Assembly of viruses

Describes how viruses are assembled inside the host cell

1. ] ASSEMBLY OF VIRUSES MOUSUMI BORA PhD SCHOLAR DIVISION OF VIROLOGY INDIAN VETERINARY RESEARCH INSTITUTE

2. ] Assembly of Viruses INTRODUCTION ASSEMBLY OF PROTEIN SHELL SELECTIVE PACKAGING OF THE NUCLEIC ACID GENOME ACQUISITION OF AN ENVELOPE RELEASE FROM THE HOST CELL VIRION MATURATION

3. Virus assembly - key step in the replication cycle Involves transportation of chemically distinct macromolecules through different pathways, to a point within the cell where they are assembled into a nascent viral particle Assembly of each virus should be at a defined point within the cell Assembly process include Interactions between proteins of viral and cellular origin Between viral proteins and nucleic acids and lipids Between the viral proteins themselves INTRODUCTION Assembly of Viruses

4. Studies on virus assembly was first carried out by Heinz Fraenkel-Conrat and Robley Williams in 1955 Tobacco Mosaic Virus (TMV) Purified tobacco mosaic virus RNA and its protein coat can assemble by themselves to form functional viruses X-ray diffraction studies HISTORY Assembly of Viruses 1. Nucleic acid (RNA), 2. Capsomer protein (PROTOMER), 3. Capsid

5. Virion assembly can be studied by Cryo Electron Microscopy Intracellular sites of assembly The nature of assembly intermediates Mechanism of envelope acquisition Release of particles Difference imaging Combination of X-ray crystallography and Electron Microscopy EM can be combined with Immunocytochemical methods Identification of individual viral proteins/ structures Binding of specific antibodies or attached to electron dense particles of gold STUDY OF VIRUS ASSEMBLY Assembly of Viruses

6. ASSEMBLY OF VIRION COMPONENTS Assembly of Viruses

7. SITES OF VIRUS ASSEMBLY Assembly of Viruses

8. SITES OF VIRUS ASSEMBLY Assembly of Viruses

9. NUCLEAR IMPORT/ EXPORT AND SECRETORY PATHWAYS OF NUCLEIC ACIDS AND PROTEINS Assembly of Viruses NUCLEAR IMPORT/EXPORT PATHWAYS SECRETORY PATHWAY

10. Provides a proteinaceous channel between the nucleus and cytosol Large structure, 50 mDa Constructed of multiple copies of approximately 30 different proteins called nucleoporins (Nups) Small molecules and proteins may be able to passively diffuse through the NPC 3,000 NPCs on the nuclear envelope of an animal cell NUCLEAR PORE COMPLEX Assembly of Viruses

11. Proteins that are actively transported into or out of the nucleus are characterized by the presence of amino acid motifs For import into the nucleus, these motifs are termed nuclear localization signals (NLS) Generally short (<20 amino acids) For export, nuclear export signals (NES) Nuclear transport signal is also short (10 amino acids) HIV-1 possess both an NLS and an NES and appear to shuttle back and forth between the cytoplasm and the nucleus NUCLEAR LOCALIZATION SIGNALS Assembly of Viruses

12. NUCLEAR IMPORT Newly synthesized NLS-containing protein interacts with cytosolic receptor proteins This complex is then translocated, in an energy- independent process, through the nuclear pore into the nucleus Best-characterized protein import receptor is importin-a (karyopherin-a) NUCLEAR EXPORT Exportins interact with their substrates only in the nucleus in the presence of RanGTP NUCLEAR IMPORT/EXPORT PATHWAY Assembly of Viruses

13. Adenoviruses Non-enveloped icosahedral viruses Capsid is composed of 252 capsomeres, of which 240 are hexons and 12 are pentons Replicates exclusively in the nucleus Depend on nuclear targeting/transport pathways to export newly synthesized mRNAs out of the nucleus and to import structural proteins back into the nucleus Nuclear import of the major capsid protein (hexon or polypeptide II) depends on the pVI (precursor) polypeptide Trimer formation , depends upon chaperone-like protein- L4 (100kDa) L4-binds to the newly synthesized hexon monomer and mediates its association with two additional monomers Precursor core proteins would be packaged into the empty capsid along with the genome to form immature virions Proteolytic cleavage of the precursor proteins by the viral proteinase yields the mature virion ASSEMBLY OF NON-ENVELOPED VIRUSES IN THE NUCLEUS Assembly of Viruses

14. ASSEMBLY OF ADENOVIRUS Assembly of Viruses

15. ASSEMBLY OF ENVELOPED VIRUSES IN THE NUCLEUS Assembly of Viruses Herpesviruses Capsid is icosahedral; 162 capsomers Assembled in the infected cell nucleus Basic assembly unit is a complex of the major capsid and scaffolding proteins MAJOR CAPSID SCAFFOLDING PROTEINS PROCAPSID ds DNA GENOME P ACKAGED RELEASE OF SCAFFOLDING PROTEINS

