5 RESULTS

5.1  Aim of performing mutations

The mutations were designed to characterize the role of electrostatic interactions for the stability of the ectodomain of the HA protein in its native conformation. The mutations were categorised as stabilising or destabilising. An effort was made to identify amino acids specifically responsible for inter-monomer and intra-monomer stability.

5.1.1  Intra-monomer destabilisation

The tetrad salt bridge involving Glu 89, Arg 109, Arg 269 of HA1 with Glu 67 of HA2 (Fig.5.1.1.A) and that of Lys 299 of HA1 with Glu 69 of HA2 (Fig.5.1.1B) were the target regions to destabilise the HA protein. In each of the instances, positively charged Arg or Lys residues were substituted either by the negative Glu or by the neutral Gly amino acid. The mutations were designed to mimic the possible protonation effects of the negatively charged Glu residues. Thus, the intended destabilisation mutations were R109G, R109E, R269G, R269E, K299G, and K299E.

	Fig.5.1.1: HA crystal structure in backbone formation (mutants R109 and K299).
	The crystal structure of trimeric HA (right hand side) is shown with respective HA1 and HA2 monomers. The amino acids in the hinge region forming salt bridges are shown as spacefilled molecules. All the three monomers are shown with the respective residues.. The insets highlight the respective amino acids of only one monomer. The inset “A” shows Arg 109 of the HA1 chain forming a complex salt bridge with Glu 89 of HA1 and with Glu 67 of HA2. Arg 269 of the HA1 chain is also involved in the salt bridge formation with Glu 67 of the HA2 chain. The inset “B” shows Glu 69 of the HA2 chain forming a simple (single) salt bridge with Lys 299 of the HA1 chain.

A destabilising mutation was introduced at the distal tip of the HA protein, at the interface of the three HA1 monomers (Fig.5.1.2). The mutant was created by substitution of Thr 212 and Asn 216 to Glu. The mutant was designated as T212E-N216E. Selection of the site for mutation was based on the disulphide linking experiment of Godley et al. (1992).

A stabilising mutant was created at the same site as mentioned earlier, but with an attempt to create a salt bridge (Fig.5.1.2). To this end, the Thr 212 to Glu mutation was retained, and Asn in position 216 was changed to Arg. The resultant mutant was designated as T212E-N216R.

	Fig.5.1.2: HA crystal structure in backbone formation (mutant T212-N216).
	The crystal structure of trimeric HA (right hand side) is shown with respective HA1 and HA2 chains. Mutated amino acids in the three monomers are highlighted. The spacefilled molecules are located at the interface region of the HA1 monomers. The inset shows Asn at 216 of one monomer in close contact with Thr at 212 position of neighbouring monomers. These amino acids were mutated for either stabilising or destabilising the HA molecule (for details refer text).

An effort was made to enhance the stability of HA protein with the introduction of a salt bridge in the stem region near the interface region of HA1 and HA2. The target site was Ile 89 of HA2 in contact with Tyr 308 of HA1 (Fig.5.1.4B). In one mutation, Ile 89 was changed to Arg to form an ionic link with the OH group of Tyr 308. In another mutation, a double mutant was created with Tyr 308 being changed to Arg and Ile 89 to Glu. The resulting mutants were designated as I89R and I89E-Y308R. In addition, Ser 110 was substituted to Asp (Fig.5.1.4A) to build up ionic interaction with His 64 in its close neighbourhood.

