Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.

BACKGROUND: Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. METHODS: Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. RESULTS: After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 (∆APD shortening 113 ms), indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). CONCLUSIONS: Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2.

AIMS: Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. CONCLUSION: A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.

Cockayne syndrome group A and ferrochelatase finely tune ribosomal gene transcription and its response to UV irradiation.

CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.

Identification of a targeted and testable antiarrhythmic therapy for long-QT syndrome type 2 using a patient-specific cellular model.

Aims: Loss-of-function mutations in the hERG gene causes long-QT syndrome type 2 (LQT2), a condition associated with reduced IKr current. Four different mutation classes define the molecular mechanisms impairing hERG. Among them, Class 2 mutations determine hERG trafficking defects. Lumacaftor (LUM) is a drug acting on channel trafficking already successfully tested for cystic fibrosis and its safety profile is well known. We hypothesize that LUM might rescue also hERG trafficking defects in LQT2 and exert anti-arrhythmic effects. Methods and results: From five LQT2 patients, we generated lines of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harbouring Class 1 and 2 mutations. The effects of LUM on corrected field potential durations (cFPD) and calcium-handling irregularities were verified by multi electrode array and by calcium transients imaging, respectively. Molecular analysis was performed to clarify the mechanism of action of LUM on hERG trafficking and calcium handling. Long-QT syndrome type 2 induced pluripotent stem cell-derived cardiomyocytes mimicked the clinical phenotypes and showed both prolonged cFPD (grossly equivalent to the QT interval) and increased arrhythmias. Lumacaftor significantly shortened cFPD in Class 2 iPSC-CMs by correcting the hERG trafficking defect. Furthermore, LUM seemed to act also on calcium handling by reducing RyR2S2808 phosphorylation in both Class 1 and 2 iPSC-CMs. Conclusion: Lumacaftor, a drug already in clinical use, can rescue the pathological phenotype of LQT2 iPSC-CMs, particularly those derived from Class 2 mutated patients. Our results suggest that the use of LUM in LQT2 patients not protected by ß-blockers is feasible and may represent a novel therapeutic option.

Xanthine Quartets on Au(111).

The quartet of xanthine (X), a purine base ubiquitously distributed in most human body tissues and fluids, has been for the first time fabricated and visualized, as the first alternative purine quartet besides the known guanine (G)-quartet. The X-quartet network is demonstrated to be the most stable phase on Au(111). Unlike guanine, the fabrication of the X-quartets is not dependent on the presence of metal atoms, which makes it the first metal-free purine quartet. The X-quartet holds great promise to potentially construct artificial new DNA quadruplexes for genetic regulation and antitumor therapy. Moreover, both the X-quartet itself and the quartet networks favor homochirality, suggesting homochiral xanthine oligomers and the networks may have been formed as the precursors of the pristine oligonucleotides on primitive Earth.

Optimized lentiviral transduction of human amniotic mesenchymal stromal cells.

Mesenchymal stromal cells are excellent candidates for regenerative medicine since they are multipotent, easy to isolate, can be expanded to obtain clinically relevant numbers and are immunoprivileged. Stable genetic modification with viral vectors can improve mesenchymal stromal cell function and enhance their therapeutic potential. However, standard viral vectors achieve sub-optimal transduction efficiency with a single infection. On the other hand, multiple transduction cycles or antibiotic-based selection methods may alter the stem cell phenotype. We hypothesized that the use of lentiviral vectors containing specific regulatory sequences may result in improved transduction efficiency. Thus, we compared two types of third generation lentiviral vectors, one of which, the pLenti7.3 vector, contains the optimized sequences for Polypurine Tract and Woodchuck Post-transcriptional Regulatory Element. We demonstrated that with the pLenti7.3 it is possible to efficiently transduce human mesenchymal stromal cells with a single transduction cycle. Additionally, we successfully showed that by using the pLenti7.3 vector it is possible to efficiently over-express different growth factors, particularly relevant for cardiac protection and differentiation, in human mesenchymal stromal cells.

The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer.

RNA-binding proteins (RBPs) are increasingly identified as post-transcriptional drivers of cancer progression. The RBP LARP1 is an mRNA stability regulator, and elevated expression of the protein in hepatocellular and lung cancers is correlated with adverse prognosis. LARP1 associates with an mRNA interactome that is enriched for oncogenic transcripts. Here we explore the role of LARP1 in epithelial ovarian cancer, a disease characterized by the rapid acquisition of resistance to chemotherapy through the induction of pro-survival signalling. We show, using ovarian cell lines and xenografts, that LARP1 is required for cancer cell survival and chemotherapy resistance. LARP1 promotes tumour formation in vivo and maintains cancer stem cell-like populations. Using transcriptomic analysis following LARP1 knockdown, cross-referenced against the LARP1 interactome, we identify BCL2 and BIK as LARP1 mRNA targets. We demonstrate that, through an interaction with the 3' untranslated regions (3' UTRs) of BCL2 and BIK, LARP1 stabilizes BCL2 but destabilizes BIK with the net effect of resisting apoptosis. Together, our data indicate that by differentially regulating the stability of a selection of mRNAs, LARP1 promotes ovarian cancer progression and chemotherapy resistance.

