Hemagglutinin glycosylation pattern-specific effects: implications for the fitness of H9.4.2.5-branched H9N2 avian influenza viruses.

Since 2007, h9.4.2.5 has emerged as the most predominant branch of H9N2 avian influenza viruses (AIVs) that affects the majority of the global poultry population. The spread of this viral branch in vaccinated chicken flocks has not been considerably curbed despite numerous efforts. The evolutionary fitness of h9.4.2.5-branched AIVs must consequently be taken into consideration. The glycosylation modifications of hemagglutinin (HA) play a pivotal role in regulating the balance between receptor affinity and immune evasion for influenza viruses. Sequence alignment showed that five major HA glycosylation patterns have evolved over time in h9.4.2.5-branched AIVs. Here, we compared the adaptive phenotypes of five virus mutants with different HA glycosylation patterns. According to the results, the mutant with 6 N-linked glycans displayed the best acid and thermal stability and a better capacity for multiplication, although having a relatively lower receptor affinity than 7 glycans. The antigenic profile between the five mutants revealed a distinct antigenic distance, indicating that variations in glycosylation level have an impact on antigenic drift. These findings suggest that changes in the number of glycans on HA can not only modulate the receptor affinity and antigenicity of H9N2 AIVs, but also affect their stability and multiplication. These adaptive phenotypes may underlie the biological basis for the dominant strain switchover of h9.4.2.5-branched AIVs. Overall, our study provides a systematic insight into how changes in HA glycosylation patterns regulate the evolutionary fitness and epidemiological dominance drift of h9.4.2.5-branched H9N2 AIVs, which will be of great benefit for the glycosylation-dependent vaccine design.

RABV induces biphasic actin cytoskeletal rearrangement through Rac1 activity modulation.

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.

Identification and characterization of circular RNAs in the A549 cells following Influenza A virus infection.

Influenza A virus (IAV) is one of the most dominant zoonotic-pathogen that causes annually recurring epidemic disease. The detailed molecular mechanism underlying IAV infection is still not fully understood. Circular RNAs (circRNAs) are generated from RNA back-splicing and involved in diverse biological processes. Here, we employed high-throughput circRNA microarray technology to profile circRNA expression in A549 cells in response to IAV infection. The analysis data revealed that 178 circRNAs expression were significantly upregulated while 137 downregulated, respectively, compared to the mock (P＜0.05, Fold Change＞2). Subsequently, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were also conducted. Moreover dysregulated circRNAs were characterized, and of which nine were verified by quantitative real-time PCR (qRT-PCR). We further confirmed that circRNA_0082633 expression was increased following IAV infection. Overexpression of circRNA_0082633 suppressed IAV infection while depletion of circRNA_0082633 promoted viral proliferation. Interestingly, the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling was involved in IAV-induced circ_0082633 expression. More importantly, we demonstrated that circ_0082633 expression enhanced type I interferon (IFN) signaling by IFN-stimulated response element (ISRE) promoter activity and Ifnb1 mRNA levels. These data firstly provided the expression profile of circRNAs in PR8-infected A549 cells and shed new light on the pathogenesis research of IAV infection. Our findings also suggest that circRNA_0082633 served an important function in IAV infection.

Long noncoding RNA TSPOAP1 antisense RNA 1 negatively modulates type I IFN signaling to facilitate influenza A virus replication.

Although the expression of thousands of host long noncoding RNAs (lncRNAs) can be regulated by viral infection, the number of lncRNAs with experimentally verified function is limited. In this study, the expression of host lncRNA TSPOAP1-AS1 was significantly induced by influenza A virus (IAV) infection in a dose- and time-dependent manner. Polyinosine-polycytidylic acid (poly (I:C)), a synthetic analog of double-stranded RNA, also increased TSPOAP1-AS1 expression. RNA fractionation revealed that TSPOAP1-AS1 was a nucleocytoplasmic lncRNA, and an increased nuclear/cytoplasmic ratio was detected after IAV infection. The nuclear factor-κB signaling acting as a critical factor in the transcription of TSPOAP1-AS1 was determined through the use of pharmacological and genetic approaches. Functionally, overexpression of TSPOAP1-AS1 resulted in a significant increase in IAV replication. In contrast, the abolition of TSPOAP1-AS1 by RNA interference restricted viral replication. Furthermore, we demonstrated that TSPOAP1-AS1 negatively modulated the IAV-induced Ifnb1 transcription, interferon-sensitive response element (ISRE) activation, and downstream interferon-stimulated genes expression. Collectively, our data provides evidence for the host lncRNA utilized by viruses to support its replication.