	Fig.5.1.4: HA crystal structure in the backbone formation (mutants S110 and I89-Y308).
	The crystal structure of trimeric HA (right hand side) is shown with respective HA1 and HA2 chains. The amino acids near the hinge region at the interface of HA1 and HA2 chains are shown as spacefilled molecules. Selected amino acids in HA1 and HA2 are in close apposition within a monomer. Thus specific mutation of those residues could lead to the formation of a salt bridge. The insets highlight the respective amino acids of only one monomer. The inset “A” shows Ser 110 of the HA1 chain in close contact with His 64 of the HA2 chain. Ser 110 is mutated to Asp to explore the possibility of a salt bridge formation. The inset “B” shows Ile 89 of the HA2 chain in close contact with Tyr 308 of the HA1 chain. In one case, Ile 89 is mutated to Arg to stabilise the HA molecule with a cation-Pi interaction with Tyr 308. In another instance, a double mutant was generated with Ile 89 mutated to Glu and Tyr 308 mutated to Arg for establishing a new salt bridge formation.

Furthermore, the study also aimed to stabilise the HA by disulfide bonds. An attempt was made to determine the role of HA2 monomers in the low pH induced conformational changes. In particular, the amino acids were selected so as to give also insight into conformational changes relevant for triggering fusion. Therefore, non-covalent interactions (salt bridges) were chosen and substituted with covalent forces (disulfide linkages). In two instances, salt bridges [Glu 74 - Arg 76 (fig.5.1.5 B); and Arg 109 - Glu 67] were substituted with disulfide linkages in an effort to stabilise the HA protein. These mutations were E74C-R76C, and R109C-E67C. Cysteine mutations were also made at two additional sites as Ile 77 (fig.5.1.5 A), and at Thr 111 being in contact with Ala 44 (fig.5.1.5 C). These mutations were designated as I77C and T111C-A44C, respectively.

	Fig.5.1.5: HA crystal structure in the backbone formation (mutants I77, E74-R76, A44-T111).
	The crystal structure of trimeric HA (right hand side) is shown with respective HA1 and HA2 chains. The highlighted (spacefilled) amino acids are from three monomers. The spacefilled residues (in the top region of HA2 chains) are located at the interface region of HA2 monomers forming a complex salt bridge. The inset “A” shows (top view) Ile 77 of HA2 chain of all the three monomers. Ile 77 was mutated to cys to explore the possibility of a disulfide bond between the three HA2 monomers. The inset “B” shows (top view) Glu 74 of HA2 chain in close contact with Arg 76 of neighbouring monomer of HA2 chains. The Arg 74 and Glu 76 were mutated to cys amino acids to covalently link the HA2 monomers by disulfide bond. The inset “C” shows Ala 44 and Thr 111 from the same HA2 chain. These amino acids were mutated to Cys residues to cross link the two helices of HA2 chain within a monomer.

The selected amino acids for mutations were searched for their conservation among all the entire influenza virus group. The consurf 3D tool from http://consurf.tau.ac.il was used to align all the available HA sequences in PDB database onto the 3D crystal structure of H3 subtype (PBD id: 1Hgd). The results were summarized in the fig.5.2 along with visual colour key for reference. It may be observed that majority of the amino acids selected for mutagenesis were highly conserved.

	Fig.5.2: Consurf 3D: the amino acid sequence of known infuenza A viruses
	Unique amino acid sequences from H1-H13 were aligned using consurf web based tool. The sequences were aligned to the 3D crystal structure of H3 subtype (PBD id: 1HGD) and visualised using protein explorer. The colour key indicating the degree of conservation is given on the bottom. Only the amino acids selected for mutagenesis (labelled in blue) and those involved in the interaction (labelled in black) with the mutants are shown. Those of the amino acids, which are not conserved, are shown with their substituted amino acids (occurring as natural variants) in parenthesis.

