The name Influenza was given during Renaissance when unknown infection hit Florence, Italy, and people have correlated the disease with the stars alignment: Influence of the Stars (it. Influenza). Formerly an avian virus, it was transmitted to humans around 10,000 years ago during the Ice Age (Collier and Oxford, 2000).
I.1.1. Epidemiology of Influenza A Virus
Remembering the pandemic Spanish flu in 1918 and the avian influenza in 1997, 2004, and the pandemic H1N1 Swine origin influenza virus (SOIV) from 2009 (Medina and Garcia-Sastre, 2011) we realise that Influenza A virus viral pathogens have the potential to lead to new pandemic viruses (Cheung and Poan, 2007).
According to World Health Organisation (WHO), influenza causes annually around three to five million cases of severe illnesses. Almost 250,000 to 500,000 cases where Influenza A virus is responsible end up in deaths. Most vulnerable to influenza are people aged 65 and over and infants (WHO, 2012).
Influenza A virus is often associated with seasonal morbidity and mortality. Influenza A viruses are subtyped according to their antigenic and genetic nature of the glycoproteins found at the surface: 15 hemagglutinin and 9 neuraminidase. So far only viruses of H1N1, H2N2 and H3N2 subtypes are associated with human epidemics. The epidemiology of these viruses is due to the antigenic variations events that are present at the surface of the two surface glycoproteins of the virus: HA and NA. These new variations have the ability to infect people despite prior vaccination. Mutations of HA and NA genes are leading to new variations of influenza A virus and these mutations are happening as a result of RNA polymerase unfunctional activity during the transcribing of the influenza A virus genome. The new strains appear as response to genetic changes that encode the amino-acids in the HA and NA. Antigenic shift is a type of variation which occurs in influenza A viruses when a new IAV is developed by the change of HA or sometimes NA that is introduced in human population. This event might happen when IAV is transmitted from an animal or bird to humans. Or when because of genetic reassortment between the animal and human a new progeny virus is arised. Some say that the avian influenza A viruses should acquire at least one gene from humans when it crosses the species barrier. Humans can also be infected with swine influenza A viruses and these sporadic infections are very dangerous for pregnant women of persons with weak immune system (Cox and Subbarao, 2000).
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I.1.2. Pathogenesis of Influenza A virus
Influenza A virus is considered to be the most pathogenic member of its family. It can infect humans of all age leading to sporadic infections in population to epidemics and pandemics (Julkunen et al., 2001).
The Orthomyxoviridae family members cause influenza in mammals and birds as it is illustrated in Table 1. The Orthomyxoviridae family consist of Influenza A, B and C virus and 2 other family members.
Number of gene segment
Humans, pigs, whales, birds, and others
Humans and seals
Hemagglutinin Esterase-Fusion (HEF)
Mostly humans, also found in swine
Table 1. Characteristics of different types of influenza virus (reupdated after Cheung and Poan, 2007).
Influenza viruses replicate in the epithelial cells of the upper respiratory tract.
I.1.3. Influenza A virus morphology and structure
As morphology, the virions are spherical and are about 100-200 nm in diameter and filamentous and measure about 100 nm in diameter and 20Âµ long. HA is known to be very important in the attachment and entry of virus to the host's cell, whereas NA is associated with the release of the new virus from cells. Following attachment to specific receptors, virions are endocytosed and the acid pH in the endosomes facilitate a change of the HA. Protons facilitate the release of M1 protein from the ribonucleoprotein complex, following RNA to enter the cell nucleus for transcription and replication (Collier and Oxford, 2000).
Influenza A Virus is an Orthomyxoviridae family member, with an enveloped negative sense RNA genome. It has 8 RNA segments that encode 11 proteins, and the recently discovered N40 protein that is expressed in the PB1 segment (Medina and Garcia-Sastre, 2011). Eight of the proteins are packaged in enveloped virions and the viral envelope is made out of two envelope glycoproteins: hemagglutin (HA) which is the receptor binding membrane fusion protein, and the enzyme neuraminidase (NA), (Enami et al, 1991).
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The following elements are found in influenza A viruses: 3 polymerases (PB1, PB2, and PA), matrix protein M1, ion channel protein M2 and non structural proteins NS1 and NS2 (Julkunen et al., 2001)
Influenza A virus is an enveloped virus with pleumorphic virions. The shapes differ from small spherical to long filamentous. HA, NA, M2 viral proteins are affecting the morphology of the influenza virus particles. The lipid envelope characteristic for influenza A virus is hosts cell derived during budding. HA, NA and M2 are found in the lipid envelope. The spike glycoproteins are rod shaped and mushroom shaped according to electron micrographs studies. Homotrimer HA involved in membrane fusion and it's a receptor binding, NA homotetramer hydrolyses sialic acid gropus in order to release the new viral particles. M2 is an integral membrane homotetramer and functions as an ion channel (Cheung and Poon, 2007).
In 1980's it was discovered that the Influenza A virus transcription was located in the nucleus. This major finding has led to the conclusion that influenza viruses uses the nuclear compartment to transcribe and replicate its genome using the host's RNA Pol II transcription and splicing machinery (Amorim and Digard, 2006).
