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We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP3R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP3R. DANGER interacts with IP3R in vitro and co-immunoprecipitates with IP3R from cellular preparations. DANGER robustly enhances Ca2+-mediated inhibition of IP3R Ca2+ release without affecting IP3 binding in microsomal assays and inhibits gating in single-channel recordings of IP3R. DANGER appears to allosterically modulate the sensitivity of IP3R to Ca2+ inhibition, which likely alters IP3R-mediated Ca2+ dynamics in cells where DANGER and IP3R are co-expressed. [ABSTRACT FROM AUTHOR]

Ca2+ is a universal signal: the dynamic changes in its release and entry trigger a plethora of cellular responses. Central to this schema are members of the phospholipase C (PLC) superfamily, which relay information from the activated receptor to downstream signal cascades by production of second-messenger molecules. Recent studies reveal that, in addition to its enzymatic activity, PLC-γ regulates Ca2+ entry via the formation of an intermolecular lipid-binding domain with canonical transient receptor potential 3 (TRPC3) ion channels. This complex, in turn, controls TRPC3 trafficking and cell-surface expression. Thus, TRPC3 ion channels are functionally linked to both lipase-dependent and -independent activities of PLC-γ. Understanding the underlying molecular mechanisms that regulate this complex will probably clarify the processes of receptor-activated Ca2+ entry. [Copyright &y& Elsevier]

Many ion channels are regulated by lipids, but prominent motifs for lipid binding have not been identified in most ion channels. Recently, we reported that phospholipase C?1 (PLC-?1) binds to and regulates TRPC3 channels, components of agonist-induced Ca2+ entry into cells. This interaction requires a domain in PLC-?1 that includes a partial pleckstrin homology (PH) domain-a consensus lipid-binding and protein-binding sequence. We have developed a gestalt algorithm to detect hitherto‘invisible’PH and PH-like domains, and now report that the partial PH domain of PLC-?1 interacts with a complementary partial PH-like domain in TRPC3 to elicit lipid binding and cell-surface expression of TRPC3. Our findings imply a far greater abundance of PH domains than previously appreciated, and suggest that intermolecular PH-like domains represent a widespread signalling mode. [ABSTRACT FROM AUTHOR]

The inositol 1,4,5 trisphosphate (IP3 ) receptor (IP3 R) is a Ca 2+ release channel that responds to the second messenger IP3 . Exquisite modulation of intracellular Ca 2+ release via IP3 Rs is achieved by the ability of IP3 R to integrate signals from numerous small molecules and proteins including nucleotides, kinases, and phosphatases, as well as nonenzyme proteins. Because the ion conduction pore composes only ∼5% of the IP3 R, the great bulk of this large protein contains recognition sites for these substances. Through these regulatory mechanisms, IP3 R modulates diverse cellular functions, which include, but are not limited to, contraction/ excitation, secretion, gene expression, and cellular growth. We review the unique properties of the IP3 R that facilitate cell-type and stimulus-dependent control of function, with special emphasis on protein-binding partners. [ABSTRACT FROM AUTHOR]

RACK1 is not a G protein but closely resembles the heterotrimeric Gβ-subunit. RACK1 serves as a scaffold, linking protein kinase C to its substrates. We demonstrate that RACK1 physiologically binds inositol 1,4,5-trisphosphate receptors and regulates Ca2+ release by enhancing inositol 1,4,5-trisphosphate receptor binding affinity for inositol 1,4,5-trisphosphate. Overexpression of RACK1 or depletion of RACK1 by interference RNA markedly augments or diminishes Ca2+ release, respectively, without affecting Ca2+ entry. These findings establish RACK1 as a physiologic mediator of agonist-induced Ca2+ release. [ABSTRACT FROM AUTHOR]

