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1、 Virus binding and entry9/9/2004+9/14/2004 General points - virus entryThe first event in any virus life-cycle - often limits infection to the “correct” cellCan be primary determinant of tropismTissue tropism - e.g. measles (skin cells) vs. mumps (salivary gland)Species tropism - e.g. togavirus (bot
2、h 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 interactionsInitial binding is often low affinity, but high avidity (tight binding) due to multiple binding si
3、tesThe virus binds to a receptor on the cell surface - can be ubiquitous/specific, with variable densityInitial binding is followed by penetration and subsequent uncoating General points II - virus entryWhether or not the virus is enveloped makes a big difference - at least for penetrationAll viruse
4、s must cross a lipid bilayer, plant and bacterial viruses must also cross a cell wallUncoating means that the stable virus stucture must become unstable -transition from extracellular (chemical) form to intracellular (biological) formThere must be some sort of “trigger” or regulated disassembly proc
5、ess T-even (T2) phage structureFrom The Biology of Viruses, Voyles, McGraw Hill, Entry of T-even bacteriophage - bindingBest 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
6、lipopolysaccharides (LPS) of the bacterium (binding can occur in vitro, and is competed by specific sugars) - a non-specific receptorBinding is “additive” until all six tail fibers are bound Binding of 3 fibers is needed to initiate infectionThe virus may “browse” the surface - looking for a suitabl
7、e site for penetration (possibly sites of cell wall synthesis where the outer and plasma membranes are close together). Note - this is a multi-valent interactionFrom Introduction to Modern Virology, Dimmock & Primrose, Blackwell The receptor binding sites of the short tail fibers are now exposed and
8、 bind (also to LPS)- now binding is essentially irreversibleConformational change in the baseplate - hexagon to extended star-shaped conformation -Initiates sheath contraction ( to 37% of its original length)Entry of T-even bacteriophage - binding II Entry of T-even bacteriophage - penetrationOften
9、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 po
10、re through which DNA entersFrom The Biology of Viruses, Voyles, McGraw Hill, DNA is under considerable pressure and seems to exit automatically once the base palte is opened upOther phage, e.g. T3, have a motor protein to reel out the DNA Entry of other phage - IPhage (DNA) - a virus with a longer,
11、but simpler tail than T4From The Bacteriophages vol 1, ed Calender, R., Plenum Pressthe single tail fiber (J protein) binds to lamB (the maltose transporter) - an example of a specific receptorlamB is inducible. This means the virus only infects in the presence of high nutrients. Also needs Mg2+ - b
12、inding is electrostatic - an example of tropismPenetration requires the bacterial pts protein (also part of the maltose transporter) - the co-receptor attachment and penetration require different viral proteins Entry of other phage - IIPRD1 - an icosahedral phage with an internal membraneFor gram -v
13、e bacteria (with two layers of lipid separated by peptidoglycan) phage entry is a challenge1) Binding2) Conformational change - dissociation and opening of 14 nm hole in the capsid3) Second conformational change converts internal envelope to tubule, which delivers the DNAPhage encodes 2 proteins (P5
14、 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 Entry of other phage - IIIEnveloped RNA phage - 6 These phage bind to pili, the pilus then retracts down to the outer membrane, the viru
15、s undergo fusion, enzymatically destroys the peptidoglycan cell wall (p5 protein) and then penetrates the plasma membraneFrom The Bacteriophages vol 2, ed Calender, R., Plenum PressThe Hershey-Chase experiment is now no longer valid, as (most of) the protein (35S) has entered the cell along with the
16、 nucleic acid (32P). Plant virusesPlants 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 commonB) mechanical injuryC) direct cell-cell transmission (via plasmadesmata and viral movement
17、 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 Binding of animal virusesOccurs via receptors on the cell surface (plasma membrane)Protein (glycoprotein)CarbohydrateLipid (glycolipid)From P
18、rinciples of Virology, Flint et al. ASM PressReceptor utilization plays a major role in virus tropism / pathogenesis Principles of virus penetrationViruses can penetrate directly at the plasma membrane, or via endosomes Penetration of Enveloped Animal VirusesEnvelope = fusionSemliki Forest virus (SF
