1. Virus Attachment, Entry and Uncoating

2. Virus Properties
Virus is defined as a nucleoprotein complex which infects cells and uses their metabolic processes to replicate
Smallest known infective agents
• Metabolically inert - no metabolic activity outside host cell; must enter host cell to replicate
• Most are highly species specific

3. Virus versus Virion
Virus is a broad general term for any aspect of the infectious agent and includes:
• the infectious or inactivated virus particle
• viral nucleic acid and protein in the infected cell
Virion is the physical particle in the extra- cellular phase which is able to spread to new host cells; complete intact virus particle

4. General points - virus entry
The first event in any virus life-cycle - often limits infection to the “correct” cell
Can be primary determinant of tropism
Tissue tropism - e.g. measles (skin cells) vs. mumps (salivary gland)
Species tropism - e.g. togavirus (both insect/mammalian cells), poliovirus (primate cells), T4 phage - (few strains of E.coli)
Binding - initially electrostatic, based on charge ± pH, specific ions - followed by local hydrophobic interactions
Initial binding is often low affinity, but high avidity (tight binding) due to multiple binding sites
The virus binds to a receptor on the cell surface - can be ubiquitous/specific, with variable density
Initial binding is followed by penetration and subsequent uncoating

5. General points II - virus entry
Whether or not the virus is enveloped makes a big difference - at least for penetration
All viruses must cross a lipid bilayer, plant and bacterial viruses must also cross a cell wall
Uncoating means that the stable virus stucture must become unstable
-transition from extracellular (chemical) form to intracellular (biological) form
There must be some sort of “trigger” or regulated disassembly process

6. Overview Name Process 1 Attachment Virus attach to cell membrane 2
Entry
By endocytosis or fusion 3
Uncoating
By viral or host enzymes, separates NA from protein coat 4
Biosynthesis
Production of NA and proteins 5
Maturation
NA and capsid proteins assemble 6 7 8
Release
Shedding
Infecting new host
By budding (enveloped viruses) or rupture

7. Viral Replication i) Adsorption (attachment): random collision
interaction between specific proteins on viral surface and specific receptors on target cell membrane (tropism)
not all cells carrying a receptor for a particular virus can be productively infected by that virus

8. Viral Replication i) Adsorption (attachment):
some viruses may use more than one host cell receptor (e.g. HIV)
able to infect a limited spectrum of cell types (host range)
most neutralizing antibodies are specific for virion attachment proteins

9. Viral Replication ii) Entry (penetration):
2 mechanisms - endocytosis fusion of virus envelope with cell membrane
iii) Uncoating:
release of viral genome
cell enzymes (lysosomes) strip off the virus protein coat
virion can no longer be detected; known as the “eclipse period”

10. Attachment: Cell Receptor
Virus may bind up to three different cell receptors in succession:
Low affinity receptor - in high abundance, virus contacts cell surface
Primary receptor - in lower concentration
Co-receptor – follows binding of primary receptor

11. Attachment: Specificity
Host Range - the organism(s) that the virus is able to infect (narrow or wide) i.e. plant, animal, human
Tissue Tropism- the cell type(s) a virus is able to infect i.e. skin, oral, GI, CNS

12. Attachment: Binding 3-D fit between viral ligand and cell receptor
Mainly weak electrostatic charges.
Evidence for this is interaction may require:
specific pH
specific ionic strength
presence of specific ions i.e. Ca++, Mg++

13. Attachment: Nonenveloped Picornavirus
Virus ligand - a deep cleft (“canyon”) in triangular face of capsid (viral proteins VP1, VP2, VP3)
Binds to cell receptor ICAM –1 (intracellular adhesion molecule 1), normal function is to bind cells i.e. WBC