Dynein- and kinesin- mediated intracellular transport on microtubules facilitates RABV infection.

Rabies, caused by rabies virus (RABV), is one of the most important neurotropic zoonoses and poses a severe threat to human and animal health. Exploration of its mechanism of neural transmission is meaningful but still insufficient. Here, we described the effects of microtubule-depolymerizing drugs and inhibitors of microtubule motor proteins on RABV infection. Colchicine, a microtubule-depolymerizing drug, significantly impeded RABV production in N2a cells. Overexpression of CC1 or p50 attenuated viral infection through the functional disruption of cytoplasmic dynein, which was consistent with the inhibitory effect of Na3VO4, a dynein activity inhibitor. Moreover, transfection with Flag-KHCct impaired RABV infection, as cytoplasmic kinesin-based motility was blocked. These results demonstrated that RABV can infect N2a cells in a manner that depends on microtubule integrity as well as dynein and kinesin function.

CircRNA_0050463 promotes influenza A virus replication by sponging miR-33b-5p to regulate EEF1A1.

Circular RNAs (circRNAs), a new class of widely expressed endogenous regulatory RNAs, are characterized by a covalently closed loop structure without a 5' cap or 3' tail. Increasing numbers of studies have shown that circRNAs play important roles in diverse physiological and pathological processes, including the dynamic interactions between viruses and hosts. However, their multifaceted roles in cellular responses to influenza A virus (IAV) infection remain largely unknown. Here, we analyzed the expression of circ_0050463, which is predominantly localized in cytoplasm, in response to IAV infection. Knockdown of circ_0050463 with siRNA in A549 cells inhibited IAV replication. In addition, the activation of nuclear factor κB (NF-κB) was involved in IAV-induced circ_0050463 expression, as revealed by assay using a NF-Kb inhibitor (Bay 11-7082). By performing biotin-labeled RNA pull-down and luciferase reporter assay, we demonstrated that circ_0050463 functioned as an endogenous microRNA-33b-5p sponge to sequester and inhibit miR-33b-5p activity, resulting in increased eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) expression with subsequent facilitation of IAV replication. Taken together, the results of our study indicate that the circ_0050463 promotes IAV replication via miR-33b-5p/EEF1A1 axis, thus providing evidence for the host circRNAs utilized by viruses to support their replication.

Entry of Challenge Virus Standard (CVS) -11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway.

BACKGROUND: Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized. METHODS: Chemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy. RESULTS: We firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MßCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy. CONCLUSIONS: Our results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.

Early events in rabies virus infection-Attachment, entry, and intracellular trafficking.

Rabies virus (RABV), an enveloped virus with a single-stranded and negative-sense RNA genome, is the type species of the Lyssavirus Genus within the Rhabdoviridae family. As the causative agent of rabies with a nearly 100% fatality, the neurotropic RABV pose a serious threat to the global public health. Though a great effort has been made toward understanding the molecular mechanism underlying virus infection cycle, there are still many aspects need to be elucidated, especially on the early events during virus replication cycle. With the application of the multiple advanced technologies, much progress has been made on these aspects. To date, multiple receptors, such as nAChR, NCAM, p75NTR, mGluR2, carbohydrates, and gangliosides, have been identified. Following initial attachment, RABV internalization occurs through clathrin-mediated endocytosis (CME) with the help of actin. After viral entry, intracellular trafficking occurs. Two retrograde trafficking models, stating that either whole virions are parceled into vesicles or only the viral capsids are transported, have been proposed. Moreover, complete enveloped virions or G-containing vesicle-associated ribonucleoproteins (RNPs) may be formed during anterograde transport, which remains poorly characterized but is important for viral budding. Combining the data elucidating the molecular mechanisms of RABV attachment, entry, and intracellular trafficking, this review provides an integrated view of the early events in the viral life cycle.

MCPIP1 attenuates the innate immune response to influenza A virus by suppressing RIG-I expression in lung epithelial cells.

The pattern recognition receptor retinoic acid-inducible gene I (RIG-I) reportedly plays a key role in sensing influenza A virus (IAV) infection and activating type I interferon (IFN) response. MCP-1-induced protein 1 (MCPIP1) can directly degrade cytokine mRNAs, such as IL-6, IL-12, IL-1ß, and IL-2, by functioning as an RNase. Here, we initially observed that MCPIP1 exhibited virus supportive functions later in the course of IAV infection in A549 cells, and negatively regulated IAV-induced RIG-I-dependent innate antiviral response. Exogenous overexpression of MCPIP1 suppressed the expression of RIG-I, whereas shRNA-mediated inhibition of endogenous MCPIP1 enhanced RIG-I expression. The results of experiments with actinomycin D and luciferase assay demonstrated that MCPIP1 reduced RIG-I expression through destabilizing its mRNA. Various mutants of functional domains of MCPIP1 further confirmed that the inhibitory effect of MCPIP1 on RIG-I expression required RNase activity but not deubiquitinase activity. Finally, the overexpression of several IAV proteins, which have the ability to inhibit the host IFN response at different levels, induced MCPIP1 expression, especially non-structural protein 1 (NS1). Conclusively, these data demonstrate the MCPIP1 contributes to attenuate IAV-induced host antiviral response by suppressing RIG-I expression.

Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

Modulation of influenza A virus replication by microRNA-9 through targeting MCPIP1.

MicroRNAs (miRNAs), a family of small non-coding RNAs controlling translation and transcription of its target genes, play important roles in the regulation of various biological processes, including viral infection. Influenza A viruses (IAV) infection alters expression of cellular miRNAs, which in turn can modify the cellular environment to facilitate efficient virus replication. In this study, we showed that IAV infection significantly induced miR-9 expression in A549 cells, which occurred earlier than drastic expression of viral matrix (M) and nucleoprotein (NP) genes. Overexpression of miR-9 enhanced viral gene expression and production of infectious progeny, while knockdown of miR-9 significantly inhibited IAV replication in A549 cells. Recent studies have revealed antiviral potential of monocyte chemoattractant protein 1-induced protein 1 (MCPIP1), a PIN-like RNase capable of targeting and degrading viral RNA. Subsequently, we comprehensively confirmed that MCPIP1 functionally inhibited viral M and NP genes expression and progeny production, and also was regulated by miR-9 in A549 cells. Furthermore, MCPIP1 overexpression abrogated miR-9-induced IAV replication. Taken together, our findings indicate a new role of miR-9 induction in IAV infection and suggest IAV may hijack cellular miR-9 to benefit the viral life cycle. J. Med. Virol. 89:41-48, 2017. © 2016 Wiley Periodicals, Inc.

Influenza a virus NS1 protein induced A20 contributes to viral replication by suppressing interferon-induced antiviral response.

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. A20 is a cytoplasmic ubiquitin-editing protein that negatively regulates the retinoic acid-inducible gene I (RIG-I)-mediated activation of interferon regulatory factors (IRF) 3. Here, we found that influenza A virus (IAV) non-structural protein (NS) 1 dramatically induced the protein level of A20 in A549 cells whose expression levels were positively associated with the viral virulence. A20 overexpression in A549 cells significantly suppressed IAV-induced the activation of IRF3 and interferon (IFN) promoter, resulted in downregulation of IFNß and IFN-stimulated genes (ISGs) mRNA. Conversely, silencing A20 expression markedly enhanced IRF3-mediated innate antiviral responses. Furthermore, we demonstrated that A20 overexpression in A549 cells obviously promoted IAV replication, and conversely, knockdown of A20 inhibited the viral replication. Overall, the findings described in this study support and extend previous results on interferon-antagonistic strategies of IAV NS1 by showing an induced host target A20, which restricts IAV-induced host innate immune antiviral responses and thereby facilitates viral replication.

Quantitative Proteome Profiling of Street Rabies Virus-Infected Mouse Hippocampal Synaptosomes.

It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying mechanisms in rabies virus (RABV) and synaptic dysfunctions, a quantitative proteome profiling was carried out on synaptosome samples from mice hippocampus. Synaptosome samples from mice hippocampus were isolated and confirmed by Western blot and transmission electron microscopy. Synaptosome protein content changes were quantitatively detected by Nano-LC-MS/MS. Protein functions were classified by the Gene Ontology (GO) and KEGG pathway. PSICQUIC was used to create a network. MCODE algorithm was applied to obtain subnetworks. Of these protein changes, 45 were upregulated and 14 were downregulated following RABV infection relative to non-infected (mock) synaptosomes. 28 proteins were unique to mock treatment and 12 were unique to RABV treatment. Proteins related to metabolism and synaptic vesicle showed the most changes in expression levels. Furthermore, protein-protein interaction (PPI) networks revealed that several key biological processes related to synaptic functions potentially were modulated by RABV, including energy metabolism, cytoskeleton organization, and synaptic transmission. These data will be useful for better understanding of neuronal dysfunction of rabies and provide the foundation for future research.

The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages.

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.

A20 promotes Brucella intracellular growth via inhibition of macrophage cell death and activation.