Formation of Hypoxanthine Tetrad by Reaction with Sodium Chloride: From Planar to Stereo.

By reacting with NaCl on Au(111), the formation of hypoxanthine (HX) tetrads is demonstrated at the atomic scale in real space. These results directly demonstrate that alternative purine tetrads can be formed in both planar and non-planar configuration, and that ionic bonding plays a crucial role for the formation and planar-to-stereo transformation of the tetrads, providing deeper insight for constructing artificial DNA/RNA quadruplexes. Moreover, both the tilted HXs and Na show strong charge transfer with the substrate in the non-planar phase. The insights gained by this work also open up new routes to tune the electrostatic nature of metal-organic interfaces and design stereo-nanostructures on surfaces.

The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage.

The effect of amidation on the behaviour of antimicrobial peptides.

Aurein 2.6-COOH and aurein 3.1-COOH were studied along with their naturally occurring C-terminally amidated analogues. Circular dichroism (CD) and molecular dynamic (MD) simulations were used to study the effects of amidation on the interaction of antimicrobial peptides (AMPs) with lipid bilayers. CD measurements and MD analysis suggested that both peptide analogues were predominantly random coil and adopted low levels of α-helical structure in solution (<30%) and in the presence of a lipid bilayer the peptides formed a stable α-helical structure. In general, amidated analogues have a greater propensity than the non-amidated peptides to form a α-helical structure. MD simulations predicted that aurein 2.6-COOH and aurein 3.1-CHOOH destabilised lipid bilayers from 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphoserine via angled bilayer penetration. They also showed that aurein 2.6-CONH2 and aurein 3.1-CONH2 formed a helix horizontal to the plane of an asymmetric interface.

The cooperative behaviour of antimicrobial peptides in model membranes.

A systematic analysis of the hypothesis of the antimicrobial peptides' (AMPs) cooperative action is performed by means of full atomistic molecular dynamics simulations accompanied by circular dichroism experiments. Several AMPs from the aurein family (2.5,2.6, 3.1), have a similar sequence in the first ten amino acids, are investigated in different environments including aqueous solution, trifluoroethanol (TFE), palmitoyloleoylphosphatidylethanolamine (POPE), and palmitoyloleoylphosphatidylglycerol (POPG) lipid bilayers. It is found that the cooperative effect is stronger in aqueous solution and weaker in TFE. Moreover, in the presence of membranes, the cooperative effect plays an important role in the peptide/lipid bilayer interaction. The action of AMPs is a competition of the hydrophobic interactions between the side chains of the peptides and the hydrophobic region of lipid molecules, as well as the intra peptide interaction. The aureins 2.5-COOH and 2.6-COOH form a hydrophobic aggregate to minimize the interaction between the hydrophobic group and the water. Once that the peptides reach the water/lipid interface the hydrophobic aggregate becomes smaller and the peptides start to penetrate into the membrane. In contrast, aurein 3.1-COOH forms only a transient aggregate which disintegrates once the peptides reached the membrane, and it shows no cooperativity in membrane penetration.

XPD mutations in trichothiodystrophy hamper collagen VI expression and reveal a role of TFIIH in transcription derepression.

Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.

APPLICANTS' STRATEGIC USE OF EXTREME OR MIDPOINT RESPONSES WHEN FAKING PERSONALITY TESTS.

Faking, the intentional distortion of answers to personality tests, is likely a complex process. In particular, participants in previous research have mentioned that they used different kind of strategies to appear more hirable, including systematically more extreme or more midpoint responses. However, quantitative evidence is still lacking. An experiment was conducted in which 327 students (173 women, 153 men, 1 not indicated; M age = 22.1 yr., SD = 2.8) were randomly assigned to two groups. Hypothetical job advertisements primed the participants into believing that the hiring company preferred a person with either a "strong" (Strong Character group) or a "well-balanced" character (Well-balanced Character group). The participants filled out 40 items that were chosen from four established questionnaires as neither socially desirable nor undesirable. The responses to these items were used to calculate two extreme response measures and one midpoint response measure. The Strong Character group used extreme scores more often than the Well-balanced Character group (and the midpoint scores less often), independently of mean differences. This suggests that fakers use more sophisticated strategies than is often assumed.

Aurein 2.3 functionality is supported by oblique orientated α-helical formation.

In this study, an amphibian antimicrobial peptide, aurein 2.3, was predicted to use oblique orientated α-helix formation in its mechanism of membrane destabilisation. Molecular dynamic (MD) simulations and circular dichroism (CD) experimental data suggested that aurein 2.3 exists in solution as unstructured monomers and folds to form predominantly α-helical structures in the presence of a dimyristoylphosphatidylcholine membrane. MD showed that the peptide was highly surface active, which supported monolayer data where the peptide induced surface pressure changes>34 mNm(-1). In the presence of a lipid membrane MD simulations suggested that under hydrophobic mismatch the peptide is seen to insert via oblique orientation with a phenylalanine residue (PHE3) playing a key role in the membrane interaction. There is evidence of snorkelling leucine residues leading to further membrane disruption and supporting the high level of lysis observed using calcein release assays (76%). Simulations performed at higher peptide/lipid ratio show peptide cooperativity is key to increased efficiency leading to pore-formation.