It has been considered that Ca2+ release is the causal trigger for Ca2+ entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (IP3R), the lack of Ca2+ entry in response to receptor activation is attributed to the absence of Ca2+ release. We reveal in this article that IP3R recognition of IP3 determines agonist-induced Ca2+ entry (ACE), independent of its Ca2+ release activity. In DT40 IP3R-/- cells, endogenous ACE can be rescued with type 1 IP3R mutants (both a ΔC-terminal truncation mutant and a D2550A pore mutant), which are defective in Ca2+ release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an IP3R-dependent manner without Ca2+ store release. Conversely, ACE cannot be rescued with mutant IP3Rs lacking IP3 binding (both the Δ90-110 and R265Q IP3-binding site mutants). We conclude that an IP3-dependent conformational change in the IP3R, not endoplasmic reticulum Ca2+ pool release, triggers ACE. [ABSTRACT FROM AUTHOR]

We report here that PLC-γ isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-γ1 or a lipase-inactive mutant PLC-γ1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-γ1 was ineffective. RNA interference to deplete either PLC-γ1 or PLC-γ2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-γ2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-γ2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-γ2 or a lipase-inactive PLC-γ2 mutant. The results indicate a lipase-independent role of PLC-γ in the physiological agonist-induced activation of Ca2+ entry. [Copyright &y& Elsevier]

Examines the coupling mechanism of endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) store-operated Ca2+ (SOC) entry channels. Role of Ca2+ signals in cellular functions; Modification of actin cytoskeleton; Trafficking model involving ER-PM interaction; Secretion-based coupling model for SOC activation.

NADH regulates the release of calcium from the endoplasmic reticulum by modulation of inositol 1,4,5-trisphosphate receptors (IP3R), accounting for the augmented calcium release of hypoxic cells. We report selective binding of IP3R to GAPDH, whose activity leads to the local generation of NADH to regulate intracellular calcium signaling. This interaction requires cysteines 992 and 995 of IP3R and C150 of GAPDH. Addition of native GAPDH and NAD+ to WT IP3R stimulates calcium release, whereas no stimulation occurs with C992S/995S IP3R that cannot bind GAPDH. Thus, the IP3R/ GAPDH interaction likely enables cellular energy dynamics to impact calcium signaling. [ABSTRACT FROM AUTHOR]

TFII-I is a transcription factor and a target of phosphorylation by Bruton's tyrosine kinase. In humans, deletions spanning the TFII-I locus are associated with a cognitive defect, the Williams-Beuren cognitive profile. We report an unanticipated role of TFII-I outside the nucleus as a negative regulator of agonist-induced calcium entry (ACE) that suppresses surface accumulation of TRPC3 (transient receptor potential C3) channels. Inhibition of ACE by TFII-I requires phosphotyrosine residues that engage the 5H2 (Src-homology 2) domains of phospholipase C-γ (PLC-γ) and an interrupted, pleckstrin homology (PH)-like domain that binds the split PH domain of PLC-γ. Our observations suggest a model in which TFII-I suppresses ACE by competing with TRPC3 for binding to PLC-γ. [ABSTRACT FROM AUTHOR]

Apoptotic stimuli augment intracellular calcium concentration through inositol 1,4,5-trisphosphate receptors (IP3R) on endoplasmic reticulum calcium stores. We previously discovered an apoptotic cascade wherein cytochrome c binds to IP3R early in apoptosis, resulting in dysregulated calcium release Here we show that cytochrome c binding to IP3R depends on a cluster of glutamic acid residues within the C terminus of the channel. A cell permeant peptide derived from this sequence displaces cytochrome c from IP3R and abrogates cell death induced by staurosporine treatment of HeLa cells and Fas ligand stimulation of Jurkat cells. Small-molecule inhibitors of cytochrome c/IP3R interactions may prove useful in treating disorders associated with inappropriate intrinsic and extrinsic apoptotic signaling. [ABSTRACT FROM AUTHOR]

The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. [ABSTRACT FROM AUTHOR]

