Download Virus Assembly/Release

Virus Assembly/Release
Goal of virion structure:
protect genome from environment
facilitate disassembly upon infection
(virions are metastable structures)
Virus Assembly relies on host cell pathways
Steps governed by structure
enveloped or non-enveloped
Steps influenced by cellular site of genome
replication
Overview of Assembly of Non enveloped viruses
1. Assembly of capsid structure
2. Insertion of genome into capsid
3. Release of virions from cell requires
cell lysis (at least partially)
4. Assembly may occur in cytoplasm or nucleus
depending upon virus family
Nonenveloped virus Assembly
Picornavirus Assembly--simplest
Replication—cytoplasm and
associated with membranes
Other non-enveloped viruses (eg Adenovirus))
assembly is more complex
(Capsomer--VP1, VP3, VP0)
RNA
60 capsomers or
12 pentamers
Packaging of RNA
Linked to replication
Only + RNA not –RNA
Mechanism?
Fields Virology
Assembled Progeny Picornaviruses in Infected Cell Cytoplasm
Release of Picornaviruses--cell lysis?
Due to cell death or specific mechanism?
Possible Role of Autophagic Vesicles in
Polio Virus Release
Overview of Assembly of Enveloped Viruses
1. Assembly of capsid or nucleocapsid with genome
to form core
2. Assembly of envelope (modified patch of cell
membrane
3. Association of core with membrane
4. Release from cells by budding from a membrane
plasma membrane or
Golgi membranes or
RER membranes or
Nuclear membranes
5. Does not require cell death
Assembly and Release
Enveloped Viruses
Assembly
Parts of virion
budding
Does not require
Cell death
Membrane scission
Influenza Virus Assembly/Release
Assembly of Influenza
Proteins in Virion
*HA
*NA
envelope
M2
*M1
PB1, PB2, PA
Core (RNP)
*NP
*major structural proteins
A. Formation of the Envelope
Envelope: HA, NA, M2
HA, NA, and M2 are transmembrane glycoproteins
Use host secretory pathway for:
membrane insertion
pottranslational modifications
transport to PM
Types of viral
glycoproteins
HA= type 1
NA= type 2
Translation and Membrane Insertion of
Glycoproteins
Signal peptide of type 2=TM
Post Translation Modification of Glycoproteins
1. Glycosylation (RER, Golgi)
N linked (signal NXT/S)
(O linked--Not flu)
2. Oligomerization (RER)
HA trimer
NA tetramer
3. Disulfide bond formation (RER)
4. Proteolytic cleavage (RER, Golgi, extracellular)
1. Glycosylation
Addition of Core oligosaccharide to nascent chain
Roles of Glycosylation
folding
protease protection
mask from immune responses
Modification of
Core sugars-N linked
Glycosylation
NST
NXS
Endo H
Mannose
N acetyl glucosamine
glucose
various (peripheral sugars:
N acetyl glucosamine, galactose,
fucose, sialic acid)
2. Oligomerization—in the RER
Structure of HA
Trimer
H1--blue
H2--colors
3. Disulfide Bond Formation
In RER
Host cell disulfide isomerases (eg PDI)
Most likely co-translational
Intramolecular disufide bonds
Sometimes intermolecular disulfide bonds
stabilize oligomers
4. Proteolytic Cleavage
Cleavage of HA required for virus entry (fusion) but not
attachment
Cleavage site
TM
HA
H1, H2, H3 Single basic amino acids
cleavage only in RT
extra-cellular protease only in
respiratory tract (RT)
H5
Furin site = sequence RXR/KR
cleavage in many tissues
Golgi enzyme
5. Transport to cell surface via secretory pathway
Problem: Acid pH--activates HA fusion activity
M2 and passage of HA through Golgi during transport
to the Plasma membrane for assembly
H+
B. Assembly of Nucleocapsids
1. NP, P proteins, NS-2 transported to nucleus
Each has an NLS
2. NP and P proteins assemble with vRNAs (-)
(NP added as RNA is synthesized)
3. M1 transported to nucleus (has NLS)
4. NS2 (NEP) binds Crm-1-RanGTP
5. M1 binds to NP and M1 binds NEP
6. RNPs-M exit nucleus (LxxxLxxLxL)
Exit of RNPs from nucleus
From Fields Virology
Further Issues in Assembly and
Release from Plasma Membrane
1. Assembly and packaging
of genome segments
2. Interactions
Virion proteins
BuddingMembrane
curvature
Membrane scission
Specific mechanism to incorporate one copy of
each segment--evidence
EM--always 8 segments
Serial sections through a single particle
Assembly of Influenza
From Noda, et al Nature 439: 490
Implications: virions
asymmetric
Electron
Tomography of
Budding virions
Domains in vRNAs required for assembly into particles
Mechanisms ??? What is role of specific
packaging sequences??
Fields Virology
Which segments
Interact??
Approach
Arrangements of Segments in Virions
Arrangements
By tomography
data
Tomography + in vitro
Interaction data
Interactions of Protein Virion Components—
Proposed but not well established
HA--M1
NA--M1
NP--M1
M2--M1
Central Role of M1—
Connect membrane
components with core
Further Issues in Assembly and
Release from Plasma Membrane
1. Assembly and packaging
of genome segments
2. Interactions
Virion proteins
BuddingMembrane
curvature
Membrane scission
C. Budding and Release
Membrane curvature?