I.1.4. Influenza A virus components
Influenza A virus has 8 segments that are encoding for 11 viral genes: hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), matrix 2 (M2), nucleoprotein (NP), non-structural proteins (NS1, NS2), polymerase acidic protein (PA), polymerase basic protein (PB1, PB2, PB1-F2). Mostly all the influenza A virus virions have been shown to be spherical, and HA, NA and M" are the three viral transmembrane proteins that are making the viral envelope lipid bilayer. The host's lipid bilayer is rich in cholesterol enriched lipid rafts. HA is found in abundance as an envelope protein and a very small percentage for NA and M2. HA and NA are affiliated with the lipid rafts. M1 is found underneath the viral lipid membrane and is called matrix proteins because that where it holds the vRNPs. The vRNPs are created out of negative stranded RNAs that are packaged around NP. The polymerase proteins PB1, PB2 and PA are found at the edge of vRNPs and together are producing the viral RNA polymerase complex (Tasleem Samji, 2009).
I.1.4. Influenza A virus life cycle
The influenza A virus has the following life cycle stages: host cell entry, vRNPs entry into the nucleus, transcription and replication of the viral genome, vRNPs export from the nucleus, assembly and budding at the host's cell plasma membrane (Tasleem Samji, 2009).
The sialic acid found at the cell surface glycoproteins are functioning as receptors for influenza A virus. The viral particle is then endocytosed thru clarithrin - dependant endocytotic pathway. The fusion is induced by the low pH in endosomes, and as a response the viral ribonucleoprotein (vRNP) is released into the cytoplasm. Viral nucleocapsid is introduced into the nucleus and mRNA synthesis is induced by viral RNA polymerase which contains PB1, PB2 and PA polymerases. M1 and NS2 proteins are also migrating to the nucleus and as a result of their interaction with the vRNPs the transport of the newly synthesised vRNPs is controlled. vRNP-M1 protein complex collaborates with HA and Na molecules and as a result the budding of the virions takes place. Once the IAV production is dynamic it destroys the hosts cell pre-mRNAs, inhibits translation of mRNA and the hosts cell is killed by apoptosis or by cytolitic mechanisms (Julkunen et al., 2001).
Host cell entry
HA plays the most important role in this first stage. HA is a homotrimer and forms spikes at the surface of the viral lipid membrane. These spikes are sialic acid bound at the host's cell. After attaching of the viral particles they are incorporated into the host's cell using the clathrin - dependent endocytosis. The pH in the endosome is low and that leads to fusion between the viral and endosomal membrane. The acidic environment in the endosomal compartment leads to the activation of the M2 ion channel. M2 is a transmembrane protein that forms a proton selective ion channel. Because of the M2 ion channel acidification the vRNPs are released (Tasleem Samji, 2009)
According to Pielak and Chou, 2010 the M2 protein is encoded on the 7th RNA segment along with M1 and has three segments: an extracellular N-terminal segment, a transmembrane segment and an intracellular C terminal segment. M2 protein is activated proton channel that mediates the uncoating of the viral particles after their fusion in endosomes by controlling the acidification of the inner parts of the endocytosed particles (Stouffer et al., 2008). M2 specific 97 amino acid membrane protein is essential in the successful release of the viral genome during influenza A virus entry. With the interaction of M1 the viral genome is released and the viral RNA segments will travel to the host's nucleus. This dynamic process is followed by influenza by influenza virus RNA replication and transcription (McCown and Pekosz, 2006).
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Research on spherical virions has shown that influenza virus enters the cell by receptor mediated endocytosis, although the mechanism on how the endocytosis is induced is still not known. A suspicion is that spherical virus binding particles are activating a receptor tyrosine kinase resulting in cellular signalling that triggers the incorporation of influenza virions. It is believed that the filamentous influenza virions may enter the cells by using the macropinocytosis. In infected cells, both forms of influenza virus use lipid raft domains located at the plasma membrane. These lipid rafts are used as the sites of virus assembly and budding (Rossman and Lamb, 2011).
Lipid rafts are located at the plasma membrane and are specific microdomains that are involved in cellular traffic and signal transduction pathway. In some studies the rafts have been identified as floating elements within the cytoplasmic fluid. Because of their small size, mobility and instability rafts are behaving as transporters of proteins from the Golgi apparatus to the cell surface (Chazal and Gerlier, 2003).
The viral fusion requires that the responsible viral protein to activate a hydrophobic fusion peptide. This change is favourised by neutral pH, where the viral envelope's glycoproteins are binding to the cellular receptors or by an acidic pH fusion protein. As a response the viral particles are first induced in endocytosis, that being the case of influenza viruses. Experimental data has shown that lipid rafts located at the membrane have role in the virus entry. First there is the collection of the viral glycoproteins in the rafts and their interaction and recrution of the cellular receptor in rafts. Finally the virus inhibition is resulted as a response to a low level of cholesterol (Chazal and Gerlier, 2003).
A crucial role in virus entry, assembly and budding has the matrix protein M2 a proton selective, ion channel domain that together with M1 provide the virion's structure and mediate the viral lipid membrane - ribonucleoproptein (RNP) interaction (Rossman and Lamb, 2011). It was shown that low pH in the endosome has effects in activating the HA mediated fusion of the viral endosomal membranes and also for the M2 activation channel. HA and NA in influenza A virus have an intrinsic relation with the lipid raft domains compared with the M2 protein that is outside of these domains. M2 is responsible for mediating the proton conduction to the virion core. This will have effects on the RNP of M1 that will trigger its transport to the nucleus and marks the start of the viral replication (Rossman et al., 2010).
vRNPs entry into the nucleus
The viral transcription and entry of influenza A virus is effectuated in the nucleus. NP, PA, PB1, PB2, are viral proteins that create the vRNPs. Because of the nuclear localisation signals (NLS) vRNPs can bind to the nucleus import machinery, therefore they enter the nucleus (Tasleem Samji, 2009).
