Mechanism And Effect On Virulence To Mammals Of H5 Subtype Avian Influenza Virus By Modulation Of HA Protein Glycosylation Sites

H5 subtype avian influenza virus(AIV)can cause high morbidity and mortality in poultry,which become endemic in China,Vietnam,Indonesia,India,Bangladesh and Egypt,resulting in great losses in the poultry industry worldwide.Although there is no evidence to prove that H5 virus can cause human to human transmission,the viruses have an ability of cross-species transmission and can infect human with high mortality,which poses a huge potential threat to humans.Hemagglutinin(HA),one of the most important pathogenic factors in AIV infection,contains several N-glycosylation sites and undergoes modification in the endoplasmic reticulum(ER)and Golgi apparatus.As H5 subtype AIVs evolve continuously,glycosylation site distribution of HA is becoming more and more complicated.H5 subtype AIVs prevailed in our country are mainly distributed in the clade 2.3.4.4 and 2.3.2.1 of the clade 2.3.In this study,AIV A/Mallard/Huadong/S/2005(H5N1,Clade 2.3.4)and its glycosylation site mutants were selected to investigate the effect of glycosylation site on AIV virulence and to explore its possible mechanisms.1.Effects of H5N1 subtype AIV HA stem glycosylation at position 10/11 on virus pathogenicityA stem glycosylation site of hemagglutinin(HA)is important to the stability of the HA trimmer.A previous study shows that the stem 10/11 overlap glycosylation site of the H5 subtype avian influenza virus may influence the cleavage of HA,whereas the exact site and its effect on virulence remain unclear.In this study,site-directed mutagenesis was used to generate single or double mutant rS-?10(10NNAT),rS-?11(10NNSA),and rS-?10/11(10NNAA)of the overlapping glycosylation site(10NNST)on the HA of A/Mallard/Huadong/S/2005(S).By using Western-blot analysis,we show that both rSY-?11 and rS-?10/11 mutant viruses had significant delay on HA cleavage and a reduced HA molecular mass compared to the wild-type virus rS,while the rS-?10 mutant virus exhibited a similar HA molecular mass to that of the wild-type virus rS.Interestingly,both rS-?11 and rS-?10/11 mutant viruses reverted their glycosylation sites at 11N after passage,indicating that 11N is a true and critical glycosylation site.Compared to the wildtype virus rS,rS-?11 and rS-?10/11 mutant viruses had decreased growth rates,reduced thermo-and pH-stability,decreased pathogenicity,and limited systemic spread.Therefore,our study suggests that the 11N glycosylation site plays a key role in HA cleavage,structural stability and pathogenicity in H5 subtype avian influenza virus.2.Effects of H5N1 subtype AIV HA head glycosylation on endoplasmic reticulum stressOur previous study shows that deletion of 158 or 169 glycosylation site on the HA head of the H5 subtype AIV strain rS-144-/158+/169+increases the viral virulence in mammals,however,the mechanism remains unknown.In this study,several AIVs with different deletions at HA head glycosylation sites 144,158 or 169 were tested for their biological characteristics to clarify the possible mechanism.The result showed that rS-144-/158-/169+and rS-144-/158+/169-viruses induced higher levels of inflammatory cytokines(IL-6?IL-8?IL-1? and TNF-?)than rS-144-/158+/169+did in the infected cells,but the TCID50,EID50 and MDT of the viruses showed no difference.Moreover,rS-144-/158-/169+and rS-144-/158+/169-viruses induced higher levels of endoplasmic reticulum(ER)stress in the cells.Inhibition of inositol-requiring enzyme 1?(IRE1?)phosphorylation reduced the inflammation induced by AIV infection.In vivo analysis of Kira6 intervention futher confirmed that ER stress played a key role in higher virulence for HA head 158 or 169 site de-glycosylation AIV Our findings reveal that deletion of additional HA head glycosylation sites 158 or 169 enhanced the AIV virulence via activating of strong ER stress and inflammation.rS-144-/158-/169+virus activated the c-Jun N-terminal kinase(JNK),X-box binding protein 1(XBP1),and nuclear factor-?B pathways by activating IREla phosphorylation under ER stress,whereas the rS-144-/158+/169-virus activated only the JNK pathway by altering IRE1? phosphorylation.3.Effects of H5N1 subtype AIV HA head glycosylation on Golgi apparatus stressGolgi apparatus is the downstream organelle of ER,which function is mainly to further modify unmatured protein from ER and transport to destination.Whether AIV can induce Golgi apparatus stress(GAS)infected cell remains unclear.In this study,H5 subtype AIV strain A/Mallard/Huadong/S/2005 was selected to test Golgi apparatus morphology and Golgi stress pathways after infection.The result showed that AIV infection caused A549 Golgi apparatus stress with morphology of swelling and broken Golgi apparatus.TFE3 pathway of GAS were strongly activated during AIV infection,HSP47 pathway of GAS was activated at early stage of infection but suppressed at late stage of infection,while CREB3-ARF4 pathway of GAS was not activated.Moreover,compared with wild type(WT)virus,the timepoint of Golgi apparatus collapse induced by rS-144-/158-/169+was early,while that of Golgi apparatus collapse induced by rS-144+/158-/169+was late.However,the timepoint and extent of Golgi apparatus collapse induced by rS-144-/158+/169-and rS-144+/158+/169-was similar as that induced by WT virus.No significant difference was found in activation of TFE3 pathway by HA glycosylation mutant viruses.While rS-144-/158-/169+virus induced relative higher HSP47 pathway activation than other viruses.These results indicate that rS-144-/158-/169+virus can induce higher GAS than WT virus,while rS-144-/158+/169-induced same GAS as WT virus.4.Effects of H5N1 subtype AIV HA head glycosylation on cell ferroptosisTo further study the effect of glycosylation site on AIV virulence.The level of cell ferroptosis was detected in A549 infected by H5N1 virus strain A/Mallard/S/2005.The results showed that AIV infection hindered the synthesis of cell antioxidant molecules.Specifically,expression of SLC7A11 and GPX4 protein gradually decreased during AIV infection,and the reduced glutathione gradually depleted with AIV infection.While the expression of COX2 gradually increased during AIV infection.Detection of cellular lipid peroxides and cellular iron aggregation proved that the cell death induced by AIV was ferroptosis.Ferroptosis inhibitor could significantly reduce LDH release and partially restore cell viability.Finally,compared to WT virus,the level of ferroptosis induced by rS-144-/158-/169+was relative higher while rS-144+/158-/169+was relative lower.However,the levels of ferroptosis induced by rS-144-/158+/169-and rS-144+/158+/169-were similar as that induced be WT strain.These data indicate that H5N1 subtype AIV infection can induce cell ferroptosis.rS-144-/158-/169+can induce higher cell ferroptosis than WT virus,while rS-144-/158+/169-induced the similar level of cell ferroptosis as WT virus.