Mitoferrin1 Transgenic Zebrafish Line Serves as a Model to Study Erythroid Cell Fate during Hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3576-3576
Author(s):  
Julio Amigo ◽  
Ming Yu ◽  
Camila O. dos Santos ◽  
Prasad N. Paradkar ◽  
Jeffrey D. Cooney ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Site Directed Mutagenesis ◽  
Developmental Expression ◽  
Luciferase Reporter ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Erythroid Cells ◽  
Egfp Reporter

Abstract We previously identified the mitochondrial solute carrier, Mitoferrin1 (Slc25a37, Mfrn1) as the principal mitochondrial iron importer essential for heme and iron-sulfur (Fe-S) cluster synthesis in developing erythroblasts. Its closely related paralog, Mitoferrin2 (Slc25a28, Mfrn2) functions in an analogous role in non-erythroid cells. Zebrafish with mutations in Mfrn1 have defects in hemoglobinization and maturation of erythroid cells caused by defective acquisition of iron into the mitochondria (GC Shaw, et al., 2006 Nature 440:96–100). Mfrn1 is highly expressed in embryonic and definitive sites of hematopoiesis in zebrafish and mouse, such as the developing blood island (ICM), fetal liver and adult bone marrow. In contrast, Mfrn2 is ubiquitously expressed, including at very low levels in erythroid cells. To understand the transcriptional regulation of Mfrn1 and Mfrn2, we used bioinformatics tools to identify potential cis-regulatory motifs (CRM) within each gene from mouse. The Gateway-modified Tol-2 vector was used to rapidly clone these conserved, minimal CRM fragments from mouse upstream of the basal promoter and an EGFP-reporter. Each construct was then introduced into zebrafish embryos for transient and stable expression. Using this strategy, we identified CRM’s that recapitulate the endogenous mRNA expression pattern of Mfrn genes during zebrafish development. Germ line stable transmission of the murine Tg(Mfrn1:EGFP) reporter in zebrafish showed robust EGFP expression in erythroid progenitors and mature erythrocytes and the remarkable conservation of function for CRM’s across species. In contrast, the mouse Tg(Mfrn2:EGFP) was expressed in skeletal muscle, heart, liver, and pronephros. The Mfrn1 enhancer is located ~35 kb upstream of the transcription start site and contains two GATA consensus motifs, which bind GATA-1 by chromatin immunoprecipitation analysis. Moreover, the ~150 bp Mfrn1 enhancer fragment exhibits transcriptional activation when coupled to the minimal γ-globin promoter driving expression of a luciferase reporter in K562 cells. Site-directed mutagenesis revealed that both GATA motifs are required for robust erythroid expression. In a complementary approach, transient knockdown of GATA-1 in zebrafish embryos using anti-sense morpholinos selectively ablated Mfrn1 mRNA expression in the ICM, consistent with the epistatic relationship of GATA-1 and Mfrn1. The zebrafish transgenic lines harboring the two murine Mfrn enhancers have proven useful in studying the regulatory and developmental expression in the Mfrn genes in erythroid and non-hematopoietic organs, such as heart and liver. Our results show that the combined use of bioinformatics, Gateway-mediated cloning, and Tol-2 mediated transgenesis in zebrafish embryos is an effective approach to functionally interrogate the transcriptional activity of putative CRM’s in vivo. The conservation and faithful expression of mouse CRM’s in zebrafish demonstrate the utility of this functional approach for analyzing mammalian CRM’s.

scholarly journals Developmental Expression of Mouse Erythrocyte Protein 4.2 mRNA: Evidence for Specific Expression in Erythroid Cells

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 695-705 ◽  
Author(s):  
Lingyun Zhu ◽  
Samir B. Kahwash ◽  
Long-Sheng Chang
Keyword(s):  
Mrna Expression ◽  
Fetal Liver ◽  
Late Gestation ◽  
Developmental Expression ◽  
Erythroid Cells ◽  
Mouse Development ◽  
Specific Expression ◽  
Protein 4.2 ◽  
Specific Regulation ◽  
In Erythroid Cells

