

translation
 Poliovirus entry

- Virion binds to cellular receptor (Pvr)

- Receptor-mediated conformational transition

results in formation of altered particles

- Not known whether the viral RNA leaves capsid at

the plasma membrane or from endosomes

- Interaction with Pvr leads to pore formation of N

termini of VP1

- RNA is released from the capsid into the cytosol

 Reovirus entry

- After attachment and binding to cellular receptor,

entry by receptor-mediated endocytosis

- Proteolysis in the late endosomes produces

infectious subviral particle (ISVP)

- ISVPs cross lysosome membrane and enter

cytoplasma as core particle
Influenza entry
 Influenza entry II
- Model of pH-induced conformational changes during the membrane fusion process

- Globular heads of HA mediate binding to sialic acid-containing cell receptors

- Virus-receptor complex is endocytosed and import of H+ ions into the endosome acidifies the interior

- HA conf. change that produces fusogenic protein

- HA loop region of native HA becomes coiled coil, moving fuison peptide to near cell membrane

- Release of vRNP by H+ flow into virion interior conducted by M2 protein

 Conformational changes of Influenza HA protein

HA1

HA0
S
S

HA2

fusionspeptid

- Structure of uncleaved HA0 precursor (left)

- Structure of low pH trimer, fusion peptide (α-helix) is orientated towards the cell membrane
 Adenovirus entry

- Adenoviruses bind cellular receptor

via fiber protein

- Interaction with integrin receptor leads

to internalization by endocytosis

- Viral capsid proteins are sequentially

removed in the endosome

- Capsid is transported in the cytoplasm

along microtubules

- Binding to nuclear pore complex

- Import of DNA
 HIV entry

- Interaction of HIV with CD4 and co-

receptor (CXCR4, CCR5)

- Conf. change when gp120 binds

CD4, then interaction with chemokine
receptor

- N-terminal fusion peptide of gp41

penetrates cell membrane

- Loop structure brings virus and cell

membrane close together allowing
fusion
 Summary

- Attachment of viruses to their target cells often determines host specificity and tissue tropism of
viruses

- Independent from metabolic processes in the host cell

- Binding of the ligand (virus) to its receptor is often accompanied by conformational changes
(mostly in the ligand)

- Binding is followed by virus penetration (= access to the cytosol)

- This step requires energy and can be slowed down by e.g. cooling

- Access of the nucleocapsid to the cytoplasm is mostly accomplished by receptor-mediated

endocytosis followed by fusion of the viral with the cellular membrane

- Endocytosis requires (beside the viral RBP) only cellular molecules; fusion relies -as a rule- on
the action of viral fusion proteins