Transcription and replication
Influenza a virus has a negative strand RNA and it needs a positive sense RNA strand for its genome to be transcribed. The positive strand RNA serves a template for the vRNAs production. Viral RNA dependent RNA polymerase (RdRp) initiates RNA synthesis and many of the host's cell machinery are used, such as host transcription machinery. The hosts cellular molecules that are involved in the RNA synthesis are: BAT1, Heat shock protein 90 (Hsp90), the mini-chromosome maintenance complex (MCM), Tat-SF1 and DNA dependent polymerase II. The vRNA are transcribed into mRNA and replicated. Cellular RNA polymerase II binds to DNA and initiates transcription. RdRp is known to bind to Pol II. Splicing factor BAT1 for mRNA interacts with NP and leads to the formation of NP-RNA complex. Tat-SF1 facilitates the NP-vRNP formation complex. Hsp90 interacts with PB2 and stimulates viral RNA synthesis (Watanabe et al., 2010).
Export of vRNPs from the nucleus
Negative sense vRNPs are exported from the nucleus using the CRM1 dependent pathway through the nuclear pores. NP is interacting with CRM1, M1 protein is interacting with the vRNPs because of its C-terminal and because if it's N-terminal of M1 binds to NEP. After the binding of M1 to the RNP complex, NEP attaches to M1-RNP complex using its C-terminal domain. Heat shock cognate (Hsc) 70 protein and MAP kinase cascade are believed to play important roles in the nuclear transport of the vRNP complexes (Tasleem Samji, 2009).
Assembly and budding
Once the vRNAs are outside of the nucleus, the virus prepares its viral particles to be released outside of the cell. Influenza A virus uses the host's plasma membrane to form the viral particles. All the viral proteins that are found in the lipid bilayer HA, NA and M2 are found at the viral particle forming. Virus particles bud from the apical side of polarised cells, triggering the transport of HA, NA, and M2 to the apical plasma membrane. Studies have shown that M2 cytoplasmatic tail (CT) plays a very important role in viral particles formation. The deletion of M2 CT led to the production of elongated viral particles. M1 is involved in final steps of budding. A very important stage occurring right before the release of the new viral particle is the cleavage of sialic acid residue from glycoproteins. Prior to bud release, NA removes these sialic acids and the newly made viral bud is released (Tasleem Samji, 2009).
Viral morphogenesis and budding need four steps: viral components assembly, bud initiation, bud developing and incorporation in the plasma membrane. The most important events of the viral budding happen during assembly, when the viral components are brought to the budding site where they initiate budding. Influenza virus particles require three elements: the viral envelope containing a lipid bilayer and the three viral transmembrane proteins: HA, NA and M2. The protein that forms a bridge between the envelope and the viral core is M1 protein, located underneath the lipid bilayer. The viral nucleocapsid or viral core consists mainly of vRNP (viral nucleocapsid) (Nayak et al., 2009).
HA associated with the lipid raft is crucial for viral replication. Nevertheless studies have shown that the activation of the antiviral protein Viperin causes an instability of the lipid raft domains that has effects in influenza A virus replication diminishing. In studies were virus like particles (VLP) have been used, the HA protein buds from cells in vesicles and it does not require any viral protein expression. VLPs lack of protein synthesis that normal viral proteins have and as a result the VLPs show aberrant assembly and budding. M1 viral protein is a good candidate as a budding completion element. M1 interacts in the virion with the plasma membrane and the NP and it was shown that M1 is polymerised at the sites of budding thus it may provide the viral elongation mechanism. When used in VLP system it was noticed that M1 together with HA induce increased efficiency of the viral budding. But what was more surprising was that the addition of M2 protein in the HA+M1+M2 complex that was used in the VLP system resulted in significant budding increasing. Thus M2 is essential for the completion of the viral budding process (Rossman and Lamb, 2011).
I.2. M2 viral protein
M2 viral protein is a proton selective ion channel incorporated within the viral envelope and allows protons to enter virus particles in endosomes. M2 is a tetramer which consists of four polypeptide chains of 97 residues. The 24th residue is an N - terminal extracellular domain that acts as a transmembrane domain (Rossman et al., 2010).
M2 is pH activated and controls the levels of acid inside the viral particles. Rossman et al., have shown that M2 amphipathic helix is conserved when studied in influenza A virus. The amphipathic helix is used by the viral proteins to modify the membrane's shape, M2 is able to modify the shape of the membrane as the cholesterol is able to modify the amphipathic helix membrane curvature. When examined the localisation of M2 by immunogold labelling and electron microscopy in infected cells it was revealed that M2 is localised predominantly at the base of budding virions. The most important finding in this study was that mutated M2 amphipathic helix blocks membrane scission indicating a major role of M2 during the final stage of viral budding (Rossman et al., sept 2010).
