Replication and Proliferation of the Influenza A Virus

8/10/2019 Replication and Proliferation of the Influenza A Virus

1/20

IntroductionReplication and Proliferation

Conclusion

Replication and Proliferation of the Influenza A

Virus

Emily Cribas

Penn State

December 15, 2014

1 / 2 0


2/20


Conclusion

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

2 / 2 0


3/20


Conclusion

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

3 / 2 0


4/20


Conclusion

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


5/20


Conclusion

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
http://wwnorton.com/common/mplay/6.5/?p=/college/biology/microbiology3/&f=influenzahttp://wwnorton.com/common/mplay/6.5/?p=/college/biology/microbiology3/&f=influenza


6/20


Conclusion

5 Basic Steps for Viral Reproduction

1 Adsorption

2 Penetration and Uncoating

3 Replication and Transcription

4 Assembly

5 Virus Release

6 / 2 0

I d i


7/20


Conclusion

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;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.7 / 2 0

I t d ti


8/20


Conclusion

1. Adsorption*

*Obtained from CDC website 8 / 2 0

Introduction


9/20


Conclusion

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


Twitchell, B., Ed.; W. W. Norton and Company: 2011.9 / 2 0

Introduction


10/20


Conclusion

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

Introduction
http://wwnorton.com/common/mplay/6.5/?p=/college/biology/microbiology3/&f=fusion_peptidehttp://wwnorton.com/common/mplay/6.5/?p=/college/biology/microbiology3/&f=fusion_peptide


11/20


Conclusion

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

Introduction


12/20


Conclusion

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.

12/20

Introduction


13/20

Replication and ProliferationConclusion

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


Twitchell, B., Ed.; W. W. Norton and Company: 2011.13/20

Introduction


14/20


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



Introduction


15/20


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

9Chutiwitoonchai, N. et al. PloS oneJan. 2014, 9, ed. by Takimoto, T., e105081.15/20

IntroductionR li i d P lif i


16/20


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.

16/20

IntroductionR li ti d P lif ti


17/20


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!

10





18/20


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

11





19/20


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

http://wwnorton.com/common/mplay/6.5/?p=/college/biology/microbiology3/&f=influenzahttp://wwnorton.com/common/mplay/6.5/?p=/college/biology/microbiology3/&f=influenza


20/20


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!

20/20

Documents

Replication and Proliferation of the Influenza A Virus