The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza

Sui, Baoquan; Bamba, Douty; Weng, Ke; Ung, Huong; Chang, Shaojing; Dyke, Jessica Van; Goldblatt, Michael; Duan, Roxanne; Kinch, Michael S.; Li, Wu-Bo

doi:10.1016/j.virol.2009.02.046

Cited by 69 publications

(66 citation statements)

References 31 publications

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“…This suggests that they have a role in limiting influenza A virus replication; thus, silencing their expression or preventing their function with small-molecule chemical inhibitors could be useful in applications where amplification of virus replication is beneficial such as in the propagation of viruses during vaccine production. It also highlighted the breadth of new information obtained from siRNA screens performed during influenza A virus infection, both in the study reported here and in those done by others (24,25,(34)(35)(36)(37)(38)(39).…”

Section: Discussionsupporting

confidence: 63%

Targeting Organic Anion Transporter 3 with Probenecid as a Novel Anti-Influenza A Virus Strategy

Perwitasari

Yan

Johnson

et al. 2013

Antimicrob Agents Chemother

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Influenza A virus infection is a major global health concern causing significant mortality, morbidity, and economic loss. Antiviral chemotherapeutics that target influenza A virus are available; however, rapid emergence of drug-resistant strains has been reported. Consequently, there is a burgeoning need to identify novel anti-influenza A drugs, particularly those that target host gene products required for virus replication, to reduce the likelihood of drug resistance. In this study, a small interfering RNA (siRNA) screen was performed to identify host druggable gene targets for anti-influenza A virus therapy. The host organic anion transporter-3 gene (OAT3), a member of the SLC22 family of transporters, was validated as being required to support influenza A virus replication. Probenecid, a prototypical uricosuric agent and chemical inhibitor of organic anion transporters known to target OAT3, was shown to be effective in limiting influenza A virus infection in vitro (50% inhibitory concentration [IC 50 ] of 5.0 ؋ 10 ؊5 to 5.0 ؋ 10 ؊4 M; P < 0.005) and in vivo (P < 0.05). Probenecid is widely used for treatment of gout and related hyperuricemic disorders, has been extensively studied for pharmacokinetics and safety, and represents an excellent candidate for drug repositioning as a novel anti-influenza A chemotherapeutic.

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Section: Discussionsupporting

confidence: 63%

Targeting Organic Anion Transporter 3 with Probenecid as a Novel Anti-Influenza A Virus Strategy

Perwitasari

Yan

Johnson

et al. 2013

Antimicrob Agents Chemother

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“…In this study, we have identified STIP1, FKBP5, and ␣-and ␤-tubulin as binding partners of the PB2 protein ( Table 1), suggesting that PB2 might be involved in multimeric complexes with several chaperones and associated proteins. These proteins were also identified in some of the recent genome-wide screening studies as important for the influenza virus life cycle (32,50,56). The association of the influenza virus RNA polymerase and vRNPs with tubulins has been described previously (28,36), and it has been suggested that this interaction maybe involved in the long-distance transport of virus RNPs from the nucleus to the vicinity of the cell membrane, prior to virion formation and budding (28).…”

Section: Discussionmentioning

confidence: 92%

“…CCT also plays a role in viral assembly of hepatitis B virus (35) and initial folding of EBNA-3 nuclear protein of Epstein-Barr virus (31). Interestingly, the CCT complex has not been identified by the recent genome-wide screening studies (2,18,29,32,50,56) as a host factor involved in the influenza virus life cycle.…”

Section: Discussionmentioning

confidence: 99%

Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

Fislová

Thomas

Graef

et al. 2010

J Virol

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The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), ␣-and ␤-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.Influenza A viruses, members of the family of Orthomyxoviridae, contain a segmented RNA genome of negative polarity. The genomic RNA segments together with the three subunits of the viral RNA-dependent RNA polymerase (PB1, PB2, and PA protein) and the nucleoprotein (NP) form viral ribonucleoprotein complexes (vRNPs). The PB1 subunit is the polymerase itself, while the PB2 and PA subunits are involved in the generation of 5Ј capped RNA primers through binding to and endonucleolytic cleavage of host pre-mRNAs (8,10,11,41,61). After the virus enters the cell via endocytosis, vRNPs are released into the cytoplasm and transported into the nucleus. In the nucleus, vRNPs catalyze the synthesis of viral mRNAs and complementary RNAs (cRNA) which, in turn, are used as templates for the synthesis of vRNAs. The newly formed vRNPs in association with other viral proteins (M1 and nonstructural protein 2/nuclear export factor [NS2/NEP]) are transported into the cytoplasm and subsequently to the cell membrane, where the assembly process takes place, followed by the release of progeny virions by budding (44).The PB1, PB2, and PA proteins are synthesized in the cytoplasm whereupon PB1 and PA form a dimeric complex that is transported into the nucleus. In the nucleus the dimer assembles with the PB2 subunit, which is transported separately (7,14). RanBP5 was identified as a factor that is involved in the import of the PB1-PA dimer into the nucleus ...

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“…Using this approach, we identified 171 cellular interactors of the viral transcription/replication machinery. This method complements previous studies that have aimed to identify critical cellular factors that are involved in the virus life cycle, including a number of genome-wide screens (11)(12)(13)(14)(15). Although informative, these genome-wide screens have failed to identify the specific stages of the virus life cycle at which particular cellular factors are critical.…”

mentioning

confidence: 95%

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

J Virol

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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...

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The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza

Cited by 69 publications

References 31 publications

Targeting Organic Anion Transporter 3 with Probenecid as a Novel Anti-Influenza A Virus Strategy

Targeting Organic Anion Transporter 3 with Probenecid as a Novel Anti-Influenza A Virus Strategy

Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

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

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