The Mechanism Of Host Proteins DDX5 And HOIP Affecting The Replication Of Avian Influenza Virus In Human Cells

Influenza A virus is a single-stranded,segmented RNA virus in the Orthomyxoviridae family that causes a highly contagious respiratory disease in humans and animals.To date,pathogenic strains of Influenza A viruses have led to several serious pandemics and caused high mortality rates,for example the pandemics of 1918,1957,1968 and 2009.Avian influenza A viruses(AIVs)do not usually infect humans,but many,typically severe cases of human infection with H5,H6,H7,H9,and H10 subtype AIVs have been reported since 1997.However,the mechanism of AIV cross-host transmission of is still unclear,and the mechanism of how AIV utilizes host factors to efficiently replicate in mammalian cells is still unknown.Therefore,studying the role of host factors in the replication process of AIV can provide scientific basis for exploring the replication mechanism of AIV in human cells and finding new targets for anti-avian influenza drugs.Firstly,CRISPR screening technology was used to screen host factors that essential for efficient H7N9 AIV replication in human cells.A549 mixed cells containing all knockout information were constructed by using A549 cell lines with stable expression of Cas9 protein and 187536 sg RNA libraries targeting 18543 genes.The cells were infected with high-dose(10 MOI)H7N9 AIV,and then the surviving cells were enriched,and the candidate genes were identified by high-throughput sequencing analysis of the surviving cells.A total of 134 host factors that essential for efficient H7N9 AIV replication in human cells were identified(p < 0.01 was the screening condition).High score genes mainly include IFNAR2,SBNO2,STAT1,PDCD10,IFNAR1,CAB39,etc.These genes are mainly involved in natural immunity,apoptosis,transcriptional regulation and other pathways,providing effective data for the study of host factors essential for efficient AIV replication.Our previous protein-interaction omics data have shown that host protein DDX5 interacts directly or indirectly with AIV PB2 protein.First,we confirmed that DDX5 protein interacts with AIV PB2,PB1 and NP through Bi FC and Co-IP experiments,and the interaction was confirmed to occur in the nucleus by confocal microscopy.It was found that AIV virus replication titer decreased significantly at 36 and 48 hours by silencing or knocking out endogenous DDX5 protein expression using si RNA interference and CRISPR/Cas9 gene knockout techniques,respectively.As RNA helicase protein,the main function of DDX5 is to participate in the process and maturation of RNA.When endogenous DDX5 function is absent,AIV polymerase activity,m RNA,and c RNA production are affected,indicating that DDX5 affects AIV RNA transcription and replication.The nuclear export of AIV v RNP(Viral ribonucleoprotein)complex was observed to be affected in knockout cell lines,indicating that DDX5 affected the nuclear output of v RNP.Take together,the above results indicate that DDX5 interacts with AIV polymerase,promoting the activity of AIV polymerase,thereby promoting the transcription and replication of AIV RNA,and thereby affecting the nuclear output of RNP.Therefore,DDX5 is one of the host proteins that AIV relies on for efficient replication.Linear ubiquitin ligase complex(LUBAC)was composed by linear ubiquitin ligase protein HOIP with HOIL-1L and SHARPIN proteins,which plays an important role in the activation of NF-kappa B and anti-apoptotic pathway.Firstly,we confirmed that HOIP interacts with AIV PB2 and PB1 through Bi FC and Co-IP experiments,and the interaction was confirmed to occur in the nucleus by confocal microscopy.When HOIP and LUBAC were overexpressed in cells,the promoter activity of NF-kappa B was significantly increased.Meanwhile,overexpression of AIV polymerase protein can down-regulate the activation of NF-kappa B caused by LUBAC.The activation of NF-kappa B was down-regulated by both si RNA interference and CRISPR/Cas9 knockout of HOIP genes.It indicated that AIV polymerase protein down-regulated the activation of NF-kappa B by interacting with HOIP protein.In addition,overexpression of PB2 and polymerase complex can promote apoptosis caused by TNF alpha and TRAIL,suggesting that AIV polymerase promotes apoptosis by interacting with HOIP protein.Further validation showed that the increase of apoptosis promoted the nuclear export and packaging of v RNP into progeny virus,which promoted the replication of influenza virus.Take together,AIV interacts with HOIP through polymerase proteins,especially PB2 and PB1,to down-regulate the activity of NF-kappa B and promote apoptosis,thereby changing the permeability of cells,facilitating the generation of progeny viruses and enabling AIV to replicate better in human cells.