CHAPTER 18: INFLUENZA VIRUS
malaise may last for several weeks.
Orthomyxoviridae: Greek ortho (correct or normal) and myxa
(mucus); virions bind to mucoproteins
VIRION
- Enveloped particles, quasi-spherical or filamentous.
- Diameter: 80–120 nm.
- Envelope is derived from host cell plasma membrane by
budding.
- Compact helical nucleocapsids.
GENOME
- Linear sSRNA, negative sense.
- 6-8 different segments.
- Total genome length 10–15 kb.
GENES AND PROTEINS
Each genome segment codes for one or two proteins
• Envelope glycoproteins: hemagglutinin (HA) and neuraminidase
(NA)
• Integral membrane protein (M2) with ion channel activity
• Matrix protein (M1)
• Nucleocapsid protein (NP)
• Three RNA polymerase proteins (PA, PB1, and PB2)
• Nonstructural protein (NS1)
• Minor structural protein (NS2)
• Additional nonstructural protein (PB1-F2)
VIRUSES AND HOSTS
- Influenza types A, B, and C: hosts include birds, various
mammals and humans.
- Thogotovirus: transmitted by tick
- Isavirus: infects fish, particularly salmon
DISEASES
- Symptoms: high fever, sore throat, cough, headache, muscular
pain.
- Can be fatal in elderly, infants, and chronically ill, often by
secondary bacterial infections.
- 1918: influenza pandemic killed 20 million people worldwide.
- H1N1 swine flu: new pandemic strain spread worldwide in 2009.
- Emerging avian influenza virus strains threaten domestic fowl
and another possible human pandemic.
DISTINCTIVE CHARACTERISTICS
- Replicate in the nucleus, unlike most RNA viruses.
- Viral mRNA synthesis is primed by stealing capped 5' ends of
cellular pre-mRNAs in the nucleus.
- Can undergo reassortment (“antigenic shift”) by exchanging
genome segments between related strains.
- Reassortment generates new viruses that can cause pandemics
due to changed surface antigens (HA and NA).
INFLUENZA VIRUSES CAUSE SERIOUS ACUTE DISEASE IN
HUMANS, AND OCCASIONAL PANDEMICS
- Responsible for one of the worst human epidemics
- Continue to kill hundreds of thousands of humans worldwide
every year.
• Reason: ability of influenza viruses to escape immune
surveillance by changing the amino acid sequences of
their surface glycoproteins.
- Flu: (the “common cold”) caused not by influenza virus but
by a number of upper respiratory viruses including
rhinoviruses and coronaviruses.
- Real influenza: severe acute disease in all age groups with
symptoms of sore throat, cough, high fever, headache, and
muscular aches and pains.
• Acute stage lasts about three days, but cough and
- More severe pandemics occur at irregular 10- to 50-year
intervals:
• “Spanish flu” pandemic in 1918–1920
• “Asian flu” in 1956 (60,000 deaths in North America)
• “Hong Kong flu” in 1967.
• “Swine flu” pandemic of 2009–2010, which caused
upward of 16,000 deaths worldwide, did not lead to
widespread serious illness as was feared.
INFLUENZA VIRUS INFECTIONS OF THE RESPIRATORY TRACT CAN
LEAD TO SECONDARY BACTERIAL INFECTIONS
- Initially infects epithelial cells in the upper respiratory tract
- Virus infection: causes loss of the ciliated epithelium, leading
to loss of the ability of the respiratory tract to clear viruses or
bacteria by mucociliary flow that normally traps these agents
in mucus and disposes of them.
- Virus replication: induces production of interferons,
cytokines, and other soluble response factors, leading to
local and systemic inflammatory responses.
• Results in the symptoms fever, headache, chills, malaise,
and muscle aches.
- Resulting secondary bacterial pneumonias cause many of the
deaths attributed to influenza virus infection.
ORTHOMYXOVIRUSES ARE NEGATIVE-STRAND RNA VIRUSES
WITH SEGMENTED GENOMES
Myxoviruses: split into two families→ orthomyxoviruses and
paramyxoviruses
Orthomyxoviridae family:
- Influenza virus types A, B, and C
- Thogotovirus: thickborne mammalian virus
- Isavirus: infects Atlantic salmon
Orthomyxoviruses
- Negative-strand RNA genome composed of bet 6 and 8
distinct gene segments.
