Orthomyxoviruses Flashcards
What are orthomyxoviruses?
Viruses with a segmented, (–) strand RNA genome.
They are responsible for the highly contagious, acute respiratory illness known as influenza, or simply, the “flu.”
How many types of influenza virus are there?
There are four types of influenza virus; A, B, C and D.
Which influenza type causes the seasonal epidemics of disease?
Influenza A and B viruses cause most of the seasonal epidemics of disease.
Which influenza virus can cause flu pandemics?
Influenza A is the only influenza virus known to cause flu pandemics.
What does influenza C cause in humans?
Influenza C causes a mild illness in humans.
What does Influenza D cause in humans?
Influenza D infects cattle and is not thought to infect humans.
How are influenza viruses typically transmitted?
Influenza viruses are typically transmitted from one person to the next by the respiratory route.
Describe the Influenza A virus particle.
The virus particle is highly pleomorphic (i.e., many shapes), most commonly spherical or ovoid in shape, and approximately 80–120 nm in diameter.
The virus has a lipid bilayer envelope with viral glycoproteins embedded in the membrane.
The glycoproteins are often called spike proteins because they protrude from the envelope
What are the two types of glycoproteins in the Influenza A virus’ envelope?
Hemagglutinin (H) and neuraminidase (N).
What is hemagglutinin?
A trimer of identical subunits.
Hemagglutinin is the anti-receptor that attaches to the host cell.
The virus attaches to the sialic acid found on host cell glycoprotein to begin the replication cycle.
Originally named because of the ability of the virus to agglutinate red blood cells.
What is neuraminidase?
Neuraminidase is a tetrameter of identical subunits that the virus uses to detach from the host cell by digesting the sialic acid after the replication cycle is completed.
Neuraminidase is important for release from the host cell in egress.
How many glycoproteins are in the lipid envelope of the influenza A virus, and what is the proportionality between H and N glycoproteins?
There are about 500 of these glycoproteins in the lipid envelope of the influenza A virus.
There are approximately 5 H glycoproteins to every N glycoprotein.
In addition to hemagglutinin and neuraminidinase, what are two other important envelope proteins for the influenza A virus?
The M2 and M1 proteins.
What is M2?
M2 is an ion channel that allows protons (H+) to enter into the interior of the virus particle.
The acidification process is important in the releasing of the virus’ genome from the M1 protein at the early stages of the replication cycle.
What do the M1 envelope proteins do?
The M1 proteins lines the inside of the lipid envelope and serves to allow the nucleocapsid, the envelope and the glycoproteins to assemble correctly during the late stages of the replication cycle.
What makes up the Influenza A genome?
10 genes distributed on 8 pieces of (–) sense RNA molecules.
Each segment is coatec with nucleoprotein and has its own RNA polymerase complex associated with it (PA, PB1, and PB2).
Six of the segments code for only one protein, and each of the other two segments code for two proteins.
Each segment is replicated and transcribed independently
Note that the segments 1, 2 and 3 are approximately the same size, segments 4, 5 and 6 are approximately the same size, and segments 7 and 8 are approximately the same size.
Which segments are approximately the same size in the Influenza A genome?
Segments 1, 2 and 3 are approximately the same size, segments 4, 5 and 6 are approximately the same size, and segments 7 and 8 are approximately the same size.
What is a RiboNucleoProtein?
The segments of the Influenza A genomes are called RiboNucleoProteins (RNPs) and consist of the (–) RNA, the nucleoprotein (NP) and an RNA-dependent RNA polymerase complex (PA, PB1, PB2).
One copy of NP binds to approximately 20 nucleotides in the RNA strand, thus there are multiple copies of this structural protein
How are Influenza A viruses classified?
By subtypes based on the properties of their H and N surface proteins.
There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1 through H18 and N1 through N11, respectively).
While there are potentially 198 different influenza A subtype combinations, only 131 subtypes have been detected in nature.
Which subtypes of influenza A viruses routinely circulate in people?
A(H1N1) and A(H3N2).
How many different H and N subtypes are there?
There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes
(H1 through H18 and N1 through N11, respectively).
How is the nomenclature to describe isolates of influenza virus strains of human origin expressed?
In this order:
- virus type,
- geographic site where it was first isolated,
- strain number,
- year of isolation,
- virus subtype (for type A viruses only)
For example, seasonal influenza A (H3N2), A/Perth/16/2019
For influenza strains of non-human origin, how is the nomenclature to describe the virus expressed?
