Host proteins interact with viral elements and affect the life cycle of highly pathogenic avian influenza A virus H7N9

Abstract

Host-virus interactions can significantly impact the viral life cycle and pathogenesis; however, our understanding of the specific host factors involved in highly pathogenic avian influenza A virus H7N9 (HPAI H7N9) infection is currently restricted. Herein, we designed and synthesized 65 small interfering RNAs targeting host genes potentially associated with various aspects of RNA virus life cycles. Afterward, HPAI H7N9 viruses were isolated and RNA interference was used to screen for host factors likely to be involved in the life cycle of HPAI H7N9. Moreover, the research entailed assessing the associations between host proteins and HPAI H7N9 proteins. Twelve key host proteins were identified: Annexin A (ANXA)2, ANXA5, adaptor related protein complex 2 subunit sigma 1 (AP2S1), adaptor related protein complex 3 subunit sigma 1 (AP3S1), ATP synthase F1 subunit alpha (ATP5A1), COPI coat complex subunit alpha (COP)A, COPG1, heat shock protein family A (Hsp70) member 1A (HSPA)1A, HSPA8, heat shock protein 90 alpha family class A member 1 (HSP90AA1), RAB11B, and RAB18. Co-immunoprecipitation revealed intricate interactions between viral proteins (hemagglutinin, matrix 1 protein, neuraminidase, nucleoprotein, polymerase basic 1, and polymerase basic 2) and these host proteins, presumably playing a crucial role in modulating the life cycle of HPAI H7N9. Notably, ANXA5, AP2S1, AP3S1, ATP5A1, HSP90A1, and RAB18, were identified as novel interactors with HPAI H7N9 proteins rather than other influenza A viruses (IAVs). These findings underscore the significance of host-viral protein interactions in shaping the dynamics of HPAI H7N9 infection, while highlighting subtle variations compared with other IAVs. Deeper understanding of these interactions holds promise to advance disease treatment and prevention strategies.

Keywords: HPAI H7N9; Host proteins; Host-viral interaction; Life cycle; RNAi.

Fig. 1

RNAi screening of host factors related to highly pathogenic H7N9 avian influenza A virus (HPAI H7N9) HP001. (A) RNAi analysis of host factors required for the HP001 life cycle. After incubation with siRNA for 48 h, cells were infected with HP001 for another 48 h. Total RNA was extracted and the absolute quantity of HP001 RNA was measured by using a H7N9 nucleic acid test kit. All qRT-PCR experiments were performed in triplicate and repeated three times independently. Cells without siRNA interference but infected with HP001 served as a positive control (H7N9 group); cells transfected with isotype siRNA and infected with HP001 served as a negative control (isotype group). SiRNAs that reduced the number of viral copies significantly compared with the H7N9 group and isotype group were identified as possibly related to the HP001 life cycle (*p < 0.05). (B–E) RNAi silencing analysis of host factors that failed to inhibit HP001 efficiently. (F) Western blotting analysis of the targeted host proteins to verify siRNA knockdown efficiency. Normal 293T cells and cells transfected with non-targeting siRNAs served as controls. RNAi, RNA interference; qRT-PCR, quantitative real-time reverse transcription PCR; siRNA, small interfering RNA.

Fig. 2

Characterization of the targeted host proteins involved in the regulation of the HP001 life cycle. Networks, interactions, and functions of the 12 host proteins were evaluated using the GeneMANIA database. The functions were connected to COPI-coated vesicle function, vesicle-mediated transport related to Golgi and ER, COP9 signalosome, and defense response to virus and lysosomal membrane. COPI, coat protein; ER, endoplasmic reticulum; COP9, constitutive photomorphogenesis 9.

Fig. 3

Co-immunoprecipitation (Co-IP) and immunoblotting analysis of the interactions between targeted host proteins and HP001 proteins. Extracts from A549 cells infected with HP001 were incubated with antibodies recognizing targeted host proteins plus Protein G beads; proteins pulled down were detected using western blotting using anti-HA, M1, NA, NP, PA, and PB1 antibodies. Extracts containing HP001 incubated with Protein G beads without any antibody, and extracts from normal cells without HP001 mixed with Protein G beads and antibodies served as controls. Isotype antibodies served as negative controls. (A) Six host proteins (ANXA2, ANXA5, ATP5A1, COPA, HSPA1A, and HSPA8) interacted with HA. (B) Seven host proteins (AP2S1, AP3S1, COPA, COPG1, HSPA1A, HSPA8 and RAB18) interacted with M1. (C) Six host proteins (ANXA2, ANXA5, AP2S1, AP3S1, COPA and HSP90AA1) interacted with NA. (D) Seven host proteins (ANXA2, AP2S1, AP3S1, COPA, HSPA1A, HSPA8, and RAB18) interacted with NP. (E) Four host proteins (ANXA2, HSPA1A, RAB11B and RAB18) interacted with PA. (F) Four host proteins (ATP5A1, HSPA1A, HSP90AA1 and RAB11B) interacted with PB1. HA, hemagglutinin; M1, matrix 1 protein; NA, neuraminidase; NP; nucleoprotein; PA, polymerase basic 1; PB1, polymerase basic 2; ANXA2, annexin A2; ANXA5 annexin A5; ATP5A1, ATP synthase F1 subunit alpha; COPA, COPI coat complex subunit alpha; HSPA1A, heat shock protein family A (Hsp70) member 1A; HSPA8, heat shock protein family A (Hsp70) member 8; AP2S1, AP3S1, COPG1, COPI coat complex subunit G1; HSP90AA1, heat shock protein 90 alpha family class A member 1.

Fig. 4

Interactive network of 12 host proteins and six HP001 proteins. To summarize the interactions, an interactive network was constructed to allow an intuitive understanding of the interactions among the targeted host proteins and HP001 proteins. The connecting lines indicate interactions confirmed by co-immunoprecipitation (Co-IP).

Fig. 5

Immunoblot analysis of the samples before and after co-immunoprecipitation (Co-IP). (A) Immunoblot analyses of the samples before Co-IP proves the specificity of the antibodies. To verify the specificity of the antibodies against the target host proteins and HP001 proteins, extracts from A549 cells infected with or without HP001 were checked by immunoblotting analyses with antibodies recognizing the targeted host proteins or HP001 proteins before Co-IP. The results indicated a high degree of antibody specificity. (B) Immunoblotting analysis of the samples after Co-IP further demonstrated the specificity of the antibodies. Extracts from A549 cells infected with HP001 were incubated with antibodies recognizing the targeted host proteins plus Protein G beads; extracts containing HP001 incubated with Protein G beads without any antibody, and extracts from normal cells without HP001 mixed with Protein G beads and antibodies served as controls. After Co-IP incubation, samples were check by immunoblotting analyses with antibodies recognizing the targeted host proteins.