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IntroductionReplication and Proliferation
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Replication and Proliferation of the Influenza A
Virus
Emily Cribas
Penn State
December 15, 2014
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The Flu
Also known as seasonal influenzaContagious respiratory illness caused by influenza viruses thatinfect the nose, throat, and lungsCan cause mild to severe illness, sometimes leading to deathSymptoms include:
FeverSore throatHeadaches
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A Global Perspective*
Specific subtypes have recently re-emerged as fatal viruses
Estimates of death range from as low as 3,000 to a high of49,000 people from 1976 to 2007
Has become a serious world-wide concern due to re-emergenceor new, deadlier emergence
Strains are becoming antiviral resistant
Influenza A is the most prominent type of flu virus in NorthAmerica
*Information obtained from the Center for Disease Control and Prevention website
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The Influenza A Virus
Belongs to the family ofOrthomyxoviridae, which consist of 8negative-sense single-stranded RNA genomes that encode fordifferent viral proteins including:1
Polymerase acidic protein (PA)
2 polymerase basic proteins (PB1,PB2)
Nucleoprotein (NP)
Matrix Protein (M1)
Proton channel protein (M2)2 non-structural proteins (NS1,NS2/NEP)
Hemagluttin (HA)
Neuraminidase (NA)
Figure: Viral Structurea
aNelson, M. I.; Holmes, E. C. Nature revie
1Chutiwitoonchai, N. et al. PloS oneJan. 2014, 9, ed. by Takimoto, T., e105081. 4 / 2 0
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Overview of Replication
Figure: Influenza A Viral Replication and Proliferation2 Link
2Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;Twitchell, B., Ed.; W. W. Norton and Company: 2011. 5 / 2 0
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5 Basic Steps for Viral Reproduction
1 Adsorption
2 Penetration and Uncoating
3 Replication and Transcription
4 Assembly
5 Virus Release
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1. Adsorption
The virus must bind, through one of its proteins, to one of thesurface receptors found on the lipid bilayer of the host cell3
Viral protein: Hemagluttinin (HA)
Surface (sugar) receptor: Sialic acid
3Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;
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1. Adsorption*
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2. Penetration
The virus enters the cell, where an endosome (vesicle) formsaround it
As the vesicles move toward the nucleus, their pH drops because a
cellular channel is constantly pumping H+
ions into the vesicle
This causes a conformational change in the HA protein, whichexposes a short hydrophobic sequence on HA that can insert intothe endosomal membrane, causing it to fuse to the viral envelope
When this happens, the viral RNAs are released into thecytoplasm4
4Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;
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2. Uncoating
M1 forms the shell of the virion, and therefore must be releasedbefore the genome is released into the cell
M2 forms a similar channel that pumps protons from the endosometo the virus, which in turn lowers the internal pH and leads to M1
release
Figure: Penetration & Uncoating5 Link
5Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third; 10/20
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3. Nuclear Localization Signals
The genome in Influenza A contains 2 well-known nuclearlocalization signals (NLSs) found on the nucleocapsid protein(NP), (one unconventional and one bipartite) that are specificpeptide sequences telling the RNA to move into the nucleus6
The unconventional NLS:It binds to karyopherins 1 and 2Found between residues 3 and 13Indispensible for NP nuclear localization (especially residues 7and 8)
The bipartite NLS:
Located between residues 198 and 216Indispensible in NP nucleolar localization
Important for vRNA transcription6Ozawa, M. et al. Journal of virology Jan. 2007, 81, 3041.11/20
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3. Entrance into the Nucleus
NLSs on the NP are recognizedby nuclear pore receptors(karyopherins) that interact withthe cytosolic fibrils that extendfrom the rim of the pore
They are pulled by the fibrilsalong with the receptor, whichguides it along the way bygrabbing onto short amino acid
sequences inside the nuclearfibrils, until the RNA is releasedinto the nucleus
The empty receptor then returns
to the cytosol via the nuclearpore for reuse
Figure: Nuclear Entry by NPsa
aAlberts Bruce; Bray, D. e. a., Essential Cell BioloGarland Science: 2014.
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3. Transcription
1. Synthesis of (+) strand mRNA
Starts with a 7-methylguanine capped RNA fragmentViral RNA polymerase obtains cap from the host by cleaving
them from host nuclear pre-mRNA, cap snatching(+) mRNA returns to cytoplasm for translation, using the capto bind to the host ribosomeRNA segments encoding envelope proteins attach to ER forprotein synthesis and transport to host membraneNewly synthesized NP proteins and RNA pol return to nucleusalong with genome-packaging proteins (M1 and NS2)7
7Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;
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3. Transcription
2. Synthesis of (+) strand and (-) strand genomic DNA
The original RNA genome (negative sense) can serve as atemplate for RNA synthesis without capThe synthesized (+) strand is then coated with new NPsimported from the cytoplasmThe NP coated (+) strand then serves as a template tosynthesize (-) strand RNA that become coated with NPNP coated (-) RNA associates with new polymerase for futurereplication cyclesRNA complexes with M1 and N2 that were imported from the
cytoplasmFully packaged (-) RNA segments exit the nucleus tocytoplasm for assembly8
8Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;
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3. Nuclear Export Signals
Similar to NLSs, Influenza A also contains 7 nuclear export signals(NESs) that tell the newly transcribed RNA to exit the nucleus
Only one of the export signals is seen as indispensable in
replication and export, and it is also found on the NP protein
NP-NES3:9
Is the most critical sequence for viral production and
replication (especially residues 2 and 3)Indispensable for CRM1 NES receptor binding
Highly conserved
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3. Exit out of the Nucleus
Both nuclear import and exportrequire energy obtained from thehydrolysis of GDP
A nuclear export receptor,CRM1, and its cofactor,
RanGTP, recognize NP-NES inthe nucleus
CRM1 binds to the N-terminusof NEP and RanGTP
The C-terminus of NEP binds tothe NLS on N-terminus of M1
The C-terminus of M1 binds tovRNP (viral ribonucleic protein)through interaction with NPand exit
Figure: Nuclear Exporta
aPaterson, D.; Fodor, E. PLoS pathogens Jan. 201e1003019.
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4. Assembly
Envelope proteins (HA and NA) are synthesized at the ER
Within the lumen, theyre glycosylated by host enzymes and
transferred to the Golgi for export to the cell membrane
Within the membrane, these proteins assemble around a groupof (-) RNA segments complexed with matrix and packaging
proteins, completing the virus!
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5. Release
To exit, the virus buds out
NA protein cuts the sialic acid link of the host glycoproteins
to release the virus into the bloodstream and the processbegins again
Tamiflu (main antiviral agent) can inhibit this host release
activity, but strains are quickly becoming resistant
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Summary of Replication and Proliferation
Figure: Influenza A Viral Replication and Proliferation12 Link
12Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;Twitchell, B., Ed.; W. W. Norton and Company: 2011. 19/20
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Replication and ProliferationConclusion
The rising resistance of strains of this Influenza A viruswarrant action to:
1 Develop new antiviral drugs and2 Understand the pathway of the virus in more depth
The influenza virus has led to pandemics (Spanish flu in 1918)and occasional epidemics, and should not be overlooked
Take your vaccines and stay alert!
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