In this order:
- virus type,
- species of host,
- geographic site where it was first isolated,
- strain number,
- year of isolation,
- virus subtype (for type A viruses only)
For example, avian influenza A (H1N1), A/duck/Alberta/35/76.
Discuss possible resevoirs of Influenza A viruses.
All known influenza A subtypes exist in the wild bird reservoir and are associated with interspecies transfer and the emergence of new influenza strains with pandemic potential.
The link between humans and pigs is significant in the development and transmission of new flu virus strains.
What will Influenza A infect?
Humans, animals and birds.
Pigs and birds are thought to be particularly important reservoirs for influenza A.
What is the host cell receptor for influenza virus?
The N-acetylneuraminic acid (also called sialic acid) that residues on mucoproteins.
Where is sialic acid found?
Sialic acid is very common on cell surfaces and in mucus, common of cells in the respiratory tract
Where is the natural environment for the influenza virus for mammals, and why?
Upper respiratory tract.
This is because the virus is dependent on a protease in respiratory secretions to activate the H protein so that the virus can release its genome into the cell.
How does the influenza virus infect birds?
Influenza viruses typically infect the gastrointestinal tract of domestic and wild birds.
The influenza RNA dependent RNA polymerase is a complex of two proteins named PB1 and PB2.
True or false?
False.
The influenza genome contains eight segments of negative sense RNA.
True or false?
True.
The influenza virus is an enveloped virus.
True or false?
True.
Influenza (and all orthomyxoviruses) have (+) RNA genomes.
True or false?
False.
The influenza virus particle contains RNA-dependent RNA polymerase.
True or false?
True
The matrix protein sticks out the furthest from the envelope bilayer.
True or false?
False.
A genome segments is called an RiboNucleoProteins (RNPs) and consists of the (–) RNA, the nucleoprotein (NP) and an RNA-dependent RNA polymerase complex.
True or false?
True.
An influenza genome can have missing segments if one of the other segments is duplicated.
True or false?
False.
Some of the influenza RNA genome segments are positive sense, whereas other segments are negative sense.
True or false?
False.
There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes.
True or false?
True.
The current subtypes of influenza A viruses that routinely circulate in people include A(H5N1) and A(H3N2).
True or false?
False.
A virus with the influenza nomenclature “A/Perth/16/2019” would have been the 16th strain isolated.
True or false?
True
The host cell receptor for influenza virus is the N-acetylneuraminic acid (also called sialic acid).
True or false?
True.
Sialic acid is found on many proteins in the human body.
True or false?
True.
In mammals, influenza A preferentially infects cells in the respiratory tract.
True or false?
True.
Pigs can be a reservoir of influenza A viruses for humans.
True or false?
True.
Where do influenza viruses replicate their genomes?
Influenza viruses are (–) sense RNA viruses that replicate their genomes in the host cell nucleus.
Assembly of the virus particle starts with what?
What completes it?
Assembly of the RNPs in the nucleus, and is completed in the cytoplasm near the plasma membrane of the cell.
How fast is the replication cycle of the influenza virus?
The replication cycle of influenza viruses, from the time of entry to the production of new virus, is very quick.
The release of the first influenza viruses from infected cells occurs after only 6 hours.
What is the affinity of the virus particle with sialic acid on mucoproteins of the epithelial cells?
Probably of low affinity, but there is high avidity because of multiple low affinity interactions.
How does the influenza virus enter the epithelial cell?
By receptor-mediated endocytosis
What happens prior to the endocytosis of the Influenza virus particle?
The virus’ hemagglutinin proteins (H, H0) are modified by cleavage by the protease “tryptase Clara.”
The Clara cells of the bronchiolar epithelium secrete this protease into the mucus layer of the respiratory tract.
The cleavage of H0into H1and H2is necessary for the full infectivity of the influenza virus.
What is needed for release of the fusion peptide?
This H protein has been cleaved by tryptase Clara to yield H1(HA1 in the diagram) and H2 (HA2 in the diagram).
The N-terminal region of the H2peptide contains the fusion peptide needed to allow the viral envelope to fuse with the endosome membrane.
What is the ‘fusion peptide’ and why is it important?
The N-terminal portion of H2 has an area called the “fusion peptide” which is critical for the subsequent fusion events of the endosome membrane and the virus’ envelope.
After the H protein has been cleaved by tryptase Clara, what happens to the resultant proteins?
H1 binds to the SA on the mucoprotein and the H2 remains integrated in the bilayer of the envelope.
What happens after endocytosis of an Influenza A particle into an epithelial cell?
