Triphosphate Incorporation Research Articles

SOX2 Modulates Reprogramming of Gene Expression in Two-Cell Mouse Embryos1

Sox2 is a key gene that controls transcriptional networks required for pluripotency. The role of Sox2 in the developmental transition of a highly differentiated oocyte to totipotent blastomeres of the early preimplantation embryo, however, is not known. We report that Sox2, which is localized in the nucleus, is first zygotically expressed during the 2-cell stage and that its expression dramatically increases between the morula and blastocyst stages. Injecting a cRNA encoding Sox2 into 1-cell embryos resulted in overexpression of SOX2 by approximately 70% and developmental arrest at the 2-cell stage, whereas injecting cRNAs encoding Pou5f1, Myc (also known as c-Myc), or Klf4 has little effect on the ability of 2-cell embryos to cleave to the 4-cell stage. Global transcription assessed by bromo uridine triphosphate incorporation is reduced by approximately 15%, and transcript profiling revealed that approximately 15% of zygotically expressed genes are dramatically repressed in 2-cell embryos overexpressing SOX2. Furthermore, overexpressing a dominant-negative SOX2 perturbs reprogramming of gene expression in 2-cell embryos, though to a much lesser extent than that observed following overexpression of SOX2, and leads to developmental failure after the 2-cell stage but before the 8-cell stage. Results of these experiments implicate Sox2 as a critical transcriptional regulator in the oocyte-to-embryo transition that entails formation of totipotent blastomeres and indicate that the amount of Sox2 is critical for successful execution of this transition.

Comment on “Deoxyuridine Triphosphate Incorporation during Somatic Hypermutation of Mouse VkOx Genes after Immunization with Phenyloxazolone”

Recently, Roche et al. ([1][1]) reported locations of uracils in the L chain genes of mice undergoing hypermutations. To accomplish this, the authors used the USER kit from New England Biolabs (Ipswich, MA) to remove the uridine nucleoside, dephosphorylate the 3′ end of DNA, and ligate an anchor

Response to Comment on “Deoxyuridine Triphosphate Incorporation during Somatic Hypermutation of Mouse VkOx Genes after Immunization with Phenyloxazolone”

We haven taken into consideration A.S. Bhagwat's comment concerning the mechanism of β-elimination by the endonuclease VIII (Nei) enzyme. Our conclusion that deoxyuridine triphosphate incorporation happens during somatic hypermutation of murine VkOx genes relies on the following results: the

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

Deoxyuridine Triphosphate Incorporation during Somatic Hypermutation of Mouse VkOx Genes after Immunization with Phenyloxazolone

Somatic hypermutation (SHM) of Ig genes is the result of a two-phase process initiated by activation-induced cytidine deaminase, relying on two different strategies for the introduction of mutations at CG pairs (phase I) and at AT pairs (phase II). To explain the selectivity of phase II, two mechanisms were proposed: AT-selective error-prone DNA-polymerases, deoxyuridine triphosphate (dUTP) incorporation, or both. However, there has been no experimental evidence so far of the possible involvement of the latter. We have developed a ligation-anchored PCR method based on the formation of single-strand breaks at uracils. In this study, we show the presence of uracil in hypermutating VkOx genes in wild type (AID(+/+)) mice, demonstrating that dUTP incorporation via DNA polymerases could be a major mechanism in SHM. Thus, error-prone DNA polymerases would participate in SHM via low-fidelity replication and incorporation of dUTP.

