A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase

Jung, Tanis E.; Brownlee, George G.

doi:10.1099/vir.0.81453-0

Cited by 44 publications

(32 citation statements)

References 29 publications

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“…As both subunits have been implicated in promoter binding 24 , we speculate that association with NEP might also lead to a stabilization of cRNPs, thereby increasing the synthesis of vRNA transcripts, as suggested by others 25 .…”

Section: Discussionsupporting

confidence: 61%

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

et al. 2012

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Infection of mammals by avian influenza viruses requires adaptive mutations to achieve high-level replication in the new host. However, the basic mechanism underlying this adaptation process is still unknown. Here we show that avian polymerases, lacking the human signature PB2-E627K, are incapable of generating usable complementary RnA templates in cultured human cells and therefore require adaptation. Characterization of the highly pathogenic human H5n1 isolate A/Thailand/1(KAn-1)/2004 that retained the avian PB2-E627 reveals that the defect in RnA replication is only partially compensated by mutations in the polymerase. Instead, mutations in the nuclear export protein are required for efficient polymerase activity. We demonstrate that adaptive mutations in nuclear export proteins of several human isolates enhance the polymerase activity of avian polymerases in human cultured cells. In conclusion, when crossing the species barrier, avian influenza viruses acquire adaptive mutations in nuclear export protein to escape restricted viral genome replication in mammalian cells.

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Section: Discussionsupporting

confidence: 61%

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

et al. 2012

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“…1a), using them to co-purify RanBP5 from 293T cells. PB1 was divided into an N-terminal fragment containing the PA-binding and promoter-binding domains (residues 1-290), a middle fragment containing the core RdRP motifs (residues 289-488) and a C-terminal fragment containing the PB2-binding site (residues 488-757) (Jung & Brownlee, 2006;Sugiyama et al, 2009;Ruigrok et al, 2010). Efficient binding of RanBP5 was observed with full-length tagged PB1 and with the N-terminal fragment of PB1, but not with the middle or C-terminal fragments of the protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…4a). Nor do the mutations overlap known promoter-binding or polymerase motifs (González & Ortín, 1999;Jung & Brownlee, 2006;Li et al, 1998; Müller et al, 1994;Poch et al, 1989). A previous study has shown that the NLS2 mutation did not substantially affect the ability of PB1 to transcribe and replicate a template RNA [a negative-sense chloramphenicol acetyltransferase (CAT) gene flanked by the untranslated regions of WSN segment 8] in an RNP reconstitution (Fodor & Smith, 2004); we confirmed this observation using full-length vRNA and cRNA of WSN segment 6 as a template (data not shown).…”

Section: E C Hutchinson and Others 1862mentioning

confidence: 99%

Characterization of the interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import factor Ran-binding protein 5

Hutchinson

Orr

Liu

et al. 2011

Journal of General Virology

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The influenza A virus RNA polymerase is a heterotrimer that transcribes and replicates the viral genome in the cell nucleus. Newly synthesized RNA polymerase subunits must therefore be imported into the nucleus during an infection. While various models have been proposed for this process, the consensus is that the polymerase basic protein PB1 and polymerase acidic protein PA subunits form a dimer in the cytoplasm and are transported into the nucleus by the betaimportin Ran-binding protein 5 (RanBP5), with the PB2 subunit imported separately to complete the trimeric complex. In this study, we characterized the interaction of PB1 with RanBP5 further and assessed its importance for viral growth. In particular, we found that the N-terminal region of PB1 mediates its binding to RanBP5 and that basic residues in a nuclear localization signal are required for RanBP5 binding. Mutating these basic residues to alanines does not prevent PB1 forming a dimer with PA, but does reduce RanBP5 binding. RanBP5-binding mutations reduce, though do not entirely prevent, the nuclear accumulation of PB1. Furthermore, mutations affecting RanBP5 binding are incompatible with or severely attenuate viral growth, providing further support for a key role for RanBP5 in the influenza A virus life cycle. INTRODUCTIONInfluenza A virus is the prototypic orthomyxovirus and a serious pathogen of humans and animals. The viral genome consists of eight segments of negative-sense, ssRNA, which are encapsidated as viral ribonucleoprotein complexes (vRNPs) by multiple copies of nucleoprotein (NP) and the trimeric RNA-dependent RNA polymerase (RdRP; consisting of the polymerase basic proteins PB1 and PB2, and the polymerase acidic protein PA) (Palese & Shaw, 2007). Unusually among RNA viruses, orthomyxoviruses replicate their genomes in the nucleus. Consequently, newly synthesized viral proteins must be imported into the nucleus to allow the assembly and export of new vRNPs, as well as to regulate host processes in the infected cell. Of the proteins expressed by influenza A virus, nuclear import is observed for the three polymerase proteins and NP, as well as matrix (M1) protein, non-structural (NS1) protein, nuclear export protein (NEP) and PB1-F2 (Chen et al., 2001;Smith et al., 1987). Nuclear import of structures larger than 20-30 kDa is selective and energy dependent, requiring binding of nuclear import factors (NIFs) through nuclear localization signals (NLSs) on import substrates (Görlich & Kutay, 1999). A number of influenza A virus proteins have been shown to contain NLSs and to bind NIFs Naito et al., 2007; Resa-Infante et al., 2008;Tarendeau et al., 2007). PB1 and PA, despite both having signals that can promote nuclear import when individually expressed (Akkina et al., 1987;Nath & Nayak, 1990;Nieto et al., 1994), do not accumulate efficiently in the nucleus unless expressed together, a requirement that appears to be particularly marked for PB1 (Fodor & Smith, 2004;Huet et al., 2010;Nieto et al., 1992). Although all possible pairwise interact...

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“…The PB1 subunit of the viral RdRP can be phosphorylated at position T223. This is close to a nuclear localization signal (NLS) and a region that is believed to be involved in promoter binding, indicating a potential role in regulating the cellular localization or RNA binding of PB1 (36,90,(117)(118)(119)(120).…”

Section: Discussionmentioning

confidence: 99%

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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A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase

Cited by 44 publications

References 29 publications

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

Characterization of the interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import factor Ran-binding protein 5

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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