INFLUENZA A VIRUS GENOME

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INFLUENZA A VIRUS GENOME
PR8
HK
PB2 PROTEIN
PB2 PROTEIN
PB1 PROTEIN
PB1 PROTEIN
PA PROTEIN
PA PROTEIN
HEMAGGLUTININ
HEMAGGLUTININ
NEURAMINIDASE
NUCLEOPROTEIN
NUCLEOPROTEIN
NEURAMINIDASE
M1, M2 PROTEINS
M1, M2 PROTEINS
NS1, NEP PROTEINS
NS1, NEP PROTEINS
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RNA viruses are classified according to their
genome structure.
Viral genome is mRNA sense. Viral genome acts as
mRNA.
ss () sense
Viral genome is in sense complementary to
mRNA. Viral genome must be copied into mRNA by
RDRP.
ss (-) sense
Viral genome is in sense complementary to some
mRNAs and is the same sense as other
mRNAs. Viral genome must be copied into mRNA by
RDRP.
ss ambisense
dsRNA viral genome must be copied into mRNA by
RDRP.
dsRNA
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RNA Synthesis by Negative-strand RNA Viruses
mRNA
An
5
(-) genome (vRNA)
3
5
Segmented Orthomyxoviridae Bunyaviridae
() anti-genome (cRNA)
5
3
3
5
(-) genome (vRNA)
An
An
mRNAs
5
An
An
(-) genome
3
5
Non-segmented Mononegavirales
() anti-genome
5
3
(-) genome
3
5
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NMR Structure of 31mer RNA Corresponding to the
Ends of an Influenza Virus RNA
From Bae et al. PNAS 98 10602, 2001.
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Influenza A virus panhandle promoter
Data from Fodor et al. J. Virol. 694012, 1995.
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Model of influenza A virus cap-snatching
Li M.L., Rao P., Krug R.M. EMBO J 2001 Apr
1720(8)2078-86
Endonuclease active site
Pol RNA active site
Binding sites for 5 and 3 ends of vRNA
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TYPE A,B, and C INFLUENZA VIRUSES
A B C
Frequency of isolation
/-
Severity of disease
/-
Animal reservoir
YES NO NO
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ANTIGENIC SHIFT ANTIGENIC DRIFT
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EPIDEMIOLOGY OF HUMAN INFLUENZA VIRUSES
B
H3N2
H2N2
A
H1N1
H1N1
40
50
60
70
80
00
90
1918
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INFLUENZA VIRUS
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PLASMID-ONLY INFLUENZA A VIRUS RESCUE
Transcription plasmids
Protein expression plasmids
pPOLI-PB2
POL I
R
pPOLI-PB1
R
POL I
pGT-h-PB1
pPOLI-PA
MLP
pA
R
POL I
pGT-h-PB2
pPOLI-HA
MLP
pA
R
POL I
pGT-h-PA
pPOLI-NP
pA
MLP
POL I
R
pGT-h-NP
pPOLI-NA
MLP
pA
R
POL I
pPOLI-M
POL I
R
pPOLI-NS
POL I
R
Transfection
RESCUED VIRUS
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Influenza virus NP mediates nuclear import of
viral RNPs
O'Neill RE, Jaskunas R, Blobel G, Palese P,
Moroianu J. J Biol Chem. 1995 Sep
29270(39)22701-4.
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Nuclear Import of Influenza Virus RNA Can Be
Mediated by Viral Nucleoprotein and Transport
Factors Required for Protein Import   Robert E.
O'Neill,  Richard Jaskunas,  Günter Blobel, 
Peter Palese,  Junona Moroianu
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NUCLEAR IMPORT ASSAY USING DIGITONIN-PERMEABILIZED
CELLS
FITC-RNA
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IMPORT OF VIRAL RNA INTO NUCLEI OF
DIGITONIN-PERMEABILIZED CELLS
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INFLUENZA VIRUS NP REQUIRES KARYOPHERIN aß FOR
DOCKING AT THE NUCLEAR PORE AND RANp10 FOR
NUCLEAR IMPORT
DOCKING OF NP AT THE NUCLEAR PORE
ADDITION OF Ran AND p10 TRANSLOCATESDOCKED NP TO
THE NUCELUS
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INFLUENZA VIRUS RNP NUCLEAR IMPORT REQUIRES THE
CELLULAR karyopherin a (NPI-1) ? (NPI-3)
PROTEINS
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NEP OF INFLUENZA A,B, AND C VIRUSES POSSESS
NUCLEAR EXPORT ACTIVITY