16. ASSEMBLY OF HERPESVIRUSES Assembly of Viruses Brown et al., 2011

17. ASSEMBLY OF HERPESVIRUSES

18. Reoviruses Segmented double-stranded RNA genome Protein capsid is organized as one, two, or three concentric capsid layers , which surround the dsRNA segments of the viral genome Outer capsid mediates viral entry to the host cell cytoplasm ( Outer capsid proteins : σ 1, σ 3, μ 1, λ 2) Outer capsid protein sigma1 forms trimers that extend from the fivefold axes of virions and mediates viral attachment to cellular receptors Another protein λ 2 forms pentameric turrets that surround the fivefold axes and bridge the inner and outer capsids λ2 is involved in viral mRNA synthesis and assembly of the outer capsid onto virus particles Reoviruses are the only animal viruses that appear to complete their assembly entirely in the cytoplasm without the involvement of membranes ASSEMBLY OF VIRUSES IN CYTOPLASM

19. ASSEMBLY OF REOVIRUSES

20. ASSEMBLY OF REOVIRUSES

21. Picornaviruses Non -enveloped, icosahedral symmetry Consisting of a protein shell surrounding the naked RNA genome Capsids of picornaviruses are composed of four structural proteins: VP4, VP2, VP3, and VP1 Viral proteins are synthesized from a polyprotein precursor, which is cleaved nascently Processing of picornavirus polyprotein Maturational cleavage of VP0 to VP2 and VP4 ASSEMBLY OF VIRUSES IN CYTOPLASM

22. ASSEMBLY OF VIRUSES IN CYTOPLASM

23. ASSEMBLY OF PICORNAVIRUSES

24. Bunyaviruses Negative-stranded, enveloped viruses; segmented genome Assembles in tube-like virus factories that are built around the Golgi complex and are connected to mitochondria and rough ER These factories appear to allow accumulation of RNPs that can associate with viral glycoproteins (Gn and Gc) and bud into the lumen of swollen Golgi stacks Gn and Gc form a Gn-Gc heterodimer that is transported to the Golgi complex ASSEMBLY IN THE GOLGI COMPLEX Gc Gn HELICAL NUCLEOPROTEINS ( 3 segments) NUCLEOCAPSID (N) PROTEIN ACCUMULATE IN THE GOLGI COMPONENT OF THE VIRUS FACTORIES

25. Poxviruses Linear double-stranded DNA genome Enveloped viruses Assembly begins with the formation of crescents by diversion of membrane from the endoplasmic reticulum Mature virion is released from the infected cell only upon lysis Acquire additional membranes by wrapping ( derived from a late or post -Golgi compartments to form the wrapped virions ) known as Intracellular Enveloped Virus (IEV) ASSEMBLY IN THE GOLGI COMPLEX Virus particle (IEV) Cellular membrane ACTIN TAILS

26. ASSEMBLY OF POXVIRUS ( Vaccinia virus )

27. Togaviruses Rhabdoviruses Paramyxoviruses Orthomyxoviruses Retroviruses ASSEMBLY AT THE PLASMA MEMBRANE

28. Togaviruses Single-stranded, positive sense RNA Enveloped; Icosahedral symmetry Best studied in Alphaviruses The major glycoproteins E1 and E2 of the Alphaviruses are translated from a subgenomic 26S RNA as a pE2, 6K, E1 precursor complex The 6K and E1 proteins are released from the precursor by signal peptidase but remain in a complex with pE2 Following transport to the Golgi, pE2 is processed to E2 and E3. Stable trimers of E1-E2 heterodimers are then transported to the plasma membrane, where they associate with nucleocapsids The 6K protein travels to the plasma membrane with the E1-E2 complex but is inefficiently incorporated into virions Cryo-electron microscopy analyses of mature alphavirus particles have revealed that both the envelope and the core display icosahedral symmetry. ASSEMBLY OF TOGAVIRUSES

29. ASSEMBLY OF TOGAVIRUSES

30. ASSEMBLY OF TOGAVIRUSES ( Alphavirus)

31. Rhabdoviruses Minus sense ssRNA genome bound to nucleoprotein Helical nucleocapsid ; Bullet shape Entire nucleocapsid is enclosed in a mono-molecular layer of the matrix protein, M Assembles by budding at the host cell cytoplasmic membrane Assembly is initiated by interaction of the nucleocapsid with a specialized region of membrane containing M and G proteins Matrix protein and the membrane binds to the nucleocapsid progressively creating helical turns beginning at the domed virion end As helical turns are created, the overall structure projects progressively further outward from the host cell Assembly is terminated with formation of the blunt end and detachment of the complete virion from the host cell ASSEMBLY OF RHABDOVIRUSES

32. ASSEMBLY OF RHABDOVIRUSES Brown et at., 2010

33. Paramyxoviruses are spherical, pleomorphic / filamentous forms Single stranded RNA genomes of negative polarity Glycoprotein spikes extend from the surface of the membrane Nucleocapsids assemble in the cytoplasm in two steps: Paramyxoviriuses bud only from the apical surface ASSEMBLY OF PARAMYXOVIRUSES