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of macrophage activation and apoptosis in tumor necrosis factor receptor1 (TNFR1) signaling pathway. Brucella infection can induce A20 expression in macrophages. Here, we hypothesize that A20 promotes Brucella intracellular growth via inhibition of activation and apoptosis of macrophages. To test this hypothesis, we stably incorporated mouse A20-shRNA into the RAW264.7 cells by lentiviral gene transfer to successfully knockdown A20. A20-deficient RAW264.7 cells were subsequently challenged with Brucella abortus and colony formation units (CFUs) of bacteria, TNFα production, NF-kB activation, macrophages apoptosis and cell death were evaluated. The A20 knockdown was shown to effectively promote B. abortus-stimulated TNFα release, NF-kB activation and macrophage cell death, which suppressed B. abortus intracellular replication. Unexpectedly, deficiency of A20 failed to lead to B. abortus-induced macrophage apoptosis. A20 deficiency coupled NF-kB inhibition promoted caspase-8 dependent B. abortus-induced macrophage apoptosis. These findings provide a novel mechanism by which Brucella intracellular growth within macrophages occurs through up-regulation of A20 thereby limiting activation and macrophages cell death.

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis.

Induction of the cellular miR-29c by influenza virus inhibits the innate immune response through protection of A20 mRNA.

Influenza A viruses (IAVs) are negative-sense, single-stranded, segmented RNA viruses, which primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. MicroRNAs (miRNAs) represent a family of small noncoding RNAs controlling translation and transcription of many genes. The human miR-29 family of miRNAs has three mature members, miR-29a, miR-29b, and miR-29c. Recent studies have revealed that miR-29 is involved in regulation of the innate and adaptive immune responses. However, the function of miR-29 in the immune response to IAV infection remains to be further explored. Our previous study has shown that miR-29 family members are up-regulated during IAV infection, especially miR-29c. Here we report that miR-29c is involved in inhibition of IAV-induced innate immune responses. We found that posttranscriptional regulation was involved in IAV-induced A20 expression in A549 cells. Consistent with a previous report, miR-29c functionally protected A20 transcripts in A549 cells. Overexpression of miR-29c with miR-29c mimic enhanced IAV-induced A20 protein expression and conversely that miR-29c inhibitor significantly blocked IAV-induced A20 protein expression in A549 cells. Furthermore, functional results showed that IAV-induced miR-29c expression correlated with decreased NF-κB activity and expression of several antiviral and proinflammatory cytokines via up-regulation of A20. Together, the findings indicate a new role of miR-29c in IAV infection and suggest its induction may contribute to counteract the innate immune response.

Street rabies virus causes dendritic injury and F-actin depolymerization in the hippocampus.

Rabies is an acute viral infection of the central nervous system and is typically fatal in humans and animals; however, its pathogenesis remains poorly understood. In this study, the morphological changes of dendrites and dendritic spines in the CA1 region of the hippocampus were investigated in mice that were infected intracerebrally with an MRV strain of the street rabies virus. Haematoxylin and eosin and fluorescence staining analysis of brain sections from the infected mice showed very few morphological changes in the neuronal bodies and neuronal processes. However, we found a significant decrease in the number of dendritic spines. Primary neuronal cultures derived from the hippocampus of mice (embryonic day 16.5) that were infected with the virus also showed an obvious decrease in the number of dendritic spines. Furthermore, the decrease in the number of dendritic spines was related to the depolymerization of actin filaments (F-actin). We propose that the observed structural changes can partially explain the severe clinical disease that was found in experimental models of street rabies virus infections.

[PB1-F2 protein of influenza A virus interacts with human MOAP-1 protein].

OBJECTIVE: To identify the interaction between influenza A virus PB1-F2 and human modulator of apoptosis 1 (MOAP-1). METHODS: The recombinant plasmid pACT2-MOAP-1 was constructed and co-transformed into yeast AH109 with pGBKT7-PB1-F2. The growth of the co-transformants on quadruple dropout medium and beta-galactosidase activity of the reporter gene were tested. We further confirmed the interaction of cellular protein MOAP-1 and PB1-F2 by glutathione S-transferase (GST) pull-down and co-immunoprecipitation (Co-IP) assays. In addition, we investigated the effect of PB1-F2 on MOAP-1 protein level by Western blot. RESULTS: The results of yeast two-hybrid assay showed that MOAP-1 specifically interacted with PB1-F2 in yeast cells. Furthermore, the binding of MOAP-1 with PB1-F2 was demonstrated by glutathione S-transferase pull-down and Co-IP assays. PB1-F2 could upregulate exogenous MOAP-1 protein level. CONCLUSION: These results suggested that influenza virus PB1-F2 interacted with MOAP-1 and it might be involved in the regulation of cell growth and apoptosis via association with MOAP-1.