scholarly journals pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Kenji Sugiyama ◽  
Atsushi Kawaguchi ◽  
Mitsuru Okuwaki ◽  
Kyosuke Nagata
Keyword(s):  
Influenza Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Free Form ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Nuclear Extracts ◽  
Virus Rna ◽  
Viral Genomic Rna

Replication of influenza viral genomic RNA (vRNA) is catalyzed by viral RNA-dependent RNA polymerase (vRdRP). Complementary RNA (cRNA) is first copied from vRNA, and progeny vRNAs are then amplified from the cRNA. Although vRdRP and viral RNA are minimal requirements, efficient cell-free replication could not be reproduced using only these viral factors. Using a biochemical complementation assay system, we found a novel activity in the nuclear extracts of uninfected cells, designated IREF-2, that allows robust unprimed vRNA synthesis from a cRNA template. IREF-2 was shown to consist of host-derived proteins, pp32 and APRIL. IREF-2 interacts with a free form of vRdRP and preferentially upregulates vRNA synthesis rather than cRNA synthesis. Knockdown experiments indicated that IREF-2 is involved in in vivo viral replication. On the basis of these results and those of previous studies, a plausible role(s) for IREF-2 during the initiation processes of vRNA replication is discussed.

scholarly journals Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins

1998 ◽  
Vol 72 (7) ◽  
pp. 5493-5501 ◽  
Author(s):  
Siddhartha K. Biswas ◽  
Paul L. Boutz ◽  
Debi P. Nayak
Keyword(s):  
Influenza Virus ◽  
Protein Complex ◽  
Rna Synthesis ◽  
Sodium Dodecyl ◽  
Direct Interaction ◽  
Critical Factor ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Replication Assay

ABSTRACT Influenza virus nucleoprotein (NP) is a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. In this study, we investigated the interaction of NP with the viral polymerase protein complex. Using coimmunoprecipitation with monospecific or monoclonal antibodies, we observed that NP interacted with the RNP-free polymerase protein complex in influenza virus-infected cells. In addition, coexpression of the components of the polymerase protein complex (PB1, PB2, or PA) with NP either together or pairwise revealed that NP interacts with PB1 and PB2 but not PA. Interaction of NP with PB1 and PB2 was confirmed by both coimmunoprecipitation and histidine tagging of the NP-PB1 and NP-PB2 complexes. Further, it was observed that NP-PB2 interaction was rather labile and sensitive to dissociation in 0.1% sodium dodecyl sulfate and that the stability of NP-PB2 interaction was regulated by the sequences present at the COOH terminus of NP. Analysis of NP deletion mutants revealed that at least three regions of NP interacted independently with PB2. A detailed analysis of the COOH terminus of NP by mutation of serine-to-alanine (SA) residues either individually or together demonstrated that SA mutations in this region did not affect the binding of NP to PB2. However, some SA mutations at the COOH terminus drastically affected the functional activity of NP in an in vivo transcription-replication assay, whereas others exhibited a temperature-sensitive phenotype and still others had no effect on the transcription and replication of the viral RNA. These results suggest that a direct interaction of NP with polymerase proteins may be involved in regulating the switch of viral RNA synthesis from transcription to replication.

scholarly journals Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription

2003 ◽  
Vol 77 (9) ◽  
pp. 5098-5108 ◽  
Author(s):  
Pablo Gastaminza ◽  
Beatriz Perales ◽  
Ana M. Falcón ◽  
Juan Ortín
Keyword(s):  
Influenza Virus ◽  
Rna Replication ◽  
Terminal Region ◽  
Viral Rna ◽  
Mrna Accumulation ◽  
Virus Rna ◽  
Transcription In Vitro ◽  
Type Mutant

ABSTRACT PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

scholarly journals Hantavirus N Protein Exhibits Genus-Specific Recognition of the Viral RNA Panhandle

2006 ◽  
Vol 80 (22) ◽  
pp. 11283-11292 ◽  
Author(s):  
M. A. Mir ◽  
B. Brown ◽  
B. Hjelle ◽  
W. A. Duran ◽  
A. T. Panganiban
Keyword(s):  
Viral Rna ◽  
Genomic Rna ◽  
N Protein ◽  
High Affinity ◽  
The Andes ◽  
The Family ◽  
Viral Genomic Rna ◽  
Negative Sense

ABSTRACT A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into “panhandle” hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5′ and 3′ ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

scholarly journals Differential splicing of ANP32A in birds alters its ability to stimulate RNA synthesis by restricted influenza polymerase

2018 ◽  
Author(s):  
Steven F. Baker ◽  
Mitchell P. Ledwith ◽  
Andrew Mehle
Keyword(s):  
Influenza Virus ◽  
Host Range ◽  
Rna Synthesis ◽  
Splice Variants ◽  
Polymerase Activity ◽  
Genomic Rna ◽  
Differential Splicing ◽  
Exon Duplication ◽  
Species Specific ◽  
Viral Genomic Rna

AbstractAdaptation of viruses to their host can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently been identified as a cellular co-factor impacting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation amongst birds differentially impacts viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

scholarly journals Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its cRNA and Viral RNA Promoters during Viral RNA Replication

2006 ◽  
Vol 80 (5) ◽  
pp. 2337-2348 ◽  
Author(s):  
Tao Deng ◽  
Frank T. Vreede ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
De Novo ◽  
Viral Rna ◽  
Virus Rna ◽  
Initiation Of Replication ◽  
Replicative Intermediates ◽  
Internal Initiation

ABSTRACT Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5′ triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

scholarly journals Association of the Influenza A Virus RNA-Dependent RNA Polymerase with Cellular RNA Polymerase II

2005 ◽  
Vol 79 (9) ◽  
pp. 5812-5818 ◽  
Author(s):  
Othmar G. Engelhardt ◽  
Matt Smith ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Influenza A ◽  
Large Subunit ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Virus Rna ◽  
Transcription Sites

ABSTRACT Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

scholarly journals 2′-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase

2019 ◽  
Vol 294 (45) ◽  
pp. 16897-16907 ◽  
Author(s):  
Alyson K. Boehr ◽  
Jamie J. Arnold ◽  
Hyung S. Oh ◽  
Craig E. Cameron ◽  
David D. Boehr
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

scholarly journals Cellular Splicing Factor RAF-2p48/NPI-5/BAT1/UAP56 Interacts with the Influenza Virus Nucleoprotein and Enhances Viral RNA Synthesis

2001 ◽  
Vol 75 (4) ◽  
pp. 1899-1908 ◽  
Author(s):  
Fumitaka Momose ◽  
Christopher F. Basler ◽  
Robert E. O'Neill ◽  
Akihiro Iwamatsu ◽  
Peter Palese ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Splicing Factor ◽  
Yeast Two Hybrid ◽  
Amino Terminal ◽  
Virus Rna ◽  
Rna Interaction ◽  
Two Hybrid

ABSTRACT Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis. A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity. Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library. In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48–NP complex was dissociated following addition of free RNA. Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase. RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding. Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.

scholarly journals De Novo Initiation of RNA Synthesis by the Arterivirus RNA-Dependent RNA Polymerase

2007 ◽  
Vol 81 (16) ◽  
pp. 8384-8395 ◽  
Author(s):  
Nancy Beerens ◽  
Barbara Selisko ◽  
Stefano Ricagno ◽  
Isabelle Imbert ◽  
Linda van der Zanden ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Reverse Genetics ◽  
De Novo ◽  
Critical Role ◽  
Comparative Sequence Analysis ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase

ABSTRACT All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3′ end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3′-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis.

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