I.3. Annexin A6
Derived from Greek "annexin" which means "bring/hold together" suggesting the property of the annexins to hold precise biological structures such as membranes. Annexin proteins are binding Ca2+ domains and contain a conserved annexin repeat of 70 amino acid sequence (Gerke and Moss, 2002).
Annexin A6 is a member of the Annexin family of calcium/ phospholipid binding proteins. Annexin A6 is expressed in mammalian cells mostly in specialised cell types such as endocrinal cells, and different types of Annexin are regulated in different ways (Smith et al., 1994).
Annexin A6 contains two domains: a Ca2+ dependent conserved core and phospholipid binding; and a variable N terminal tail (Enrich et al., 2010).
The known functions of annexin A6 associated with the functions of the annexin family member are: phopholipase - A2 inhibition, anticoagulation, regulation of the sarcoplasmic reticulum calcium channel, and Annexin A6 alone is involved in growth regulation, vesicular trafficking mechanisms such as endocytosis and exocytosis. Annexin A6 interacts with the cytoskeleton and plasma membrane and isolated Annexin A6 from sciatic nerve binds to actin. The human Annexin A6 is localised on chromosome 5q (Edwards and Moss, 1995).
Annexin A6 localisation studies have revealed that the Annexin A6 is present in mitochondria's inner membrane of different tissues by using immunogold electron microscopy. The localisation is associated with the Ca2+ high levels found in mitochondria (Rainteau et al., 1995).
Annexin A6 lowers the levels of cholesterol in the Golgi apparatus at the plasma membrane due to the fact that Annexin A6 sequesters cholesterol in endosomes. Annexin A6 also is targeting proteins for some signalling pathways, the most important being p120GAP, that downregulates Ras. It is believed that the regulators recruiting potential of Annexin A6 involved in the EGFR/Ras pathway is promoted by the Annexin A6 actin remodelling. There are characteristics that lead us to another attribute: Annexin A6 organiser of the membrane domains. Annexin A6 is a modulator of cholesterol homeostasis, scaffold formation by the signalling complexes and membrane - actin adjuster in the endocytic and exocytic transport (Enrich et al., 2010).
It was also demonstrated that Annexin A6 had no effects in the virus entry stages (Ma et al., 2012).
The group of Dr. Beatrice Nal has hypothesised in 2011 that M2 viral protein may interact with host factors that will help lead to suppression or have an enhancing effect in the influenza A virus infection process. A good candidate for this hypothesis is Annexin A6 human protein that was identified as novel cellular factor that interacts with M2.
As hypothesised, the results showed that M2CT interacts with human Annexin A6 using yeast two hybrid screen as seen in table 2.
M2 CT (cytoplamic tail)
Random primed human placenta cDNA
Numbers of interactions tested
Numbers of positive clones for Annexin A6
Table2. Yeast two - hybrid screening, which have led to the identification of the Annexin A6 - M2 CT interaction (Reupdated after Ma et al., 2011).
M2 was used as target (bait) was used to screen a random primed cDNA library from human placenta in a yeast-two-hybrid assay. This experiment has tested 63.89 million interactions and 15 positive results were identified that have shown interaction between M2 CT and Annexin A6. Further experiments have showed that M2 binds to Annexin A6 in human cells HEK293T infected with influenza a virus A/WSN/33. A549 cells were used to study the relative subcellular localisation of M2 and Annexin A6 interaction, A549 cells were transfected with an Annexin A6 encoding plasmid and infected with A/WSN/33. In non infected samples Annexin A6 was distributed in the same proportion in the cytosol, the infected but not transfected samples showed M2 localised at the endoplasmic reticulum and Golgi apparatus and also at the plasma membrane. The transfected infected cells showed same M2 level as the non transfected ones, but after using a confocal microscope it was revealed colocalisation of M2 and Annexin A6 at the plasma membrane in the infected cells. Silencing Annexin A6 led to significant progeny virus titer increasing, thus Annexin A6 negatively modulated influenza A virus infection. Nevertheless further experiments showed that negative modulation induced by the absence of Annexin A6 happens only in late stages of virus life cycle. And the most important discovery was that human protein Annexin A6 negatively modulates influenza A virus replication by not allowing the virus budding process to complete. Following Dr. Beatrice Nal group's finding that Annexin A6 is a negative modulator of influenza A virus infection at the budding stage, indicated by the number drop of the virus progeny PFU titer in supernatants and the released virions in A431-Anx A6 cells that were showing an unspecific elongated shape instead of the spherical shape which is characteristic for influenza A virus. Annexin A6 is involved in biological development of the host's mechanisms, and these mechanisms are also used by the influenza virus in important viral steps such as assembly or budding, for example cholesterol and cortical actin (Ma et al., 2012).
In another study, the cytoplasmic part of M2 influenza A virus was found to interact with caveolin-1, that is cholesterol binding hosts protein and it is found abundantly in the lipid raft (Wang et al., 2011).
The current treatment used against influenza A virus infection is Amantadine drug that targets M2. Drug resistance and side effects of this drug are increasing so science is in need of a new antiviral drug. As ion channels are essential for the viral life cycle, studies are focusing on these membrane proteins with aim to find new possible interactions that are taking place between the viral protein channels and the host's protein channels.
Cellular factors that are interacting with M2 are potential candidates for negatively modulating its activity. As it was demonstrated that Annexin A6 is restricting influenza a virus infection at the budding stage new revolutionary interaction discoveries will lead to better drug and treatment to be developed (Nieva et al., 2012).
