1. Assembly, Maturation, and Release (Getting it all together and leaving!)

2. Assembly  The final phase of the viral life cycle is FUNDAMENTALLY DIFFERENT than that of any other type of organism.  Viruses are assembled from component parts, not from division of a pre-existing virus.  For naked viruses:  Spontaneous self assembly can occur in vitro by combining pre-formed component parts.  Assembly may require specific virus encoded, nonstructural proteins.  The particle may be assembled from precursor proteins that are subsequently modified to form the infectious virion.  The final phase of the viral life cycle is FUNDAMENTALLY DIFFERENT than that of any other type of organism.  Viruses are assembled from component parts, not from division of a pre-existing virus.  For naked viruses:  Spontaneous self assembly can occur in vitro by combining pre-formed component parts.  Assembly may require specific virus encoded, nonstructural proteins.  The particle may be assembled from precursor proteins that are subsequently modified to form the infectious virion.

3. Assembly  Enveloped viruses can’t assemble in vitro because their envelope is derived from a host cell membrane.  Assembly of naked or enveloped viruses always requires protein-protein interactions and protein-nucleic acid interactions. The order of assembly could occur in 2 different ways:  The genomic nucleic acid serves as a focus for assembly of the capsid surrounding it.  A hollow capsid is formed and is then filled with the genomic nucleic acid.  Enveloped viruses can’t assemble in vitro because their envelope is derived from a host cell membrane.  Assembly of naked or enveloped viruses always requires protein-protein interactions and protein-nucleic acid interactions. The order of assembly could occur in 2 different ways:  The genomic nucleic acid serves as a focus for assembly of the capsid surrounding it.  A hollow capsid is formed and is then filled with the genomic nucleic acid.

4. Assembly  The choice of which strategy to use is a function of the capsid architecture  Helical viruses use the first strategy  Icosahedral viruses use the second strategy  Rigid Helical viruses (Tobacco mosaic virus)  Composed of RNA plus identical capsomers arranged in a helix surrounding it.  TMV capsid proteins only recognize TMV RNA. This means that the protein-nucleic acid interactions are very specific.  The choice of which strategy to use is a function of the capsid architecture  Helical viruses use the first strategy  Icosahedral viruses use the second strategy  Rigid Helical viruses (Tobacco mosaic virus)  Composed of RNA plus identical capsomers arranged in a helix surrounding it.  TMV capsid proteins only recognize TMV RNA. This means that the protein-nucleic acid interactions are very specific.

5. TMV

6. TMV assembly  First, 34 capsid proteins assemble into a pair of disks.  The outer portions interact to hold the two disks together, while the inner portion has a gap where RNA binds.  When the RNA enters, the gap is closed to hold the RNA in place.  First, 34 capsid proteins assemble into a pair of disks.  The outer portions interact to hold the two disks together, while the inner portion has a gap where RNA binds.  When the RNA enters, the gap is closed to hold the RNA in place.

7. TMV assembly

8. Simple assembly model

9. TMV assembly  RNA interacts with the disks beginning at the “pac site” which is about 1000 bases from the 3’ end of the genome.  The pac site consists of ~ 500 bases that can form a series of hairpin loops.  Loop 1 contains the active residues (GGG) which are highlighted on the following picture.  RNA interacts with the disks beginning at the “pac site” which is about 1000 bases from the 3’ end of the genome.  The pac site consists of ~ 500 bases that can form a series of hairpin loops.  Loop 1 contains the active residues (GGG) which are highlighted on the following picture.

10. TMV assembly

11.  Pac sequence loop 1 enters the disks and becomes intercalated into the gap.  When the flexible residues close upon the RNA, the disks shift their conformation to a lock-washer arrangement.  This is the beginning of the helical conformation.  Assembly proceeds in the 5’ to 3’ direction as RNA is drawn up through the hole in the helix and intercalated into additional disks as they are added.  Pac sequence loop 1 enters the disks and becomes intercalated into the gap.  When the flexible residues close upon the RNA, the disks shift their conformation to a lock-washer arrangement.  This is the beginning of the helical conformation.  Assembly proceeds in the 5’ to 3’ direction as RNA is drawn up through the hole in the helix and intercalated into additional disks as they are added.

12. TMV assembly

13. Assembly  Flexible helical capsids  The helical nucleocapsids of enveloped viruses are flexible.  Since the virus has an envelope to shield the nucleic acid from the elements (environment), the capsids don’t have the job of shielding the nucleic acid.  Therefore, they are organized in a looser arrangement and the RNA may actually be wrapped around the outside of the nucleocapsid.  The 5’ ends of the genomic RNA may have pac- like regions to assure that only the correct RNA is assembled in the nucleocapsid.  Flexible helical capsids  The helical nucleocapsids of enveloped viruses are flexible.  Since the virus has an envelope to shield the nucleic acid from the elements (environment), the capsids don’t have the job of shielding the nucleic acid.  Therefore, they are organized in a looser arrangement and the RNA may actually be wrapped around the outside of the nucleocapsid.  The 5’ ends of the genomic RNA may have pac- like regions to assure that only the correct RNA is assembled in the nucleocapsid.

