Reprogramming the cell by RNA viruses

1
Lecture 3 Reprogramming the cell by RNA
viruses
2
Shut-off of host-cell translation
3
Picornaviruses
5NCR
3NCR
poly(A)
single, long ORF
IRES
Picornavirus polyproteins
Cardiovirus
pol
L
pro
1A
1B
1C
1D
2B
2C
3A
3C
3D
2A
3B
pro
pro
Aphthovirus
pol
1A
L
1B
1C
1D
2B
2C
3A
3C
3D
3B
2A
1-3
Enterovirus
pro
pro
pol
1A
1B
1C
1D
3A
2C
3C
3D
2A
2B
3B
4
Eukaryotic Initiation Factor 4G (eIF4G)
m7GpppN
eIF4E
PABP
eIF3
eIF4A
eIF4A
Entero-, Rhinovirus 2A Proteinase
635 / 636
Aphthovirus L Proteinase 642 / 643
shut-off of host-cell cap-dependent mRNA
translation
eIF4E - cap binding eIF4A -
bidirectional RNA helicase eIF3 - ribosome
binding PABP poly(A) binding protein
5
m7GpppN
eIF3
eIF4A
eIF4A
eIF4E
PABP

IRES can bind this portion of the (cleaved)
initiation complex directly to initiate virus
translation
6
Eukaryotic Initiation Factor 4G (eIF4G)
m7GpppN
eIF4E
PABP
eIF3
eIF4A
eIF4A
MnkI
eIF4E-BP (eIF4E binding protein)
eIF4E (soluble form)
phosphorylation / dephosphorylation
eIF4E-BP
Cardiovirus 2A protein increases phosphorylation
of 4EBP
eIF4E
phosphorylated form binds eIF3
7
Re-modelling the Cytoskeleton
8
Cytopathic Effect in FMDV-infected Cells
0 hrs
1.25 hrs
2.5 hrs
Armer,H., Moffat,K., Wileman,T., Belsham,G.J.,
Jackson,T., Duprex,W.P., Ryan,M.D. Monaghan, P.
(2008). Foot-and-Mouth Disease Virus, but Not
Bovine Enterovirus, Targets the Host Cell
Cytoskeleton via the Nonstructural Protein 3Cpro.
J. Virol. 82, 1055610566.
9
Actin
FMDV protein 2C
FMDV
Tubulin
2.5 3 hours
10
FMDV re-models microtubules
FMDV
BEV
anti-?-tubulin (red) virus capsid proteins - green
11
FMDV (3Cpro) re-localises, but does not degrade,
?-tubulin
FMDV
BEV
anti- ? -tubulin (red)
12
Re-modelling the Endomembrane system
13
Re-modelling cytoplasmic membranes
All positive-stranded RNA viruses situate their
replication complexes on membranous surfaces.
Many different viruses have been shown to induce
changes in membrane structures. Picornaviruses
poliovirus-infected cells contain virus-induced
vesicles derived from
the ER. Flaviviruses convoluted
membranes and vesicle packets containing
cellular protein
markers from the intermediate compartment (IC)
and the trans- Golgi
network (TGN). Hepatitis C virus A membranous
web of vesicles is formed associated with,
and derived from, the
ER. Pestiviruses BVDV-infected cells
contain tubules and vesicles derived from the
rough ER. Alphaviruses
Sindbis virus (SV) and Semliki Forest Virus
(SFV)-infected cells show
membrane invaginations and spherules
associated with lysosome and
endosomes. Rubella
Replication complexes are situated on
lysosomes. Nodaviruses Flock House Virus
(FHV) replicates in spherules formed by
invaginations of
mitochondrial membranes. Plant ve strand RNA
viruses also use the surface of vacuoles and
chloroplasts
14
Site of FMDV Replication
5-BrUTP incorporation (RNA replication)
anti-capsid antibodies
merge
Monaghan, P., Cook, H., Jackson,T., Ryan,M.D.
Wileman, T. (2004). The ultrastructure of the
developing replicationsite in foot-and-mouth
disease virus-infected BHK-38 cells. J.
Gen.Virol. 85, 933946
15
The Cytopathic Effect Picornavirus infections
Replication complexes (poliovirus)
Golgi apparatus
(disappears)
16
FMDV Replication Site
predominantly single-membrane vesicles
occasional double-membrane vesicles
17
40C
32C
32C
Moffat,K., Knox,C., Howell,G., Clark,S.J.,
Yang,H., Belsham,G.J.,Ryan,M.D. Wileman, T.
(2007). Inhibition of the Secretory Pathway by
Foot-and-Mouth Disease Virus 2BC Protein Is
Reproduced by Coexpression of 2B with 2C, and the
Site of Inhibition Is Determined by the
Subcellular Location of 2C. J. Virol. 81,
1129-1139.
18
(No Transcript)
19
Coat proteins assemble on the membrane surface
and curve the membrane into a vesicle
COP II coated vesicles
COP I coated vesicles
inactive,soluble ARF-GDP
active,membrane- bound Arf-GTP
Sar I
Arf
20
GBF1, BIG1 BIG2 are guanine nucleotide
exchange factors for Arf1 These GEFs rapidly
cycle between cytosolic and membrane-bound
forms The drug Brefeldin A (BFA), an
non-competitive inhibitor of the exchange
reaction that binds to an Arf-GDP-Arf GEF
complex, stabilizes GBF1 on Golgi membranes
collapses Golgi back onto the ER
GBF1 BIG1 BIG2
21
3Cpro
3Dpol
2C
2Apro
1B
1C
1D
1A
2B
3A 3B
Picornavirus 2C proteins are AAATPase
proteins 2C proteins hydrolyse ATP to
ADP Transfection of cells with plasmids
expressing 2C leads to alterations in membrane
structures
22
3Cpro
3Dpol
2C
2Apro
1B
1C
1D
1A
2B
3A 3B
Enterovirus 3A protein inhibits endoplasmic
reticulum (ER)-to-Golgi transport. Suggested to
be important for viral suppression of immune
responses. 3A inhibits the activation of the
GTPase ADP-ribosylation factor 1 (Arf1), which
regulates the recruitment of the COP-I coat
complex to membranes. 3A specifically inhibits
the function of GBF1, a guanine nucleotide
exchange factor for Arf1, by interacting with its
N-terminus.
23
3Cpro
3Dpol
2C
2Apro
1B
1C
1D
1A
2B
3A 3B