The endosome is acidified, resulting in conformational change in the H protein that allows the fusion peptide to be inserted into the endosome membrane.
The fusion of the two lipid bilayer structures of the endosome membrane and virus envelope is required for the release of the virus’ genome into the cytoplasm of the cell
In addition to the endosome acidification, how else is the virus particle acidified?
Why is this necessary?
The interior of the virus particle is also acidified by the movement of protons through the M2 channel in the envelope.
This acidification is necessary for the release of the RNPs from the M1 protein lining the inside of the virus envelope.
The released RNPs are transported into the nucleus.
What happens once the Influenza A genome has been released into the cytoplasm?
The intact RNPs enter the nucleus through the nuclear pore.
NPs have a special targeting sequence that gets the genome into the nucleus.
NPs also protect the genome from any RNA nucleases.
The transport of RNP into the nucleus does not seem to involve the cell’s cytoskeleton structures.
Where does the synthesis of influenza virus mRNA?
The synthesis of virus mRNA occurs in the nucleus of the cell.
How is the influenza virus transcribed?
The (–) strand of RNA is used as a template by the viral RNA polymerase complex to synthesize viral mRNA.
Each of the 8 RNA segments of the influenza genome is transcribed independently.
RNA splicing of the mRNA of two segments yields two mRNA products (for each segment)
Where does the influenza RNA polymerase complex anchor for transcription?
The influenza RNA polymerase complex anchors the 5’ end of the genomic (–) strand RNA.
What is ‘cap-stealing’?
The RNA polymerase binds to a host cell mRNA molecule near the 5’ end and uses its endonuclease to cleave the 5’ cap with about 10 – 13 nucleotides from the mRNA (the rest of the host cell mRNA is released).
This process is called “cap-stealing ”
The influenza RNA polymerase complex requires a primer for the synthesis of viral mRNA.
True or false?
True
The influenza virus polymerase complex binds to the cells mRNA can cleaves the RNA 5’ Cap to use as a primer for the synthesis of its mRNA.
Why is the viral mRNA often called ‘incomplete’?
The entire sequence of the genomic (–) RNA is not copied into mRNA.
For this reason, the mRNA is often called the “incomplete” (+) strand of RNA in the virus’ replication cycle.
The mRNA is incomplete because of the “panhandle” structure that is formed when the complex anchors to the 5’ end of the viral (–) strand RNA.
This results in the last 15–22 nucleotides of the genomic (–) RNA not being copied into the mRNA.
What does the influenza RNA polymerase use as a primer for the synthesis of viral mRNA?
Uses a fragment of host cell mRNA as a primer for the synthesis of the viral mRNA.
How does the viral RNA polymerase complex complete the synthesis of the (+) strand of RNA?
By using the (–) strand as the template and adding the complementary nucleotides to the cap/host cell derived nucleotides
Describe the synthesis of Influenza mRNAs.
RNA dependent RNA polymerase complex initiates transcription by binding to the leader sequence in 3’ of the genomic negative strand RNA.
The RdRp transcribes a 5’ triphosphate-leader RNA, then stop and restart on a transcription initiation signal.
The RNA initiated on this signal is capped.
At the end of viral genes is a transcription stop signal on which the RdRp will produce a polyadenylation signal by stuttering on a U stretch before releasing the mRNA.
How does the RDRP add a polyA tail to viral mRNA?
Near the 5’ end of the (–) RNA template, there is a polyadenylation sequence that is read multiple times by the RNA polymerase (i.e.,the polymerase stutters at this sequence), resulting in the addition of a poly(A) tail.
Describe polyadenylation of the influenza mRNA.
The stop signal present at the end of each gene comprises a stretch of U on which the viral polymerase acquires a stuttering behaviour: after each Adenine (A) inserted, it moves back one nucleotide along with the mRNA.
It resumes transcription adding a new A, then again moves back.
In this way a U on the genomic template is copied hundreds of times at the end of viral mRNA thereby producing a polyA tail.
Eventually the polymerase will release the polyAdenylated mRNA and stop transcription.
What is the function of newly synthesized PA, PB1, PB2, and NP proteins?
Newly synthesized PA, PB1, PB2 and NP proteins are transported back to the nucleus where they catalyze the synthesis of the genomes for the progeny virus.
Initially they catalyze the synthesis of full-size (+) strand RNAs (anti-genomic RNA, used as a template for making genomic (–) RNA), then the synthesis of (–) strand RNAs (genomic RNA, used as the nucleic acid for progeny viruses).
