Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter

Gerlach, Piotr; Malet, Hélène; Cusack, Stephen; Reguera, Juan

doi:10.1016/j.cell.2015.05.006

Cited by 171 publications

(262 citation statements)

References 34 publications

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“…The RNA-dependent RNA polymerase catalytic domain of protein L Sequence analysis suggests that the N-terminal half of protein L functions as a RNA-dependent RNA polymerase (RdRP), and is responsible for both DNA replication and transcription. HHpred 45 detects the Bunyavirus RdRP (PDB id: 5AMR 83 ) as a structural template (Probability: 84%). The alignment between Ebolavirus RdRP and Bunyavirus RdRP includes both the catalytic domain and a helical bundle connected to its C-terminus.…”

Section: The Zinc-finger Domain Of Vp30mentioning

confidence: 99%

Predictive and comparative analysis of Ebolavirus proteins

2015

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Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus.

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Section: The Zinc-finger Domain Of Vp30mentioning

confidence: 99%

Predictive and comparative analysis of Ebolavirus proteins

2015

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“…The binding of NP to the virus genome is required to form the vRNP template for an efficient elongation reaction by RdR Pol (8)(9)(10)(11)(12). A recent structural model of RdR Pol of a segmented negative-strand RNA virus led to the proposal that the disruption of the vRNP structure during template reading is restricted around the active center due to the proximity of the entry and exit channels of template (42). Based on these findings, it is possible that Prp18 functions as a molecular chaperone for NP around the active center of RdR Pol to maintain the structural integrity of vRNP for processive template reading.…”

Section: Discussionmentioning

confidence: 99%

Pre-mRNA Processing Factor Prp18 Is a Stimulatory Factor of Influenza Virus RNA Synthesis and Possesses Nucleoprotein Chaperone Activity

Minakuchi

Sugiyama

Kato

et al. 2017

J Virol

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The genome of influenza virus (viral RNA [vRNA]) is associated with the nucleoprotein (NP) and viral RNA-dependent RNA polymerases and forms helical viral ribonucleoprotein (vRNP) complexes. The NP-vRNA complex is the biologically active template for RNA synthesis by the viral polymerase. Previously, we identified human pre-mRNA processing factor 18 (Prp18) as a stimulatory factor for viral RNA synthesis using a Saccharomyces cerevisiae replicon system and a single-gene deletion library of Saccharomyces cerevisiae (T. Naito, Y. Kiyasu, K. Sugiyama, A. Kimura, R. Nakano, A. Matsukage, and K. Nagata, Proc Natl Acad Sci USA, 104:18235-18240, 2007, https://doi.org/10.1073/pnas.0705856104). In infected Prp18 knockdown (KD) cells, the synthesis of vRNA, cRNA, and viral mRNAs was reduced. Prp18 was found to stimulate in vitro viral RNA synthesis through its interaction with NP. Analyses using in vitro RNA synthesis reactions revealed that Prp18 dissociates newly synthesized RNA from the template after the early elongation step to stimulate the elongation reaction. We found that Prp18 functions as a chaperone for NP to facilitate the formation of NP-RNA complexes. Based on these results, it is suggested that Prp18 accelerates influenza virus RNA synthesis as an NP chaperone for the processive elongation reaction. IMPORTANCETemplates for viral RNA synthesis of negative-stranded RNA viruses are not naked RNA but rather RNA encapsidated by viral nucleocapsid proteins forming vRNP complexes. However, viral basic proteins tend to aggregate under physiological ionic strength without chaperones. We identified the pre-mRNA processing factor Prp18 as a stimulatory factor for influenza virus RNA synthesis. We found that one of the targets of Prp18 is NP. Prp18 facilitates the elongation reaction of viral polymerases by preventing the deleterious annealing of newly synthesized RNA to the template. Prp18 functions as a chaperone for NP to stimulate the formation of NP-RNA complexes. Based on these results, we propose that Prp18 may be required to maintain the structural integrity of vRNP for processive template reading.

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“…In contrast, RdRps that initiate RNA synthesis at the NP gene start site and then cap the nascent NP mRNA become committed to transcription (23,24). Structural studies of both sNSV and nsNSV RdRps suggest that both can be found in a number of alternative states or conformations, each of which may be important during different steps of RNA synthesis (25)(26)(27)(28).…”

Section: Discussionmentioning

confidence: 99%

A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity

Matsumoto

Ohta

Kolakofsky

et al. 2017

J Virol

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The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA.IMPORTANCE We examined the importance of amino acids in the putative RNAbinding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression. KEYWORDS human parainfluenza virus type 2, encapsidation, nucleoprotein, polymerase H uman parainfluenza virus type 2 (hPIV2) is a major respiratory pathogen and a member of the Rubulavirus genus of the family Paramyxoviridae, which includes simian virus 41, hPIV4, mumps virus, and PIV5. The hPIV2 genome RNA contains six tandemly linked genes (3=-NP-P/V-M-F-HN-L-5=) and is tightly coated by the nucleoprotein (NP) to form a helical nucleocapsid that serves as the template for viral RNA synthesis. The phosphoprotein (P) and large protein (L) together form the viral RNAdependent RNA polymerase (RdRp), which is responsible for both transcription to produce mRNAs and replication to produce genomes and antigenomes (1). hPIV2 NP contains 543 amino acids, whose functional domains are partially identified. The N-terminal 294 amino acids are required for NP self-assembly, and two C-terminal

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Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter

Cited by 171 publications

References 34 publications

Predictive and comparative analysis of Ebolavirus proteins

Predictive and comparative analysis of Ebolavirus proteins

Pre-mRNA Processing Factor Prp18 Is a Stimulatory Factor of Influenza Virus RNA Synthesis and Possesses Nucleoprotein Chaperone Activity

A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity

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