Annexin A6 known as the member of the Annexin family and a Ca2+ dependent membrane binding protein, known to "bring together" calcium signalling and membrane functions. Annexin A6 interacts with AP-1-clathrin complex at the Âµ1 and Âµ2 subunits that are involved in endocytosis (Ma et al., 2011).
The molecular mechanism involving Annexin A6 and responsible for the negative modulation of virions' release from infected cells and/or morphogenesis defects is not known.
Having as starting point the study made by Dr. Nal's group in Hong Kong, there are still pending questions that need to be answered. Further studies should clarify whether or not Annexin A6 is a linker to stabilised cortical F-actin. This linkage could be related to M2-mediated late stage of virus budding and be a possible factor of the virus budding completion delay or defect that was found in A431 cells Annexin A6 overexpression. Annexin A6 induced changes in cortical actin cytoskeleton which may also affect other actin dependent steps of virus assembly and budding (Ma et al., 2011). In order to find an answer to this question, further studies should focus on viral life cycle stages changes in F-actin depleted cells.
Knowing that Anx A6 is also a regulator of cholesterol and Anx A6 is also a regulator of cholesterol and Anx A6 overexpression led to decreased levels of condensed membrane domains, a new study should clarify if the viral budding is depleted as a result of Anx A6 expression or because of low levels of cholesterol and decreased levels of condensed membrane domains induced by the Anx A6 depletion or overexpression.
And at last but not least new experiments should clarify the molecular mechanism that underlies the Anx A6 mediated depletion of influenza virus budding.
In this project we have used MDCK cells as a control and A431- human vulval squamous epithelial and A549- human alveolar basal epithelial stable cells as experimental materials.
The hypothesys of my work is that Annexin A6 localises with M2 at the neck of budding in influenza A virus at the surface of infected epithelial cells. Following this hypothesys a further objective of the study is to show that annexin A6 affects virus morphogenesis through its interaction with influenza A virus M2.
The main objective of this project is to optimise the conditions for the Immunofluorescence assay by labelling M2 viral protein and Annexin A6 human protein in influenza A virus A/WSN/33 (H1N1) infected cells. The optimised conditions for the Immunofluorescence assay will be applied in further assays that will investigate the Anx A6 - M2 interaction and the defective viral budding phenotype.
Another objective of this study is to find the optimal conditions for scanning electron microscopy (SEM) protocol. The SEM experiment was done on MDCK influenza A virus A/WSN/33 (H1N1) infected cells to observe the viral buds morphology. Further SEM experiments should have the optimised conditions for detailed investigation by labelling M2 and Annexin A6. Also these conditions could be applied for a future electron microscopy experiment on influenza A virus infected cells.
Chapter II. Materials and Methods
Human alveolar basal epithelial (A549), human vulval squamous epithelial (A431) and Madin-Darby Canine Kydney (MDCK) cells were sent by our collaborators Francois Kien from the Pasteur Institute in Hong Kong, and grown in Dulbecco's modified Eagle's medium (DMEM) (Thermo Scientific, Rockford, USA) supplemented with 10% fetal bovine serum (FBS)(Invitrogen, New-York, USA), 100Âµg/ml penicillin and 100 Âµg/ml streptomycin.
II.2.1. IAV amplification
MDCK cells were grown in 150 cm2 at a concentration of 1 x 105. Before infection, the cells were washed twice with PBS and 10 ml of pure DMEM was added to the flasks. Serial virus dilutions were prepared starting from a virus titer of 1.8 x 106 pfu/ml to 1.8 x 105 pfu/ml and finally the required viral dilution per flask of 1.8 x 104 pfu/ml. We added 1ml of virus MOI: 0.001 into the flask and left it to be absorbed by the cells for 1 h at 37oC. After 1 h we removed the unabsorbed virus and rinsed the cells with PBS and added 25 ml of infection medium: DMEM with final concentration of 1% P/S, 1 Âµg/ml TPCK - Trypsin, 0.3% BSA(Invitrogen, California, USA). After 3 days at 37oC the medium of the cell culture became partly cloudy due to cell death.
II.2.2 IAV Titration Hemagglutination assay
An IAV titration was performed by using hemagglutination assay in order to an accurate virus titer. The test was performed using 4ml of Chicken red blood cells (TCS Biosciences, Buckingham, UK) and Sheep red blood cells that have been topped up to 9 ml with PBS and then centrifuged at 2000 rpm, 4oC for 10 min. The cell pellet is diluted 1:10 in PBS using a 96 well plate we will add to each well 50 Âµl PBS and 50Âµl RBC resuspended at a concentration of 0.5%. Starting from left to right we added 50Âµl of IAV A/WSN/33 virus to the first well mixed well then transferred 50Âµl to the next well and so on. After 30 minutes we could read the positive results that should have formed uniform reddish colour and the negative ones that will appear as dots in the centre of the wells.
II.2.3 IAV Plaque Assay
Several plaque assays have been performed to find the accurate virus titer of the stock. Different conditions have been tested, in the first place the MDCK cells have been seeded on 6 well plates at a concentration of 3 x 105 , then we prepared the viral dilution (0 to 8) in the 0 well was added only virus and in the rest of the wells was added infection media as well. After 1 h incubation the unabsorbed virus was removed and the 2% Agar overlay in PBS was mixed with the infection medium (DMEM, 2%PS, 1Âµg/ml Trypsin-TPCK () and 0.6% BSA), to result a 1:1 dilution. We added 2-3 ml of overlay medium to each well and allowed to solidify at room temperature. The plates were then turned over and left 3 days for incubation. To see any plaques we had to remove the agar with the forceps and stain the cells with 0.1% Cristal violet in PBS.