Both strands are synthesized in the forms of nucleocapsids
What happens after the viral genome has been transcribed and the mRNA has been translated?
Several proteins return to the nucleus to initiate the replication of the genome and the assembly of the virus particle.
Describe the replication of the viral Influenza A genome.
This round of RNA synthesis is for the purpose of creating a (+) strand of RNA (i.e., antigenome) that will be used as a template for the synthesis of (–) strand RNA for the virus progeny.
This process would use the RNA polymerase complex to copy the (–) strand into a (+) strand, but the process of replicating the genome does not require a primer and does not involve cap-stealing.
Each of the eight segments of the genome is replicated independently.
The switch to the production of full-sized (+) RNA may be regulated by the amount of “free” NP protein molecules present in the cell.
The genome/antigenome ratio is about 10.
Describe replication of the viral genome again!
>:)
The RNA polymerase complex binds to the leader sequence on the encapsidated (–) RNA genome, and starts replication.
This process is unprimed.
The (+) RNA (antigenome) is also coated with NP during replication.
The RNA polymerase complex ignores all transcription signals when in replication mode.
The (+) RNA is then replicated under the same process, the RNA polymerase complex binding first to the trailer sequence.
What results in the cessation of viral mRNA synthesis and induces the export of the progeny nucleocapsids into the cytoplasm?
Some of the newly made (–) strand RNA can be used as a template for the synthesis of additional viral mRNA.
The binding of M1 to the newly synthesized (–) strand RNA.
What drives the formation of the ‘bud’ at the plasma membrane?
The H and N proteins (synthesized by ER ribosomes) are transported to the cell surface and become incorporated into the plasma membrane of the cell.
It is the H and N proteins that drives the formation of the “bud” at the plasma membrane.
The matrix protein M1 associates with the cytoplasmic tails of the H and N proteins.
In turn, the M1 protein recruits the virion RNPs and the second matrix protein M2 to this site of virus assembly.
Briefly describe the budding of the Influenza virus particle at the plasma membrane.
The virus particle assembles at the plasma membrane.
The virus envelope proteins attract other proteins which in turn attract the virus RNPs.
Describe the two models for the packagin of the segmented genome of influenza.
The random model implies that the virus acquires a complete genome purely through chance, as there is no mechanism to distinguish between the different genome segments.
The specific packaging model suggests a mechanism ensures that only one copy of each different segment is specifically selected during viral assembly.
There is evidence to support both models, so the aspects of genome packaging are still unclear.
What is the random model?
The random model suggests that during packaging, more than eight RNPs are packaged to ensure that some virions contain a complete genome (i.e.,at least one copy of each segment). in a segment non-specific manner such that a reasonable proportion of virions contain at least one copy of each segment.
What is the specific model?
The specific model proposes that unique segment-specific packaging signals operate to form a defined array of eight RNPs containing one copy of each segment.
Describe influenza virus egress.
The new virus then buds from the plasma membrane of infected cells.
Host cell membrane proteins appear to be excluded from virion particles.
Once the particle has budded from the plasma membrane, the M2 protein promotes membrane fission resulting in the detachment or release of the virus bud.
The neuraminidase (N) removes the sialic acid residues so that the virus particle can be freed from the cell.
The removal of the sialic acid ensures that the virus particle won’t re-infect the same cell.
What removes the sialic acid residues so that the influenza virus particle can be freed from the cell?
What does this ensure?
The neuraminidase (N) removes the sialic acid residues so that the virus particle can be freed from the cell.
The removal of the sialic acid ensures that the virus particle won’t re-infect the same cell.
How do H and N glycoproteins change?
H and N glycoproteins can change in two ways: antigenic drift or antigenic shift
What is antigenic drift?
These are minor changes in the H and/or N glycoproteins due to the accumulation of changes in the amino acid sequence and result in minor antigenic differences.
These changes are mutations caused by the viral RNA dependent RNA polymerase.
These changes result in H and N that are immunologically similar to the previous strain.
Therefore, the existing antibodies might still be (but it is also possible they may not be) effective at neutralizing the virus.
What is antigenic shift?
This results in a new virus strain with novel H and/or N proteins that are immunologically distinct
(i.e., the existing antibodies will not be able to neutralize the virus).
These changes arise from the genetic re-assortment of previously circulating human and animal Influenza viruses.
The H and N are encoded on different RNA segments of the Influenza genome.
If a host cell is infected with two different influenza viruses (simultaneously), there can be a high frequency of re-assortment producing different combinations of H and N variants.
Describe prevalence of antigenic shift and antigenic drift in Influenza A viruses.