The gene of the wild type HA protein (HA-wt) was a generous gift from Dr. Judith White (Department of Cell Biology, University of Virginia Health System, USA). The gene contained the entire HA1 and HA2 sequence cloned behind the T7 promoter in pTM1 plasmid vector. The gene sequence in the plasmid was sequenced and found to be 100 % homologous with that of the published literature. All the mutants were generated using this gene sequence as the source material. Special care was taken to design oligos for easy screening of mutants as described in the section 4.2. The list of the primers with their restriction sites was given in the section 4.2. The PCR technique (as schematically outlined in fig 4.1) synthesises the gene sequence of the whole plasmid (7026 bp) while incorporating the mutant in the desired position. The PCR product was treated with Dpn I. The Dpn I endonuclease is specific for methylated and hemimethylated DNA and was used to digest parental DNA (DNA from most of the E.coli strains is dam methylated). Only the parental DNA is susceptible to Dpn I treatment. The resultant gene sequence was subsequently transformed, where in the nicked DNA was repaired and ligated into a circular plasmid by E.coli DNA polymerase. The recombinant colonies picked from the YT-ampicillin agar plates were extracted for DNA by mini prep method (section 4.3.1). The resultant DNA was analysed by RE analysis for the incorporation of the new restriction enzyme site. The oligos besides containing desired mutation also had a new RE site as a silent mutation. Hence, the DNA that is proven positive for a new RE site also contained the desired mutant sequence. Finally, the desired sequence of the mutant gene was also confirmed by gene sequencing. Sequencing was commercially done at Invitek GmbH, Berlin.

The HA-wt and the associated mutant proteins were expressed in either CV-1 or COS-7 cells (both derivatives of African green monkey kidney; fibroblast-like; monolayers) using T7 RNA polymerase vaccinia system (section 4.5.1). The expression was carried using lipofectin and cells were analysed on incubation for 12-14 hrs with or without metabolic labelling. The HA-wt and its mutants were analysed for surface expression, trimer formation, and conformational changes and further for fusion efficiency with fluorescent labelled RBC’s.

HA-wt and its associated mutant proteins (R109E, R109G, R269E, R269G, K299E, K299G, S110D, T212E-N216R, T212E-N216E, I89R, I89E-Y308R) were expressed in CV-1 or COS-7 cells using T7 RNA polymerase vaccinia virus system. The cells after being metabolically labelled with [³⁵S]cysteine /methionine and incubated for 12-14 hrs, were then processed for cleavability of HA0 to HA1 and HA2 (section 4.5.3). The protein was further immunoprecipitated using N2 antibody (conformation-specificmAb, which specifically recognizes HA trimers at neutral pH) as described in section 4.6 and analysed on SDS-PAGE under reducing conditions. The epitope recognized by this antibody is located close tothe interface between the HA1 top domains of the native HA trimer(Copeland et al., 1986). The fluorogram of a typically expressed wt-HA in CV-1 cells is shown in Fig.5.5a. It shows the HA protein before and after TPCK treatment. In addition, it is seen that after TPCK treatment not all the HA0 is cleaved into HA1 and HA2. The uncleaved HA0 in lane 2, Fig. 5.5a, indicates that the protein was still under processing and remained within the cell at the point of TPCK treatment. A similar pattern was observed for all the mutants (Fig 5.5b and 5.5c) except that of R109E (lane: 2; Fig 5.5c), which was not expressed on cell surface by immunoprecipitation assays. The DNA concentration used in immunoprecipitation was 6 µg for HA-wt and all mutants except for R109E for which the DNA concentration was increased up to 10 µg. The expression level and its pattern of all mutants except of R109E were found to be comparable to that of HA-wt. Figure 5.5 only shows HA-wt and destabilising mutants. The surface expression of the other mutants will be shown along with the conformational assay.

	Fig 5.5: Surface expression of HA-wt and destabilising mutants.
	“ + ” denotes TPCK treatment and “ - ” denotes without TPCK treatment Fig 5.5 a: shows the HA-wt without (lane 1) or with TPCK trypsin treatment (lane 2). Fig 5.5 b: shows R269G (lane 1and 2), K299G (lane 3 and 4) and K299E (lane 5 and 6). Lanes 1, 3 and 5 show the respective mutant HA protein without TPCK trypsin treatment. Lanes 2, 4 and 6 show the mutant HA protein after TPCK trypsin treatment. Fig 5.5 c: shows R269E, R109E and R109G mutant HA proteins after TPCK trypsin treatment. Lane 2 shows R109E mutant being resistant to TPCK trypsin treatment indicating the lack of surface expression.