641 DEVELOPMENT OF IN VITRO AND CELL-BASED ASSAYS FOR ASSESSING NUCLEOTIDE BIOSYNTHESIS INHIBITION

[1] D.G. Streeter, J. T. Witkowski, G.P. Khare, R.W. Sidwell,r;j. Bauer, R.K. Robins, L. N. Simon (1973) Mechanism of Action of 1-i3-DRibofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole), A New Broad-Spectrum Antiviral Agent Proc. Nat. Acad. Sci. USA Vol. 70, No. 4, pp. 1174-1178 [2] R. C. Willis, D. A. Carson, and J. E. Seegmiller (1973) Adenosine Kinase Initiates the Major Route of Ribavirin Activation in a Cultured Human Cell Line. PNAS USA, 75: 3042-3044. [3] Wu JZ, Larson G, Walker H, Shim JH, Hong Z. Phosphorylation of ribavirin and viramidine by adenosine kinase and cytosolic 5'nucleotidase II: Implications for ribavirin metabolism in erythrocytes. (2005) Antimicrob Agents Chemother. 49(6):2164-71 References Background Numerous nucleoside analogues (NAs) are currently used to treat viral hepatitis. They are usually designed to inhibit one viral target. This remains in contrast with the observation that ribavirin (1-β-D-ribofuranosyl1,2,4-triazole-3-carboxamide), a purine nucleoside analogue currently used as a part of bi-therapy of hepatitis C infection, has multiple modes of action: (i) depletion of intracellular GTP pools by inhibition of the cellular IMPDH, (ii) inhibition of viral polymerase activity by the 5’-triphosphate metabolite of ribavirin, (iii) induction of error catastrophe as a result of accumulation of mutations in the viral genome. Even if no direct relationship between ribavirin antiviral action and IMPDH inhibition has been demonstrated, the depletion of cellular GTP should result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to lower intracellular concentration of its natural competitor.

642 CHARACTERISATION OF HEPATITIS C VIRUS ENTRY AND RNA REPLICATION IN CELLS OF CNS ORIGIN

(ii) inhibition of viral polymerase activity by the 5’-triphosphate metabolite of ribavirin, (iii) induction of error catastrophe as a result of accumulation of mutations in the viral genome. Even if no direct relationship between ribavirin antiviral action and IMPDH inhibition has been demonstrated, the depletion of cellular GTP should result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to lower intracellular concentration of its natural competitor. Aims: Relation between therapeutic potential of a nucleoside analogue and its anti-metabolite action remains difficult to demonstrate mainly because of the lack of investigation tools. The purpose of this study was to develop a range of assays to reveal nucleotide biosynthesis inhibition. Results: For a rapid evaluation of new nucleoside analogues as IMPDH inhibitors, we have developed an in vitro enzymatic assay where the synthesis of monophosphorylated form of nucleoside analogue (NA-MP) is provided by cloned human nucleoside kinases, and NA-MP is immediately tested for inhibition of human recombinant IMPDH. This assay has been validated with nucleoside analogues ribavirin and mizoribine. We have also developed original cell-based analytical approach in which 27 cellular riboand deoxyribonucleotides are extracted from cultured cells, separated by ion-pairing chromatography and quantified. This cellular assay, validated with several NA (ribavirin, aracytidine, gemcitabine) and known anti-metabolites (mycophenolic acid, leflunomide, hydroxyurea), provides a powerful tool for studying the effect of new nucleoside analogues on whole spectra of cellular purine and pyrimidine deoxyand ribonucleotides. In addition, in regards with new antiviral molecules identified in HCV cell culture systems, our cellular assay allows to distinguish the molecules that directly acts on the viral proteins from others that inhibits the cell nucleotide biosynthesis.

Evaluation of Cell Surface Expression of Phosphatidylserine in Ovarian Carcinoma Effusions Using the Annexin-V/7-AAD Assay

Phosphatidylserine cell surface exposure during apoptosis can be detected by its binding to the protein annexin-V. We investigated annexin-V expression in 76 ovarian carcinoma effusions using flow cytometry. Results were analyzed for association with clinicopathologic parameters and survival. Annexin-V expression was additionally compared with the previously studied apoptotic markers cleaved caspase-3, cleaved caspase-8, and deoxyuridine triphosphate (dUTP) incorporation into DNA fragments. Annexin-V was expressed in all specimens and was more frequently detected compared with cleaved caspases and dUTP incorporation (P < .001). Annexin-V expression was higher in grade 3 vs grades 1 and 2 tumors (P = .014). A higher percentage of annexin-V-expressing cells in postchemotherapy specimens was associated with poor overall (P = .005) and progression-free (P = .013) survival. We present the first evidence of annexin-V expression in ovarian carcinoma effusions. The higher annexin-V expression compared with other apoptosis parameters and its association with high-grade disease and poor survival in postchemotherapy patients suggest a role in cell survival rather than apoptosis in effusions.