19、V) 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)The very high particle:pfu ratio (approachi
20、ng 1:1) of SFV ensures that all the virus particles are part of the “real” entry pathwayFigure courtesy of A. Helenius Endosomes are used by cells for nutrient and growth factor uptakeThe virus “hijacks” the cellular pathwayOne key feature of endosomes is their progressive acidification - due the th
21、e action of the vacuolar H+/vATPaseEndosomes do much more than provide low pHDeliver through cortical actin and microtubule-mediated transport in the cytosolSpecific redox/ionic environmentDefined lipids for fusion/penetrationEndosomes and virus entryFrom Cell Biology, Pollard and Earnshaw, Saunders
22、 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 b
23、e 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 pHFusion can be induced at the cell surface by exposure
24、to low pH Influenza virus binding - IBinds to cell surface carbohydrate - sialic acidUbiquitous/non-specific receptorIn principle, this can be present as part of glycoprotein or glycolipidFrom Principles of Virology, Flint et al. ASM PressSpecific requirement for 2-3 and 2-6 linkages - gives differe
25、nt tropism for avian vs. human cells (pigs have both)The first virus receptor to be identified - principally due to the fact that there is a receptor-destroying enzyme associated with the virus Influenza virus binding - IIThe major influenza glycoprotein, hemagglutinin (HA) has a specific sialic aci
26、d-binding site on its “top domain” -From Principles of Virology, Flint et al. ASM Press HA mediates both binding and penetration Penetration of influenza virusInfluenza virus requires a lower pH (5.0-5.5) and enters the “late” endosome, but fusion occurs before entry into the lysosome (this avoids d
27、egradation) The acid-triggered fusion event is well understood - a conformational tail forms a rigid “six-helix bundle” or “coiled-coil” of -helices, which flips the fusion peptide out and allows insertion into the membraneNote the fusion peptide is “external”From Principles of Virology, Flint et al
28、, ASM PressThe “trigger” is irreversible - this means that exposure of virions to low extracellular pH will destroy infectivity From Fields Virology. 4th Ed Lippicott Williams and Wilkins The low pH has a second very important role for influenza entry - the virus contains an ion channel in its envel
29、ope (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+/vATPaseFrom Principles of Virology, Flint et al, A
30、SM Press Fusion of an enveloped virusFrom Dimitrov (2004) Nature Reviews Microbiology 2:109-122 Retrovirus (HIV)A classic example of a receptor/co-receptor requirementA specific receptorBinds initially to CD4 - present on immune system cells (T-cells) - gives the virus tropism for the immune systemT
31、his 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 Penetration of retrovirus (HIV) - IHIV enters by a quite different ro
32、uteEntry is not low pH-dependent (no inhibition by NH4Cl etc), and fusion occurs directly with the plasma membraneFrom Principles of Virology, Flint et al, ASM Press Penetration of retrovirus (HIV) - IIIf pH is not required for fusion, what is the trigger ?Following receptor binding a conformational
33、 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 PressHIV has a receptor (CD4) and a co-receptor (CCR5 or CXCR4) Entry of avian leuko
34、sis virus (a model, simple, retrovirus)Classically all retroviruses were thought to be pH-independentMore recently ALV has been proposed to require low pH, but in addition to its receptor-induced conformational changeEntry is occurring via endosomes in this case Entry of vesicular stomatitis virus (
35、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 thereFusion etc is similar to influenza.Both VSV G and influenza HA are referred to as type I fusion proteinswith two main differencesThe t
36、rigger is reversibleThe pH threshold is less stringent (approx. pH 6.5). Fusion is though to occur from the “early” endosome Type I and type II fusion proteinsType I is the most common and understood fusion proteinInfluenza, VSV, retrovirus Type II fusion proteins are not proteolytically activated,
37、have internal fusion peptides and no “coiled-coil” form; they are principally -sheet Flavivirus (TBE), and Alphavirus (SFV) Comparison of type I and type II fusion proteinsFrom Principles of Virology, Flint et al, ASM Press SFV and TBE - alternative ways to expose fusion peptidesIn SFV the fusion pe