Secreted glycoproteins are synthesized on endoplasmic reticulum (ER) associated ribosomes and core oligosaccharide chains are attached co-translationally. HA is trimerised, and glycosylated in ER and transported to the Golgi complex on its route to cell surface, as described in section 1.2.3. During passage of the HA through ER and Golgi complex, trimming of oligosaccharides is done. Therefore, the glycosylation pattern of R109E on comparison with that of R109G and HA-wt would give more insight into the location (stacking) of the mutant protein in the cell. The glycosylation of HA protein was assessed using endoglycosidases (Endo H and PNGase F) as described in 4.8. Endo H removes simple, high-mannose N-linked oligosaccharides characteristic of ER localization but cannot remove N-linked glycans typical for processing in the Golgi (Maley et al., 1989). PNGase F digests all N-linked oligosaccharides regardless of their state of processing (Maley et al., 1989).

The fluorograph (Fig.5.6) shows glycosylation patterns for HA-wt (Fig.5.6, lanes 4-6) and R109G (Fig.5.6, lanes 7-9). It may be noticed that, both HA-wt and R109G have similar profile. In contrast, a significant difference was observed for the glycosylation pattern for R109E. The fig. 5.6 shows that HA-wt protein without trypsin treatment (precursor protein: HA0) was Endo H resistance due to loss of high mannose residues in the processed glycosyl chains on its transport to Golgi complex. The Endo-H resistance is a characteristic of proteins that have passed through medial Golgi compartment. A rather similar profile was observed for both HA-wt and R109G proteins even after trypsin treatment (HA0 cleaved into HA1 and HA2). The mutant protein from R109E on the other hand shows sensitivity to Endo H (Fig 5.6; lanes 11 & 14). The PNGase F treatment also showed similar profile (Fig.5.6; lanes 12 & 15) indicating similar activity for both the enzymes. The pattern of R109E was found to be similar irrespective of trypsin treatment in contrast to those for R109G and HA-wt. This shows that the R109E mutant protein was held up either in the ER or in cis-Golgi compartment and therefore not transported to the surface.

	Fig 5.6: Glycosylation assay for HA-wt, R109G and R109E.
	“ + ” denotes TPCK treatment and “ - ” denotes without TPCK treatment. “H” stands for Endo H enzyme and “F” stands for PNGase F

Trimerisation of HA molecules is an important step occurring in the ER. A homobifunctional cross-linker DSP was used to assess the trimer formation ability of all the mutants. The lysates as described in 4.9 were incubated with or without DSP reagent for 15 min at 15°C to covalently link the three monomers within each HA trimer. Samples without DSP reagent were taken as controls for the respective protein. The samples were immunoprecipitated and analysed by SDS-PAGE followed by fluorography. Fig. 5.7 reveals that the samples treated with DSP contained a higher molecular weight band when compared to their respective controls. Fig 5.7 shows that almost all mutants form a trimer similar to HA-wt (analysed on a 6% SDS-PAGE gel). The other mutants T212E-N216R, T212E-N216E, and R109E also showed trimer formation with DSP reagent, but the figures are not enclosed.

	Fig 5.7 : Trimer formation with DSP on a 6 % SDS-PAGE gel with non-reducing dye.
	“ + ” denotes TPCK treatment and “ - ” denotes without TPCK treatment The figure shows HA-wt (lanes 1, 1a); R269G (lanes 2, 2a); K299G (lanes 3, 3a); K299E (lanes 4, 4a); I89R (lanes 5, 5a); I89E-Y308R (lanes 6, 6a); and R269E (lanes 7, 7a). Lanes 1, 2, 3, 4, 5, and 6 show the respective HA protein band without DSP treatment. Lanes 1a, 2a, 3a, 4a, 5a, and 6a show the respective HA protein bands after treating with DSP. The difference in molecular weights between HA proteins with and without DSP show that trimer is formed.

