Introduction

Replication and Proliferation

Conclusion

Replication and Proliferation of the Influenza A

Virus
Emily Cribas
Penn State

December 15, 2014

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Introduction
Replication and Proliferation
Conclusion

The Flu
Also known as seasonal influenza
Contagious respiratory illness caused by influenza viruses that
infect the nose, throat, and lungs
Can cause mild to severe illness, sometimes leading to death
Symptoms include:
Fever
Sore throat
Headaches

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Introduction
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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 of
49,000 people from 1976 to 2007
Has become a serious world-wide concern due to re-emergence
or new, deadlier emergence
Strains are becoming antiviral resistant
Influenza A is the most prominent type of flu virus in North
America
*Information obtained from the Center for Disease Control and Prevention website
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Introduction
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The Influenza A Virus

Belongs to the family of Orthomyxoviridae, which consist of 8
negative-sense single-stranded RNA genomes that encode for
different 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)

Figure: Viral Structurea

a Nelson,

M. I.; Holmes, E. C. Nature review

Neuraminidase (NA)
1 Chutiwitoonchai,

N. et al. PloS one Jan. 2014, 9, ed. by Takimoto, T., e105081.

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Introduction
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Overview of Replication

Figure: Influenza A Viral Replication and Proliferation2

Link

2 Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.

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Introduction
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5 Basic Steps for Viral Reproduction

Adsorption

Penetration and Uncoating

Replication and Transcription

Assembly

Virus Release

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Introduction
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Conclusion

1. Adsorption

The virus must bind, through one of its proteins, to one of the
surface receptors found on the lipid bilayer of the host cell3
Viral protein: Hemagluttinin (HA)
Surface (sugar) receptor: Sialic acid

3 Slonczewski

Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.
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Introduction
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1. Adsorption*

*Obtained from CDC website

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Introduction
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2. Penetration
The virus enters the cell, where an endosome (vesicle) forms
around 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, which
exposes a short hydrophobic sequence on HA that can insert into
the endosomal membrane, causing it to fuse to the viral envelope
When this happens, the viral RNAs are released into the
cytoplasm4
4 Slonczewski

Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.
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Introduction
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2. Uncoating
M1 forms the shell of the virion, and therefore must be released
before the genome is released into the cell
M2 forms a similar channel that pumps protons from the endosome
to the virus, which in turn lowers the internal pH and leads to M1
release

Figure: Penetration & Uncoating5

5 Slonczewski

Link

Joan; Foster, J., Microbiology: An Evolving Science, Third;

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Introduction
Replication and Proliferation
Conclusion

3. Nuclear Localization Signals

The genome in Influenza A contains 2 well-known nuclear
localization signals (NLSs) found on the nucleocapsid protein
(NP), (one unconventional and one bipartite) that are specific
peptide sequences telling the RNA to move into the nucleus6
The unconventional NLS:
It binds to karyopherins 1 and 2
Found between residues 3 and 13
Indispensible for NP nuclear localization (especially residues 7
and 8)
The bipartite NLS:
Located between residues 198 and 216
Indispensible in NP nucleolar localization
Important for vRNA transcription
6 Ozawa,

M. et al. Journal of virology Jan. 2007, 81, 3041.

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Introduction
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3. Entrance into the Nucleus

NLSs on the NP are recognized
by nuclear pore receptors
(karyopherins) that interact with
the cytosolic fibrils that extend
from the rim of the pore

They are pulled by the fibrils

along with the receptor, which
guides it along the way by
grabbing onto short amino acid
sequences inside the nuclear
fibrils, until the RNA is released
into the nucleus
Figure: Nuclear Entry by NPsa
The empty receptor then returns
a Alberts Bruce; Bray, D. e. a., Essential Cell Biolog
to the cytosol via the nuclear
Garland Science: 2014.
pore for reuse
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Introduction
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Conclusion

3. Transcription

1. Synthesis of (+) strand mRNA

Starts with a 7-methylguanine capped RNA fragment
Viral 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 cap
to bind to the host ribosome
RNA segments encoding envelope proteins attach to ER for
protein synthesis and transport to host membrane
Newly synthesized NP proteins and RNA pol return to nucleus
along with genome-packaging proteins (M1 and NS2)7

7 Slonczewski

Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.
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Introduction
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Conclusion

3. Transcription
2. Synthesis of (+) strand and (-) strand genomic DNA
The original RNA genome (negative sense) can serve as a
template for RNA synthesis without cap
The synthesized (+) strand is then coated with new NPs
imported from the cytoplasm
The NP coated (+) strand then serves as a template to
synthesize (-) strand RNA that become coated with NP
NP coated (-) RNA associates with new polymerase for future
replication cycles
RNA complexes with M1 and N2 that were imported from the
cytoplasm
Fully packaged (-) RNA segments exit the nucleus to
cytoplasm for assembly8
8 Slonczewski

Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.
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Introduction
Replication and Proliferation
Conclusion

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

9 Chutiwitoonchai,

N. et al. PloS one Jan. 2014, 9, ed. by Takimoto, T., e105081.

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Introduction
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3. Exit out of the Nucleus

Both nuclear import and export
require energy obtained from the
hydrolysis of GDP
A nuclear export receptor,
CRM1, and its cofactor,
RanGTP, recognize NP-NES in
the nucleus
CRM1 binds to the N-terminus
of NEP and RanGTP
The C-terminus of NEP binds to
the NLS on N-terminus of M1
Figure: Nuclear Exporta
The C-terminus of M1 binds to
vRNP (viral ribonucleic protein)
a Paterson, D.; Fodor, E. PLoS pathogens
through interaction with NP
e1003019.
and exit

Jan. 201
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Introduction
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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 group
of (-) RNA segments complexed with matrix and packaging
proteins, completing the virus!10

10 Slonczewski

Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.
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Introduction
Replication and Proliferation
Conclusion

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 process
begins again
Tamiflu (main antiviral agent) can inhibit this host release
activity, but strains are quickly becoming resistant11

11 Slonczewski

Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.
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Conclusion

Summary of Replication and Proliferation

Figure: Influenza A Viral Replication and Proliferation12

Link

12 Slonczewski Joan; Foster, J., Microbiology: An Evolving Science, Third;

Twitchell, B., Ed.; W. W. Norton and Company: 2011.

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Introduction
Replication and Proliferation
Conclusion

The rising resistance of strains of this Influenza A virus

warrant action to:
1
2

Develop new antiviral drugs and

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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