38、ptide is protected by E2In TBE the flat E protein rotates and twists Surface representation of dengue virus Clathrin vs. non-clathrin internalizationMost viruses were originally assumed to use clathrin as a route into the cellUsed by SFV, VSV, adenovirus etcOther routes of entry exist and can be use
39、dCaveolae (as used by SV40) are the best characterized)Influenza and rotavirus are other examplesIn most cases non-clathrin pathways are ill-defined 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 (K4
40、4A) inhibits endocytosisEps15 binds to AP-2, the clathrin adaptor proteinIt is specific to clathrin-coated vesiclesDominant-negative mutant (Eps15delta95-295) inhibits endocytosisFrom Biochem. J. (2004) Immediate Publication, doi:10.1042/BJ20040913 Cargo- and compartment-selective endocytic scaffold
41、 proteins Iwona Szymkiewicz, Oleg Shupliakov and Ivan Dikic Detergent-resistant domains in cell membranes Enriched in cholesterol and sphingomyelinLipid raftsPlay a very important role in virus buddingCan also be important for virus entry , esp non-clathrin endocytosis e.g SV40From Munro S. Cell. 20
42、03 Nov 14;115(4):377-88. Lipid rafts: elusive or illusive? HerpesvirusesA complex systemHerpesviruses have 10-12 surface glycoproteinsBinds initially to heparan sulfate (via gC) used by a multitude of different viruses - non-specificAn attachment or “capture” receptorSubsequently binds to a co-recep
43、tors that allows entry (via gD) - herpesvirus entry mediator - specificA fusion receptorHveATNF-RHveBNectin2 (Prr 2)HveCNectin1 (Prr 1)HveDPVR Different herpesviruses use different receptors But very different viruses can use the same receptor e.g. pseudorabies virus and polio virus Another example
44、= CAR - the coxsackie/adenovirus receptor Poliovirus/Rhinovirus (Picornaviridae)Picornaviruses bind to a variety of specific cell surface molecules - these are specific proteinsBinding occurs via canyons (depressions) in the virus surfaceSimilar viruses can have quite distinct receptorsFrom Principl
45、es of Virology, Flint et al. ASM Press Penetration of non-enveloped virusesRhinovirus/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 t
46、he A particle -physically swollen (less dense) From Principles of Virology, Flint et al, ASM Press A particles are now hydrophobic. Viruses have apparently lost VP4, and the hydrophobic core is exposed on the virus surfaceWith a non-enveloped virus, fusion is not possible. Instead picornaviruses for
47、m a membrane poreFrom Principles of Virology, Flint et al, ASM PressPenetration might be controlled by sphingosine, a lipid present in the “pocket” - or (more likely) by the pocket allowing “breathing” of the capsidParvoviruses may contain a phospholipase A2 activity in their capsid proteinThe speci
48、fic lipid composition of endosomes may be crucial for some viruses Picornaviruses as enzymes ?Virus entry as thermodynamics ? AdenovirusA relatively complex systemReceptor and co-receptorClathrin-mediated endocytosisInstead of forming a discrete pore, adenovirus ruptures or lyses the endosomal membr
49、aneThe trigger is low pH, via the penton base proteinThe virus undergoes proteolytic cleavage - by virus-encoded proteases SV40Entry occurs via endocytosis but in a clathrin-independent mannerEntry does not depend on low pHThe virus enters through “caveolae” - a specialized endocytic vesicle that fo
50、rms upon specific cellular signaling induced by virus bindingReceptor is combination of a protein (MHCI) and a glycolipid (sialic acid)?The “caveosome” containing the virus is delivered to the ERCaveolae are specialized lipid rafts ReovirusThe rare example of a virus requiring the lysosome Reoviruse
51、s 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 proteasesThe subsequent penetration step is unknownFrom Principles of Virology, Flint et al, A
52、SM Press Rotavirus entryTrypsin cleavage of VP4 (= spike protein)VP4 becomes VP8* and VP5*Transient exposure of hydrophobic peptideTrimeric coiled coil formationFrom Dormitzer et al (2004) Nature 430:1053Comparable to influenza HA ? The problem of cytoplasmic transportAssume the virus in question ha
53、s 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 neuronsFrom Sodeik, Trends Microbiol 8: 465mmTable box 5.21 cmpolio 6
54、1 yrHSV 231 yr Microtubules and virus entry To facilitate transport viruses often bind to the cytoskeleton and use microtubule-mediated motor proteins for transport, i.e. dyneinVSV/Rabies, influenzaAdenovirusHerpesvirusFrom Sodeik, Trends Microbiol 8: 465 Nuclear Import Why replicate in the nucleus?