Lowering the pH triggers the conformational change of the native state HA into its fusogenic state as described in section 1.3.2. It is known that the ectodomain of HA upon triggering the conformational change becomes sensitive to proteinase K (Godley et al., 1992). Thus, resistance of the HA protein to proteinase K treatment reflects its stability. The assay was performed as outlined in section 4.10, and the stability was probed stepwise decreasing the pH using the fusion buffer. The assay was done for HA-wt and for all mutants. The assay confirmed that HA-wt was sensitive to conformational changes at pH of ≤5.4 (Fig.5.8).

	Fig. 5.8: pH triggered conformational change of HA-wt.
	Upon incubation at the desired pH HA-wt protein was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. The HA1 domain becomes sensitive to proteinase K upon a conformational change of the HA ectodomain (Section 4.10). Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 8 corresponds to preincubation at pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0, respectively. HA1 becomes sensitive below pH 5.4 (lane 6). HA2 protein band also diminished at pH ≤ 5.4.

5.8.1  Destabilisation of intra-monomer interactions

The destabilising mutants (Fig 5.8.1a – 5.8.1f) were found to be sensitive to proteinase K enzyme at a higher pH (ranging from pH 5.6 till pH 7.0) as compared to HA-wt (Fig 5.8). The pH threshold for proteinase K sensitivity was always at higher pH for mutants carrying “Glu” (R109E; R269E; R299E) with respect to mutants carrying “Gly” (R109G; R269G; K299G) at the same position.

	Fig 5.8.1a: pH triggered conformational change of R109E.
	Upon incubation at the desired pH, R109E was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. The plasmid DNA of R109E used for transfection was 10 µg as against regular 6 µg for all other mutants. Lane 1 shows the protein at neutral pH without proteinase K enzyme treatment (control). It may be observed that the HA0 was not cleaved completely (faint bands of HA1 and HA2 could be seen). Lanes from 2 till 6 corresponds to preincubation at pH 7, 6, 5.8, 5.6, and 5.4 respectively. Very faint bands could be seen at pH 7.0 and pH 6.0 (lanes 2 and 3 respectively).

	Fig 5.8.1b: pH triggered conformational change of R109G.
	Upon incubation at the desired pH, R109G was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. The HA1 domain becomes sensitive to proteinase K upon a conformational change of the HA ectodomain (Section 4.10). Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 7 corresponds to preincubation at pH 7, 6, 5.8, 5.6, 5.4, and 5.2 respectively. HA1 becomes sensitive below pH 5.8 (lane 4).

	Fig 5.8.1c: pH triggered conformational change of R269E.
	Upon incubation at the desired pH, R269E was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 8 corresponds to preincubation at pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes sensitive below pH 5.8 (lane 4).

	Fig 5.8.1d: pH triggered conformational change of R269G.
	Upon incubation at the desired pH, R269G was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 8 corresponds to preincubation at pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes sensitive below pH 5.8 (lane 4).

	Fig 5.8.1e: pH triggered conformational change of K299E.
	Upon incubation at the desired pH, K299E was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 8 corresponds to preincubation at pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes sensitive below pH 5.8 (lane 4). But still the protein bands were visible till pH 5.4 (lane 6).

	Fig 5.8.1f: pH triggered conformational change of K299G.
	Upon incubation at the desired pH, K299G was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 7 corresponds to preincubation at pH 7, 6, 5.8, 5.6, 5.4, and 5.2 respectively. HA1 becomes sensitive below pH 5.6 (lane 5). Still the protein band was visible till pH 5.4 (lane 6).

