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Assembly
Lecture 11
Virology W3310/4310
Spring 2012
“Anatomy is des.ny.”
-­‐-­‐SIGMUND FREUD
1
All virions complete a common set of assembly reac3ons
*
common to all viruses
common to many viruses
2
The structure of a virus par3cle determines
• How it is formed
• Hos it enters a new cell
• How it replicates
3
A tale of two picornaviruses
•
Poliovirus virions survive passage through the stomach to replicate in the gut: acid resistant capsid
•
Rhinovirus virions are inacEvated in the gut and only replicate in the respiratory tract: acid sensi3ve capsid
4
Despite varia3ons in structure and biological proper3es, all infec3ous virions must be METASTABLE
•
•
•
Stable enough to survive in the wild
Unstable enough to come apart during infecEon
The assembly pathway is irreversible in the cell that makes the virions, but reversible in the uninfected cell receiving the virion
5
Produc3on of virus par3cles depends on host cell machinery
•
Cellular proteins catalyze or assist the folding of individual protein molecules
•
Transport systems move viral proteins and nucleic acids to and from sites of assembly
•
Secretory pathway processes and moves viral membrane proteins
•
Nuclear import and export machinery moves viral nuclear proteins and nucleic acid in and out of the nucleus
6
Concentra3ng components for assembly: Nothing happens fast in dilute solu1ons
•
Viral components oOen are concentrated so much that they are visible in the microscope (so-­‐called ‘factories’ or ‘inclusions’
•
Non-­‐enveloped viruses oOen use internal membranes to concentrate proteins (poliovirus)
7
Viral proteins achieve high concentra3ons by several methods
•
Viral replicaEon and translaEon occur in a compartment (‘factory’ or localized site)
•
Localized producEon of viral proteins and protein-­‐
protein interacEons enable formaEon of independent sub-­‐assemblies
•
Local concentraEons of viral structural components can be boosted by lateral interac4ons between membrane-­‐associated proteins (e.g. membrane ‘patches’)
8
GeFng things to the right place
9
Viral proteins have ‘addresses’ built into their structure
• Membrane proteins go to the appropriate membranes
-­‐
Signal sequences, faWy acid modificaEons
• Membrane proteins stay in the appropriate membranes
-­‐
RetenEon signals
• Nuclear proteins go to the nucleus
-­‐
Nuclear localizaEon sequences (NLS)
• Viral mRNA or ribonucleoprotein complexes move into the cytoplasm
-­‐
Nuclear export signals
• Capsids and protein complexes exhibit directed moEon, not diffusion
-­‐
Microtubules and intermediate filaments are the tracks; dyneins, kinesins, myosins are the motors
10
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CHAPTER 12
Localiza3on of viral proteins to the nucleus Plasma
membrane
Golgi apparatus
Ribosome
Rough endoplasmic reticulum
Py(VP1)5 + VP2 /3
Ad hexon +
100 kDa
Nuclear envelope:
Outer nuclear membrane
Inner nuclear membrane
Nucleus
Nuclear pore complex
Mitochondrion
Cytoskeleton:
Influenza virus NP
Intermediate filament
Microtubule
Actin filament bundle
Extracellular matrix
Figure 12.1 Localization of viral proteins to the nucleus. The nucleus and major membrane-bound
compartments of the cytoplasm, as well as components of the cytoskeleton, are illustrated schematically
and not to scale. Viral proteins destined for the nucleus are synthesized by cytoplasmic polyribosomes, as
illustrated for the influenza virus NP protein. They engage with the cytoplasmic face of the nuclear pore
11
Figure 12.2 Localization of viral proteins to the plasma membrane. Viral envelope glycoproteins (red)
are cotranslationally translocated into the ER lumen and folded and assembled within that compartment.
They travel via transport vesicles to and through the Golgi apparatus and from the Golgi apparatus to the
plasma membrane. The internal proteins of the particle (purple) and the genome (green) are also directed
to plasma membrane sites of assembly.
