Receptor Tyrosine Kinase Inhibitors Block Multiple Steps of Influenza A Virus Replication

The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA ( Viruses are obligate intracellular parasites that have been compelled by evolutionary processes to encode a minimal number of proteins for the completion of an infectious life cycle, in order to infect naive hosts and to persist in nature. This is exemplified by RNA viruses, the genomes of which are generally smaller than the genomes of DNA viruses, and therefore they greatly rely on the exploitation and subversion of host cellular proteins, structures, and pathways to facilitate virus replication (1). Transcription and replication of the influenza A virus genome are performed by the virally encoded RNA-dependent RNA polymerase (RdRP), a major determinant of species tropism and pathogenicity and a key player in the adaptation of avian influenza A viruses to mammalian hosts. These characteristics of the viral RdRP are thought to be governed by numerous interactions with cellular factors (reviewed in references 2 and 3). It is therefore of great importance to understand the relationship between the viral transcription/replication machinery and host cellular factors in order to understand how the host cell can influence the function of the viral transcription/replication machinery and vice versa. Insight into the molecular biology of these relationships could lead to novel antiviral strategies and has the potential to identify host-specific interactions that would act as a barrier to pandemic emergence.The genome of influenza A virus, like that of other members of the Orthomyxoviridae, is segmented and consists of eight individual viral ribonucleoprotein (vRNP) complexes. Each vRNP contains single-stranded viral RNA (vRNA) that is en...

Section: Discussionsupporting

confidence: 75%

Interactome Analysis of the Influenza A Virus Transcription/Replication Machinery Identifies Protein Phosphatase 6 as a Cellular Factor Required for Efficient Virus Replication

York

Hutchinson

Fodor

2014

“…We and others have shown that host signaling pathways play important roles in the influenza viral life cycle (13)(14)(15)(16)(17)(18)(19)(20). Increasing evidence suggests that diverse receptor tyrosine kinases (RTKs) and downstream signaling pathways, such as epidermal growth factor receptor (EGFR), TrkA, Raf/MEK/ERK, and phosphatidylinositol 3-kinase (PI3K)/Akt, are involved in discrete steps of the influenza viral life cycle (13)(14)(15)(16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

“…Increasing evidence suggests that diverse receptor tyrosine kinases (RTKs) and downstream signaling pathways, such as epidermal growth factor receptor (EGFR), TrkA, Raf/MEK/ERK, and phosphatidylinositol 3-kinase (PI3K)/Akt, are involved in discrete steps of the influenza viral life cycle (13)(14)(15)(16)(17)(18)(19)(20). We have previously shown that TrkA activation is required for optimal viral RNA synthesis, vRNP export, and virus assembly and budding (17,18). Eierhoff et al have demonstrated that EGFR is activated by influenza A virus infection and facilitates its efficient cell entry (15).…”

mentioning

confidence: 99%

PLC-γ1 Signaling Plays a Subtype-Specific Role in Postbinding Cell Entry of Influenza A Virus

Zhu

Liang

2014

Self Cite

Host signaling pathways and cellular proteins play important roles in the influenza viral life cycle and can serve as antiviral targets. In this study, we report the engagement of host phosphoinositide-specific phospholipase ␥1 (PLC-␥1) in mediating cell entry of influenza virus H1N1 but not H3N2 subtype. Both PLC-␥1-specific inhibitor and short hairpin RNA (shRNA) strongly suppress the replication of H1N1 but not H3N2 viruses in cell culture, suggesting that PLC-␥1 plays an important subtype-specific role in the influenza viral life cycle. Further analyses demonstrate that PLC-␥1 activation is required for viral postbinding cell entry. In addition, H1N1, but not H3N2, infection leads to the phosphorylation of PLC-␥1 at Ser 1248 immediately after infection and independent of viral replication. We have further shown that H1N1-induced PLC-␥1 activation is downstream of epidermal growth factor receptor (EGFR) signaling. Interestingly, both H1N1 and H3N2 infections activate EGFR, but only H1N1 infection leads to PLC-␥1 activation. Taking our findings together, we have identified for the first time the subtype-specific interplay of host PLC-␥1 signaling and H1N1 virus that is critical for viral uptake early in the infection. Our study provides novel insights into how virus interacts with the cellular signaling network by demonstrating that viral determinants can regulate how the host signaling pathways function in virally infected cells.

“…Interestingly, EGFR signaling and trafficking are also regulated by a number of other viruses, including hepatitis C virus (40), hepatitis E virus (7), human papillomavirus (55,58), and herpes simplex virus type-1 (38), and this pathway may regulate postentry steps in the viral life cycle, including viral RNA synthesis, nuclear export of vRNP, or release of virions (36). This finding underscores the notion that influenza virus may be susceptible to inhibition by smallmolecule inhibitors of EGFR tyrosine kinase activity (36), possibly including antitumor drugs such as gefitinib (37).…”

Section: Vol 85 2011mentioning

confidence: 99%

Comprehensive Proteomic Analysis of Influenza Virus Polymerase Complex Reveals a Novel Association with Mitochondrial Proteins and RNA Polymerase Accessory Factors

Bradel-Tretheway

Mattiacio

Krasnoselsky

et al. 2011

The trimeric RNA polymerase complex (3P, for PA-PB1-PB2) of influenza A virus (IAV) is an important viral determinant of pathogenicity and host range restriction. Specific interactions of the polymerase complex with host proteins may be determining factors in both of these characteristics and play important roles in the viral life cycle. To investigate this question, we performed a comprehensive proteomic analysis of human host proteins associated with the polymerase of the well-characterized H5N1 Vietnam/1203/04 isolate. We identified over 400 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), of which over 300 were found to bind to the PA subunit alone. The most intriguing and novel finding was the large number of mitochondrial proteins (ϳ20%) that associated with the PA subunit. These proteins mediate molecular transport across the mitochondrial membrane or regulate membrane potential and may in concert with the identified mitochondrion-associated apoptosis inducing factor (AIFM1) have roles in the induction of apoptosis upon association with PA. Additionally, we identified host factors that associated with the PA-PB1 (68 proteins) and/or the 3P complex (34 proteins) including proteins that have roles in innate antiviral signaling (e.g., ZAPS or HaxI) or are cellular RNA polymerase accessory factors (e.g., polymerase I transcript release factor [PTRF] or Supt5H). IAV strain-specific host factor binding to the polymerase was not observed in our analysis. Overall, this study has shed light into the complex contributions of the IAV polymerase to host cell pathogenicity and allows for direct investigations into the biological significance of these newly described interactions.The trimeric RNA (3P) influenza virus polymerase complex (PA-PB1-PB2) assembles in the nucleus, where influenza virus replication occurs. Several events inside the host cell must take place in order to support viral replication, such as the regulation of early antiviral host responses, host protein shutoff, and nuclear transport of viral RNA and proteins (21,29,63). These events require the viral RNA polymerase to interact with cellular factors, some of which may also be important for host range specificity but have yet to be identified (17,44). A range of RNA interference (RNAi)-based, genome-wide screens have been published recently that identified host factors involved in influenza virus replication (4,22,31,35,52,56). Although the degree of overlap between these studies was minimal, similar biological pathways and functional classifications were identified, suggesting their importance in the influenza virus infectious cycle. Watanabe et al. (67) compiled a list of 128 proteins that affected influenza virus replication in at least two out of the six screens. These included proteins involved in the nuclear import of viral ribonucleoproteins (vRNPs), such as importin family members and nuclear pore complex proteins.Other studies have attempted to directly identify proteins that interact with either the influenza virus po...