• JULIE TALON, JASON PARAGAS, ROBERT E. ONEILL,
  AND PETER PALESE

ADOLFO GARCIA-SASTRE
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THE INFLUENZA VIRUS NEP PROMOTES RNP NUCLEAR
EXPORT BY INTERACTING WITH THE CELLULAR NUCLEAR
EXPORT RECEPTOR Crm1
Crm1
M1
NEP
vRNA
NP
Nucleus
Cytoplasm
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THE FUNCTION OF NS2(NEP) AS A NUCLEAR EXPORT
FACTOREXPERIMENTAL EVIDENCE

• NEP BINDS TO THE NUCLEAR EXPORT RECEPTOR CRM1 AND
  NUCLEOPORINS
• NEP CAN REPLACE THE HIV-1 Rev EFFECTOR DOMAIN
• ANTI-NEP ANTIBODY BLOCKS RNP EXPORT
• AA 11-23 PROMOTES EXPORT OF A REPORTER PROTEIN
• NEP IS REQUIRED FOR FUNCTIONAL VIRUS-LIKE
  PARTICLES (VLP)

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EXPRESSION OF CAT FROM pDM128 REQUIRES FUNCTIONAL
REV
RRE
SD
SA
CAT
SV40 promotor
NES
SD
SA
Rev

CAT
CAT EXPRESSED
NO REV--NO CAT
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REV AND NEP FUSION GENES
RRE binding
Activation domain
Rev RevM10 RevM10-NEP NEP
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NEP CONTAINS A REV-LIKE EFFECTOR DOMAIN
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MICROINJECTED ANTIBODIES DIRECTED AGAINST
INFLUENZA VIRUS NEP INHIBIT RNP NUCLEAR EXPORT
x
NUCLEAR EXPORT
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COMPARISON OF NES SEQUENCES FROM VIRAL AND
CELLULAR PROTEINS
Influenza virus proteins NEP (A/PR/8/34) NEP
(B/Yamagata/1/73) NEP (C/California/78)
12ILLRMSKMQL21 10IEWRMKKMAI19
95LWLPMKSLSL105 122MKHQILTRLKL132
Lentivirus proteins HIV-1 Rev VISNA
Rev HTLV-1 Rev
75LPPLERLTL83 106MVGMEMLTL114
81LSAQLYSSLSL91
Cellular proteins PKI Gle1
31LALKLAGLDI46 351LPLGKLTL358
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FUNCTIONS OF INFLUENZA VIRUS PROTEINS

• Ps POLYMERASE
• HA ATTACHMENT, FUSION
• NA NEURAMINIDASE ACTIVITY, RELEASE OF VIRUS
• NP STRUCTURAL PROTEIN,
• IMPORT OF RNA
• M1 STRUCTURAL PROTEIN,BUDDING NUCLEAR EXPORT
• M2 ION CHANNEL
• NEP NUCLEAR EXPORT
• NS1 IFN ANTAGONIST, PLEIOTROPIC
  FUNCTIONS

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NATURE MEDICINE
December 2001 Volume 7 Number 12 pp 1306 -
1312
A novel influenza A virus
mitochondrial protein that
induces cell death
Weisan Chen1, 5, Paul A. Calvo1, 5, Daniela
Malide1, James Gibbs1, Ulrich
Schubert1, 2, Igor Bacik1,
Sameh Basta1, Robert O'Neill3, Jeanne
Schickli3, Peter Palese3, Peter
Henklein4, Jack R.
Bennink1 Jonathan W. Yewdell1
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PB1-F2 induces apoptosis