II.3.1. Primary antibodies
Primary antibodies used in this study were monoclonal mouse anti-M2 (Santa Cruz Biothechnology, California, USA); polyclonal rabbit anti-annexin antibody (Abnova, Taipei, Taiwan); monoclonal mouse anti-annexin antibody (Abcam, Cambridge, UK); rabbit anti-myc antibody (Sigma, Dorset, UK).
II.3.2. Secondary antibodies
The secondary antibodies used in this study were: goat anti-mouse FITC (Jackson Immunosearch, Pennsylvania, USA); Phalloidin (Life Technologies, California, USA); goat anti-rabbit Texas-Red (Invitrogen, California, USA).
II.4. Immunofluorescence assay
II.4.1. Immunofluorescence assay on non permeabilised infected cells was done to study the co localization of viral protein M2 on MDCK and A431, A431 Annexin A6 cells. The cells were seeded on coverslips prior to infection at 3x105 was on coverslips and infected with influenza A/WSN/33 virus, using different MOI of 1, 0.1, 0.01. The cells were fixed in 4% paraformaldehyde (PFA) (VWR International, Lutterworth, UK) in PBS for 15 minutes. Quenching in 50mM NH4Cl (Fisher, Leicestershire, UK) in PBS for 15 minutes and then the cells were washed 2 times in PBS. For blocking the unspecific binding of antibodies the cells were incubated for 30 min in 10% normal goat serum (NGS) (Thermo Scientific, Rockford, USA) in PBS. The cells were incubated in monoclonal mouse anti-M2 (Santa Cruz Biotechnology, California, USA) diluted in 5% NGS in PBS and then conjugated with secondary antibody Goat anti-mouse - FITC (Jackson Immunosearch, Pennsylvania, USA) diluted in 5% NGS in PBS for 1 hour at room temperature. DAPI was used to stain the nuclei and the images were captured with an Olympus microscope BX 41.
II.4.2. Immunofluorescence assay on non permeabilised, permeabilised A/WSN/33 influenza A virus infected MDCK was done to study the co localization on M2 viral protein in MDCK cells. The cells have been grown on coverslips at a concentration of 3 x 105 infected with influenza A/WSN/33 virus at MOI. After 8h incubation the cells have been fixed in 4% paraformaldehyde in PBS pH 7.4 overnight. To try different conditions in different stages of infection some coverslips have been fixed after 24 hours post infection for 10 minutes in 50mM NH4CL. Some coverslips have been permeabilised in 0.08% Triton (Fisher Scientific, Leicestershire, UK) x 100 in PBS for 5 min, or in Tween20 (Fisher, New Jersey, USA) 0.1% in PBS. The non permeabilised samples were labelled for M2 viral protein with monoclonal mouse anti-M2 (Santa Cruz Biotechnology, California, USA) followed by incubation with goat anti-mouse FITC (Jackson Immunosearch, Pennsylvania, USA), the staining of F-Actin was done using Phalloidin (Life Technologies, California, USA) that directly stains actin in blue without adding a primary antibody. Staining after permealisation was done for 5 min in 0.08 % Triton in PBS at 4oC. Nuclei were stained for non-permeabilised cells with DAPI and the images were captured with Olympus microscope BX 41.
II.4.3. Immunofluorescence assay on permeabilised non infected A431 Annexin A6-myc, A431, A549 Annexin A6-myc and MDCK cells was done to study different antibodies on order to co localise Annexin A6. The cells were first grown on coverslips at a concentration of 3 x 105. Then they were fixed in 4% PFA in PBS for 15 minutes and quenched for 15 min in 50mM NH4CL. The cells were permeabilised in 0.08% Triton in PBS for 5 min at 4oC. After blocking for 30 min in 10% NGS in PBS the cells were incubated using the polyclonal rabbit anti annexin antibody (Abnova, Taipei, Taiwan), and the monoclonal mouse anti annexin antibody (Abcam, Cambridge, UK) diluted in 5% NGS and then conjugated with the secondary antibodies Goat anti-rabbit Texas Red (Invitrogen, California, USA) and Goat anti-mouse FITC (Jackson Immunosearch, Pennsylvania, USA). To identify myc we incubated the coverslips in rabbit anti myc antibody (Sigma, Dorset, UK) diluted in 5% NGS. The nuclei were stained with DAPI and the images were captured with Zeiss - Axioplan 2 microscope.
II.5. SEM assay on WSN infected MDCK cells
Scanning electron microscopy was performed on MDCK cells infected with A/WSN/33 virus to analyse the virus budding. Cells were primary fixed at 8 and 24 hours with 2.5% glutaraldehyde (Agar Scientific, Stansted, UK) in 0.1 Phosphate buffer (Fisher Scientific, Leicestershire, UK) pH 7.2 for 2 hours. Cells were washed in 0.1M Phosphate buffer pH 7.2 twice for 10 min following a secondary fixation with 1% Osmium tetroxide (Agar Scientific, Essex, UK) in 0.1 Phosphate buffer pH 7.2 for 1h at 4oC. The cells were washed twice with distilled water and then they were dehydrated in increasing concentration of ethanol. Critical point dry stage followed in CO2 using dryer Quorum K 859. After drying the coverslips were mounted onto aluminium holders and then coated with gold to increase their electrical conductivity. The samples were examined with Zeiss Supra 35 field emission scanning electron microscope.