Background: The recA/RAD51 gene family encodes a diverse set of recombinase proteins that affect homologous recombination, DNA-repair, and genome stability. The recA gene family is expressed across all three domains of life - Eubacteria, Archaea, and Eukaryotes - and even in some viruses. To date, efforts to resolve the deep evolutionary origins of this ancient protein family have been hindered by the high sequence divergence between paralogous groups (i.e. ~30% average pairwise identity). Results: Through large taxon sampling and the use of a phylogenetic algorithm designed for inferring evolutionary events in highly divergent paralogs, we obtained a robust, parsimonious and more refined phylogenetic history of the recA/RAD51 superfamily. Conclusions: In summary, our model for the evolution of recA/RAD51 family provides a better understanding of the ancient origin of recA proteins and the multiple events that lead to the diversification of recA homologs in eukaryotes, including the discovery of additional RAD51 sub-families. [ABSTRACT FROM AUTHOR]

:One of the goals in neuroscience is to obtain tractable laboratory cultures that closely recapitulate in vivo systems while still providing ease of use in the lab. Because neurons can exist in the body over a lifetime, long-term culture systems are necessary so as to closely mimic the physiological conditions under laboratory culture conditions. Ideally, such a neuronal organoid culture would contain multiple cell types, be highly differentiated, and have a high density of interconnected cells. However, before these types of cultures can be created, certain problems associated with long-term neuronal culturing must be addressed. We sought to develop a new protocol which may further prolong the duration and integrity of E18 rat hippocampal cultures. We have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. [ABSTRACT FROM AUTHOR]

Both multiple sequence alignment and phylogenetic analysis are problematic in the "twilight zone" of sequence similarity (≤25% amino acid identity). Herein we explore the accuracy of phylogenetic inference at extreme sequence divergence using a variety of simulated data sets. We evaluate four leading multiple sequence alignment (MSA) methods (MAFFT, TCOFFEE, CLUSTAL, and MUSCLE) and six commonly used programs of tree estimation (Distance-based: Neighbor-Joining; Character-based: PhyML, RAxML, GARLI, Maximum Parsimony, and Bayesian) against a novel MSA-independent method (PHYRN) described here. Strikingly, at "midnight zone" genetic distances (∼7% pairwise identity and 4.0 gaps per position), PHYRN returns high-resolution phylogenies that outperform traditional approaches. We reason this is due to PHRYN's capability to amplify informative positions, even at the most extreme levels of sequence divergence. We also assess the applicability of the PHYRN algorithm for inferring deep evolutionary relationships in the divergent DANGER protein superfamily, for which PHYRN infers a more robust tree compared to MSA-based approaches. Taken together, these results demonstrate that PHYRN represents a powerful mechanism for mapping uncharted frontiers in highly divergent protein sequence data sets. [ABSTRACT FROM AUTHOR]

Accurately assigning folds for divergent protein sequences is a major obstacle to structural studies. Herein, we outline an effective method for fold recognition using sets of PSSMs, each of which is constructed for different protein folds. Our analyses demonstrate that FSL (Fold-specific Position Specific Scoring Matrix Libraries) can predict/relate structures given only their amino acid sequences of highly divergent proteins. This ability to detect distant relationships is dependent on low-identity sequence alignments obtained from FSL. Results from our experiments demonstrate that FSL perform well in recognizing folds from the "twilight-zone" SABmark dataset. Further, this method is capable of accurate fold prediction in newly determined structures. We suggest that by building complete PSSM libraries for all unique folds within the Protein Database (PDB), FSL can be used to rapidly and reliably annotate a large subset of protein folds at proteomic level. The related programs and fold-specific PSSMs for our FSL are publicly available at: http://ccp.psu.edu/download/FSLv1.0/. [ABSTRACT FROM AUTHOR]