The stabilising mutants (Fig.5.8.2a – 5.8.2c) were found to be resistant to proteinase K enzyme at all pH (from pH 5.0 –7.0) in contrast to HA-wt (Fig 5.8) and to destabilised mutants (Fig 5.8.1a- 5.8.1f). The mutants studied were I89R, I89E-Y308R, and S110D. The only exception among the stabilising mutants was S110D. The Ser 110 being in close proximity to His 64 of the HA2 chain was mutated to Asp in order to introduce a salt bridge. However, as revealed by proteinase K this mutation lead to a destabilisation of the HA ectodomain as indicated by Fig 5.8.2c.

	Fig 5.8.2a pH triggered conformational change of I89R.
	Upon incubation at the desired pH, I89R was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. The HA1 domain becomes sensitive to proteinase K upon a conformational change of the HA ectodomain (Section 4.10). Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 8 corresponds to preincubation pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes sensitive at 5.0 (lane 8).

	Fig 5.8.2b: pH triggered conformational change of I89E-Y308R.
	Upon incubation at the desired pH, I89E-Y308R was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lanes 1 till 8 shows the expressed protein (TPCK trypsin treated) corresponds to preincubation pH 7, 6, 5.8, 5.6, 5.4, 5.2, 5.0, 4.8, and 4.4 respectively. It may be observed that HA1 protein is resistant at the entire range of pH.

	Fig 5.8.2c: pH triggered conformational change of S110D.
	Upon incubation at the desired pH, S110D was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lane 1 shows the protein at neutral pH without proteinase K treatment (control). Lanes from 2 till 8 corresponds to preincubation pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes sensitive below pH 6.0 (lane 3) on contrary to other stabilising mutants

A destabilising mutant was created to study the inter-monomer interactions in the distal region. The mutant T212E-N216E (Fig.5.8.3) was found to be similar to HA-wt in its sensitivity to proteinase K.

	Fig 5.8.3: pH triggered conformational change of T212E-N216E.
	Upon incubation at the desired pH, T212E-N216E was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. The HA1 domain becomes sensitive to proteinase K upon a conformational change of the HA ectodomain (Section 4.10). Lane 1 shows the protein at neutral pH without proteinase K treatment. Lanes from 2 till 8 corresponds to preincubation pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes sensitive below pH 5.4 (lane 3) similar to HA-wt.

A destabilising mutant was created to study the inter-monomer interactions in the distal region. The mutant T212E-216R (Fig.5.8.4) was found to be similar to other stabilizing mutants in its resistance to proteinase K. The mutant was resistant to conformational change at the entire range of pH studied.

	Fig 5.8.4: pH triggered conformational change of T212E-N216R.
	Upon incubation at the desired pH, T212E-N216R was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions.. Lanes 1 till 7 corresponds to preincubation pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes resistant at the entire range of pH.

Two Cytseine mutants (E74C-R76C and I77C) were studied with respect to the low pH triggered conformational change. The proteianse K sensitivity of mutant I77C (Fig 5.8.5b) was very similar to that of HA-wt (Fig 5.8). The other mutant E74C-R76C (Fig 5.8.5a) shows only low surface expression and its pattern of proteinase K digestion was very indistinct (not clearly visible).

	Fig 5.8.5a: pH triggered conformational change of E74C-R76C.
	Upon incubation at the desired pH, E74C-R76C was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lane 1 shows the protein at neutral pH without proteinase K treatment. Lanes from 2 till 8 corresponds to preincubation pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes resistant at all the pH. A clear distinct HA2 was not observed at any of the pH.

	Fig 5.8.5b: pH triggered conformational change of I77C.
	Upon incubation at the desired pH, I77C was subjected to proteinase K conformational assay. Proteins were analysed on a 12 % SDS-PAGE gel under reducing conditions. Lanes 1 till 7 corresponds to preincubation pH 7, 6, 5.8, 5.6, 5.4, 5.2, and 5.0 respectively. HA1 becomes sensitive below pH 5.6, similar to HA-wt.