16. T-even (T2) phage structure
From The Biology of Viruses, Voyles, McGraw Hill,

17. Entry of T-even bacteriophage - binding
Best understood = T4 phage (the virus in the Hershey-Chase experiment)
Initial binding is reversible and electrostatic - the outer-most part of the long tail fiber binds to surface lipopolysaccharides (LPS) of the bacterium (binding can occur in vitro, and is competed by specific sugars) - a non-specific receptor
Binding is “additive” until all six tail fibers are bound
Binding of 3 fibers is needed to initiate infection
The virus may “browse” the surface - looking for a suitable site for penetration (possibly sites of cell wall synthesis where the outer and plasma membranes are close together).
Note - this is a multi-valent interaction
From Introduction to Modern Virology, Dimmock & Primrose, Blackwell

18. Entry of T-even bacteriophage - binding II
The receptor binding sites of the short tail fibers are now exposed and bind (also to LPS)- now binding is essentially irreversible
Conformational change in the baseplate - hexagon to extended star-shaped conformation -
Initiates sheath contraction ( to 37% of its original length)

19. Entry of T-even bacteriophage - penetration
Often referred to as a “hypodermic syringe”
• Sheath of the helical tail slips and forms a shorter helix.
• The tube itself stays the same with the end result that the tube is pushed down and contacts the membrane - note the tail does not directly punch through
• Lysozyme molecules are releases which forms a pore through which DNA enters
From The Biology of Viruses, Voyles, McGraw Hill,
DNA is under considerable pressure and seems to exit automatically once the base palte is opened up
Other phage, e.g. T3, have a motor protein to reel out the DNA

20. Entry of other ‘phage - I
Phage λ (DNA) - a virus with a longer, but simpler tail than T4
From The Bacteriophages vol 1, ed Calender, R., Plenum Press
the single tail fiber (J protein) binds to lamB (the maltose transporter) - an example of a specific receptor
lamB is inducible. This means the virus only infects in the presence of high nutrients. Also needs Mg2+ - binding is electrostatic - an example of tropism
Penetration requires the bacterial pts protein (also part of the maltose transporter) - the co-receptor
attachment and penetration require different viral proteins

21. Entry of other ‘phage - II
PRD1 - an icosahedral phage with an internal membrane
For gram -ve bacteria (with two layers of lipid separated by peptidoglycan) phage entry is a challenge
1) Binding
2) Conformational change -> dissociation and opening of 14 nm hole in the capsid
3) Second conformational change converts internal envelope to tubule, which delivers the DNA
Phage encodes 2 proteins (P5 and P17) that have peptidoglycan-hydrolyzing activity
- equivalent protein in T-even phage = gp5 (lysozyme activity)
From Rydman and Bamford (2002) ASM News

22. Entry of other ‘phage - III
Enveloped RNA phage - φ6 (Phi 6)
These phage bind to pili, the pilus then retracts down to the outer membrane, the virus undergo fusion, enzymatically destroys the peptidoglycan cell wall (p5 protein) and then penetrates the plasma membrane
From The Bacteriophages vol 2, ed Calender, R., Plenum Press
The Hershey-Chase experiment is now no longer valid, as (most of) the protein (35S) has entered the cell along with the nucleic acid (32P).

23. Plant viruses Plants have a thick, rigid cell wall
Generally plant viruses do not have specific entry mechanisms, but rely on
A) introduction into the cell by a vector (insect) - most common
B) mechanical injury
C) direct cell-cell transmission (via plasmadesmata and viral movement protein)
This is fine if you are a non-enveloped virus, but enveloped plant viruses do exist (bunya-, rhabdo- families
These viruses must fuse their envelope

24. Binding of animal viruses
Occurs via receptors on the cell surface (plasma membrane)
Protein (glycoprotein)
Carbohydrate
Lipid (glycolipid)
From Principles of Virology, Flint et al. ASM Press
Receptor utilization plays a major role in virus tropism / pathogenesis

25. Entry / Uncoating
Entry is the mechanism used by the virus to penetrate into the host cell
Uncoating is the separation of the nucleic acid from the capsid, and refers to changes that occur to make the viral nucleic acid ready for expression