Membrane scission?
Model:
General mechanism of enveloped
virus budding and release
Observations in HIV system
mutation in gag--particles assemble but
fail to release from cell membrane
Mutation called late domain mutation
At least 5 types of late domains
PTAP
PPXY
YPXL
ΦYX0/2(PΦ)
FPIV
Late domains bind host proteins
For example:
PTAP binds Tsg 101
Tsg 101 member of VPS system
VPS=vacuolar protein sorting system
VPS involved in sorting proteins to
Multivesicular Body--compartment
destined for lysozome
What is the VPS System?
VPS system for
handling proteins
for degradation
VPS and virus budding
Viral budding
And release
Host
pathway
ESCRT mediated
budding
Virology 372: 221 (2008)
Mechanisms for HIV release
Inhibition VPS4 blocks release
Some viral late domains
Phenotype of
Late domain mutations
HIV
MLV
Late domains in RNA viruses usually in M proteins
Expression of M alone=particle release
From Bieniasz, Virology 344: 55
Also see Votteler and Sundquist
Cell Host and Microbe 14: 232
Problem with Influenza Budding and the VPS
No late domains in M1
M1 expressed alone—no particles
VPS 4 not required for virus release
What drives influenza budding?
M1 protein alone—no budding unless targeted
to membranes
NA or M2 expressed alone will release
particles (inefficiently)
HA alone will drive budding
HA+NA+M+M2 best for budding
Mutation of helix in
CT domain of M2
M2 expressed alone forms particles
Cell 142: 902 (2010)
M2 is localized at base of budding virus
Cell 142: 902 (2010)
Model of Influenza Budding
Virology 411: 229 (2011)
Predicts Asymmetry of Virions
1. Segment organization
2. Localization of M2
Implications for:
Envelope-Core Interactions
Glycoprotein-M1 interactions
Organization of Glycoproteins
Asymetry of Virions
Lab strains are basically spherical
Virus from infected lungs are filamentous
Growth of a human isolate in eggs selects
spherical virions
Questions—structure of filamentous virus
implications for assembly
(and mechanisms of entry)
Issues in Assembly
Filamentous virions
RNP
More spherical virions
RNPs contact membrane
only at top
NA tends to cluster at one end
(opposite from RNP)
HA
X-31
Udorn
NA
M2
PNAS 107: 10685
What drives budding??
RNP envelope (HA-RNP?, M1-RNP?) interactions??
What drives membrane scission?
M2 likely but mechanism unclear
budding
Membrane
scission
Model of Influenza Budding
Virology 411: 229 (2011)
D. Assembly and Reassortment
Infection of a cell with two different influenza viruses
results in reassortment of the gene segments
generating new viruses
Likely origin of new pandemic viruses/Antigenic Shift
Classification of Influenza A Viruses
Divided into subtypes or species
Based on HA and NA
In nature
15 different HA genes (H1--H15)
9 different NA genes (N1-N9)
Subtypes designated by which HA and which NA
are present
eg. H1N1 or H3N2
All exist in wild birds
Humans--usually one or two Influenza A strains
Influenza A and Pandemics
1890
H2N2
1900
H3N8
1918
H1N1
1957
H2N2
1968
H3N2
(1977
H1N1)
2009
H1N1
Antigenic Shift
Responsible for periodic
Appearance of new virus
Mechanism of Antigenic Shift
Humans
Birds
H5N8
Pig
H1N1
supports
replication
of both human
and avian viruses
Progeny Virus
H5N8, H1N1, H5N1, H1N8
And different combinations of other segments
1957--HA,NA,PB1 avian
1968--HA, PB1 avian
Pandemic due to new virus H5N1 or
H1N8
Internal genes also reassort with consequences
Requirements for a pandemic virus
1. New HA subtype
2. HA bind to human respiratory epithelium
3. Human to human transmission
4. Replication efficient in human cells
(PB2 implicated)
Pathogenicity varies with particular combination
of genes (varies with pandemic virus—1918 vs
others)
Determinants of pathogenicity incompletely
understood (HA and PB2 definitely implicated)
Originof
ofSegments
Segments ofinNew
Pandemic
H1N1
virus***
Origin
2009
Pandemic
Virus
H1N1
From Garten, et al Science 325: 197 (2009)
Remaining Questions in Influenza Assembly
Role of segment interactions and generation of
reassortants (implications for potential
types of antigenic shift viruses that are possible)
Specifics of interactions between components of
virions
(glycoproteins with M1, with RNP structures)
(M1 with RNP structures)
Mechanisms involved in budding
Mechanisms of membrane scission
Role of NA in release
NA releases
reattached virus
(Tamiflu
Relenza--sialic
Acid analogues)
Consequences of NA defective influenza:
Aggregation and failure to release from
Infected cells
Note location of NA in virions
Budding from specific cell domains--polarized budding
implications for pathogenesis
flu
Mechanism?
Reading:
Fields Virology
Chapter 6 (General discussion of assembly)
Relevant sections of Chapters 24, 47
Also:
Polio assembly PlosPathogens 8: e1003046
Influenza:
PlosPathogens 10:e1003971
Nucleic Acids Research 40: 2197
PNAS 110:e3840