Death-associated protein kinase (DAPK) is a key player in multiple cell death signaling pathways. We report that DAPK is regulated by DANGER, a partial MAB-21 domain-containing protein.DANGERbinds directly toDAPKand inhibitsDAPKcatalytic activity. DANGERdeficient mouse embryonic fibroblasts and neurons exhibit greater DAPK activity and increased sensitivity to cell death stimuli than do wild-type control cells. In addition, DANGER-deficient mice manifest more severe brain damage after acute excitotoxicity and transient cerebral ischemia than do control mice. Accordingly, DANGER may physiologically regulate the viability of neurons and represent a potential therapeutic target for stroke and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Multiple studies suggest that phospholipase C-γ (PLC-7) contributes to regulation of sodium/hydrogen exchanger 3 (NHE3) in the small intestine, although the mechanism(s) for this regulation remain unknown. We demonstrate here that PLC-γ binds directly to the C terminus of NHE3 and exists in similar sized multiprotein complexes as NHE3. This binding is dynamic and decreases with elevated [Ca2+]i. The PLC-γ-binding site in NHE3 was identified (amino acids 586-605) and shown to be a critical regulatory domain for protein complex formation, because when it is mutated, NHE3 binding to PLC-γ as well as NHERF2 is lost. An inhibitory peptide, which binds to the Src homology 2 domains contained in PLC-γ without interrupting binding of PLC-γ to NHE3, was used to probe a non-lipase-dependent role of PLC-γ. In the presence of this peptide, carbachol-stimulated calcium inhibition of NHE3 was lost. These results mirror previous studies with the transient receptor potential channel and suggest that PLC-γ may play a common role in regulating the cell-surface expression of ion transporters. [ABSTRACT FROM AUTHOR]

We recently modeled transient receptor potential (TRP) channels using the Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST), which derives structural, functional, and evolutionary information from primary amino acid sequences using phylogenetic profiles (Ko, K. D., Hong, Y., Chang, G. S., Bhardwaj, G., van Rossum, D. B., and Patterson, R. L. (2008) Physics Arch. Quant. Methods arXiv:0806.2394v1). Herein we test our functional predictions for the TRP_2 domain of TRPC3; a domain of unknown function that is conserved in all TRPC channels. Our functional models of this domain identify both lipid binding and trafficking activities. In this study, we reveal: (i) a novel structural determinant of ion channel sensitivity to lipids, (ii) a molecular mechanism for the difference between diacylglycerol (DAG)-sensitive and DAG-insensitive TRPC subfamilies, and (iii) evidence that TRPC3 can comprise part of the vesicle fusion machinery. Indeed, the TRPC3 TRP_2 domain mediates channel trafficking to the plasma membrane and binds to plasma membrane lipids. Further, mutations in TRP_2, which alter lipid binding, also disrupt the DAG-mediated fusion of TRPC3-containing vesicles with the plasma membrane without disrupting SNARE interactions. Importantly, these data agree with the known role of DAG in membrane destabilization, which facilitates SNARE-dependent synaptic vesicle fusion (Villar, A. V., Goni, F. M., and Alonso, A. (2001) FEBS Lett. 494, 117-120 and Goni, F. M., and Alonso, A. (1999) Prog. Lipid Res. 38, 1-48). Taken together, functional models generated by GDDA-BLAST provide a computational platform for deriving domain functionality, which can have in vivo and mechanistic relevance. [ABSTRACT FROM AUTHOR]

Inferring evolutionary relationships among highly divergent protein sequences is a daunting task. In particular, when pairwise sequence alignments between protein sequences fall <25% identity, the phylogenetic relationships among sequences cannot be estimated with statistical certainty. Here, we show that phylogenetic profiles generated with the Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST) are capable of deriving, ab initio, phylogenetic relationships for highly divergent proteins in a quantifiable and robust manner. Notably, the results from our computational case study of the highly divergent family of retroelements accord with previous estimates of their evolutionary relationships. Taken together, these data demonstrate that GDDA-BLAST provides an independent and powerful measure of evolutionary relationships that does not rely on potentially subjective sequence alignment. We demonstrate that evolutionary relationships can be measured with phylogenetic profiles, and therefore propose that these measurements can provide key insights into relationships among distantly related and/or rapidly evolving proteins. [ABSTRACT FROM AUTHOR]