• Individually wrapped within helical nucleocapsids, which
are packaged in a lipid envelope derived from the
plasma membrane to form the virion.
• Virions can be either roughly spherical or filamentous
- Influenza type A viruses: responsible for most flu epidemics
and are widespread in many avian and mammalian species.
- Influenza type B: limited to humans
- Influenza type C: can infect both pigs and humans.
Multiple subtypes are distinguished within each influenza virus
type by means of variation in the structure of their two surface
glycoproteins.
Filamentous virions:
- Can grow as long as the diameter of an epithelial cell
- Responsible for cell-to-cell transmission of virus
- Could infect a neighboring cell while still being extruded.
- Seen on fresh isolates
Quasi-spherical virions:
- Smaller, elongated and may be more readily incorporated
into tiny droplets in respiratory aerosols
- responsible for person-to-person transmission.
- Seen on virus repeatedly passaged in eggs or cell cultures
EIGHT INFLUENZA VIRUS GENOME SEGMENTS CODE FOR A
TOTAL OF 11 DIFFERENT VIRAL PROTEINS
- 9 out of 11 viral proteins are packaged in virions
- Genome segments 1 to 6 code for a single protein:
• Three RNA polymerase subunits (PB2, PB1, and PA)
• Envelope glycoprotein hemagglutinin (HA)
• Nucleocapsid protein (NP)
• Second envelope glycoprotein, neuraminidase (NA).
- PB1-F2: additional short protein coded by second RF on RNA
2 by influenza A
- Messenger RNAs: can be spliced and gives rise to two distinct
viral proteins:
• Genome segment 7: codes for the matrix protein (M1)
and a third envelope protein (M2).
• Genome segment 8: codes for two nonstructural
proteins (NS1 and NS2),
HEMAGGLUTININ PROTEIN BINDS TO CELL RECEPTORS AND
MEDIATES FUSION OF THE ENVELOPE WITH THE ENDOSOMAL
MEMBRANE
Hemagglutinin protein (HA):
- Type I transmembrane protein w/c has the ability to
agglutinate (clump) RBC.
- Binds to sialic acid-containing receptors found on RBC and
other cell types.
- Also responsible for entry of the virus genome into the cell
via fusion pathway
Synthesis of HA:
- Synthesized as a fusion-incompetent protein w/c is inserted
into the membrane of the ER and transported via the Golgi
apparatus to the PM
- Activated by the cleavage of cellular proteases into two
subunits (HA1 and HA2)
• HA1: surface subunit w/c contains the sialic acid binding
domain.
• HA2: anchored in the virus envelope and contains a
hydrophobic fusion peptide near the amino terminus
created by protease cleavage.
- Virions will then enter cell w/in an endosomal vesicle after
binding to cell receptor
- HA will undergo a conformational change as the pH of the
endosome drops.
• N-terminal part of HA2 forms a rigid alpha-helical
structure, moving the fusion peptide out of the
hydrophobic region of HA and toward the endosomal
membrane, where it inserts itself into the membrane
• One end of HA1 is now bound to the endosomal
membrane, and the other end is bound to the virus
envelope
M2 IS AN ION CHANNEL THAT FACILITATES RELEASE OF
NUCLEOCAPSIDS FROM THE VIRION
M2 protein:
- Small (97 aa protein); has N-terminal external domain, a
single transmembrane domain, and a larger internal domain
- Has the ability to form a highly selective transmembrane ion
channel that allows H+ ions to penetrate the membrane.
- Creates a small pore in the viral envelope due to the
formation of tetramers by lining up four parallel
transmembrane α-helices
M2 H+ion channel:
- Facilitates the release of viral nucleocapsids from the virion
- Allows protons to enter the interior of the virion during
acidification within the endosome
• Low pH within virions weakens the interaction of M1
protein w/ viral nucleocapsids, facilitating their release
into the cytoplasm upon membrane fusion.