Cleaved caspase-3 and nuclear factor- κB p65 are prognostic factors in metastatic serous ovarian carcinoma

Tumor progression and treatment failure in ovarian carcinoma are frequently associated with metastasis to effusions. The present study analyzed the expression and clinical role of nuclear factor-kappaB p65, nuclear factor-kappaB inhibitor alpha, and parameters of apoptosis in serous carcinoma. Cleaved caspase-3 and caspase-8 levels and deoxyuridine triphosphate incorporation were measured in 65 effusions using flow cytometry. Effusions (n = 209) and corresponding primary carcinomas and solid metastases (n = 114) were immunohistochemically analyzed for nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expression. Effusions (n = 75) were further analyzed for nuclear factor-kappaB phospho-p65 (Ser536) levels using immunoblotting. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. Caspase cleavage and deoxyuridine triphosphate incorporation were limited to less than 10% of cells in most effusions. Nuclear factor-kappaB p65 expression was frequently detected at all anatomic sites, with less frequent cytoplasmic nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expressions. Immunoblotting showed nuclear factor-kappaB p65 phosphorylation in 72 (96%) of 75 effusions. Higher than median cleaved caspase-3 levels correlated with improved overall and progression-free survival in univariate analysis of all patients (P = .024 and P = .046, respectively) and of those with postchemotherapy effusions (P = .042 and P = .036, respectively). Cleaved caspase-3 expression was an independent predictor of longer progression-free survival for patients with postchemotherapy effusions (P = .029). Nuclear factor-kappaB p65 expression correlated with poor progression-free survival for all patients (P = .048) and for those with postchemotherapy effusions (P = .025). Ovarian carcinoma cells in effusions undergo little apoptosis, but high levels of cleaved caspase-3 are associated with improved survival. Nuclear factor-kappaB p65 is frequently expressed in advanced-stage serous ovarian carcinoma, and its nuclear localization is associated with poor progression-free survival.

Genistein inhibits proliferation and functions of hypertrophic scar fibroblasts

Hypertrophic scarring is abnormal proliferation of dermal fibroblasts and excessive deposition of extracellular matrix. To date, despite many studies, treatments have not been satisfactory. Genistein, a potent, specific inhibitor of tyrosine protein kinases (TPKs), has been proved to inhibit many kinds of tumour and some fibrotic diseases. The purpose of this study was to investigate the effects of genistein on the proliferation and functions of hypertrophic scar fibroblasts (HSFBs) and the mechanism by which genistein inhibits TPK signal transduction. The first effect was observed by methyl-thiazol-diphenyl-tetrazolium assay and the second by [γ- 32p] adenosine triphosphate incorporation assay. The results demonstrated that genistein inhibits the proliferation and function of HSFBs and changes the TPK signal transduction pathway, which can provide an experimental basis for treating HS with genistein.

Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

The Histone Acetyltransferase PCAF Associates with Actin and hnRNP U for RNA Polymerase II Transcription

Actin is a key regulator of RNA polymerase (pol) II transcription. In complex with specific hnRNPs, it has been proposed that actin functions to recruit pol II coactivators during the elongation of nascent transcripts. Here, we show by affinity chromatography, protein-protein interaction assays, and biochemical fractionation of nuclear extracts that the histone acetyltransferase (HAT) PCAF associates with actin and hnRNP U. PCAF and the nuclear actin-associated HAT activity detected in the DNase I-bound protein fraction could be released by disruption of the actin-hnRNP U complex. In addition, actin, hnRNP U, and PCAF were found to be associated with the Ser2/5- and Ser2-phosphorylated pol II carboxy-terminal domain construct. Chromatin and RNA immunoprecipitation assays demonstrated that actin, hnRNP U, and PCAF are present at the promoters and coding regions of constitutively expressed pol II genes and that they are associated with ribonucleoprotein complexes. Finally, disruption of the actin-hnRNP U interaction repressed bromouridine triphosphate incorporation in living cells, suggesting that actin and hnRNP U cooperate with PCAF in the regulation of pol II transcription elongation.