Chapter III. Results
In this study we are trying to confirm that Annexin A6 is a host cellular regulator of influenza A virus by negatively influencing the viral budding. Nevertheless this modulation will not be taking place without the interaction of M2 with Annexin A6. The molecular mechanism that underlies the Annexin A6 - M2 interaction still needs to be investigated.
III.1.Following our objective to determine the best conditions of infectivity for A431 and A431 Annexin A6, an immunofluorescence assay was performed on non permeabilised infected cells.
Analysis of M2 expression at the surface of infected cells was undertaken and the objective of this experiment was to determine the best conditions for the infection of A431 and A431 annexin A6 cells with A/WSN/33 (H1N1) influenza virus. Another objective was the visualisation of M2 viral protein at the surface of infected cells. To try different conditions the cells were infected with influenza A virus at different MOI, respective 1, 0.1, 0.01 (only for MDCK cells). After 8h p.i. the cells were fixed and labelled without any permeabilisation step. MDCK cells were used as positive control. Monoclonal mouse anti-M2 conjugated with goat anti-mouse FITC were used as primary and secondary antibody.
In A431 cells M2 is distributed in the cytoplasm at MOI 0.1 (Figure 1, panel A), at MOI 1, M2 is mostly found at the plasma membrane noticeable in Figure 1, panel B. In A431 Annexin A6, M2 is visible at the plasma membrane (Figure 2, panel A) but can be noticed in intracellular compartments as well, close to the perinuclear region. This region is expressive for the M2 localisation at the endoplasmic reticulum and the Golgi apparatus. Panel B confirms that M2 is abundantly distributed in the intracellular compartments.
These results are confirmed by the MDCK control cells in Figure 3, that at 0.01 MOI (panel A) there is visible staining of M2 at the plasma membrane. At higher MOI 0.1 (panel B) and MOI 1 (panel C) M2 is visible at the plasma membrane but it is also visible in the cytosol.
These findings indicate that M2 viral protein and Annexin A6 human protein are modulating the infectivity of influenza A virus as shown that at the cells infected at the same influenza A virus MOI have resulted in M2 overexpression in the A431 Annexin A6 cells.
III.2. Following our objective to confirm that M2 has an important role during virus budding and release at late stages we performed an immunofluorescence assay on non permeabilised, permeabilised A/WSN/33 influenza A virus infected MDCK. This experiment has has confirmed that influenza A virus M2 viroporin plays a major role in virus particle release.
This experiment was done to analyse the influenza A virus M2 expression at the surface of MDCK cells. The objective of this experiment was to colocalise and describe the influenza A virus M2 expression at different post infection times 8h and 24 h p.i. in different conditions: permeabilised and non permeabilised. For better colocalisation of M2 we stained F-actin in MDCK influenza A virus infected cells using Phalloidin. Another objective of this experiment was the optimisation of the infection conditions in MDCK cells using A/WSN/33 (H1N1) influenza virus. MDCK cells were infected with A/WSN/33 (H1N1) and fixed at 8h p.i. and 24h p.i. M2 was labelled with monoclonal mouse anti-M2 and conjugated with goat anti-mouse FITC in non permeabilised cells. Two types of permeabilisation have been tried before labelling with 0.08% Triton x100 and 0.1% Tween 20. after permeabilisation the samples were labelled for M2 using the same antibodies mentioned above, and F-actin was labelled using Phalloidin. As control we used non infected MDCK cells that have been tested for all the conditions.
When permeabilised with 0.08% Triton the M2 protein staining done on influenza A virus infected cells was stronger. As seen in Figure 4, panel A, at 24h p.i. with influenza A virus MOI 0.5 - 5, M2 viral protein is abundantly present at the plasma membrane where some filamentous formations can be seen. In panel B the MDCK 24h p.i. with influenza A virus cells were not permeabilised and the image analysis reveals that M2 is visible at the plasma membrane, but M2 is abundantly found in the fiolopodia formed structures.
At 8h p.i with influenza A virus the MDCK cells visible in Figure 5, panel A show also M2 distribution at the plasma membrane but in addition M2 is found in the cytosol as well. Distinct punta-like areas are visible in the cytosol, where M2 and F-actin are expressed. In panel B, M2 was labelled before permeabilisation and the intensity of the labelling is not as strong. As observed in panel A, M2 labelling has the same pattern and in addition there are some rich F-actin areas that coincide with with the M2 rich areas.
Again the permeabilisation with 0.08% Triton resulted in better labelling as seen in Figure 6, panel A. Apart from the same distribution pattern some rich M2 areas can be observed. These punta-like patterns correspond with the punta-like F-actin rich areas. Panel B does not show a strong labelling, this might be due to the fact that Tween20 was used to permeabilise the cell, the image reveals some filopodia projections.
The results of this experiment leads us to conclude that permeabilisation with 0.08% Triton leads to better results, nevertheless further investigation is required.
