HACD3 Prevents PB1 from Autophagic Degradation to Facilitate the Replication of Influenza A Virus.

Influenza A virus (IAV) continues to pose serious threats to the global animal industry and public health security. Identification of critical host factors engaged in the life cycle of IAV and elucidation of the underlying mechanisms of their action are particularly important for the discovery of potential new targets for the development of anti-influenza drugs. Herein, we identified Hydroxyacyl-CoA Dehydratase 3 (HACD3) as a new host factor that supports the replication of IAV. Downregulating the expression of HACD3 reduced the level of viral PB1 protein in IAV-infected cells and in cells that were transiently transfected to express PB1. Silencing HACD3 expression had no effect on the level of PB1 mRNA but could promote the lysosome-mediated autophagic degradation of PB1 protein. Further investigation revealed that HACD3 interacted with PB1 and selective autophagic receptor SQSTM1/p62, and HACD3 competed with SQSTM1/p62 for the interaction with PB1, which prevented PB1 from SQSTM1/p62-mediated autophagic degradation. Collectively, these findings establish that HACD3 plays a positive regulatory role in IAV replication by stabilizing the viral PB1 protein.

M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes.

Influenza A virus (IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor (M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR, the nuclear accumulation of viral nucleoprotein (NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking, or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin (HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes, thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR-HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.

A Single Amino Acid Residue R144 of SNX16 Affects Its Ability to Inhibit the Replication of Influenza A Virus.

Influenza A virus (IAV) is an important zoonotic pathogen, posing a severe burden for the health of both animals and humans. Many host factors are involved in the life cycle of IAV to regulate its replication. Herein, we identified sorting nexin-16 (SNX16) as a new host factor that negatively modulates the replication of IAV. When transiently overexpressed in cells, SNX16 appears to be expressed as two obvious bands. Mutagenesis analysis indicated that the amino acid residue R144 of SNX16 was responsible for its two-band expression phenotype. We found that the R144A mutation of SNX16 changed its cellular distribution in A549 cells and partially weakened the inhibitory effect of SNX16 on IAV replication. Further investigation revealed that SNX16 could negatively regulate the early stage of the replication cycle of IAV. Taken together, our results demonstrated that SNX16 is a novel restriction host factor for the replication of IAV by engaging in the early stage of IAV life cycle, and a single amino acid residue at position 144 plays an important role in the cellular distribution and anti-influenza function of SNX16.