Influenza A virus M2 protein:
- contains a His- XXX-Trp motif (where X can be any amino acid)
that appears to constitute the main functional element of the
M2 channels.
- only conserved region shared with the similarly functioning
M2 protein of influenza B virus
- also found in the p7 proteins of some genotypes of hepatitis
C virus (aflavivirus).
NUCLEOCAPSIDS ENTER THE NUCLEUS, WHERE mRNA
SYNTHESIS AND RNA REPLICATION OCCUR
Nucleocapsids
- Contain multiple copies of the NP protein that wrap the
ssRNAs in an unusual twin helical conformation with a central
loop
- Also contain a trimer of RNA polymerase proteins PA, PB1,
and PB2.
Orthomyxoviruses replicate in the nucleus
- Transport and transcription of virions into nucleus → delivery
and translation of viral mRNA to cytoplasm → delivery of viral
proteins to nucleus → direct genome replication and
formation of nucleocapsid → nucleocapsid transported back
to cytoplasm for assembly
- Nucleocapsids are imported from the cytoplasm to the
nucleus via the nuclear pore complexes
- NP protein and polymerase proteins contain nuclear
localization signals that mediate interactions with cellular
importin-α.
**importin-α: docks with nuclear pore complexes and promotes entry
of its cargo into the nucleus.
CAPPED 5' ENDS OF CELLULAR PREMESSENGER RNAS ARE USED
AS PRIMERS FOR SYNTHESIS OF VIRAL mRNAS
Capped cellular premessenger RNAs are synthesized in the
nucleus by cellular RNA polymerase II and undergo
polyadenylation and splicing before being transported as mature
mRNAs to the cytoplasm.
PB2 protein:
- Contains a domain that recognizes the 5' cap structure found
on all eukaryotic mRNAs.
- Binds to the capped end of a cellular premessenger RNA
PB1:
- Acts as a nuclease, cleaving the bound premessenger RNA at
an A or G residue
- Also acts as an RNA polymerase by adding ribonucleotides
complementary to the genome RNA sequence, proceeding
toward its 5' end.
PA: role in transcription is not well understood
- Capped RNA fragment bound to PB2 is used as a primer to
begin copying the genome RNA.
- Resulting capped viral mRNAs have heterogeneous
sequences
- Orthomyxoviruses can make capped messenger RNAs even
though they do not code for a capping enzyme.
- Reason why the replication of influenza virus is blocked by
treatment of infected cells with inhibitors of cellular RNA
synthesis such as actinomycin D or α-amanitin.
- Cellular premessenger RNAs are rapidly spliced and exported
to the cytoplasm
- Influenza virus cannot make its own mRNAs.
VIRAL mRNAS TERMINATE IN POLY(A) TAILS GENERATED BY
“STUTTERING” TRANSCRIPTION
Transcription of the viral RNA polymerase stutters or pauses when
poly(U) sequence is reached
- Stuttering reads through the poly(U) sequence a number of
times and repeatedly adding complementary A residues to
the transcript to generate a poly(A) tail
- RNA polymerase eventually terminates transcription at that
position.
- Results from blockage of further movement of the RNA
polymerase by PB1 itself since it remains bound to the
terminal 12 to 13 nt at the 5' end of the same genome RNA it
is copying.
TWO INFLUENZA A mRNAS UNDERGO ALTERNATIVE SPLICING
IN THE NUCLEUS
Transcription generates a complete set of 8 viral mRNAs
- 6 of these are exported directly to the cytoplasm where
they are translated by ribosomes.
- RNAs generated from genome segments 7 and 8 contain
splicing consensus sequences that are recognized by the
cellular splicing machinery in the nucleus.
- Only a fraction of these two viral mRNAs are spliced, and
both unspliced (M1 and NS1) mRNAs and spliced (M2 and
NS2) mRNAs are exported from the nucleus.
- Ensures that M2 and NS2 are made in lesser amounts than
the highly abundant M1 and NS1 proteins.