Influenza A viruses continually undergo antigenic drift.
This process accounts for most of the changes that occur in viruses from one “flu season” to the next.
In contrast, antigenic shift occurs only occasionally and accounts for major pandemics.
However, after a new pandemic influenza strain has evolved, it may undergo antigenic drift and evolve into a seasonal influenza strain.
Describe the drift of influenza virus.
After a re-assortment process has occurred, if the virus became established in humans, the virus would begin to drift like any other seasonal influenza virus.
During drift, small antigenic changes in the H protein generated by mutation are selected to increase immune evasion of the virus particle.
Why are pigs susceptible to both human and avian influenza viruses?
Avian viruses bind preferentially to α2-3-linked sialic acids moieties whereas influenza viruses adapted to humans have a binding preference for cell receptors that contain α2-6-linked sialic-acid moieties.
The respiratory epithelial cells of pigs have receptors that express both α2-3- and α2-6-linked sialic-acid moieties, thus pigs are susceptible to both human and avian influenza viruses.
Dual infections can occur and re-assortment of gene segments can give rise to new strains.
The pigs serve as a “mixing bowl” for the generation of new strains of influenza viruses; these strains can then be transmitted back to humans
What is the mode of action of neuraminidinase inhibitor drugs?
N inhibitors prevent viruses from being released from infected cells, thus halting virus replication.
What is a ‘plug drug’?
Zanamivir and Oseltamivir are sialic acid analogues and are referred to as “plug drugs” because they block the active site of the N protein.
Without active N, the virus is unable to cleave sialic acid and escape from the cell in order to infect a new host cell.
These drugs are effective for both Influenza A and B infections.
Describe the manufacture of Influenza A virus vaccine.
- The circulating strain of influenza is co-infected with a vaccine strain into eggs.
- A virus that has the H and N derived from the circulating strain and the remaining genetic elements from the vaccine strain is selected from the pool of re-assortments.
- The new virus strain is amplified in eggs and collected from the allantoic fluid.
- The virus is inactivated, treated for with detergents to solubilize the envelope and subjected to partial purification to enrich for the H and N components for the final injectable vaccine.
Describe the host range of Influenza A.
A has a broad host range (humans, other mammals, birds).
Where does the Influenza virus derive its envelope?
Envelope derived from the plasma membrane of the cell it previously infected.
Where do most new influenza strains emerge?
The solid line between humans and pigs is also significant - most new influenza strains emerge from pigs.
Genetic and biologic observations suggest that pigs may serve as ‘mixing vessels’ for the generation of human-avian influenza A virus re-assortants with pandemic potential.
What must each virus particle have in order to have a complete genome?
Each virus particle must have 1 of each of the 8 (–) RNA segments in order to have a complete genome.
No duplications, no missing ones!
Where does assembly of the new influenza viral particles take place?
The assembly of the new viral particles takes place at the plasma membrane of the host cell.
The RNPs and M1 proteins are directed towards the plasma membrane of the cell where the envelope proteins that had been inserted (H, N and M2).
What promotes bending of the membrane and final budding of the influenza viruses?
The accumulation of the viral subunits promotes bending of the membrane and final budding of the viruses.
What promotes membrane fission so that the virus particle can detach from the host cell?
M2 (the ion channel)
What controls the release of the virus particle?
The release of the virus particles is controlled by the glycoprotein neuraminidase (A) that cleaves the sialic acid( SA) residues both from the particles and from the host cell.
What are the three important sites in the RDRP complex?
Site that binds the 5’ end of the (–) RNA template and does not release it.
Site that acts as an endonuclease.
Site that reads the (–) RNA template and catalyzes the synthesis of the mRNA.
How does the RDRP convert between its two conformations?
When the concentration of NP protein is sufficiently high enough, the RDRP complex dissociates, then reassembles into the alternate conformation for replication of the viral genome.
Use this image to describe the Influenza A replication cycle.
(Long answer)
- The binding of influenza virus to the host cell (1) is mediated by the spike protein hemagglutinin (HA).
- The internalization of the virus occurs in endosomes (2) where, upon acidification (3), HA undergoes a conformational change which leads to the fusion between the viral envelope and the endosomal membrane (4).
- Thus the viral genome enters the nucleus (5) where it is both replicated and duplicated (6).
- Despite of HA, NA and M2, whose synthesis occurs in the ER, the other viral components are synthesized in the cytosol, where also RNPs are formed (7).
- The viral subunits accumulate at the budding site for the subsequent assembly and release of the new viral progeny (9)