Localiza3on of viral proteins to plasma membrane Other viral
proteins
Cell surface
viral protein
Viral RNA
Nascent viral protein
Golgi
Fusion
Nucleus
Transport
vesicle
Mitochondrion
Microtubule
ER
Cytoskeleton
Extracellular matrix
12
E X P E R I M E N T S
Movement of vesicular stomatitis virus nucleocapsids
within the cytoplasm requires microtubules
stomatitis virus nucleocapsids
be transported to the plasma
e for assembly and budding of
cles contain the (−) strand RNA
nd several viral proteins, includrotein (see the text). To examine
ar trafficking of nucleocapsids,
e coding for a green fluorescent
GFP) was inserted into that for
region of the P protein. Coniments established that the Pon protein catalyzed both viral
nthesis and genome replication,
t exhibited somewhat reduced
urthermore, mutant particles
P-eGFP in place of the P proinfectious.
cted cells, P-eGFP colocalized
ly synthesized viral RNA, as
h the N and L proteins, in cytoructures of the size predicted
capsids. Time-lapse imaging of
GFP-containing structures indit nucleocapsids move toward
eriphery and, unexpectedly, do
e association with mitochonignificance of this association is
ear. However, as mitochondria
n to move on microtubules, it
kely that viral nucleocapsids
o. Indeed, nucleocapsids were
to be distributed throughout
asm in close association with
les (panel A below). Treatment
d cells with drugs that disrupt
les, such as nocodazole, dramatred this pattern: nucleocapsids
became clustered in the absence of microtubules in large aggregates and did not
reach the plasma membrane (panel B).
Such drugs also reduced virus yield significantly, confirming the importance of
microtubules in the transport of vesicular
somatitis virus nucleocapsids to sites of
assembly.
Movement of VSV nucleocapsids in cytoplasm requires microtubules
Das, S. C., D. Nayak, Y. Zhou, and A. K. Pattnaik.
2006. Visualization of intracellular transport of
vesicular stomatitis virus nucleocapsids in living
cells. J. Virol. 80:6368–6377.
Localization of P-eGFP-containing nucleocapsids (green) and microtubules (red) in cells
infected by the mutant virus VSV-PeGFP and untreated (A) or treated with nocodazole prior to
infection (B). Nuclei are in blue. Courtesy of Asit Pattnaik, University of Nebraska—Lincoln.
A
B
Box 12.11
13
Three strategies for making sub-­‐assemblies
14
viral chaperone
15
Sequen3al capsid assembly: Poliovirus
16
Assembly intermediates and the assembly-­‐line concept
• Assembly-­‐line mechanisms ensure orderly formaEon of viral parEcles and virion subunits
• FormaEon of discrete intermediate structures
• Can’t proceed unless previous structure is formed: quality control
17
{sequenEal}
Viral scaffolding proteins • establish transient intermediate structures
• viral proteases packaged in these intermediate structures become acEvated to finalize structure
18
Sequen4al Assembly
Adenovirus genome packaged into a preformed shell in the nucleus
19
Self-­‐assembly versus assisted assembly reac3ons
• Self-­‐assembly
-­‐ HIV capsid proteins can form empty shells
-­‐ Influenza HA glycoprotein can bud and form virus-­‐like parEcles
-­‐ HBV surface anEgen can assemble into virus-­‐like parEcles
• Assisted assembly
-­‐ Can’t assemble on their own
-­‐ Proteins and nucleic acid genomes are required as scaffolds or chaperones to form structure
20
Concerted Assembly
Influenza virus parCcles form by budding
21
22
23
Concerted Assembly
Retrovirus parCcles form by budding
Mature a6er release
24
434
434
CHAPTER 12
A
A
CHAPTER 12
Signal
Signal
sequence peptidase
Signal
Signal
sequence peptidase
ALV
Fusion
Extracellular domain
Fusi
Cytoplasmic
domain
Transmembrane
Signal
peptidase
ALV
Signal
peptidase
HIV
HIV
Variable regions
Variable regions
B
S S
TM
B
Fusion peptide
SU
SU
Viral membrane
Table 12.4
Figure 12.13 Modification and processing of retroviral Env polyproteins.
