Passage
The influenza A virus is an enveloped RNA virus with potential for zoonosis (transmission from animals to humans) . It obtains its plasma membrane from its host cell, but the composition of the viral envelope and vertebrate plasma membrane are distinct. The envelope of H6-G225D consists mostly of phosphatidylethanolamine as well as some negatively charged glycerophospholipids. The outer membrane contains hemagglutinin (HA) proteins that bind to the carbohydrate known as sialic acid (SA) on host cell receptor proteins, facilitating viral entry. It also contains neuraminidase enzymes that break the bond between SA and galactose in the host membrane glycans, allowing new virions to be released.Minute changes in the genes that code for HA proteins can alter the virulence, host, and attachment mechanism. In general, influenza A infects avian species by attaching to tracheal tissue, which displays SA groups bound to receptors through an α-2,3-glycosidic linkage to galactose (Figure 1) . Avian viruses generally cannot attack human cells, in which SA is linked to galactose through an α-2,6-glycosidic linkage instead of an α-2,3 linkage.
Figure 1 Structure of the influenza A receptor in avian cell membranesIn a recent study, however, a G225D substitution in HA, caused by a GGC to GAU codon mutation, enabled avian virions to infect human cells. The stability of G225D HA proteins was compared to that of H6-P186L, which is created by a single CCC to CUC mutation and exclusively infects avian cells, and H1N1, which targets only humans. The viral RNA of H6-P186L, H6-G225D, and H1N1 was cloned using cDNA and expressed in embryonic kidney cells. The melting curve of each purified HA protein was determined using differential scanning calorimetry, with peaks representing melting temperatures (Figure 2) .
Figure 2 Hemagglutinin melting curves for H6N1 and H1N1 influenza A strains
Adapted from De vries RP, Tzarum N, Peng W, et al. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors. EMBO Mol Med. 2017;9(9) :1314-1325.
-Compared to H6-P186L virions, H6-G225D virions exhibit:

Question

Passage
The influenza A virus is an enveloped RNA virus with potential for zoonosis (transmission from animals to humans) . It obtains its plasma membrane from its host cell, but the composition of the viral envelope and vertebrate plasma membrane are distinct. The envelope of H6-G225D consists mostly of phosphatidylethanolamine as well as some negatively charged glycerophospholipids. The outer membrane contains hemagglutinin (HA) proteins that bind to the carbohydrate known as sialic acid (SA) on host cell receptor proteins, facilitating viral entry. It also contains neuraminidase enzymes that break the bond between SA and galactose in the host membrane glycans, allowing new virions to be released.Minute changes in the genes that code for HA proteins can alter the virulence, host, and attachment mechanism. In general, influenza A infects avian species by attaching to tracheal tissue, which displays SA groups bound to receptors through an α-2,3-glycosidic linkage to galactose (Figure 1) . Avian viruses generally cannot attack human cells, in which SA is linked to galactose through an α-2,6-glycosidic linkage instead of an α-2,3 linkage.

Figure 1 Structure of the influenza A receptor in avian cell membranesIn a recent study, however, a G225D substitution in HA, caused by a GGC to GAU codon mutation, enabled avian virions to infect human cells. The stability of G225D HA proteins was compared to that of H6-P186L, which is created by a single CCC to CUC mutation and exclusively infects avian cells, and H1N1, which targets only humans. The viral RNA of H6-P186L, H6-G225D, and H1N1 was cloned using cDNA and expressed in embryonic kidney cells. The melting curve of each purified HA protein was determined using differential scanning calorimetry, with peaks representing melting temperatures (Figure 2) .

Figure 2 Hemagglutinin melting curves for H6N1 and H1N1 influenza A strains
Adapted from De vries RP, Tzarum N, Peng W, et al. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors. EMBO Mol Med. 2017;9(9) :1314-1325.
-Compared to H6-P186L virions, H6-G225D virions exhibit:

Quizplus · Accepted Answer

11ecba2d_0eb4_d805_a3bf_59701895fe6c_MD0008_00 The stability of a protein can be analyzed by measuring the effect of temperature on its folded three-dimensional structure. Increasing temperature provides heat energy to disrupt intermolecular forces that promote folding, such as hydrogen bonds and hydrophobic interactions. As a result, proteins exposed to high temperatures lose their secondary, tertiary, and quaternary structures and can no longer fulfill their function in the cell. This process, called denaturation, is used to detect the melting point, at which there are equal amounts of folded and unfolded protein in a sample. In techniques such as differential scanning calorimetry, the rate at which proteins denature is correlated with the melting temperature T_m, shown as a peak. Proteins with lower melting temperatures are more susceptible to heat, and therefore denature faster. Figure 2 shows the melting curves for the HA proteins of three different subtypes of influenza A. The H6-G225D has the lowest melting temperature at 50.2°C (Choice C), and therefore it is more susceptible to denaturation. In contrast, the H6-P186L virion is more stable with a melting temperature of 53.5°C, so a greater amount of heat energy will be required to disrupt its folded structure. The gap between the T_m of H6-G225D and H6-P186L indicates that within the temperature range shown, the HA of H6-G225D will denature at a faster rate than the HA of H6-P186L. (Choices A and D) Intermolecular forces make proteins more stable and increase their T_m. H6-G225D melts at a lower temperature than H6-P186L, so the intermolecular forces in H6-G225D must be weaker, making the overall structure less stable. Educational objective: Increasing temperatures can cause proteins to unfold (denature) by disrupting the intermolecular forces that maintain secondary, tertiary, and quaternary structures. When the melting temperature of a protein is achieved, there are equal amounts of folded and unfolded proteins. A lower melting temperature indicates lower stability and a higher denaturation rate.