GENOME REPLICATION BEGINS WHEN NEWLY SYNTHESIZED NP
PROTEIN ENTERS THE NUCLEUS
Viral proteins (NP and the RNA polymerase subunits) are
transported from their site of synthesis in the cytoplasm back
into the nucleus
- Enables the replication machinery to begin to produce full-
length, plus-strand copies of the 8 viral genome segments
- full-length transcripts contain 5' triphosphate groups and
result from unprimed transcription initiation.
Mechanism of switching from primed to unprimed RNA
synthesis:
Interaction of with RNA polymerase complex or with the 5' and
3' ends in nucleocapsid → Begins copying the genome RNA
directly from its 3' terminus, plus strand copy synthesized →
Plus-strand copy complexed with NP protein, forming
nucleocapsids containing plus-strand antigenome RNAs.
- RNA polymerase does not stutter and terminate at the
poly(U) stretch
- Occurs because the PB1 subunit has been displaced
- Plus-strand antigenome RNAs are copied in a similar fashion
by the RNA polymerase complex to generate nucleocapsids
containing full-length, minus-strand genomes
- Minus strand genomes can be transcribed to produce more
viral mRNAs or can generate more full-length, plus-strand
RNAs for genome replication
- NP does not bind to influenza virus mRNA since they are
capped and contain cellular mRNA sequences, not viral
sequences, at their 5' ends.
NUCLEOCAPSIDS ARE EXPORTED FROM THE NUCLEUS IN A
COMPLEX WITH MATRIX PROTEIN AND NS2
Nucleocapsids must be transported back out of the nucleus to the
cytoplasm, so that they can be incorporated into progeny virions
- Matrix protein (M1): imported into the nucleus and binds to
nucleocapsids to form a complex
- NS2:
• Piggybacks onto nucleocapsid/matrix protein complexes
and signals the cellular protein export system to
transport the complexes through the nuclear pores to
the cytoplasm
• Contains a nuclear export signal, and it can also bind to
the matrix protein.
THE NS1 PROTEIN INTERFERES WITH POLYADENYLATION OF
CELLULAR mRNAS
NS1 protein: most abundant viral protein in virus-infected cells
- can bind to both single- and double-stranded RNA and to two
host cell proteins including:
• Cleavage and polyadenylation specificity factor (CPSF):
responsible for the cleavage of cellular pre-mRNAs
shortly downstream of the consensus AAUAAA
polyadenylation signal
• poly(A)-binding protein II (PABII): facilitates production
of full-length poly(A) chains on the 3' terminus created
by cleavage.
- Binding of NS1 inhibits the normal 3' end cleavage and
polyadenylation of cellular mRNAs; synthesis of viral mRNA is
not affected
Influenza PB2 and PB1: its cap-binding and endonuclease
activities leads to the degradation of many cellular pre-mRNAs in
virus-infected cells.
- Most cellular pre-mRNAs are degraded in the nucleus, and
cellular mRNA production is therefore suppressed.
THE NS1 PROTEIN ALSO SUPPRESSES A VARIETY OF HOST CELL
ANTIVIRAL RESPONSE PATHWAYS
NS1:
- Binds and inhibits signaling by RIG-1
**RIG-1: responds to RNA virus infections by the induction of
interferon synthesis via interferon response factor-3.
- Activates the phosphatidylinositol 3-kinase (PI3K)/Akt
pathway w/c leads to caspase-9 activity inhibition and
suppression of apoptosis.
- Binds and sequesters dsRNA w/c suppresses the activity of
two major interferon stimulated antiviral mechanisms:
double-stranded, RNA-dependent protein kinase (PKR) and
2', 5'-oligo(A) synthetase/Ribonuclease L.
- Prevents the maturation of human primary dendritic cells,
thereby limiting host T-cell activation considered as a
potential target for chemotherapeutic agents to suppress
influenza virus infection
- Its four C-terminal constitutes PDZ ligand domain of the
consensus sequence X-Ser/Thr-X-Val (where X is any amino
acid).
• Interacts w/ PDZ-binding protein(s) and modulate
pathogenicity through additional mechanisms
• PDZ domains: protein–protein recognition modules
PB1-F2 MAY CONTRIBUTE IN SUPPRESSION OF THE HOST
IMMUNE RESPONSE
PB1-F2 protein:
- Recently discovered in the PB1 polymerase gene segments of
certain influenza A virus strains.