(A) Sequence features and modifications of the Env proteins of avian leukosis
virus (ALV) and human immunodeficiency virus type 1 (HIV) are depicted as
in Fig. 12.3. The variable
Env
TM regions of human immunodeficiency virus type 112.13
Modific
differ greatlySin Ssequence among viral isolates. The translocation Figure
products shown
here are cleaved by the ER signal peptidase (red arrows) and (A)
by furin
family
Sequence
features
proteases in the trans-Golgi network (orange arrowheads). The latter liberates
(ALV) and hum
Fusion
peptidefrom thevirus
the transmembrane (TM) and surface unit
(SU) subunits
Env precursor.
in maintain
Fig. 12.3.
(B) Both noncovalent interactions and a disulfide bond (-S-S-) can
the The variab
association of the SU and TM proteins.
differ greatly in sequen
Examples of acylated or isoprenylated viral proteins
Virus
Protein
Lipid
Viral membrane
Probable function
here are cleaved by th
proteases in the transthe transmembrane (T
(B) Both noncovalent
association of the SU a
Envelope proteins
Alphavirus
Sindbis virus
Coronavirus
E2
Palmitate
Required for efficient budding of virus particles
25
• AddiEon of lipid to viral proteins allows targeEng to membranes independent of signal sequence
• Viral proteins are synthesized in the cytoplasm, and modified with lipids post-­‐
translaEonally
• Also added -­‐ geranylgeranol (C20) or palmitate
26
• Changes at the myristoylaEon sequence prevent interacEon of Gag with the cytoplasmic face of the plasma membrane
• Virus assembly and budding are inhibited
27
Genome packaging
•
Viral genomes must be disEnguished from cellular DNA or RNA molecules where assembly takes place
•
Requires discriminaEon among similar nucleic acid molecules
•
Example: retrovirus genomes <1% cytoplasmic RNA, yet is the RNA packaged in majority of retrovirus parEcles
•
DiscriminaEon is the result of packaging signals in the viral genome -­‐ sequences necessary for incorporaEon of nucleic acid into virions; geneEcally defined
28
Packaging signals -­‐ DNA genomes
Adenovirus
• Packaging signal near leO inverted repeat and origin
• Signal is complex: a set of repeated sequences; overlapping with enhancers that sEmulate late transcripEon
• Structure is recognized by viral protein IV2a (also a transcripEon acEvator)
29
•Herpesvirus genome replicaEon produces concatemers with head-­‐to-­‐tail copies of viral genome
•HSV-­‐1 packaging signals pac1 and pac2 needed for recogniEon of viral DNA and cleavage within DR1
30
Packaging signals -­‐ RNA genomes
350 nt; necessary and sufficient
env mRNAs not packaged
Necessary but not sufficient for HIV-­‐1 genome packaging
31
Packaging signals -­‐ RNA genomes
•
NC of Gag mediates selecEve encapsidaEon of genomic RNA during assembly
•
Central region binds RNAs with Ψ sequences
•
Structure of HIV-­‐1 NC bound to Ψ SL3 shows protein contacts with RNA
32
Packaging signals -­‐ RNA genomes
• Packaging limits -­‐ upper limit on size of viral nucleic acid that can be accommodated in icosahedral capsid or nucleocapsid; ~5 -­‐ 10% larger than viral genome
• Coupling of encapsidaEon of viral nucleic acid with its synthesis may contribute to specificity, e.g. poliovirus RNA replicaEon on membrane vesicles; no packaging signal idenEfied in genome
33
Packaging of segmented genomes
• How to ensure that virions receive one copy of each segment? Random or selec4ve mechanisms for influenza virus (8 segments) cannot be disEnguished
• Random mechanism would yield 1 infecEous parEcle per 400 assembled -­‐ within the known parEcle to pfu raEo
• If 12 segments are packaged, 10% of parEcles would contain complete viral genome