26. Principles of virus penetration
Viruses can penetrate directly at the plasma membrane, or via endosomes

27. Penetration of Enveloped Animal Viruses
Envelope = fusion
Semliki Forest virus (SFV) a togavirus - the classic virus for entry studies
Early experiments (early 1980s) by electron microscopy showed entry into vesicles - now known to be clathrin-coated (clathrin-coated vesicles, or CCVs)
The virus then enters the endosome (“early” endosome)
Figure courtesy of A. Helenius
The very high particle:pfu ratio (approaching 1:1) of SFV ensures that all the virus particles are part of the “real” entry pathway

28. Endosomes and virus entry
Endosomes are used by cells for nutrient and growth factor uptake
The virus “hijacks” the cellular pathway
One key feature of endosomes is their progressive acidification - due the the action of the vacuolar H+/vATPase
Endosomes do much more than provide low pH
Deliver through cortical actin and microtubule-mediated transport in the cytosol
Specific redox/ionic environment
Defined lipids for fusion/penetration
From Cell Biology, Pollard and Earnshaw, Saunders

29. • In most cases a pH of around 6.2-6.5 is sufficient for fusion
• The lowered pH causes conformational changes in the spike glycoprotein, and the exposure of a fusion peptide
• This is the “trigger” needed for virus entry
• In most cases a pH of around is sufficient for fusion
- fusion occurs in the early endosome
• Entry and infectivity (in cell culture) can be blocked by :
– 1) addition of a weak base (e.g. NH4Cl) that neutralize the endosome
– 2) drugs that target the vH+/ATPase (e.g bafilomycin A)
– 3) drugs that break down the proton gradient (e.g. monensin)
– 4) exposure of the virus to a low external pH
Fusion can be induced at the cell surface by exposure to low pH

30. Influenza virus binding - I
Binds to cell surface carbohydrate - sialic acid
Ubiquitous/non-specific receptor
In principle, this can be present as part of glycoprotein or glycolipid
Specific requirement for α2-3 and α2-6 linkages - gives different tropism for avian vs. human cells (pigs have both)
From Principles of Virology, Flint et al. ASM Press
The first virus receptor to be identified
- principally due to the fact that there is a receptor-destroying enzyme associated with the virus

31. Influenza virus binding - II
The major influenza glycoprotein, hemagglutinin (HA) has a specific sialic acid-binding site on its “top domain” -
From Principles of Virology, Flint et al. ASM Press
HA mediates both binding and penetration

32. Penetration of influenza virus
Influenza virus requires a lower pH ( ) and enters the “late” endosome, but fusion occurs before entry into the lysosome (this avoids degradation)
The acid-triggered fusion event is well understood - a conformational tail forms a rigid “six-helix bundle” or “coiled-coil” of α-helices, which flips the fusion peptide out and allows insertion into the membrane
Note the fusion peptide is “external”
The “trigger” is irreversible - this means that exposure of virions to low extracellular pH will destroy infectivity
From Principles of Virology, Flint et al, ASM Press

33. From Principles of Virology, Flint et al, ASM Press
• The low pH has a second very important role for influenza entry - the virus contains an ion channel in its envelope (M2).
• The presence of M2 allows acidification of the virus interior, and promotes uncoating of the M1/vRNPs
• Drugs that block M2 block infection - amantadine. This is highly specific for the viral M2 ion channel, with no effect on the cellular H+/vATPase

34. Fusion of an enveloped virus
Model for viral membrane fusion mediated by class I fusion roteins. Influenza virus, which is internalized into an endosome, is shown as an example. In the native state of the fusion protein — which is a trimer — most of the surface subunit (green) is exposed. Part of the transmembrane subunit, including the fusion peptide, is not exposed. Following fusion-activating conditions, conformational changes occur to 'free' the fusion peptide (red) from its previously unexposed location. In the case of influenza HA, this occurs by a 'spring-loaded' mechanism. The 'pre-hairpin' intermediate spans two membranes — with the transmembrane domain positioned in the viral membrane and the fusion peptide inserted into the host-cell membrane. The pre-hairpin intermediate forms a trimer of hairpins, and membrane fusion occurs, which leads to pore formation and release of the viral genome into the cytoplasm.
From Dimitrov (2004) Nature Reviews Microbiology 2:

