5. The Pathogenicity of Viruses - Influenza virus

Question 1

What are the symptoms of the influenza virus?

Answer 1

Fever, headache, cough, sore throat, nasal congestion, sneezing and body aches.

Question 2

What family of viruses does the influenza virus belong to?

Answer 2

Orthomyxoviridae

Question 3

What does the ‘H’ and ‘N’ stand for in the naming of Influenza A subtypes? e.g. H1N1

Answer 3

Cell surface structures:

H: Haemagglutinin

N: Neuraminidase

Question 4

How many subtypes of influenza A virus have been identified?

Answer 4

17 HA subtypes and 9 NA subtypes.

Question 5

How many RNA segments are present in influenza virus A, B and C?

Answer 5

8 segments are present in influenza virus A and B while only 7 is present in the Influenza C virus.

Question 6

Name and describe the 8 RNA segments of influenza virus A.

Answer 6

1. PB2: Component of RNA polymerase complex associated with 5’ cap binding in cap snatching.
2. PB1: Component of RNA polymerase elongation during translation.
3. PA: Codes for a protease associated with endonuclease activity during cap snatching.
4. HA: Surface glycoprotein for receptor binding to host cell and fusion activity as well as assembly and budding.
5. NP: RNA binding, RNA synthesis, RNPS nuclear export
6. NA: surface glycoprotein
7. M: produces matrix proteins M1 and M2. M1: Interacts with RNPs and glycoproteins, nuclear export, assembly and budding,M2: Membrane protein, ion channel activity, assembly and budding.
8. NS: alternatively spliced to produce NS1 and NEP/NS2NS1: Important in host immune response evasion among other functions (multi-functional)NEP/NS2: RNP nuclear export and regulation of RNA synthesis

Question 7

What structures do the influenza vRNA associate with to form the viral ribonucleoproteins (vRNPs)?

Answer 7

The 8 viral RNA segments associate with four viral proteins which include nucleocaspid protein (NP) and three polymerase proteins PB1, PB2 and PA.

Question 8

What cell surface structures do influenza viruses bind to?

Answer 8

Neuraminic acids (sialic acids)

Question 9

What type of neuraminic acid does a human-infecting strain of influenza virus bind to?

Answer 9

N-acetylneuraminic acid, preferentially attached via an alpha-2,6 linkage (and to a lower affinity those attached via an alpha-2,3 linkage) to the penultimate galactose.

Question 10

What must be present for host cells deficient in sialic acid to support the influenza virus?

Answer 10

Cells deficient in sialic acid can be made to support influenza virus if they excpress one of the C-type lectins DC-SIGN or L-SIGN.

Question 11

What mutation in the HA gene of the influenza virus responsible for the 1918 pandemic, has been found to alter the preferential binding of HA from the alpha-2,6 linked sialic acid to the alpha-2,3 linked sialic acid?

Answer 11

D190E

Question 12

What does this phenotypic change resulting from this single mutation suggest?

Answer 12

This suggests that the HA gene originally bound to alpha-2,3 linked sialic acids to cause infection in avian species however the E190D mutation allowed it to bind to alpha-2,6 linked sialic acids in order to infect humans

Question 13

How does influenza virus enter the cell once attachment has occured?

Answer 13

Clathrin-mediated endocytosis is the most often described mechanism for influenza viral entry however a non-clathrin, non caveolae-mediated internalization has also been described to result in entry of the influenza virus in an endosome. The importance of epidermal growth factor receptor (EGFR) has also been identified in the entry of influenza virus and it is suspected to be activated during influenza virus attachment in order to initiate signalling cascades such as phosphatidylinositol-3-kinase (PI3K) which is known to be associated with influenza virus internalisation.

Question 14

Once the influenza virus has entered in an endosome, how are the contents of the viroid released into the cytoplasm of the host cell?

Answer 14

the HA molecule on the cell surface of the influenza virus is split into two subunits - HA1 and HA2. Under the acidic environment of the endosome, HA2 undergoes a conformational change which results in the exposure of a fusion peptide at the N terminal end of the structure. This fusion protein associated with the endosomal membrane and once numerous HA2 subunits have associated with the endosomal membrane, a pore is formed allowing for release of the viral contents into the cytoplasm of the host cell. The M2 protein is also thought to be important in this process by acting as an ion channel which fascilitates the internalisation of protons into the virus from the endosome in order to dissociate the RNP complex from other viral components.

Question 15

How are the influenza virus RNPs transported into the nucleus from the cytoplasm?

Answer 15

A nuclear localisation signal (NLS) encoded by the amino acid sequence on the nucleocaspid protein binds to Karyophern-alpha which in turn then proceeds to recruit Karyopherin-beta to form a trimeric complex which docks at a nuclear pore to allow passage of vRNPs into the nucleus.

Question 16

Describe the process of transcription initiation in the case of the influenza virus.