14. Assembly  Icosahedral RNA viruses  Remember that an icosahedron has 20 faces and each face is composed of 3 subunits (or multiples of 3). The subunits may be identical or different. There are also 12 vertices or corners.  All vertices are surrounded by 5 identical capsomers (pentamer) and the intersections between faces are formed by six capsomers (hexamers)  Icosahedral RNA viruses  Remember that an icosahedron has 20 faces and each face is composed of 3 subunits (or multiples of 3). The subunits may be identical or different. There are also 12 vertices or corners.  All vertices are surrounded by 5 identical capsomers (pentamer) and the intersections between faces are formed by six capsomers (hexamers)

15. Icosahedral viruses

16. Icosahedral RNA viruses  The capsomer proteins all appear to share common structural themes which are related to their roles in assembly:  Eight beta sheets linked together by alpha helices or random coils  An arm of variable length is found at the amino terminus  The tertiary structure looks like a cheese wedge with an arm extending away.  The capsomer proteins all appear to share common structural themes which are related to their roles in assembly:  Eight beta sheets linked together by alpha helices or random coils  An arm of variable length is found at the amino terminus  The tertiary structure looks like a cheese wedge with an arm extending away.

17. Icosahedral RNA viruses

19.  Assembly of the icosahedral capsid requires intermediate forms and may involve several steps:  Poliovirus will be used as an example  Proteolytic cleavage of the polyprotein occurs  The basic building block (protomer) of the capsid is made with Vp0, VP1, and VP3.  Five protomers combine to form a pentamer  Twelve pentamers combine to form an empty procapsid  RNA enters the procapsid (the arrangement is not clear, but it is not random)  Assembly of the icosahedral capsid requires intermediate forms and may involve several steps:  Poliovirus will be used as an example  Proteolytic cleavage of the polyprotein occurs  The basic building block (protomer) of the capsid is made with Vp0, VP1, and VP3.  Five protomers combine to form a pentamer  Twelve pentamers combine to form an empty procapsid  RNA enters the procapsid (the arrangement is not clear, but it is not random)

20. Icosahedral RNA virus Assembly  A maturation cleavage converts VP0 into VP2 and VP4  The loops and carboxy termini face the outside of the capsid, while the amino termini and VP4 face the inside of the capsid.  Remember: After attachment of the virus to the host cell, VP4 is released and this exposes a hydrophobic domain on VP1 which interacts with the either the plasma membrane of the host cell or the membrane of an endocytic vesicle following endocytosis, to create a pore through which the viral nucleic acid is released into the cytoplasm.  A maturation cleavage converts VP0 into VP2 and VP4  The loops and carboxy termini face the outside of the capsid, while the amino termini and VP4 face the inside of the capsid.  Remember: After attachment of the virus to the host cell, VP4 is released and this exposes a hydrophobic domain on VP1 which interacts with the either the plasma membrane of the host cell or the membrane of an endocytic vesicle following endocytosis, to create a pore through which the viral nucleic acid is released into the cytoplasm.

21. Poliovirus Assembly

23. Icosahedral DNA Virus Assembly  SV40  The capsid of SV40 virus has an unusual arrangement that appears to violate the rules for icosahedral shapes.  Rather than having the classic 12 pentamers and 60 hexamers, all capsomers are grouped into pentamers.  SV40  The capsid of SV40 virus has an unusual arrangement that appears to violate the rules for icosahedral shapes.  Rather than having the classic 12 pentamers and 60 hexamers, all capsomers are grouped into pentamers.

24. SV40 Assembly

25. SV40 structure

26. Assembly  There is very little known about the assembly events of other icosahedral DNA animal viruses.  It is clear, however, that the assembly takes place in a very ordered sequence of events.  There is very little known about the assembly events of other icosahedral DNA animal viruses.  It is clear, however, that the assembly takes place in a very ordered sequence of events.

