1. Virus binding and entry Virology lecture 3 Dr. Sadia Anjum

2. 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

3. 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

4. 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)

5. Comparison of type I and type II fusion proteins From Principles of Virology, Flint et al, ASM Press

6. 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

7. Virus entry

8. 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

9. Clathrin mediated internalization

10. 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

11. Clathrin vs. non-clathrin internalization

12. Influenza Entry

13. Herpes viruses A complex system Herpesviruses have 10-12 surface glycoproteins Binds initially to heparan sulfate (via gC) – It is - non-specific a nd is attachment or “capture” receptor Subsequently binds to a co-receptors that allows entry (via gD) - herpesvirus entry mediator - specific 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

15. 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

16. 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 Similar viruses can have quite distinct receptors From Principles of Virology, Flint et al. ASM Press

17. Rhinovirus/Poliovirus

18. 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 From Principles of Virology, Flint et al, ASM Press Penetration might be controlled by sphingosine, a lipid present in the “pocket” -- or (more likely) by the pocket allowing “breathing” of the capsid Parvoviruses may contain a phospholipase A2 activity in their capsid protein The specific lipid composition of endosomes may be crucial for some viruses

19. Adenovirus

20. 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

21. 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

22. 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 ?

23. Detergent-resistant domains in cell membranes Enriched in cholesterol and sphingomyelin Lipid rafts 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?

24. 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

25. 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 From Sodeik, Trends Microbiol 8: 465 μmμm μmμm Table box 5.2 1 cm polio 61 yr HSV 231 yr

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

27. 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)

28. 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

29. Parvovirus 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 But, the NLS is hidden on the inside of the capsid Therefore a conformational change must occur to expose the NLS From Flint et al Principles of Virology ASM Press

30. 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

31. 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 Herpesvirus Note the capsid is “empty” - no dark center on EM From Whittaker Trends Microbiol 6: 178

32. 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, http://www- ermm.cbcu.cam.ac.uk/01002447h.htm

33. 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

34. HIV Once in the cytoplasm the RNA genome is reverse transcribed into a DNA copy - the pre-integration complex (PIC) There is (probably) a role for microtubules in cytoplasmic transport The PIC is a large (Stokes radius = 28nm) nucleoprotein complex that contains several proteins, including: – integrase (IN) matrix (MA) and Vpr Each of these three proteins seems to play a role in transporting the very large PIC to and across the nuclear pore Also a role for the “central DNA flap”

35. For more detail Chapter 5 of Flint et al. Chapter 4 of Fields Virology “Brief overview on cellular virus receptors”, Mettenleiter TC, Virus Research 82 (2002) 3-8 Cool movies -- http://trimeris.com/science/hivfusion.html