Answer 16

Once in the nucleus, a host pre-mRNA is essentially “stolen” for its 5’ cap by a process termed ‘cap-snatching’ and used a primer for influenza virus RNA transcription. This occurs when the 5’ end of the vRNA binds to the PB1 subunit to cause an allosteric change in the polymerase complex which allows for PB2 protein to recognise and bind the cap structure of host pre-mRNAs. This change in conformation also allows for PB1 to bind the vRNAs with greater affinity causing stabilisation of the polymerase complex and activation of the endonuclease activity of PA. PA then proceeds to ‘trim’ the host pre-mRNA in order to remove excess RNA which is not required for viral RNA transcription.

Question 17

Where on the host pre-mRNA does cleavage occur by PA of the influenza virus RNP complex during transcription initiation?

Answer 17

10-13 residues after the 5’ cap , usually after a purine residue

Question 18

Describe the process of transcription elongation during the transcription of the influenza virus RNA.

Answer 18

Once cleavage of the host pre-mRNA occurs the 5’ cap is used as a primer to build upon in order to form an mRNA transcript of the influenza virus RNA. Translation elongation occurs via the polymerase activity of PB1. The first nucleotide residue to be added onto the 5’ cap is most often a Cysteine due to the penultimate Guanine residue at the 3’ end of the vRNA transcript, however it is also possibly a gianine due to the presence of a cysteine residue on the vRNA trancript at position 3. Translation elongation continues until a stretch of Uridine residues are encountered approximately 16 residues before the 5’ end of the vRNA transcript. This is a signal for polyadenylation to begin.

Question 19

Which protein of the influenza virus RNA polymerase complex is associated with polyadenylation?

Answer 19

PB1 catalyses the polyadenylation, when it encounters a stretch of 5-7 U residues. Due to steric hindrance resulting from the association of the polymerase complex proteins and the vRNA, the polymerase stutters on this stretch of 5-7 uridine residues repeatedly copying to produce the poly(A) tail.

Question 20

Which vRNA genes produces splicable transcripts?

Answer 20

Influenza A: 7 and 8

Influenza B: 8

Influenza C: 6 and 7

Question 21

How does splicing occur in the mRNA transcripts of influenza vRNA?

Answer 21

Using host cell machinery to a low efficiency producing a percentage of spliced transcripts of 10%.

Question 22

Why does splicing of influenza mRNA transcripts occur at low efficiency?

Answer 22

Proteins must be expressed from both spliced and unspliced mRNAs.

Question 23

How are influenza virus mRNA converted into protein products?

Answer 23

Influenza virus mRNA leaves the nucleus via nuclear pores to associate with host cell ribosomes in order to produce protein products.

Question 24

How does the influenza affect the host cell to result in the preferential formation of its own protein products? (4 points)

Answer 24

1. Degradation of host pre-mRNAs by cap snatching and trimming.
2. Inhibition of mRNA processing by competitive inhibition of splice machinery.
3. Degradation of cellular RNA Polymerase II associated with host cell transcription.
4. Preferential translation of viral mRNAs

Question 25

How does the influenza virus manipulate host translation machinery to result in the preferential translation of its own mRNA over host mRNA?

Answer 25

The influenza virus NS1 protein associates with the 5’ noncoding region of viral mRNA transcripts as well as host cell proteins important in translation initiation including translation initiation factor eIF4GI and poly(A)-binding protein 1. Together this protein complex recruits ribosomes to viral mRNA preferentially over host mRNA.

Question 26

How is a vRNA replicated to created more vRNAs?

Answer 26

The machinery associated with this process is not very well understood however it is strongly suggested that the transcription competent polymerase is structurally different from the replication competent polymerase. It is known that the cap binding activity of PB2 is not required, nor is the endonuclease activtity of the PA protein. No capped primer is used in the replication of vRNA which is initially used to create a full length cRNA molcule in order to be used as a transcript to synthesise new vRNAs.

Question 27

What proteins are responsible for the nuclear export of newly formed influenza virus RNPs?

Answer 27

The M1 protein and the Nuclear export protein (NEP/NS2)

Question 28

How does the M1 and NEP/NS2 protein work to export influenza vRNPs from the nucleus?

Answer 28

M1 binds to vRNA and NP and is thought to also bind to nucleosomes causing the dissociation of RNPs from the nuclear matrix. NP is also thought to bind to the export receptor Crm1 aiding in nuclear export.NEP/NS2: Interacts with export receptor Crm1 and several nucleoporins. It also associates with M1 and initiates nuclear export.

Question 29

How is nuclear export of influenza virus RNPs controlled to occur only after replication has occured?

Answer 29

M1 inhibits replication and translation and initiates nuclear export. M1 has also been shown to be expressed later on during the replication cycle, allowing time for replication to occur before M1 initiates export

Question 30

How do exported influenza virus RNPs prevent re entering the nucleus?

Answer 30

It is thought that when NS2/NEP is bound to M1 it blocks the NLS on M1 preventing the return of the exported influenza virus RNPs into the nucleus. NP is also thought to contribute to this role by binding to filamentous actin in the cytoplasm to retain the influenza RNPs there.