34. ASSEMBLY OF PARAMYXOVIRUSES

35. INFLUENZA VIRUS Enveloped virus; segmented negative strand RNA genome Assembly and budding complex, multistep process that occurs in lipid raft domains on the apical membrane of infected cells The spike glycoproteins Hemagglutinin (HA) : mediates viral entry into cells and has receptor binding and membrane fusion activity Neuraminidase (NA) : NA mediates enzymatic cleavage of the viral receptor Integral membrane protein (M2): multi-functional, proton-selective, ion channel which has roles both in virus entry as well as in assembly and budding (Rossman and Lamb, 2011) ASSEMBLY OF ORTHOMYXOVIRUSES

36. ASSEMBLY OF ORTHOMYXOVIRUSES Virus replication Newly formed RNP Assembled in nucleus Exported to cytoplasm Matrix protein (M1) Nuclear export protein (NEP/NS2) Viruses assemble and bud from the apical plasma membrane of polarized cells

37. ASSEMBLY OF ORTHOMYXOVIRUSES

38. Retroviruses are enveloped viruses Assembly by budding through the plasma membrane of the infected cell The immature capsid of the virus is assembled from polyprotein precursors The gag protein of all retroviruses contains the MA, CA and NC proteins linked by spacer peptides that are variable in length and position. The association of gag molecules with the plasma membrane with one another and with the RNA genome initiates assembly at the inner surface of the plasma membrane Betaretroviruses , complete assembly of their core in the interior of the cell prior to its association with the plasma membrane Cleavage of Gag and Gag- Pol proteins by the viral protease (PR) produces infectious particles ASSEMBLY OF RETROVIRUSES

39. ASSEMBLY OF RETROVIRUSES

40. MECHANISM OF ASSEMBLY OF THE STRUCTURAL UNITS OF PROTEIN SHELLS

41. ASSEMBLY FROM INDIVIDUAL PROTEIN MOLECULES Mechanism Virus Structural unit Association of individual protein molecules Adenovirus (adenovirus type 2) Protein IV trimer (fiber) and protein III pentamer (penton base) that forms pentons Hepadnavirus (hepatitis B virus) C (capsid) protein dimers Papovavirus (simian virus 40) VP1 pentamer, with one molecule of VP2 or VP1 in its central cavity Reovirus (reovirus type 1) λ, σ2 ( inner capsid protein) homo- oligomers ; σ3-μ, ( outer capsid protein) hetero- oligomers

42. ASSEMBLY FROM INDIVIDUAL PROTEIN MOLECULES

43. ASSEMBLY FROM A POLYPROTEIN PRECURSOR Mechanism Virus Structural unit Assembly from polyprotein precursors Alphavirus (Sindbis virus) Capsid (C) protein folds in, and cleaves itself from, a nascent polyprotein also containing glycoprotein sequences Picornavirus (poliovirus) Immature 5S structural units, VP0-VP3-VP1 Retrovirus (avian sarcoma virus) NC, CA, and MA protein shells assembled via Gag polyprotein

44. ASSEMBLY FROM A POLYPROTEIN PRECURSOR

45. CHAPERONE-ASSISTED ASSEMBLY Assembly of viral proteins into structural units is assisted by cellular chaperons Facilitate protein folding by preventing non-specific, improper association among exposed, sticky patches on nascent and newly synthesized proteins First chaperone to be identified – the product of E. coli gro EL gene ; essential for reproduction of bacteriophage T4 and lambda Adenoviral L4 100-kDa protein, which is required for formation of the hexon trimer from the protein II monomer

46. CHAPERONE-ASSISTED ASSEMBLY

47. ACQUISITION OF AN ENVELOPE Enveloped viruses assemble by virtue of specific interactions among virion components at a cellular membrane before budding and pinching off of a new virus particle Enveloped viruses assemble by one of two mechanisms : Sequential Assembly of Internal Components and Budding from a Cellular Membrane The assembly of internal structures of the virion and their interaction with a cellular membrane Modified by insertion of viral proteins are spatially and temporally separated Exemplified by (−) strand RNA viruses Influenza viruses B. Coordination of the Assembly of Internal Structures with the Acquisition of the Envelope Assembling cores of the majority first appear as crescent-shaped patches at the inner surface of the plasma membrane Extend to form a closed sphere as the plasma membrane wraps around and eventually pinches off the assembling particle Retroviruses

48. MATURATION OF PROGENY VIRUS Virus-encoded proteolytic enzymes – helps in process of assembly and post assembly maturation of viruses Proteolytic cleavage in Alphaviruses - allow protein domains to enter different pathways I n Herpesviruses , proteolytic cleavage of the scaffolding protein occurs after assembly of the procapsid is complete and is a prerequisite for DNA packaging Cleavage of the P1 precursor of Picornaviruses appears to be a prerequisite for entry of the capsid proteins into the assembly pathway Cleavage of the Gag precursors in the immature capsid of Retroviruses help in maturation of the virions ADENOVIRUES

49. RELEASE OF NASCENT PARTICLES Non enveloped viruses Lysis of infected cell Except Picornaviruses and Polioviruses E nveloped viruses Bud growth Bud formation Fusion of the bud membrane

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