55、What are the “benefits?” DNA viruses - need cellular DNA polymerase and/or accessory proteins (eg topoisomerase) - All DNA viruses replicate in the nucleusexception = Pox viruses (even these will not replicate in an enucleated cells or cytoplast)Almost all RNA viruses replicate in the cytoplasm, and
56、 most will replicate in a cytoplastPrincipal exceptions = retroviruses (these have a DNA intermediate)andinfluenzavirus(hasasplicedgenome) What are the “problems” with nuclear replication?An additional barrier during genome transportThe nucleus of a eukaryotic cell is surrounded by a double lipid bi
57、layer - the nuclear envelope. The nuclear envelope is studded with transport channels - the nuclear poresFrom Flint et al Principles of Virology ASM Press ParvovirusPossibly the simplest example of nuclear entrySmall icosahedral DNA virus (18-26nm diameter)Enters through endosomes (pH-dependent)VP1
58、contains a nuclear localization signal (NLS) Basic amino acids The NLS binds to cellular receptors (karyopherins or importins) that carry proteins into the nucleusBut, the NLS is hidden on the inside of the capsid Therefore a conformational change must occur to expose the NLSFrom Flint et al Princip
59、les of Virology ASM Press AdenovirusContains NLSs on its capsids, binds microtubulesBut, The functional size limit of the nuclear pore is 26 nmThe 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
60、nucleus - with DNA binding proteins attachedSpecific importins help disassemble the capsid 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 nuc
61、lear pore and then undergoes final disassembly, and the DNA is “injected” into the nucleus HerpesvirusNote the capsid is “empty” - no dark center on EMFrom Whittaker Trends Microbiol 6: 178 Influenza virusThe nucleoprotein (NP) contains NLSs and the RNPs are small enough to translocate across the nu
62、clear poreThe 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 amantadineFrom Whittaker Exp. Rev. Mol. Med. 8 February, http:/www-ermm.cbcu.cam.ac.uk/01002447h.htm Retrovi
63、rusesSimple + complexSimple 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 rela
64、tively inefficient and restrictive for virus tropismComplex retroviruses (lentiviruses) have evolved mechanism for nuclear entry in non-dividing cells, e.g. HIV HIVOnce in the cytoplasm the RNA genome is reverse transcribed into a DNA copy - the pre-integration complex (PIC)There is (probably) a rol
65、e for microtubules in cytoplasmic transportThe PIC is a large (Stokes radius = 28nm) nucleoprotein complex that contains several proteins, including:integrase (IN) matrix (MA) and VprEach of these three proteins seems to play a role in transporting the very large PIC to and across the nuclear poreAlso a role for the “central DNA flap” Further readingChapter 5 of Flint et al.Chapter 4 of Fields Virology - not particularly good“Brief overview on cellular virus receptors”, Mettenleiter TC, Virus Research 82 (2002) 3-8Cool movies - http:/