Question 31

What happens to the integral membrane proteins HA, NA and M2 once they are synthesised by host ribosomes?

Answer 31

The three integral membrane proteins HA, NA and M2 enter the endoplasmic reticulum to be folded. HA and NA are also glycosylated here. After this they are trnasported to the Golgi where cysteine resiudes on HA and M2 are palmitoylated.

Question 32

How do influenza vRNPs and other influenza virus components assemble at the assembly site?

Answer 32

HA, NA and M2 leave the golgi to be directed to the apical plasma membrane via their apical sorting signals, which are located for HA and NA in their transmembrane domainsRelatively little is known about how the other viral components assemble at the apical plasma membrane.

Question 33

What two different models have been proposed to explain the packaging of the 8 RNA segments of the influenza A virus?

Answer 33

1. The random incorporation model: assumes a common structural feature is present in all vRNPs which enables them to be randomly incorporated into budding virions. It suggests that while all 8 vRNPs will be present in select virions, more than 8 vRNPs will be present to increase the chance of producing a virion than contains all 8 vRNPs
2. The selective incorporation model: each vRNA segment is selected for and packaged independently.

Question 34

What evidence supports the random incorporation model?

Answer 34

Mathematical analysis of the number of infectious influenza particles out of total influenza particles supports this model

Question 35

What evidence supports the selective incorporation model?

Answer 35

Electron microscopy of serially sectioned budding virions suggest there is exactly 8 vRNPs in specific positions.

Question 36

Describe the budding process of the influenza virus.

Answer 36

1. Outward curvature of the plasma membrane.
2. The virus bud extrudes to envelope to inner core.
3. The membranes fuse around the bud.

Question 37

What viral proteins are suspected to play a role in the budding process?

Answer 37

HA, NA and M2.

Question 38

How is the new influenza virus released from the host cell, once budding is complete?

Answer 38

As HA anchors to host cell via its binding to sialic acid, the NA protein removes sialic acid to release the virus from the host cell.

Question 39

What is the role of NA?

Answer 39

NA removes sialic acid which is important in release of a new virion from a host cell and in preventing binding of adjacent viral particles.

Question 40

How is the influenza virus able to avoid host cell immune response?

Answer 40

NS1 acts to suppress the virus indiced host IFN response.

Question 41

How was the role of NS1 identified?

Answer 41

Influenza A virses lacking NS1 displayed unusual growth properties as it was notably less virulent in IFN-competent systems but grew normally in IFN-deficient systems.

Question 42

How is the influenza virus suspected to affect IFN?

Answer 42

It is thougth that either the influenza virus prevents IFN mRNA synthesis or destabilises IFN RNA.

Question 43

What is the process of genetic ‘reassortment’?

Answer 43

Reassortment occurs when a host cell is infected with two or more different influenza viruses allowing for the rearrangement of viral gene segments into new virions.

Question 44

Describe the process of the genetic recombination of the influenza virus.

Answer 44

Recombination occurs when RNA segments are inserted into the vRNA.

Question 45

What are the effects of recombination of the influenza virus? Give an example.

Answer 45

Phenotypic changes are common, often in terms of pathogenicity. Avian influenza viruses have switched from low to high pathogenicity after 21 nucleotides of the M segment or 30 nucleotides from the NP segment were inserted into the HA segment.

Question 46

How does the influenza virus reinfect humans to cause disease?

Answer 46

Antigenic drift and Antigenic shift

Question 47

What is antigenic drift?

Answer 47

Antigenic drift occurs as a result of point mutations in the influenza A and B viruses and refers to minor antigenic changes in the HA and NA proteins.

Question 48

What is antigenic shift?

Answer 48

Antigenic shift involves major antigenic changes in which an HA or NA is antigenically distinct from a circulating variant.

Question 49

Give two examples where antigenic drift affects the pathology of the influenza virus.

Answer 49

H7N7: A143T in HA protein –> increased attachment to bronchial epithelial cells and alveolar macrophages in humans H5N1: T271A in PB2 protein –> increased polymerase activity in mammalian cells.

Question 50

Give two example where antigenic shift affected the pathology of the influenza virus.

Answer 50

1918 spanish influenza pandemic where a novel H1N1 virus emerged. 1957 the H1N1 influenza virus was replaced with the H2N2 subtype causing Asian influenza. 1968 the H2N2 wa replaced with H3N2 leading to the Hong Kong influenza.1977 the H1N1 subtype reapreared causing russion influenza2009 an antigenically distinct H1N1 influenza emerged causing a pandemic.

Question 51

Which influenza virus proteins are known determinants of pathogenicity?

Answer 51

HA, PB2, NS1 and PB1-F2

Question 52

What does the influenza virus do at the cellular level to cause disease?

Answer 52

Influenza effectively shuts off cell protein synthesis and induced apoptosis as an additional mechanism of cell destruction.