Melatonin prevents oxidized low‐density lipoprotein‐induced increase of myosin light chain kinase activation and expression in HUVEC through ERK/MAPK signal transduction

Melatonin, the main secretary product of the pineal gland, is potentially effective in the prevention of a number of diseases in which free radical processes are involved. The development of hypercholesterolemia is a multifactorial process in which elevated oxidized low-density lipoprotein (ox-LDL) levels play a central role. The purpose of this study was to test whether melatonin prevents ox-LDL-induced increase of myosin light chain kinase (MLCK) activation and expression in human umbilical vein endothelial cells (HUVECs) through extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signal transduction. HUVEC were cultured in vitro and treated with ox-LDL, melatonin, and PD98059 (a selective inhibitor of ERK), respectively. The expression, transcription, and activity of MLCK were measured by western blot, immunohistochemistry, reverse transcription-polymerase chain reaction and gamma-(32)P-adenosine triphosphate (ATP) incorporation, respectively. The results showed that the expression and activity of MLCK were increased in ox-LDL-treated HUVECs and this was decreased by melatonin and PD98059. The expression and activity of MLCK induced by ox-LDL was associated with the phosphorylation of ERK. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and the activity which can be reduced by melatonin through ERK/MAPK signal transduction.

Structural Motifs of Importance for the Constitutive Activity of the Orphan 7TM Receptor EBI2: Analysis of Receptor Activation in the Absence of an Agonist

The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through Galphai, as shown, for instance by guanosine 5'-O-(3-thio)triphosphate incorporation. Two regions of importance for the constitutive activity were identified by a systematic mutational analysis of 29 residues in EBI2. The cAMP response element-binding protein transcription factor was used as a measure of receptor activity and was correlated to the receptor surface expression. PheVI:13 (Phe257), and the neighboring CysVI:12 (Cys256), in the conserved CW/FxP motif in TM 6, acted as negative regulators as Ala substitutions at these positions increased the constitutive activity 5.7- and 2.3-fold, respectively, compared with EBI2 wild type (wt). In contrast, ArgII:20 (Arg87) in TM-2 acted as a positive regulator, as substitution to Ala, but not to Lys, decreased the constitutive activity more than 7-fold compared with wt EBI2. IleIII:03 (Ile106) is located only 4 A from ArgII:20, and a favorable electrostatic interaction with ArgII:20 was created by introduction of Glu in III:03, given that the activity increased to 4.4-fold of that wt EBI2. It is noteworthy that swapping these charges by introduction of Glu in II:20 and Arg in III:03 resulted in a 2.7-fold increase compared with wt EBI2, thereby rescuing the two signaling-deficient single mutations, which exhibited a 3.8- to 4.5-fold decrease in constitutive activity. The uncovering of these molecular mechanisms for EBI2 activation is important from a drug development point of view, in that it may facilitate the rational design and development of small-molecule inverse agonists against EBI2 of putative importance as antiviral- or immune modulatory therapy.

Deoxynucleoside Triphosphate Incorporation Mechanism of Foamy Virus (FV) Reverse Transcriptase: Implications for Cell Tropism of FV

Here, we investigated the pre-steady-state deoxynucleoside triphosphate (dNTP) incorporation kinetics of primate foamy virus (PFV) reverse transcriptase (RT) in comparison with those of HIV-1 and MuLV RTs. PFV RT displayed a drastic reduction in primer extension at low dNTP concentrations where HIV-1 RT remains highly active, indicating a low dNTP binding affinity in the case of PFV RT. Indeed, kinetic analysis showed that, as observed with MuLV RT, PFV RT exhibits approximately 10 to 80 times lower dNTP binding affinity than HIV-1 RT. These three RTs, however, show similar catalytic activities. In conclusion, PFV RT displays mechanistic distinctions in comparison to HIV-1 RT and shares close similarity to MuLV RT.

Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry

We describe here a protocol for the detection of epithelial cells in effusions combined with quantification of apoptosis by flow cytometry (FCM). The procedure described consists of the following stages: culturing and induction of apoptosis by staurosporine in control ovarian carcinoma cell lines (SKOV-3 and OVCAR-8); preparation of effusion specimens and cell lines for staining; staining of cancer cells in effusions and cell lines for cell surface markers (Ber-EP4, EpCAM and CD45) and intracellular/nuclear markers of apoptosis (cleaved caspase-3 and caspase-8, and incorporated deoxyuridine triphosphates); and FCM analysis of stained cell lines and effusions. This protocol identifies a specific cell population in cytologically heterogeneous clinical specimens and applies two methods to measure different aspects of apoptosis in the cell population of interest. The cleaved caspase and deoxyuridine triphosphate incorporation FCM assays are run in parallel and require (including sample preparation, staining, instrument adjustment and data acquisition) 8 h. The culturing of cell lines requires 2-3 days and induction of apoptosis requires 16 h.