The formation of disulfide bond among the three HA2 monomers in the mutants E74C-R76C and I77C was explored. The respective mutants were expressed in CV-1 cells, and metabolically labelled as per the standard procedure. The cells were then lysed, and the lysate without being treated with TPCK trypsin was immunoprecipitated. The protein samples were analysed on SDS-PAGE using both reducing and non-reducing loading dyes. The disulfide linkage if formed should be intact in the samples treated with non-reducing dye, due to absence of β-mercaptoethanol in the loading buffer. Those samples treated with reducing buffer were considered as controls for the respective mutant. The Cys mutants were expected to harbour newly formed inter-monomer disulfide linkages in addition to the regular intra-monomer disulfide bonds in the HA-wt. The HA-wt does not have any inter-monomer disulfide linkages. Hence, a typical disulfide linkage in E74C-R76C would show a profile similar to that of DSP cross-linked HA-wt (Fig 5.7).

As may be seen from Fig.5.9, lane 2 the E74C-R76C formed a perfect disulfide bond between all the three monomers, while the I77C (Fig 5.9, lane 4) does not seem to have formed a disulfide bond.

	Fig 5.9: Cysteine bond formation for E74C-R76C and I77C.
	HA protein samples of E74C-R76C and I77C were analysed on a 9 % SDS-PAGE gel under reducing or non-reducing dye. Lanes 1 and 2 represent E74C-R76C and lanes 3 and 4 represent I77C. Lanes 1 and 3 correspond to reducing conditions while lanes 2 and 4 to non-reducing conditions.

The mutants were tested for their ability to promote fusion between CV1 cells and bound human RBCs. The RBCs were labelled using fluorescent-labelled dyes; the membrane bound red fluorescent R18 and the green fluorescent cytoplasmic marker calcein-AM. The fusion activity was monitored under fluorescence microscope by the transfer of lipid dyes from labelled RBC to HA expressing CV-1 cells.

To this end, HA-wt and all mutants were expressed in CV-1 cells (section 4.12.1). HA expressing cells were processed with trypsin and additionally with neuraminidase. The cells were bound to labelled RBC. The fusion was triggered at desired pH by incubating the cells in fusion buffer at 37°C with 5 % CO₂ for 5 min. Subsequently, cell media were re-neutralised with PBS. Fusion activity was monitored at neutral pH (7.4), at pH 5.0, and furthermore, at intermediate pH (5.4 and 5.6). Monitoring the behaviour of R18 alone would not allow differentiating between hemifusion and full membrane fusion and pore formation. To probe for the latter, the redistribution of the cytoplasmic label calcein to was followed. As a control, CV-1 cells transfected with vTF7-3 virus, but without any other DNA, was used.

Visually, binding of RBCs to all mutants was comparable to that of HA-wt at all the pH’s, but fusion efficiency varied among mutants (except S110D which was not taken for fusion studies). At pH 5.0, all destabilising mutants showed transfer of R18 and calcein dyes to the CV-1 cells. Although pH dependence of fusion paralleled the pH dependence of proteinase K sensitivity, a difference of ±0.2 pH units was observed between proteinase K assay and fusion experiments (in all destabiliing mutants, other than R269G). The other exception being T212E-N216E, which did not show any fusion activity, while its profile in proteinase K assay was similar to that of HA-wt, i.e., sensitivity below pH 5.4. The results from proteinase K and fusion experiments were summarised in Table .5.10.

None of the stabilising mutants showed any fusion activity at any of the pH, with the only exception being I89R, which showed, only transfer of R18 dye at pH 5.0, indicative ofhemifusion. The cysteine mutants (E74C-R76C, I77C, R109C-E67C, T111C-A44C) were not tested for their fusion efficiency.

As revealed by at least four to five independent experiments, the results from all the mutants were consistent and reproducible. Notably, leakage of calcein was observed to be very low.