Abstract Erythrocyte protein 4.2 (P4.2) is an important component of the erythrocyte membrane skeletal network with an undefined biologic function. Presently, very little is known about the expression of the P4.2 gene during mouse embryonic development and in adult animals. By using the Northern blot and in situ hybridization techniques, we have examined the spatial and temporal expression of the P4.2 gene during mouse development. We show that expression of the mouse P4.2 gene is temporally regulated during embryogenesis and that the P4.2 mRNA expression pattern coincides with the timing of erythropoietic activity in hematopoietic organs. P4.2 transcripts are first detected in embryos on day 7.5 of gestation and are localized exclusively in primitive erythroid cells of yolk sac origin. These erythroid cells remain to be the only source for P4.2 expression until the switch of the hematopoietic producing site to fetal liver. In mid- and late-gestation periods, P4.2 mRNA expression is restricted to the erythroid cells in fetal liver and to circulating erythrocytes. Around and after birth, the site for P4.2 expression is switched from liver to spleen and bone marrow, and P4.2 transcripts are only detected in cells of the erythroid lineage. These results provide the evidence for specific P4.2 expression in erythroid cells. In addition, the timing and pattern of expression of the P4.2 gene suggest the specific regulation of the P4.2 gene.

scholarly journals Transcriptional activation of zebrafish cyp11a1 promoter is dependent on the nuclear receptor Ff1b

2009 ◽  
Vol 43 (3) ◽  
pp. 121-130 ◽  
Author(s):  
Sue Ing Quek ◽  
Woon Khiong Chan
Keyword(s):  
Transcriptional Activation ◽  
Bioinformatic Analysis ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Egfp Expression ◽  
Synthesis Rate ◽  
Zebrafish Embryos ◽  
Mobility Shift ◽  
Cytochrome P450scc

The cytochrome P450scc (cholesterol side-chain cleavage enzyme) encoded by CYP11A1 catalyzes the first step in steroidogenesis by converting cholesterol to pregnenolone, and thus, controls the synthesis rate of steroid hormones. In mammals, steroidogenic factor 1 (SF1) has been implicated in the cAMP-mediated transcriptional activation of CYP11A1 promoter. In zebrafish, Ff1b has been established as the homolog of SF1. To assess the dependency of cyp11a1 expression on Ff1b, the putative promoter of zebrafish cyp11a1, spanning 1.7 kb, was isolated and bioinformatic analysis revealed two conserved FF1 response elements (FREs) that potentially bind Ff1b. Transfection studies in cell lines of different lineages confirmed that this promoter fragment contained the necessary regulatory elements required for its basal transcription. Truncation and mutagenesis studies performed in Y1 adrenocortical cells revealed that only the proximal FRE was essential for transcriptional activation. Electrophoretic mobility shift assay, however, indicated that Ff1b bound to both FREs, while their in vivo occupancy was confirmed using a chromatin immunoprecipitation assay. Lastly, the cyp11a1 promoter was able to direct EGFP expression specifically to the interrenal gland and genital ridge when transiently expressed in microinjected zebrafish embryos, and the promoter activity is potentiated by ff1b overexpression as measured from luciferase reporter activity in zebrafish embryos.

scholarly journals Development of High-Content Assays for Kidney Progenitor Cell Expansion in Transgenic Zebrafish

2013 ◽  
Vol 18 (10) ◽  
pp. 1193-1202 ◽  
Author(s):  
Subramaniam Sanker ◽  
Maria Cecilia Cirio ◽  
Laura L. Vollmer ◽  
Natasha D. Goldberg ◽  
Lee A. McDermott ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Image Analysis ◽  
Time Course ◽  
Kidney Development ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Kidney Regeneration ◽  
Kidney Organogenesis

Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence–based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence–based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

scholarly journals Developmental Expression of Mouse Erythrocyte Protein 4.2 mRNA: Evidence for Specific Expression in Erythroid Cells

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 695-705
Author(s):  
Lingyun Zhu ◽  
Samir B. Kahwash ◽  
Long-Sheng Chang
Keyword(s):  
Mrna Expression ◽  
Fetal Liver ◽  
Late Gestation ◽  
Developmental Expression ◽  
Erythroid Cells ◽  
Mouse Development ◽  
Specific Expression ◽  
Protein 4.2 ◽  
Specific Regulation ◽  
In Erythroid Cells