Cn-AMP2 from green coconut water is an anionic anticancer peptide.

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn-AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti-proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC50 values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included ß-type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N-terminal sequence (TES) of Cn-AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C-terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn-AMP2 across the cancer cell membrane to attack intracellular targets and induce anti-proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers.

A novel form of bacterial resistance to the action of eukaryotic host defense peptides, the use of a lipid receptor.

Host defense peptides show great potential for development as new antimicrobial agents with novel mechanisms of action. However, a small number of resistance mechanisms to their action are known, and here, we report a novel bacterial resistance mechanism mediated by a lipid receptor. Maximin H5 from Bombina maxima bound anionic and zwitterionic membranes with low affinity (Kd > 225 µM) while showing a strong ability to lyse (>55%) and penetrate (π > 6.0 mN m(-1)) these membranes. However, the peptide bound Escherichia coli and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) membranes with higher affinity (Kd < 65 µM) and showed a very low ability for bilayer lysis (<8%) and partitioning (π > 1.0 mN m(-1)). Increasing levels of membrane DMPE correlated with enhanced binding by the peptide (R(2) = 0.96) but inversely correlated with its lytic ability (R(2) = 0.98). Taken with molecular dynamic simulations, these results suggest that maximin H5 possesses membranolytic activity, primarily involving bilayer insertion of its strongly hydrophobic N-terminal region. However, this region was predicted to form multiple hydrogen bonds with phosphate and ammonium groups within PE head-groups, which in concert with charge-charge interactions anchor the peptide to the surface of E. coli membranes, inhibiting its membranolytic action.

Formation mechanism for a hybrid supramolecular network involving cooperative interactions.

A novel mechanism of hybrid assembly of molecules on surfaces is proposed stemming from interactions between molecules and on-surface metal atoms which eventually got trapped inside the network pores. Based on state-of-the-art theoretical calculations, we find that the new mechanism relies on formation of molecule-metal atom pairs which, together with molecules themselves, participate in the assembly growth. Most remarkably, the dissociation of pairs is facilitated by a cooperative interaction involving many molecules. This new mechanism is illustrated on a low coverage Melamine hexagonal network on the Au(111) surface where multiple events of gold atoms trapping via a set of so-called "gate" transitions are found by kinetic Monte Carlo simulations based on transition rates obtained using ab initio density functional theory calculations and the nudged elastic band method. Simulated STM images of gold atoms trapped in the pores of the Melamine network predict that the atoms should appear as bright spots inside Melamine hexagons. No trapping was found at large Melamine coverages, however. These predictions have been supported by preliminary STM experiments which show bright spots inside Melamine hexagons at low Melamine coverages, while empty pores are mostly observed at large coverages. Therefore, we suggest that bright spots sometimes observed in the pores of molecular assemblies on metal surfaces may be attributed to trapped substrate metal atoms. We believe that this type of mechanism could be used for delivering adatom species of desired functionality (e.g., magnetic) into the pores of hydrogen-bonded networks serving as templates for their capture.

Influence of PB2 host-range determinants on the intranuclear mobility of the influenza A virus polymerase.

Avian influenza A viruses often do not propagate efficiently in mammalian cells. The viral polymerase protein PB2 is important for this host restriction, with amino-acid polymorphisms at residue 627 and other positions acting as 'signatures' of avian- or human-adapted viruses. Restriction is hypothesized to result from differential interactions (either positive or inhibitory) with unidentified cellular factors. We applied fluorescence recovery after photobleaching (FRAP) to investigate the mobility of the viral polymerase in the cell nucleus using A/PR/8/34 and A/Turkey/England/50-92/91 as model strains. As expected, transcriptional activity of a polymerase with the avian PB2 protein was strongly dependent on the identity of residue 627 in human but not avian cells, and this correlated with significantly slower diffusion of the inactive polymerase in human but not avian nuclei. In contrast, the activity and mobility of the PR8 polymerase was affected much less by residue 627. Sequence comparison followed by mutagenic analyses identified residues at known host-range-specific positions 271, 588 and 701 as well as a novel determinant at position 636 as contributors to host-specific activity of both PR8 and Turkey PB2 proteins. Furthermore, the correlation between poor transcriptional activity and slow diffusional mobility was maintained. However, activity did not obligatorily correlate with predicted surface charge of the 627 domain. Overall, our data support the hypothesis of a host nuclear factor that interacts with the viral polymerase and modulates its activity. While we cannot distinguish between positive and inhibitory effects, the data have implications for how such factors might operate.