- Shown to overcome host defense mechanisms and to
enhance pathogenicity by inducing increased apoptosis in
host immune cells responding to influenza virus infection.
VIRAL ENVELOPE PROTEINS ASSEMBLE IN THE PLASMA
MEMBRANE AND DIRECT BUDDING OF VIRIONS
- The cellular protein trafficking machinery translocates HA,
neuraminidase and M2 ion channel via the Golgi network to
the cell surface, where virus assembly takes place.
- Three proteins accumulate in lipid rafts (membrane enriched
in cholesterol), which are the site of virion formation by
budding.
- The cytoplasmic tails of HA, NA, and M2 are crucial to virion
formation since they interact with matrix protein (M1) bound
to virus nucleocapsids and transported from the nucleus to
the cytoplasm.
- Artificial virus-like particles (VLPs) containing influenza
surface proteins can be assembled in the absence of M1.
• However, M1 is required for the production of infectious
VLPs.
M1:
- Functions to package the genome in virions.
- Interact directly with the cytoplasmic tail of M2 to promote
recruitment of internal viral proteins and viral nucleocapsids
to the plasma membrane, facilitates
- Orthomyxovirus virions must contain at least one copy of
each genome segment in order to be able to initiate an
infection.
- 7 nucleocapsids are arranged parallel to each other as a
cylindrical barrel and an 8th nucleocapsid lies in the center of
the bundle.
• One copy of each of the eight different nucleocapsids is
incorporated into a bundle either before or during virus
budding.
- Viral proteins recognize and interact with specific RNA
sequences in the nucleocapsids to incorporate them, one by
one, into the bundles that are packaged into virions during
budding.
NEURAMINIDASE CLEAVES SIALIC ACID, THE CELLULAR
RECEPTOR THAT BINDS TO HA
Influenza neuraminidase (NA):
- type II transmembrane protein w/ a short cytoplasmic N-
terminus, a membrane-spanning domain, and a long C-
terminal domain that extends outward from the virus
envelope.
- Has the ability to cleave terminal N-acetyl neuraminic acid
(sialic acid) from oligosaccharides located on mucoproteins,
cell-surface glycoproteins, and glycolipids.
- Can reverse binding of HA to sialic acid residues by cleaving
the bound sialic acid residue from the remainder of the
oligosaccharide
INFLUENZA VIRUS STRAINS VARY IN BOTH TRANSMISSIBILITY
AND PATHOGENICITY
- Most influenza A viruses that circulate in wild or domestic
animals do not readily infect humans.
- This host specificity resides in HA, NA, PA, PB1, and PB2 that
have adapted to function optimally in a particular species.
Example:
- HA proteins of avian influenza A: bind preferentially to α-2,3
sialic acid
• In contrast w/ human influenza A w/c bind
preferentially to α -2,6 sialic acid
- Some HA proteins are cleaved by furin proteases which
infects many cell types since their fusion mechanism is
activated upon cleavage.
- Other HA proteins are not cleaved intracellularly during virus
formation but require activation by trypsin cleavage after
release of virions from an infected cell.
• Trypsin expression is limited to the upper respiratory
tract in humans,
• Less likely to infect lung and other tissues in the body;
leads to milder and more self-limiting infections.
GENETIC VARIABILITY GENERATES NEW VIRUS STRAINS THAT
CAN CAUSE PANDEMICS
Protective immunity after infection with influenza A virus does not
last for a lifetime as a result from the antigenic change in the
domains of virus envelope glycoproteins, HA and NA.
Antigenic change occurs in two ways:
1. Antigenic drift:
- Slowly but continuously
- Results from the accumulation of point mutations w/in
regions of the envelope glycoprotein genes
- Variants in antigenic domain of envelope glycoprotein as a
result of errors made by RNA polymerase during replication
since it does not possess proofreading mechanisms
- Can selectively replicate and propagate
- Common to a number of RNA viruses, including HIV, and is a
major complicating factor in production of vaccines.