Erythrocyte protein 4.2 (P4.2) is an important component of the erythrocyte membrane skeletal network with an undefined biologic function. Presently, very little is known about the expression of the P4.2 gene during mouse embryonic development and in adult animals. By using the Northern blot and in situ hybridization techniques, we have examined the spatial and temporal expression of the P4.2 gene during mouse development. We show that expression of the mouse P4.2 gene is temporally regulated during embryogenesis and that the P4.2 mRNA expression pattern coincides with the timing of erythropoietic activity in hematopoietic organs. P4.2 transcripts are first detected in embryos on day 7.5 of gestation and are localized exclusively in primitive erythroid cells of yolk sac origin. These erythroid cells remain to be the only source for P4.2 expression until the switch of the hematopoietic producing site to fetal liver. In mid- and late-gestation periods, P4.2 mRNA expression is restricted to the erythroid cells in fetal liver and to circulating erythrocytes. Around and after birth, the site for P4.2 expression is switched from liver to spleen and bone marrow, and P4.2 transcripts are only detected in cells of the erythroid lineage. These results provide the evidence for specific P4.2 expression in erythroid cells. In addition, the timing and pattern of expression of the P4.2 gene suggest the specific regulation of the P4.2 gene.

Dynamic CO-Localization of GATA1, NFE2, and EKLF and Changes in Gene Expression During Hematopoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 741-741 ◽  
Author(s):  
Laurie A. Steiner ◽  
Vincent P Schulz ◽  
Yelena Maksimova ◽  
Milind Mahajan ◽  
David M. Bodine ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Mrna Expression ◽  
Erythroid Cell ◽  
Common Finding ◽  
P Value ◽  
Erythroid Cells ◽  
Three Factor ◽  
In Erythroid Cells ◽  
Proximal Promoters

Abstract Abstract 741 Regulation of lineage choice during the development and differentiation of erythroid cells in hematopoiesis is a complex process. GATA1, NFE2, and EKLF are transcription factors critical for erythropoiesis. Focused studies, including detailed analyses of the human beta globin gene locus and a select group of erythrocyte membrane protein genes, have revealed that these three transcription factors may co-localize at common regulatory sites in erythroid-expressed genes. To address the hypothesis that GATA1, NFE2, and EKLF frequently co-localize on critical regulatory elements responsible for cell-type specific gene expression during erythropoiesis, chromatin immunoprecipitation coupled with ultrahigh throughput sequencing (ChIP-seq) was used to identify sites of GATA1, NFE2, and EKLF occupancy in human primary hematopoietic stem and progenitor cells (HSPCs) and human primary erythroid cells. ChIP was done using CD34+ HSPCs prepared by immunomagnetic bead selection and cultured CD71+/GPA+ erythroid cells (R3/R4 population) using antibodies against GATA1, NF-E2, and EKLF. The MACS algorithm (Zhang et al. Genome Biol, 2008) was used to identify regions of DNA-protein interaction, with a p-value ≤10e-5. Sites identified by MACS were ordered by p-value, and the 7000 sites with the most stringent p-values were selected for further analysis. Sites which occurred within 200bp of each other were treated as a single site. Unexpectedly, sites of GATA1, NFE2, and EKLF occupancy were common in HSPCs, with 6643 GATA1, 6657 NFE2, and 6579 EKLF sites identified, respectively. Sites identified in HSPCs were primarily in enhancers (>1kb from a RefSeq gene; 44% of GATA1, 49% of NFE2, and 51% of EKLF sites) and in introns (32% of GATA1, 34% of NFE2, and 34% of EKLF sites), with only a few sites at proximal promoters (within 1kb of a TSS; 7% of GATA1, 6% of NFE2, and 7% EKLF sites.) In erythroid cells, 6895 GATA1, 6907 NF-E2, and 6874 EKLF sites were identified. For all 3 factors, binding site occupancy varied greatly from that observed in HSPCs. Proximal promoter binding was much more common in erythroid cells than in HSPCs, with 19% of GATA1, 28% of NFE2 and 38% of EKLF sites found at promoters. Binding was frequently found at enhancers (41% of GATA, 38% NFE2, and 32% EKLF sites) and in introns (29% of GATA1, 26% of NFE2, and 21% of EKLF). To gain insight into three factor co-occupancy on a genome-wide scale, GATA1, EKLF, and NFE2 binding sites were compared using the Active Region Comparer (http://dart.gersteinlab. org/). Surprisingly, co-localization of all three factors was common in HSPCs, occurring at 2666 sites (40%, 40% and 45% of GATA1, NFE2, and EKLF sites). Sites of GATA1-NFE2-EKLF co-localization in HSPCs were located primarily at enhancers (51% of sites), in introns (32% of sites), and rarely at proximal promoters (6% of sites). In erythroid cells, co-localization of all three transcription factors was also common, occurring at 2445 sites (35%, 35%, and 36% of GATA1, NFE2, and EKLF sites, respectively). In contrast to HSPCs, sites of GATA1-NFE2-EKLF co-localization in erythroid cells were located primarily at proximal promoters (35% of sites) and enhancers (34% of sites), with co-localization in introns accounting for 20% of sites. A limited subset of sites, 1429 GATA1, 921 NFE2, and 1038 EKLF sites, were present in both HSPC and erythroid cells. Throughout the genome, there were only 233 sites of three factor co-localization in common in both HSPC and erythroid cells. Gene expression in HSPC and erythroid cell was analyzed via RNA hybridization to Illumina HumanHT-12 v3 Expression BeadChip arrays. In erythroid cells, genes with GATA1-NFE2-EKLF co-localization from 5kb upstream to 2kb downstream had significantly higher levels of mRNA expression than genes without GATA1-NFE2-EKLF co-localization (p<2.2e-16). The reverse was observed in HSPCs, where genes with GATA1-NFE2-EKLF co-localization had significantly lower levels of mRNA expression than genes without GATA1-NFE2-EKLF co-localization (p<7.3e-05). These data support the hypothesis that co-localization of GATA1, NFE2, and EKLF is a common finding in hematopoietic cells. Significant differences in factor co-localization and gene expression in HSPC and erythroid cells suggest that this coordinated binding orchestrates different patterns of gene expression during hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cytochrome P450-dependent biotransformation capacities in embryonic, juvenile and adult stages of zebrafish (Danio rerio)—a state-of-the-art review