Sphingosine kinase 1 gene transfer reduces postoperative peritoneal adhesion in an experimental model

Recovery of the surgically damaged mesothelial cell layer is a major process in reducing postoperative peritoneal adhesions. Sphingosine kinase (SPK) 1 is a signalling molecule involved in the regulation of proliferation and migration of various cell types. This study determined the effect of SPK-1 gene transfer on the recovery of damaged mesothelial cells and on peritoneal adhesion formation after surgery. Rat mesothelial cells were isolated and characterized by their expression of cytokeratin and vimentin. Their migration was determined by scratch wound motility assay. Cellular SPK-1 activity was measured by [gamma-32P]adenosine 5'-triphosphate incorporation. Wistar rats underwent laparotomy with subsequent caecum or uterine horn abrasion. Rats were randomized to either SPK-1 gene (Ad-SPK-1) transfer or control groups. The animals were killed 14 days after operation and peritoneal adhesions were graded. Adenovirus-mediated SPK-1 gene transfer increased the cellular SPK-1 activity of mesothelial cells, leading to enhanced migration. Median adhesion scores were significantly lower in the Ad-SPK-1 group than in controls in both rat caecum (0.98 versus 2.60; P < 0.001) and rat uterine horn (0.28 versus 1.83; P < 0.001) models. Adenovirus-mediated SPK-1 gene transfer promotes recovery of the surgically damaged mesothelial cell layer and prevents postoperative peritoneal adhesion formation.

Three-Dimensional Analysis of a Viral RNA Replication Complex Reveals a Virus-Induced Mini-Organelle

Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)–infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of ∼10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of ∼100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

Molecular mechanisms of bidirectional antagonism between K65R and thymidine analog mutations in HIV-1 reverse transcriptase

The K65R mutation in HIV-1 reverse transcriptase (RT) decreases susceptibility to all approved nucleoside reverse transcriptase inhibitors (NRTI) except zidovudine by selectively decreasing the incorporation of the NRTI triphosphate compared with the natural deoxyribonucleotide triphosphate substrate. Thymidine analog mutations (TAMs) confer high-level resistance to zidovudine and cross-resistance to other NRTI by increasing excision of the chain-terminating NRTI monophosphate via a phosphorolytic cleavage reaction. Recent virology and genetic studies have shown bidirectional antagonism between K65R and TAMs. The aim of this study was to elucidate the biochemical and structural mechanisms responsible for this antagonism. Steady-state and pre-steady-state kinetic analyses of NRTI triphosphate incorporation and NRTI monophosphate excision by RT containing K65R or TAMs were conducted and complemented by molecular modeling. The addition of K65R to two clinically relevant combinations of TAMs (M41L/L210W/T215Y or D67N/K70R/T215F/K219Q) significantly reduced the recombinant enzymes' ability to excise all chain-terminating NRTI monophosphate. Transient kinetic analyses showed that TAMs decreased the extent to which RT containing K65R could discriminate against D-nucleotide analogs, but not L-nucleotide analogs, by partly restoring the maximum rate of NRTI triphosphate incorporation. In addition, the TAMs combination D67N/K70R/T215F/K219Q decreased susceptibility to the L-nucleotide lamivudine by a discrimination mechanism, whereas the M41L/L210W/T215Y combination had little effect on susceptibility to lamivudine. K65R antagonizes the NRTI monophosphate excision activity of RT containing TAMs. TAMs antagonize the ability of K65R RT to discriminate against the nucleotide analog. Therapies including NRTI that select for both TAMs and K65R may prolong treatment response through the mutually antagonistic interactions between these resistance mutations.

Inhibition of Gαi2 Activation by Gαi3 in CXCR3-mediated Signaling

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the G alpha(i/o) protein family.