Heat-shock proteins (HSP5) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. lnositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate 1P7 [diphosphoinositol pentakisphosphate (5-PP-IP5)]. mediates apoptosis. Increased 1P6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds 1P6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90-1P6K2 binding elicit activation of 1P6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy. [ABSTRACT FROM AUTHOR]

The impact of calcium signalling on so many areas of cell biology reflects the crucial role of calcium signals in the control of diverse cellular functions. Despite the precision with which spatial and temporal details of calcium signals have been resolved, a fundamental aspect of the generation of calcium signals — the activation of 'store-operated channels' (SOCs) — remains a molecular and mechanistic mystery. Here we review new insights into the exchange of signals between the endoplasmic reticulum (ER) and plasma membrane that result in activation of calcium entry channels mediating crucial long-term calcium signals. [ABSTRACT FROM AUTHOR]

Receptor-coupled [Ca2+]i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca2+-signaling proteins. However, localization of Ca2+ channels that are regulated by such Ca2+-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca2+ uptake. Endogenously and exogenously expressed TRPC3 was localized apically in polarized Madin­Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 was localized basolaterally, whereas TRPC6 was detected in both locations. Localization of Gαq/11, inositol 1,4,5-trisphosphate receptor-3, and phospholipase Cβ1 and -β2 was also predominantly apical. TRPC3 co-immunoprecipitated with endogenous TRPC6, phospholipase Cβs, Gαq/11, inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical 45Ca2+ uptake was higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical 45Ca2+ uptake and transepithelial 45Ca2+ flux were also higher in TRPC3-expressing cells. Consistent with this, OAG induced [Ca2+]i increase in the apical, but not basal, region of TRPC3-MDCK cells that was blocked by EGTA addition to the apical medium. Most importantly, (i) TRPC3 was detected in the apical region of rat submandibular gland ducts, whereas TRPC6 was present in apical as well as basolateral regions of ducts and acini; and (ii) OAG stimulated Ca2+ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/RPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca2+] and secretion. [ABSTRACT FROM AUTHOR]

Abstract: Ca2+ signals in response to receptors mediate and control countless cellular functions ranging from short-term responses such as secretion and contraction to longer-term regulation of growth, cell division and apoptosis. The spatial and temporal details of Ca2+ signals have been resolved with great precision in many cells. Ca2+ signals activated by phospholipase C-coupled receptors have two components: Ca2+ release from endoplasmic reticulum (ER) stores mediated by inositol 1,4,5-trisphosphate (InsP3) receptors, and Ca2+ entry from outside the cell. The latter remains largely a molecular and mechanistic mystery. The activation of “store-operated” Ca2+ channels is believed to account for the entry of Ca2+. However, debate now focuses on how much of a contribution emptying of stores plays to the activation of Ca2+ entry in response to physiological activation of receptors. Here we discuss recent information and ideas on the exchange of signals between the plasma membrane (PM) and ER that results in activation of Ca2+ entry channels following receptor stimulation and/or store emptying. [Copyright &y& Elsevier]

Phospholipase C-γ1 (PLC-γ1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. We demonstrate that PLC-γ1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-γ1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-γ1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF- dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-γ1, but knockdown of PLC-γ1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-γ1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-γl-dependent proliferation. [ABSTRACT FROM AUTHOR]

Mitochondrial cytochrome c release and inositol (1,4,5) trisphosphate receptor (InsP3R)-mediated calcium release from the endoplasmic reticulum mediate apoptosis in response to specific stimuli. Here we show that cytochrome c binds to the InsP3R during apoptosis. Addition of 1 nM cytochrome c blocks calcium-dependent inhibition of InsP3R function. Early in apoptosis, cytochrome c translocates to the endoplasmic reticulum where it selectively binds InsP3R, resulting in sustained, oscillatory cytosolic calcium increases. These calcium events are linked to the coordinate release of cytochrome c from all mitochondria. Our findings identify a feed-forward mechanism whereby early cytochrome c release increases InsP3R function, resulting in augmented cytochrome c release that amplifies the apoptotic signal. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle. [ABSTRACT FROM AUTHOR]