Aartjan J. W. te Velthuis

Molecular evolution of the MAGUK family in metazoan genomes

BackgroundDevelopment, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.ResultsOur results indicate that at least four of the seven MAGUK subfamilies were present in early metazoan lineages, such as Porifera. We employed domain sequence and structure based methods to infer a model for the evolutionary history of the MAGUKs. Notably, the phylogenetic trees for the guanylate kinase (GK)-, the PDZ- and the SH3-domains all suggested a matching evolutionary model which was further supported by molecular modeling of the 3D structures of different GK domains. We found no MAGUK in plants, fungi or other unicellular organisms, which suggests that the MAGUK core structure originated early in metazoan history.ConclusionIn summary, we have characterized here the molecular and structural evolution of the large MAGUK family. Using the MAGUKs as an example, our results show that it is possible to derive a highly supported evolutionary model for important multidomain families by analyzing encoded protein domains. It further suggests that larger superfamilies encoded in the different genomes can be analyzed in a similar manner.

Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis

The genomes of influenza viruses consist of multiple segments of single-stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, which form viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors. Furthermore, we discuss how advances in our understanding of the structure and function of polymerases could help in identifying new antiviral targets.

Common and unique features of viral RNA-dependent polymerases

Eukaryotes and bacteria can be infected with a wide variety of RNA viruses. On average, these pathogens share little sequence similarity and use different replication and transcription strategies. Nevertheless, the members of nearly all RNA virus families depend on the activity of a virally encoded RNA-dependent polymerase for the condensation of nucleotide triphosphates. This review provides an overview of our current understanding of the viral RNA-dependent polymerase structure and the biochemistry and biophysics that is involved in replicating and transcribing the genetic material of RNA viruses.Eukaryotes and bacteria can be infected with a wide variety of RNA viruses. On average, these pathogens share little sequence similarity and use different replication and transcription strategies. Nevertheless, the members of nearly all RNA virus families depend on the activity of a virally encoded RNA-dependent polymerase for the condensation of nucleotide triphosphates. This review provides an overview of our current understanding of the viral RNA-dependent polymerase structure and the biochemistry and biophysics that is involved in replicating and transcribing the genetic material of RNA viruses.

Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

ABSTRACT The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome. IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes

Abstract Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales. Nidoviruses are characterized by their polycistronic plus-stranded RNA genome, the production of subgenomic mRNAs and the conservation of a specific array of replicase domains, including key RNA-synthesizing enzymes. Coronaviruses (26–34 kilobases) have the largest known RNA genomes and their replication presumably requires a processive RNA-dependent RNA polymerase (RdRp) and enzymatic functions that suppress the consequences of the typically high error rate of viral RdRps. The arteriviruses have significantly smaller genomes and form an intriguing package with the coronaviruses to analyse viral RdRp evolution and function. The RdRp domain of nidoviruses resides in a cleavage product of the replicase polyprotein named non-structural protein (nsp) 12 in coronaviruses and nsp9 in arteriviruses. In all nidoviruses, the C-terminal RdRp domain is linked to a conserved N-terminal domain, which has been coined NiRAN (nidovirus RdRp-associated nucleotidyl transferase). Although no structural information is available, the functional characterization of the nidovirus RdRp and the larger enzyme complex of which it is part, has progressed significantly over the past decade. In coronaviruses several smaller, non-enzymatic nsps were characterized that direct RdRp function, while a 3′-to-5′ exoribonuclease activity in nsp14 was implicated in fidelity. In arteriviruses, the nsp1 subunit was found to maintain the balance between genome replication and subgenomic mRNA production. Understanding RdRp behaviour and interactions during RNA synthesis and subsequent processing will be key to rationalising the evolutionary success of nidoviruses and the development of antiviral strategies.

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3′ terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3′ terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3′ cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3′ termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3′ terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3′ vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

Uncoupling of Influenza A Virus Transcription and Replication through Mutation of the Unpaired Adenosine in the Viral RNA Promoter

ABSTRACT Transcription and replication of the influenza A virus RNA genome are mediated by the viral RNA polymerase from a promoter consisting of the partially base-paired 3′ and 5′ termini of viral genome segments. Here we show that transcription and replication can be uncoupled by mutation of an unpaired adenosine in the 5′ strand of the promoter. This residue is important for transcription but not replication by being essential for the cap-binding activity of the RNA polymerase.

Initiation, Elongation, and Realignment during Influenza Virus mRNA Synthesis

ABSTRACT The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp “snatches” capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide in vitro evidence for the existence of this transcriptional prime-realign mechanism but show that it functions efficiently only for primers that are short or cannot stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how influenza A virus initiates transcription correctly and efficiently. IMPORTANCE Influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome by using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterize a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.

Correct And Efficient Initiation Of Viral RNA Synthesis By The Influenza A Virus RNA Polymerase

The influenza A virus uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. To ensure that the genome is copied correctly, the RdRp has been proposed to realign the initiation product of viral RNA (vRNA) synthesis from the internal initiation site to the 3′ end of the complementary RNA (cRNA) template before extending the nascent vRNA. Here we show that this process is essential for viral RNA synthesis and that the RdRp uses a mechanism involving the priming loop to enforce realignment during replication. We also find that realignment plays a role in viral mRNA synthesis, enabling the RdRp to handle sequence variation among the primers that it snatches from host mRNAs to prime viral mRNA synthesis. Overall, these observations advance our mechanistic understanding of how the influenza A virus initiates replication and transcription correctly and efficiently.The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2 and PA. To copy the viral RNA (vRNA) genome segments and the complementary RNA (cRNA) segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3’ terminus, while on the cRNA promoter the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into prime-realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly. Importance Influenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

A Mechanism for Priming and Realignment during Influenza A Virus Replication

ABSTRACT The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2, and PA. To copy the viral RNA (vRNA) genome segments and the cRNA segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3′ terminus, while on the cRNA promoter, the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular, the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into priming and realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly. IMPORTANCE Influenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome by using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and that abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.