3CDpro
3A 3B
Proteins 3C and 3D are produced by 3C proteolytic
processing of the 3ABCD ( or P3) precursor, but
the uncleaved form ( 3CD ) is also produced,
and is a stable product (is not processed into 3C
3D). 3CD is a proteinase and processes the
1ABCD (or P1) capsid proteins precursor. In
poliovirus-infected cells, there is a dramatic
redistribution of cellular pools of Arf1 that
coincides with the reorganization of membranes
used to form viral RNA replication
complexes. 3CD promotes binding of Arf1 to
membranes by initiating recruitment to membranes
of guanine nucleotide exchange factors (GEFs),
BIG1 and BIG2.
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Viroporins
25
Viroporins alter membrane permeability a
physical pore is formed upon oligomerisation
within membranes.
26
VIROPORINS
Picornaviridae Poliovirus 2B 97aa Togaviridae
SFV 6K 60aa Sindbis virus 6K
55aa Ross River virus 6K 62aa Flaviviridae
HCV p7 63aa Retroviridae HIV-1 Vpu
81aa Paramyxoviridae HRSV SH
64aa Orthomyxoviridae Influenza A virus M2
97aa Reoviridae ARV p10 98aa Phycodnaviri
dae PBCV-1 Kcv 94aa Rhabdoviridae BEFV alpha
10p 88aa
27
Picornavirus protein 2B
3Cpro
3Dpol
2C
2Apro
1B
1C
1D
1A
2B
3AB
amphipathic ?-helix
viroporin 2B localises to the ER, releasing Ca
ions stored in ER into the cytoplasm Ca ions
act as second signals within the cell
28
The M2 Proton Channels of Influenza A and B
Viruses.
Influenza virus RNA segment 7 encodes two proteins
1027
M1 ORF
1
1027
1
M2 ORF
RNA splicing
29
The M2 Proton Channels of Influenza A and B
Viruses.
M2 proteins are homo-tetrameric, type III
integral membrane proteins containing a small
N-terminal ectodomain, a single transmembrane
domain, and C-terminal cytoplasmic tail. The
A/M2 ion channel protein of influenza A virus is
found in the virion. The transmembrane domain
acts as a signal sequence to target the nascent
polypeptide domain to the membrane of the
endoplasmic reticulum as it emerges from the
ribosome, and this hydrophobic domain also serves
as the stop-transfer sequence to anchor the
protein in the membrane. The transmembrane
domain becomes the pore of the channel. The
predicted membrane spanning domains of A/M2 and
BM2 are 20 amino acids long and the N-terminal
domain of the BM2 protein (7 residues) is shorter
than that of the A/M2 protein (23 residues).
30
Life cycle of influenza virus showing the steps
in which the M2 ion channel functions.
The M2 proton channels function while the virion
is contained in the endosome (step 3) to permit
subsequent uncoating. For certain subtypes
of influenza A virus the M2 protein also shunts
the pH gradient of the trans-Golgi apparatus to
prevent premature conformational change in
haemagglutinin (step 6)
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HIV-1 vpu
32

• Vpu is an 81 amino acid type 1 integral membrane
  protein composed of three discrete ?-helices.
• The N-terminal helix constitutes the
  transmembrane anchor and is followed by a
  cytoplasmic tail containing two amphipathic
  ?-helices.
• The Vpu protein has two main roles in the viral
  life cycle
• It promotes the efficient release of viral
  particles from the cell surface
• It induces the degradation of CD4, and possibly
  other transmembrane proteins, in the endoplasmic
  reticulum (ER).