III.3. The immunofluorescence assay on permeabilised non infected A431 Annexin A6-myc, A431, A549 Annexin A6-myc and MDCK cells has revealed that Annexin A6 is distributed in the cytoplasm.
Analysis of Annexin A6 was done at the surface of A431 Annexin A6, A431, A549 Annexin A6 and MDCK cells. The purpose of this experiment was to study the colocalisation of Annexin A6 - myc using different antibodies against annexin A6 and myc. After fixation the cells were permeabilised in 0.08% Triton x100. the cells were incubated in polyclonal rabbit anti-annexin antibody or monoclonal mouse anti-annexin antibody in order to label annexin A6, and they were conjugated with goat anti-rabbit Texas Red and goat anti-mouse FITC. To label myc we incubated the cells in rabbit anti-myc antibody conjugated with goat anti-rabbit Texas Red. As control we used A431 Annexin A6, A431, A549 Annexin A6 and MDCK labelled only with the secondary antibodies.
In Figure 7, panel A it is visible that for the conditions used the mouse anti-annexin works better that the rabbit anti-annexin. The image analysis revealed that Annexin A6 is evenly distributed in the cytoplasm. Panel B shows that Annexin A6 is mostly found at the plasma membrane.
When Annexin A6 is labelled in Figure 8, panel A using rabbit anti-annexin, it was revealed that Annexin A6 is distributed within the cytoplasm and some punta-like Annexin A6 rich regions are visible. In panel B myc and Annexin A6 was labelled using both mouse anti-annexin and rabbit anti-myc, and the results showed that Annexin A6 is rich at the plasma membrane and myc is distributed in the cytoplasm.
When used a different type of cell A549 Annexin A6 in Figure 9, Annxexin A6 distribution turned out to be throught the cytoplasm (panel A) and dinstict Annexin A6 rich zones seen around the nuclear envelope. In the panel B the II only labelled control was used to show that the signal revealed in the other cells have specific signal. In panel C we can see that mouse anti-annexin gave better results than the rabbit anti-annexin. Annexin is found distributed in the cytoplasm.
Figure 10, panel A rabbit anti-myc seems to work better than the rabit anti-annexin (Pannel B). Myc is found in the cytoplasm and distinct puncta-like myc zone rich are visible. Annexin A6 is found in the cytoplasm and again pattern of puncta-like zone rich Annexin A6 is visible.
Concluding these results we noticed that mouse anti-annexin antibody gave a more intense labelling than the rabbit anti-annexin antibody and when used rabbit anti-myc antibody resulted in nice specific labelling. Further investigations need to be done to reveal if the result of my experiment is accurate.
III.4. SEM assay on WSN infected MDCK cells
Analysis of the influenza A virus A/WSN/33 (H1N1) morphology at the surface of MDCK infected cells was determined by scanning electron microscopy (SEM). The purpose of this experiment was to describe SEM observations on the surface ultra structure of influenza A virus A/WSN/33 (H1N1) and study the morphology of the virus at different stages of infection. MDCK cells were infected with A/WSN/33 (H1N1) and fixing was done at 8h p.i and 24h p.i in glutaraldehyde followed by second fixation in 1% osmium tetroxide. After critical point drying the coverslips were mounted onto aluminium holders and coated with gold. As control we used non infected MDCK cells.
In Figure 11, panel A, the most obvious difference after analising the non infected, the 8h p.i and 24h p.i. influenza A virus MDCK infected cells is the cell count. In the non infected we can see numerous interconnected cells. In the 8h p.i. the cells lost their morphology and some debris are visible. The cells loose their cellular connection. In panel B under x1000 magnification does not reveal more than the panel A, and the image 12 panel confirms the observations made. In the panel B under the x5000 magnification in the non infected image numerous debris can be observed, and filopodia and lamellipodia structures are revealed. In the 8h p.i debris are abundant, no more lamellipodia is present only some filopodia spike filaments. In the 24h p.i cells, the cytoskeleton of the cells is still visible and some projections are starting to take form.
Figure 13, panel A reveals cytoplasmic projections or filopodia in the non infected sample. In the 8h p.i debris and filopodia are present but less that in the non infected. The 24h p.i. reveals under the x10000 magnification small projections at the plasma membrane. The morphological changes of the cells cytoskeleton are also visible. In panel B the 24h p.i reveals viral particles under x20000 magnification. Under the x50000 magnification there are visible viral particles and their sizes coincide with the influenza A virus general size of approximative 100nm. These viral particles also show the same morphology as the influenza A virus: spherical. Some viral particles are showing elongated morphology. Most all of the viral particles are released at the plasma membrane and some spherical budding observed that is connected to the cell surface by a neck.
Concluding these findings, it can be said that the conditions used for scanning electron microscopy can be applied in future experiments where M2 viral protein and Annexin A6 human protein will be labelled to analyse further their expression.
Chapter IV. Discussion
The distribution of Annexin A6 at low intracellular Ca2+ is diffuse, but as the Ca2+ levels are increased Annexin A6 is found at the plasma membrane, endosomes and in the secretory vesicles. This leads us to believe that because of the Ca2+ levels dysregulation after influenza A virus infection, Annexin A6 is only found in different subcellular membrane areas.
Overexpressing Annexin A6 might results in stabilisation of the cortical cytoskeleton as Annexin A6 interacts and rearranges the F-actin.