- Resulted in the generation of numerous influenza A subtypes
with antigenically distinct HA and NA surface glycoproteins
2. Antigenic shift:
- Suddenly but episodically
- Results from reassortment of influenza virus genes during
mixed infections with two or more virus subtypes.
- Cells infected simultaneously with two virus strains can
produce reassortant virions
- Viruses with many combinations of the different HA and NA
subtypes, as well as other viral genes, are generated in
multiply infected animals in the wild
- Reassortment bet. animal and human strains can create a
virus that can replicate in humans but has acquired an HA or
NA subtype derived from the animal virus w/c can cause a
pandemic
THE 1918 PANDEMIC INFLUENZA A VIRUS WAS PROBABLY NOT
A REASSORTANT VIRUS
Subtype H1N1:
- 1918 pandemic virus strain arose from an avian virus by
mutation (“drift”), rather than by reassortment accdng to
analysis of its genome sequence
- showed extremely high pathogenicity for mice and for
embryonated chicken eggs as shown in an experiment
PA, PB1, and PB2 of the 1918 human virus: Acquired the ability
to bind to the α -2,6 sialic acid present in the human respiratory
tract by virtue of a single amino acid change from an HA that binds
to the avian α -2,3 sialic acid.
HA: lacks a furin cleavage site, NA protein enhances the cleavage
of HA in a trypsin-independent fashion, increasing the infectivity
and spread of this virus.
GENOME SEQUENCES FROM SOME PREVIOUS INFLUENZA A
VIRUS STRAINS CONFIRM THE ANTIGENIC SHIFT HYPOTHESIS
subtype H2N2 and subtype H3N2: subtype H2N2: both
reassortant viruses
- subtype H2N2:
• New influenza A virus w/c appeared in Southeast China
in 1957
• “Asian flu” pandemic.
- subtype H3N2
• Appeared in China in 1968,
• “Hong Kong” influenza pandemic, had a new HA
Up to the present, influenza viruses that causes widespread
human disease have been limited to strains that contain HA types
1, 2, or 3.
HIGHLY PATHOGENIC AVIAN INFLUENZA A H5N1 STRAINS IN
POULTRY FARMS ARE A POTENTIAL THREAT BUT ARE POORLY
TRANSMITTED AMONG HUMANS
avian H5N1 strains:
- Highly pathogenic strains w/c spread through commercial
poultry farms in Hong Kong (1997); causing 6 deaths
- Resulted in serious and often fatal disease in a number of
people directly exposed to infected birds.
- Further spread probably via infected migrating waterfowl.
- High density of domestic animals and migratory wildfowl
increase the likelihood of generation of mutants or
reassortant viruses that will acquire the ability to be
transmitted among humans.
- Neuraminidase inhibitors zanamivir and oseltamivir could
slow down the spread of influenza A virus during a pandemic
or reduce symptoms in infected people.
A NEW PANDEMIC STRAIN OF INFLUENZA A VIRUS AROSE BY
GENETIC SHIFT AND SPREAD WORLDWIDE IN 2009
New strain of human H1N1 influenza A virus was identified in the
United States and Mexico in 2009
- Genomic analysis showed that it was related to common
reassortant swine influenza A viruses isolated in North
America, Europe, and Asia,
- HA protein of this virus strongly resembles the HA protein of
the original 1918 pandemic virus.
- Drifted during > 40 years when it dominated human influenza
strains
- WHO raised the warning level for this virus to the highest
available—Phase Six—indicating that influenza had entered a
pandemic stage of worldwide spread on June 11, 2009.
• 16,713 deaths.
• Occurred in young, healthy people
• Most patients had only mild symptoms and a short
course of disease without severe complications or
mortality.
- Initially susceptible to the neuraminidase inhibitor
oseltamivir (Tamiflu), which helped to shorten the fever
period and quickly improved disease symptoms.
• Strains resistant to the drug were reported within
months, leading to controversy about its use in patients
with mild disease.
- A massive vaccine development campaign allowed the first
human vaccination campaigns against pandemic H1N1 virus
to begin in October 2009.
- Seasonal strains of influenza A H3N1 and H1N1 as well as
influenza B virus began to cocirculate with pandemic H1N1
virus.