27. Adenovirus assembly

28. Assembly  How do viruses with segmented genomes ensure that virions contain a copy of each segment?  The answer is simple for some – they don’t.  For others, there appear to be specific mechanisms for packaging their segmented genomes. Each segment may have its own unique pac site.  For influenza virus the ratio of virus particles to actual infectious units is comparable to the ratio predicted for random packaging.  However recent evidence suggests that during budding, viral proteins recognize and interact with specific RNA sequences in each of the eight nucleocapsids.  They then incorporate them, one by one, into bundles that are packaged into virions during budding  How do viruses with segmented genomes ensure that virions contain a copy of each segment?  The answer is simple for some – they don’t.  For others, there appear to be specific mechanisms for packaging their segmented genomes. Each segment may have its own unique pac site.  For influenza virus the ratio of virus particles to actual infectious units is comparable to the ratio predicted for random packaging.  However recent evidence suggests that during budding, viral proteins recognize and interact with specific RNA sequences in each of the eight nucleocapsids.  They then incorporate them, one by one, into bundles that are packaged into virions during budding

29. Release  How do viruses exit their host cells?  Naked viruses  If the virus lyses the host cells, it is said to be cytocidal or cytolytic.  For animal viruses lysis is due to the cumulative metabolic damage to the cell caused by the virus.  Disruption of lysosomes may be involved.  How do viruses exit their host cells?  Naked viruses  If the virus lyses the host cells, it is said to be cytocidal or cytolytic.  For animal viruses lysis is due to the cumulative metabolic damage to the cell caused by the virus.  Disruption of lysosomes may be involved.

30. Release  Enveloped viruses  The envelopes are derived from host cell membranes that have been modified by the insertion of viral proteins and glycoproteins  Maturation and release via the process of budding (exocytosis) involves 4 steps  Synthesis and insertion of viral glycoproteins in host cell membranes (RER, Golgi, PM, nuclear membrane)  Assembly of the viral nucleocapsid  The nucleocapsid and the modified membrane are brought together (the C terminal domain of the envelope proteins may interact directly with the nucleocapsid or the interaction may be via the matrix (M) protein)  Exocytosis or budding which may or may not kill the host cell  Enveloped viruses  The envelopes are derived from host cell membranes that have been modified by the insertion of viral proteins and glycoproteins  Maturation and release via the process of budding (exocytosis) involves 4 steps  Synthesis and insertion of viral glycoproteins in host cell membranes (RER, Golgi, PM, nuclear membrane)  Assembly of the viral nucleocapsid  The nucleocapsid and the modified membrane are brought together (the C terminal domain of the envelope proteins may interact directly with the nucleocapsid or the interaction may be via the matrix (M) protein)  Exocytosis or budding which may or may not kill the host cell

31. Budding

32.  How does a virus target a particular membrane region as the site of budding?  They utilize the transport machinery of the host cells  Some viruses bud from the plasma membrane  As they exit the cell they acquire their membrane.  How does a virus target a particular membrane region as the site of budding?  They utilize the transport machinery of the host cells  Some viruses bud from the plasma membrane  As they exit the cell they acquire their membrane.

33. Influenza virus budding from the plasma membrane

34. Budding  Some viruses bud from the RER.  Some viruses bud from the Golgi.  Some viruses bud from the nuclear envelope.  How do viruses that don’t bud from the plasma membrane exit the cell?  Some viruses bud from the RER.  Some viruses bud from the Golgi.  Some viruses bud from the nuclear envelope.  How do viruses that don’t bud from the plasma membrane exit the cell?

35. Budding From RER or Golgi

36. Budding  The plasma membranes of some host cells are polarized.  If the cell is polarized, the virus may bud from either the apical or the basolateral domain.  Viruses that bud apically tend to cause localized infections.  Viruses that bud basolaterally tend to cause systemic infections.  The envelope proteins of these viruses contain apical or basolateral transport signals that are recognized and utilized by the transport machinery of the host cell.  The site of envelope protein transport determines the site of budding.  The plasma membranes of some host cells are polarized.  If the cell is polarized, the virus may bud from either the apical or the basolateral domain.  Viruses that bud apically tend to cause localized infections.  Viruses that bud basolaterally tend to cause systemic infections.  The envelope proteins of these viruses contain apical or basolateral transport signals that are recognized and utilized by the transport machinery of the host cell.  The site of envelope protein transport determines the site of budding.

37. Polarized Budding

38. Maturation of virus particles  For most viruses, formation of the infectious virions requires the cleavage of precursor proteins into functional proteins.  The cleavage may occur before assembly.  The HA of influenza virus is cleaved into HA1 and HA2 during transit of the protein to the host cell plasma membrane.  The cleavage may occur after assembly  Remember the cleavage of polio virus VP0 into VP2 and VP4?  For most viruses, formation of the infectious virions requires the cleavage of precursor proteins into functional proteins.  The cleavage may occur before assembly.  The HA of influenza virus is cleaved into HA1 and HA2 during transit of the protein to the host cell plasma membrane.  The cleavage may occur after assembly  Remember the cleavage of polio virus VP0 into VP2 and VP4?