Alternative models of Vpu structure
33
CD4 Degradation In Vpu expressing cells, CD4 is
rapidly degraded in the ER and its half-life
drops from 6 hours to 15 minutes. Co-immunoprecip
itation experiments showed that CD4 and Vpu
physically interact in the ER and that this
interaction is essential for targeting CD4 to the
degradation pathway. It is not clear how CD4
goes from this targeted state to physical
degradation in the cytosolic proteasome. Virus
Release Vpu forms ion-conductive pores in the
plasma membrane. How an ion channel activity of
Vpu could lead to enhanced viral particle
production is still unclear. local modification
of the electric potential at the plasma membrane,
leading to facilitated formation and release of
membrane budding structures? Vpu channel could
induce cellular factors involved in the late
stages of virus formation or exclude cellular
factors inhibitory to the viral budding process.
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Re-modelling nuclear pores
35
The nucleus is bounded by a double membrane, the
nuclear envelope, that is continuous with the
endoplasmic reticulum (ER).
(NPC)
36
Nuclear Pore Complex (NPC)
cytoplasm
nucleoplasm
Proteins that comprise the structure of the
nuclear pore are nucleoporins (Nups 100s!)
37
Small molecules can diffuse freely through the
nuclear pore. Larger molecules require active
transport.
38
The GTP binding protein Ran regulates Nuclear
Transport The Ran-GTP/GDP cycle
39
nuclear import
nuclear export
Fully processed mRNAs must interact with nuclear
export receptors for nuclear export
40
Viruses may alter nuclear trafficking mRNAs
and/or proteins
41
Vesicular Stomatitis Virus (VSV) Rhabdovirus
5 major virus proteins nucleoprotein
(N) phosphoprotein (P) matrix protein
(M) glycoprotein (G) (large) polymerase protein
(L)
single-stranded, -ve sense RNA genome, 11kb
Nuclear export of mRNAs is a central step in
eukaryotic gene expression. A defect in bulk
poly(A) RNA export can be caused either by a
direct disruption of the mRNA export machinery or
by an indirect effect on mRNA biogenesis. One
example of interference with the mRNA export
pathway is viral-host interactions involving mRNA
export factors. VSV M protein binds the mRNA
nuclear export factor Rae1 that is in complex
with the nucleoporin Nup98, resulting in nuclear
retention of cellular mRNAs. (Rae1 attaches
cytoplasmic mRNPs to the cytoskeleton)
42
Influenza Virus NS1 Protein. Multifunctional
protein - localized in
both the cytoplasm and in the nucleus of infected
cells. In the cytoplasm, NS1 inhibits innate
immune response by forming a complex with the
pathogen sensor RIG-I and by targeting PKR and
the RNase L pathway. In addition, NS1 has been
shown to activate phosphatidylinositol-3-kinase
signalling, which may be important for promoting
viral replication. In the nucleus, NS1 inhibits
host gene expression this effect includes mRNA
processing, which is mediated by interaction of
NS1 with polyadenylation factors - cleavage and
polyadenylation specificity factor (CPSF) and
poly(A) binding protein II (PABII) and a
putative splicing factor (NS1-BP). The binding
of NS1 to CPSF and PABII inhibits polyadenylation
of host mRNAs, contributing to nuclear retention
of these messages.
43
A transcript RNA is cleaved 15-25 nts past a
conserved AAUAAA sequence - a polyadenylation
signal sequence
AAUAAA
15-25nts
cleavage and polyadenylation specificity factor
(CPSF)
AAUAAA
AAAAAAn
poly(A) polymerase ATP
- template independent
44
The mRNA export receptors TAP-NXT are key
constituents of the mRNA export machinery that is
responsible for nuclear exit of 70 of cellular
mRNAs. This heterodimer interacts with both
messenger ribonucleoprotein particles and nuclear
pore complex proteins (nucleoporins Nups) to
direct mRNAs through the nuclear pore complex
(NPC). The mRNA export factor Gle2
(Rae1/mrnp41), which shuttles between the nucleus
and the cytoplasm, forms a complex with RNPs,
TAP, and the nucleoporin Nup98. It has been
proposed that Rae1 may recruit TAP to Nup98 to
mediate transport through the NPC. Influenza
virus NS1 forms an inhibitory complex with TAP
/ NXT / Gle2
Influenza