	Fig 5.10a: Fusion assay for HA-wt.
	Fusion assay was done at 37°C, for 5 min. Transfer of fluorescent lipid dyes (R18 and Calcein) was observed only at preincubated pH 5.0 and 5.4. In comparison, Protinase K conformation assay showed that HA-wt is sensitive below pH 5.2.

	Fig 5.10b: Fusion assay for R109E.
	This mutant protein could not be expressed on surface and hence the failure of the fusion with labelled RBC was justified. Fusion was not observed at any of the preincubated pH.

	Fig 5.10c: Fusion assay for R109G.
	Fusion assay was done at 37°C, for 5 min. Transfer of fluorescent lipid dyes (R18 and Calcein ) was observed at pH 5.0 and 5.4. No fusion activity was noticed at pH 5.6 and 7.0. In comparison, Protinase K conformation assay showed that HA-wt is sensitive below pH 5.8.

	Fig 5.10d: Fusion assay for R269E.
	Fusion assay was done at 37°C, for 5 min. Transfer of R18 and Calcein fluorescent lipid dyes was observed at pH 5.0 and 5.4. In comparison, Protinase K conformation assay showed that HA-wt is sensitive below pH 5.8, but no fusion activity was noticed at pre-incubated pH of 5.6 and 7.0.

	Fig 5.10e: Fusion assay for R269G.
	Transfer of R18 and calcein fluorescent lipid dyes was observed at pH 5.0 and 5.4. Fusion activity was not observed at either pre incubated pH 5.6 (not shown) or at pH 7.0. Proteinase K assay showed that R269G was sensitive below 5.6.

	Fig 5.10f: Fusion assay for K299E.
	Transfer of fluorescent lipid dyes (R18 and Calcein) was observed at preincubated pH 5.0 and 5.4. No dye transfer was observed at pH values of 5.6 and 7.0. In comparision, proteinase K assay showed that protein was sensitive to conformational change below pH 5.8.

	Fig 5.10g: Fusion assay for R299G.
	Transfer of fluorescent lipid dyes (R18 and Calcein) was observed at pH 5.0, 5.4, and 5.6. No dye transfer was not observed at pH 7.0 (not shown). Proteinase K assay showed that mutant protein was sensitive to conformational change below preincubated pH 5.6.

	5.10h: Fusion assay for I89R.
	Transfer of fluorescent lipid dyes (R18 and calcien ) was observed only at pH 5.0. No fusion activity was seen at either pre-incubated pH of 5.4 and 7.0. Protainase K assay showed that mutant protein was sensitive to conformational change below pH 5.2-

	Fig 5.10i: Fusion assay for Y308E-I89R.
	Transfer of fluorescent dyes (R18 and Calcein) was not observed at any of pre incubated pH values studied indicating the stability of the mutant protein. Proteinase K assay showed that mutant protein was resistant to conformational change at all pre-incubated pH within a range of pH 5.0 to 7.0.

	Fig 5.10j: Fusion assay for T212E-N216E.
	Transfer of fluorescent dyes (R18 and Calcein) was not observed at any of pre incubated pH values within the pre-incubated pH range of 5.0 till 7.0. On contrary, proteinase K assay showed that the mutant protein was found to be very similar to that of HA-wt in its profile and was sensitive to conformational change below pH 5.4.

	Fig 5.10k: Fusion assay for T212E-N216R.
	Transfer of fluorescent dyes (R18 and Calcein) was not observed at any of pre incubated pH values studied indicating the stability of the mutant protein. Proteinase K assay showed that mutant protein was resistant to conformational change at all pre-incubated pH within a range of pH 5.0 to 7.0.

Table. 5.10: The summary of the pH threshold for HA-wt and all the mutants with respect to proteinase K assay and also for fusion assays.

The fusion assay was not performed for S110D and for the cysteine mutants. An attempt was made to quantify the fusion activity of the mutants and accordingly the efficiency was shown in the form of “+” symbols. The pH’s at which no fusion activity was observed are marked as “NO”.