2021 ◽  
Author(s):  
Ann-Kathrin Loerracher ◽  
Thomas Braunbeck
Keyword(s):  
Cytochrome P450 ◽  
Phase I ◽  
Mrna Expression ◽  
Danio Rerio ◽  
Developmental Stages ◽  
State Of The Art ◽  
Adverse Outcome Pathway ◽  
Zebrafish Embryos ◽  
Toxicological Studies ◽  
Cyp Isoforms

AbstractGiven the strong trend to implement zebrafish (Danio rerio) embryos as translational model not only in ecotoxicological, but also toxicological testing strategies, there is an increasing need for a better understanding of their capacity for xenobiotic biotransformation. With respect to the extrapolation of toxicological data from zebrafish embryos to other life stages or even other organisms, qualitative and quantitative differences in biotransformation pathways, above all in cytochrome P450-dependent (CYP) phase I biotransformation, may lead to over- or underestimation of the hazard and risk certain xenobiotic compounds may pose to later developmental stages or other species. This review provides a comprehensive state-of-the-art overview of the scientific knowledge on the development of the CYP1-4 families and corresponding phase I biotransformation and bioactivation capacities in zebrafish. A total of 68 publications dealing with spatiotemporal CYP mRNA expression patterns, activities towards mammalian CYP-probe substrates, bioactivation and detoxification activities, as well as metabolite profiling were analyzed and included in this review. The main results allow for the following conclusions: (1) Extensive work has been done to document mRNA expression of CYP isoforms from earliest embryonic stages of zebrafish, but juvenile and adult zebrafish have been largely neglected so far. (2) There is insufficient understanding of how sex- and developmental stage-related differences in expression levels of certain CYP isoforms may impact biotransformation and bioactivation capacities in the respective sexes and in different developmental stages of zebrafish. (3) Albeit qualitatively often identical, many studies revealed quantitative differences in metabolic activities of zebrafish embryos and later developmental stages. However, the actual relevance of age-related differences on the outcome of toxicological studies still needs to be clarified. (4) With respect to current remaining gaps, there is still an urgent need for further studies systematically assessing metabolic profiles and capacities of CYP isoforms in zebrafish. Given the increasing importance of Adverse Outcome Pathway (AOP) concepts, an improved understanding of CYP capacities appears essential for the interpretation and outcome of (eco)toxicological studies.

scholarly journals Slc7a11 Modulated by POU2F1 is Involved in Pigmentation in Rabbit