• New evidence for specific packaging sequence on each RNA segment
34
Influenza virus RNA packaging
hWp://www.virology.ws/2009/06/26/packaging-­‐of-­‐the-­‐segmented-­‐influenza-­‐rna-­‐genome/
35
Selec3ve packaging
•
Bacteriophage ϕ6 -­‐ 3 dsRNA segments S, M, L
•
S segment enters alone; entry of M depends on presence of S; entry of L depends on presence of S + L
•
•
Serial dependence of packaging
ParEcle:pfu raEo ~1
36
Acquisi3on of an envelope •
May follow assembly of internal structures (most enveloped viruses)
•
May be simultaneous with assembly of internal structures (retroviruses)
37
Four budding strategies
Envelope glycoproteins and capsid are essenEal for budding -­‐ alphaviruses
Internal matrix or capsid proteins drive budding -­‐ retroviruses
Envelope proteins drive budding -­‐ coronavirus
Matrix proteins drive budding, but addiEonal components (glycoproteins, RNP) needed for efficiency or accuracy
38
Internal structure assembly and budding spaEally & temporally separated
39
40
When synthesized alone, Gag directs budding of virus-­‐like parEcles
Internal structure assembly and budding are coincident in space & Eme
41
•
Amino acid change in Gag cause arrest of budding at a late stage (late or L domains)
•
Three different classes of L domains, found in + and -­‐ strand enveloped viruses
•
L domains bind cell proteins, involved in vesicle trafficking, needed for virus release
42
L domain mo3fs
43
Involvement of the ESCRT machinery in three topologically equivalent types of membrane abscission
44
45
Exocytosis: reverse of endocytosis; used by viruses that assemble within vesicular compartments of the ER or Golgi
Pathways of herpesvirus assembly and egress
46
The majority of viruses leave an infected cell by one of two general mechanisms
•
•
Release from the cell by budding or lysis
Movement from cell to cell
47
HIV-­‐1 infected T-­‐cell
Polarized release
Of HIV from Infected T-­‐cell
Epithelial cell
48
Egress (exit) in vivo is a controlled process, oVen polarized
• Via apical surface places virus in the outside world (sneezing)
• Via basal surface of epithelial cells places virus in contact with blood, lymph, nerves (systemic spread, bad news)
• Can occur at sites of cell contact (lungs, gut, synapses)
• DirecEon and mode of egress influence pathogenesis
49
Polarized spread of infec4on by an alpha herpesvirus: Release from axon terminals and infec4on of epithelial cells in vitro
Single Axon
Purple: nuclei of
epithelial cells
Green: GFP expressing virus 50
Why do infected cells lyse?
• InhibiEon of cell macromolecular processes and transport
• ApoptoEc lysis
• Specific mechanisms
-­‐ Adenovirus L3 protease cleaves intermediate filament proteins
-­‐ Damages structural integrity of cell, facilitates virus release
-­‐ Adenovirus glycoprotein required for cell lysis, mechanism not known
51
439
Intracellular Trafficking
B
Inhibition of secretion
Induction of autophagy
Endoplasmic
reticulum
Model for nonly3c release of poliovirus par3cles
Transport
vesicle
Cytosolic
contents
Intermediate
compartment
Protein 3A
Endosomal
fusion
cis
Medial
trans
Golgi
Lysosomal
fusion
2BC
3A
Lc3-pe
Poliovirus polymerase
Lamp-1/2
llular secretory pathway in poliovirus-infected cells. (A) Electron
lls (left) and HeLa cells 5 h after poliovirus infection (right) preserved
fected cell-specific vesicles can been seen in the infected cells. G, Golgi
virus particles. The bars indicate 1 µm. Adapted from A. Schlegel
with permission. Courtesy of Karla Kirkegaard, University of Colorado,
52
Extracellular virion matura3on
Acidianus convivator virus, from acidic hot spring (pH 1.5, 85-­‐93°C)
53