ASSEMBLY OF VIRUSES

MOUSUMI BORAPhD SCHOLAR

DIVISION OF VIROLOGYINDIAN VETERINARY RESEARCH

INSTITUTE

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

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

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 virusesX-ray diffraction studies

HISTORY

Assembly of Viruses

1. Nucleic acid (RNA), 2. Capsomer protein (PROTOMER), 3. Capsid

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

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

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

SITES OF VIRUS ASSEMBLY

Assembly of Viruses

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

•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

•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

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

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

ASSEMBLY OF ADENOVIRUS

Assembly of Viruses

ASSEMBLY OF ENVELOPED VIRUSES IN THE NUCLEUS

Assembly of Viruses

Herpesviruses Capsid is icosahedral; 162 capsomersAssembled in the infected cell nucleusBasic assembly unit is a complex of the major capsid and

scaffolding proteins

MAJOR CAPSIDSCAFFOLDING

PROTEINS

PROCAPSID

ds DNA GENOME PACKAGED

RELEASE OF SCAFFOLDING

PROTEINS

ASSEMBLY OF HERPESVIRUSES

Assembly of Viruses

Brown et al., 2011

ASSEMBLY OF HERPESVIRUSES

ReovirusesSegmented 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

ASSEMBLY OF REOVIRUSES

ASSEMBLY OF REOVIRUSES

PicornavirusesNon -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

ASSEMBLY OF VIRUSES IN CYTOPLASM

ASSEMBLY OF PICORNAVIRUSES

BunyavirusesNegative-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) PROTEINACCUMULATE

IN THE GOLGI COMPONENT OF THE VIRUS FACTORIES

PoxvirusesLinear 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

ASSEMBLY OF POXVIRUS (Vaccinia virus )

Togaviruses

Rhabdoviruses

Paramyxoviruses

Orthomyxoviruses

Retroviruses

ASSEMBLY AT THE PLASMA MEMBRANE

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

ASSEMBLY OF TOGAVIRUSES

ASSEMBLY OF TOGAVIRUSES ( Alphavirus)

RhabdovirusesMinus 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

ASSEMBLY OF RHABDOVIRUSES

Brown et at., 2010

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 PARAMYXOVIRUSESAssembly 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

ASSEMBLY OF PARAMYXOVIRUSES

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

ASSEMBLY OF ORTHOMYXOVIRUSESVirus 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

ASSEMBLY OF ORTHOMYXOVIRUSES

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

ASSEMBLY OF RETROVIRUSES

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

ASSEMBLY FROM INDIVIDUAL PROTEIN MOLECULES

Mechanism Virus Structural unit

Association of individualprotein molecules

Adenovirus (adenovirus type 2) Protein IV trimer (fiber) and protein III pentamer (penton base) thatforms 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

ASSEMBLY FROM INDIVIDUAL PROTEIN MOLECULES

ASSEMBLY FROM A POLYPROTEIN PRECURSORMechanism Virus Structural unit

Assembly from polyproteinprecursors

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

ASSEMBLY FROM A POLYPROTEIN PRECURSOR

CHAPERONE-ASSISTED ASSEMBLYAssembly 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

CHAPERONE-ASSISTED ASSEMBLY

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 membraneModified by insertion of viral proteins are spatiallyand 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

MATURATION OF PROGENY VIRUSVirus-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

PICORNAVIRUSESSPUMARETROVIRUSES B.

CLEAVAGE OF PRECURSOR PROTEINS

C.

ADENOVIRUES

RELEASE OF NASCENT PARTICLES

Non enveloped viruses

Lysis of infected cell

Except Picornaviruses

andPolioviruses

Enveloped viruses

Bud growth

Bud formation

Fusion of the bud membrane

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