2019 ◽  
Vol 20 (10) ◽  
pp. 2493 ◽  
Author(s):  
Yang Chen ◽  
Shuaishuai Hu ◽  
Lin Mu ◽  
Bohao Zhao ◽  
Manman Wang ◽  
...  
Keyword(s):  
Skin Color ◽  
Site Directed Mutagenesis ◽  
Molecular Regulation ◽  
Luciferase Reporter ◽  
Directed Mutagenesis ◽  
Regulation Mechanism ◽  
Solute Carrier Family ◽  
Depth Analysis ◽  
Solute Carrier ◽  
Fur Color

Solute carrier family 7 member 11 (Slc7a11) is a cystine/glutamate xCT transporter that controls the production of pheomelanin pigment to change fur and skin color in animals. Previous studies have found that skin expression levels of Slc7a11 varied significantly with fur color in Rex rabbits. However, the molecular regulation mechanism of Slc7a11 in pigmentation is unknown. Here, rabbit melanocytes were first isolated and identified. The distribution and expression pattern of Slc7a11 was confirmed in skin from rabbits with different fur colors. Slc7a11 affected the expression of pigmentation related genes and thus affected melanogenesis. Meanwhile, Slc7a11 decreased melanocyte apoptosis, but inhibition of Slc7a11 enhanced apoptosis. Furthermore, the POU2F1 protein was found to bind to the −713 to −703 bp region of Slc7a11 promoter to inhibit its activity in a dual-luciferase reporter and site-directed mutagenesis assay. This study reveals the function of the Slc7a11 in melanogenesis and provides in-depth analysis of the mechanism of fur pigmentation.

scholarly journals Expression of ferrochelatase mRNA in erythroid and non-erythroid cells

1993 ◽  
Vol 292 (2) ◽  
pp. 343-349 ◽  
Author(s):  
R Y Y Chan ◽  
H M Schulman ◽  
P Ponka
Keyword(s):  
Sequence Similarity ◽  
Erythroid Differentiation ◽  
Polyadenylation Signal ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Erythroleukemia Cells ◽  
Haem Biosynthesis ◽  
Polyadenylation Sites ◽  
Dna Fragment ◽  
Polyadenylation Signals

Ferrochelatase, which catalyses the last step in haem biosynthesis, i.e. the insertion of Fe(II) into protophorphyrin IX, is present in all cells, but is particularly abundant in erythroid cells during haemoglobinization. Using mouse ferrochelatase cDNA as a probe two ferrochelatase transcripts, having lengths of 2.9 kb and 2.2 kb, were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in the non-erythroid tissues and the 2.2 kb transcript more predominant in spleen. In mouse erythroleukemia cells the 2.9 kb ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by dimethyl sulphoxide there is a preferential increase in the 2.2 kb transcript, which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Since there is probably only one mouse ferrochelatase gene, the occurrence of two ferrochelatase transcripts could arise from the use of two putative polyadenylation signals in the 3′ region of ferrochelatase DNA. This possibility was explored by using a 389 bp DNA fragment produced by PCR with synthetic oligoprimers having sequence similarity with a region between the polyadenylation sites. This fragment hybridized only to the 2.9 kb ferrochelatase transcript, indicating that the two transcripts differ at their 3′ ends and suggesting that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal. The preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival

2014 ◽  
Vol 459 (3) ◽  
pp. 455-466 ◽  
Author(s):  
Neli S. Slavova-Azmanova ◽  
Nicole Kucera ◽  
Alison Louw ◽  
Jiulia Satiaputra ◽  
Adley Handoko ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Signalling Pathways ◽  
Erythroid Cells ◽  
Src Family Kinase ◽  
Receptor Signalling ◽  
Lyn Kinase ◽  
Control Survival

In erythroid cells both positive viability signals and feedback inhibitory signalling require the Src family kinase Lyn, influencing cell survival and their ability to differentiate. This illustrates that Lyn is critical for normal erythropoiesis and erythroid cell development.

scholarly journals Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

2012 ◽  
Vol 86 (16) ◽  
pp. 8375-8387 ◽  
Author(s):  
Marie Galloux ◽  
Bogdan Tarus ◽  
Ilfad Blazevic ◽  
Jenna Fix ◽  
Stéphane Duquerroy ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Interaction ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Interaction Domain ◽  
Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

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