Virus Attachment, Entry and Uncoating

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

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 • 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 • Often referred to as a “hypodermic syringe” 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 68 330

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. • 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 6.2-6.5 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 (5.0-5.5) 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 2:109-122

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 gp120to CD4 results in conformational change of gp120, which then binds to chemokineco-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. 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:10.1042/BJ20040913 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. 2003 Nov 14;115(4):377-88. Lipid rafts: elusive or illusive?

49. Herpesviruses • A complex system • Herpesviruses have 10-12 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