35. Retrovirus (HIV)
A classic example of a receptor/co-receptor requirement
A specific receptor
Binds initially to CD4 - present on immune system cells (T-cells) - gives the virus tropism for the immune system
This is not enough - the virus also binds to a chemokine co-receptor (eg CCR5, CXCR4) present on a sub-set of cells (macrophages / T-cells)
Gives even more precise tropism
The virus binds to both receptors via the gp120 glycoprotein

36. Penetration of retrovirus (HIV) - I
HIV enters by a quite different route
Entry is not low pH-dependent (no inhibition by NH4Cl etc), and fusion occurs directly with the plasma membrane
From Principles of Virology, Flint et al, ASM Press

37. Attachment: Enveloped HIV Virus
Host cell protein in virus envelope (cyclophilin A) initially binds HIV to low affinity receptor (heparin sulfate) of the cell
Followed by binding of viral ligand (gp120) to primary receptor (CD4) on T helper cells, macrophages, and glial cells
Binding of gp120 to CD4 results in conformational change of gp120, which then binds to chemokine co- receptor CXCR4 on T lymphocytes or CCR5 on macrophages

38. Penetration of retrovirus (HIV) - II
If pH is not required for fusion, what is the trigger ??
Following receptor binding a conformational change (also the formation of a coiled coil) occurs in the HIV-1 gp120 molecule - exposes its fusion peptide (present on gp41 - the second half of the gp160 Env protein)
From Principles of Virology, Flint et al, ASM Press
HIV has a receptor (CD4) and a co-receptor (CCR5 or CXCR4)

39. HIV virions are able to gain access to their host cells by way of viral host-cell membrane fusion (Gallo et al., 2003).
The viral envelope gp120 first recognizes its primary receptor on host cells, CD4 (Dalgleish et al., 1984; Ugolini et al., 1999), using a binding motif contained within its second constant (C2) region (Kwong et al., 1998).
This interaction gives rise to a conformational change in gp120 which exposes its third variable loop (V3) which contains a consensus amino acid motif that allows for binding to a seven transmembrane-spanning chemokine co-receptor (Jones et al., 1998; Kwong et al., 1998; Berger et al., 1999).
Both interactions are necessary for viral fusion and entry (Sattentau and Moore, 1991).

40. Entry of avian leukosis virus (a model, simple, retrovirus)
Classically all retroviruses were thought to be pH-independent
More recently ALV has been proposed to require low pH, but in addition to its receptor-induced conformational change
Entry is occurring via endosomes in this case

41. Entry of vesicular stomatitis virus (VSV)
Virus receptor is a lipid (phosphatidyl serine; PS)
a unique example ??
Very wide infection range (all cells have PS) - one of the most promiscuous viruses out there
Fusion etc is similar to influenza…..
Both VSV G and influenza HA are referred to as type I fusion proteins
with two main differences
The trigger is reversible
The pH threshold is less stringent (approx. pH 6.5). Fusion is though to occur from the “early” endosome

42. Type I and type II fusion proteins
Type I is the most common and understood fusion protein
Influenza, VSV, retrovirus
• Type II fusion proteins are not proteolytically activated, have internal fusion peptides and no “coiled-coil” form; they are principally β-sheet
• Flavivirus (TBE), and Alphavirus (SFV)

43. SFV and TBE - alternative ways to expose fusion peptides
In SFV the fusion peptide is protected by E2
In TBE the flat E protein rotates and twists

44. Entry: Nonenveloped Virus
Receptor-mediated endocytosis
Clathrin coated pits (seen by EM)
Invagination and pinching off of the membrane
Forms an intracellular endosome containing the virus
Endosome becomes acidified

45. Uncoating: Nonenveloped Virus
Low pH causes conformational changes in capsid protein (hydrophobic region interacts with membrane forming a pore)
Viral nucleic acid released

