Virus assembly is a key step in the viral replication cycle that involves transporting viral proteins and nucleic acids through the cell and assembling them into new viral particles. There are three main steps: 1) Assembly of the protein shell or capsid from individual proteins or polyprotein precursors, sometimes with the aid of chaperone proteins. 2) Selective packaging of the viral genome. 3) Some viruses acquire an envelope by budding through cellular membranes during the assembly process.

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
CYTOPLASM / NUCLEUS
TIGHTLY ASSEMBLED
ICOSAHEDRAL SHELL
PREVENTS DEGRADATION
OF THE GENOME
NON
ENVELOPED
VIRUSES
MUST ACQUIRE A LIPID
BILAYER FROM ONE OF THE
CELL’S MEMBRANE DURING
THE PROCESS OF ASSEMBLY
INTEGRITY OF THE
NUCLEOCAPSID IS LESS
CRITICAL
ENVELOPED
VIRUSES

7. SITES OF VIRUS ASSEMBLY
Assembly of Viruses
ASSEMBLY OF VIRUSES
NUCLEUS
STRONG DEPENDENCE
ON NUCLEAR
TARGETING/TRANSPORT
PATHWAYS
ADENO, HERPES
ASSEMBLY OF VIRUSES
GOLGI COMPLEX
OLIGOSACCHARIDE
MATURATION IN GOLGI
COMPARTMENTS
BUNYA, HERPES, POX
ASSEMBLY OF VIRUSES
CYTOPLASM
ASSOCIATION WITH
MEMBRANES OF THE
SECRETORY OR
ENDOCYTIC PATHWAYS
REOVIRUSES, PICORNA
ASSEMBLY OF VIRUSES
PLASMA MEMBRANE
ASSEMBLED VIRUS
NAVIGATE
ADDITIONAL
COMPARTMENTS OF THE
SECRETORY PATHWAY
TOGA, RHABDO,
ORTHOMYXO,
PARAMYXO, RETRO

8. SITES OF VIRUS ASSEMBLY
Assembly of Viruses

9. NUCLEAR IMPORT/ EXPORT AND SECRETORY PATHWAYS
OF NUCLEIC ACIDS AND PROTEINS
Assembly of Viruses
1
• THE NUCLEAR PORE
COMPLEX
2
• NUCLEAR LOCALIZATION
SIGNALS
3
• NUCLEAR TRANSPORT
PATHWAYS
1
• TRANSLOCATION
2
• POST TRANSCRIPTIONAL
MODIFICATIONS
3
• PROTEIN LOCALIZATION
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 PACKAGED
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 : σ1, σ3, μ1, λ2)
 Outer capsid protein sigma1 forms trimers that extend from the fivefold axes of virions and mediates viral
attachment to cellular receptors
 Another protein λ2 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
Assembly of
nucleocapsid
Association of free
N subunits with the
genome or template
RNA to form the
helical RNP
structure
Assembly of
nucleocapsid
Association of
the PL
complex

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
1
• ASSEMBLY FROM INDIVIDUAL PROTEIN
MOLECULES
2
• ASSEMBLY FROM A POLYPROTEIN
PRECURSOR
3
• CHAPERONE-ASSISTED ASSEMBLY

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 :
A. 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
 In 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
PROTEOLYTIC
PROCESSING OF
VIRION PROTEINS
RETROVIRUSES
A.
CLEAVAGE OF
POLYPROTEINS
PICORNAVIRUSES
SPUMARETROVIRUSES
B.
CLEAVAGE OF
PRECURSOR
PROTEINS
C.
ADENOVIRUES

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

50. THANK YOU