46. Clathrin vs. non-clathrin internalization
Most viruses were originally assumed to use clathrin as a route into the cell
Used by SFV, VSV, adenovirus etc
Other routes of entry exist and can be used
Caveolae (as used by SV40) are the best characterized)
Influenza and rotavirus are other examples
In most cases non-clathrin pathways are ill-defined

47. It is not specific to clathrin-coated vesicles
Dynamin is a GTPase that acts to “sever” the necks of the endocytic vesicle
It is not specific to clathrin-coated vesicles
Dominant-negative mutant (K44A) inhibits endocytosis
Eps15 binds to AP-2, the clathrin adaptor protein
It is specific to clathrin-coated vesicles
Dominant-negative mutant (Eps15delta95-295) inhibits endocytosis
From Biochem. J. (2004) Immediate Publication, doi: /BJ
Cargo- and compartment-selective endocytic scaffold proteins
Iwona Szymkiewicz, Oleg Shupliakov and Ivan Dikic

48. Lipid rafts Detergent-resistant domains in cell membranes
Enriched in cholesterol and sphingomyelin
Play a very important role in virus budding
Can also be important for virus entry , esp non-clathrin endocytosis e.g SV40
From Munro S.
Cell Nov 14;115(4):
Lipid rafts: elusive or illusive?

49. Herpesviruses A complex system
Herpesviruses have surface glycoproteins
Binds initially to heparan sulfate (via gC)
used by a multitude of different viruses - non-specific
An attachment or “capture” receptor
Subsequently binds to a co-receptors that allows entry (via gD) - herpesvirus entry mediator - specific
A fusion receptor
HveA TNF-R
HveB Nectin2 (Prr 2)
HveC Nectin1 (Prr 1)
HveD PVR
• Different herpesviruses use different receptors
• But very different viruses can use the same receptor
– e.g. pseudorabies virus and polio virus
– Another example = CAR - the coxsackie/adenovirus receptor

50. Poliovirus/Rhinovirus (Picornaviridae)
Picornaviruses bind to a variety of specific cell surface molecules - these are specific proteins
Binding occurs via canyons (depressions) in the virus surface
From Principles of Virology, Flint et al. ASM Press
Similar viruses can have quite distinct receptors

51. Penetration of non-enveloped viruses
Rhinovirus/Poliovirus (Picornavirus)
Although not pH dependent, poliovirus may still enter through the endosome
• Interaction of poliovirus with PVR causes major conformational changes in the virus - leads to the formation of the A particle - physically swollen (less dense)
From Principles of Virology, Flint et al, ASM Press

52. A particles are now hydrophobic
A particles are now hydrophobic. Viruses have apparently lost VP4, and the hydrophobic core is exposed on the virus surface
With a non-enveloped virus, fusion is not possible. Instead picornaviruses form a membrane pore
Penetration might be controlled by sphingosine, a lipid present in the “pocket” -- or (more likely) by the pocket allowing “breathing” of the capsid
From Principles of Virology, Flint et al, ASM Press
Parvoviruses may contain a phospholipase A2 activity in their capsid protein
The specific lipid composition of endosomes may be crucial for some viruses

53. Adenovirus A relatively complex system Receptor and co-receptor
Clathrin-mediated endocytosis
Instead of forming a discrete pore, adenovirus ruptures or lyses the endosomal membrane
The trigger is low pH, via the penton base protein
The virus undergoes proteolytic cleavage - by virus-encoded proteases

54. SV40 Entry occurs via endocytosis but in a clathrin-independent manner
Entry does not depend on low pH
The virus enters through “caveolae” - a specialized endocytic vesicle that forms upon specific cellular signaling induced by virus binding
Receptor is combination of a protein (MHCI) and a glycolipid (sialic acid)?
The “caveosome” containing the virus is delivered to the ER
Caveolae are specialized lipid rafts

55. Reovirus The rare example of a virus requiring the lysosome
Reoviruses have a complex double capsid, which is very stable to low pH (gastro-intestinal viruses; rotavirus)
The lysosomal proteases degrade the outer capsid to form a subviral particle i.e degradation by cellular proteases
The subsequent penetration step is unknown
From Principles of Virology, Flint et al, ASM Press

56. Rotavirus entry Trypsin cleavage of VP4 (= spike protein)
VP4 becomes VP8* and VP5*
Transient exposure of hydrophobic peptide
Trimeric coiled coil formation
From Dormitzer et al (2004) Nature 430:1053
Comparable to influenza HA ?

57. The problem of cytoplasmic transport
Assume the virus in question has undergone receptor binding and penetration - ie the virus/capsid in the the cytoplasm.
The cytoplasm is viscous and the nucleus is often a long distance from the site of entry.
This is especially true for specialized cells such as neurons μm μm
1 cm
polio 61 yr
HSV 231 yr
Table box 5.2
From Sodeik, Trends Microbiol 8: 465

58. Microtubules and virus entry
VSV/Rabies, influenza
Adenovirus
Herpesvirus
From Sodeik, Trends Microbiol 8: 465
• To facilitate transport viruses often bind to the cytoskeleton and use microtubule-mediated motor proteins for transport, i.e. dynein

59. Nuclear Import Why replicate in the nucleus? What are the “benefits?”
DNA viruses - need cellular DNA polymerase and/or accessory proteins (eg topoisomerase) -
All DNA viruses replicate in the nucleus
exception = Pox viruses (even these will not replicate in an enucleated cells or cytoplast)
Almost all RNA viruses replicate in the cytoplasm, and most will replicate in a cytoplast
Principal exceptions = retroviruses (these have a DNA intermediate and influenza virus (has a spliced genome)

60. What are the “problems” with nuclear replication?
An additional barrier during genome transport
The nucleus of a eukaryotic cell is surrounded by a double lipid bilayer - the nuclear envelope.
The nuclear envelope is studded with transport channels - the nuclear pores
From Flint et al Principles of Virology ASM Press

61. Parvovirus But, the NLS is hidden on the inside of the capsid
Possibly the simplest example of nuclear entry
Small icosahedral DNA virus (18-26nm diameter)
Enters through endosomes (pH-dependent)
VP1 contains a nuclear localization signal (NLS)
Basic amino acids
The NLS binds to cellular receptors (karyopherins or importins) that carry proteins into the nucleus
From Flint et al Principles of Virology ASM Press
But,
the NLS is hidden on the inside of the capsid
Therefore a conformational change must occur to expose the NLS

62. Adenovirus Contains NLSs on its capsids, binds microtubules But,
The functional size limit of the nuclear pore is 26 nm
The virus is therefore transported as far as the pore.
It docks to the nuclear pore and then undergoes final disassembly, and the DNA is “injected” into the nucleus - with DNA binding proteins attached
Specific importins help disassemble the capsid

63. Herpesvirus
• After fusion the tegument (most of it) is shed - phosphorylation dependent
• Contains NLSs on its capsids, binds microtubules via dynein
• The virus is therefore transported as far as the pore.
• It docks to the nuclear pore and then undergoes final disassembly, and the DNA is “injected” into the nucleus
Note the capsid is “empty” - no dark center on EM
From Whittaker Trends Microbiol 6: 178

64. Influenza virus
The nucleoprotein (NP) contains NLSs and the RNPs are small enough to translocate across the nuclear pore
The key to influenza nuclear import is the pH-dependent dissociation of the matrix protein (M1) from the vRNPs.
This relies of the M2 ion channel in the virus envelope, the target of amantadine
From Whittaker Exp. Rev. Mol. Med. 8 February,

65. Retroviruses Simple + complex
Simple retroviruses (oncoretroviruses) can only replicate in dividing cells, e.g. Rous sarcoma virus (RSV), avian leukosis virus (ALV).
Nuclear entry occurs upon mitosis - the nuclear envelope breaks down and the virus is “passively” incorporated into the new nucleus
This is relatively inefficient and restrictive for virus tropism
Complex retroviruses (lentiviruses) have evolved mechanism for nuclear entry in non-dividing cells, e.g. HIV