Intracellular Half-life Research Articles

APOBEC3G complexes decrease human immunodeficiency virus type 1 production.

APOBEC3G (A3G) is packaged into human immunodeficiency virus type 1 (HIV-1) virions unless HIV-1 virion infectivity factor (Vif) counteracts it. Virion A3G restricts HIV-1 reverse transcription and integration in target cells. Some A3G in producer cells colocalizes with specific cytoplasmic structures, in what are called "A3G complexes" here. Functional effects of producer cell A3G complexes on HIV-1 replication were studied. HeLa cells were cotransfected with HIV-1 constructs producing pseudoviruses, as well as either wild-type (WT) A3G or a mutant A3G (C97A, Y124A, W127A, or D128K A3G). Pseudovirus particle production was decreased from cells expressing any of the A3Gs that formed complexes by 24 h after transfection, relative to cells with C97A A3G that did not form detectable A3G complexes by 24 h or A3G-negative cells. The intracellular HIV-1 Gag half-life was shorter in cells containing A3G complexes than in those lacking complexes. HIV-1 virion output was decreased in a single round of replication from a T cell line containing A3G complexes (CEM cells) after infection with Vif-negative HIV-1, compared to Vif-positive HIV-1 that depleted A3G. Levels of production of Vif-negative and Vif-positive virus were similar from cells not containing A3G (CEM-SS cells). Knockdown of the mRNA processing body (P-body) component RCK/p54, eliminated A3G complex formation, and increased HIV-1 production. We conclude that endogenous A3G complexes in producer cells decrease HIV-1 production if not degraded by Vif.

Pharmacokinetic Modeling of Plasma and Intracellular Concentrations of Raltegravir in Healthy Volunteers

Raltegravir is a potent inhibitor of HIV integrase. Persistently high intracellular concentrations of raltegravir may explain sustained efficacy despite high pharmacokinetic variability. We performed a pharmacokinetic study of healthy volunteers. Paired blood samples for plasma and peripheral blood mononuclear cells (PBMCs) were collected predose and 4, 8, 12, 24, and 48 h after a single 400-mg dose of raltegravir. Samples of plasma only were collected more frequently. Raltegravir concentrations were determined using liquid chromatography-mass spectrometry. The lower limits of quantitation for plasma and PBMC lysate raltegravir were 2 nmol/liter and 0.225 nmol/liter, respectively. Noncompartmental analyses were performed using WinNonLin. Population pharmacokinetic analysis was performed using NONMEM. Six male subjects were included in the study; their median weight was 67.4 kg, and their median age was 33.5 years. The geometric mean (GM) (95% confidence interval shown in parentheses) maximum concentration of drug (C(max)), area under the concentration-time curve from 0 to 12 h (AUC(0-12)), and area under the concentration-time curve from 0 h to infinity (AUC(0-∞)) for raltegravir in plasma were 2,246 (1,175 to 4,294) nM, 10,776 (5,770 to 20,126) nM · h, and 13,119 (7,235 to 23,788) nM · h, respectively. The apparent plasma raltegravir half-life was 7.8 (5.5 to 11.3) h. GM intracellular raltegravir C(max), AUC(0-12), and AUC(0-∞) were 383 (114 to 1,281) nM, 2,073 (683 to 6,290) nM · h, and 2,435 (808 to 7,337) nM · h (95% confidence interval shown in parentheses). The apparent intracellular raltegravir half-life was 4.5 (3.3 to 6.0) h. Intracellular/plasma ratios were stable for each patient without significant time-related trends over 48 h. Population pharmacokinetic modeling yielded an intracellular-to-plasma partitioning ratio of 11.2% with a relative standard error of 35%. The results suggest that there is no intracellular accumulation or persistence of raltegravir in PBMCs.

Context‐dependent resistance to proteolysis of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.

Visualization of the nanospring dynamics of the IκBα ankyrin repeat domain in real time

IκBα is a crucial regulator of NFκB transcription. NFκB-mediated gene activation is robust because levels of free IκBα are kept extremely low by rapid, ubiquitin-independent degradation of newly synthesized IκBα. IκBα has a weakly folded ankyrin repeat 5-6 (AR5-6) region that is critical in establishing its short intracellular half-life. The AR5-6 region of IκBα folds upon binding to NFκB. The NFκB-bound IκBα has a long half-life and requires ubiquitin-targeted degradation. We present single molecule FRET evidence that the native state of IκBα transiently populates an intrinsically disordered state characterized by a more extended structure and fluctuations on the millisecond time scale. Binding to NFκB or introduction of stabilizing mutations in AR 6 suppressed the fluctuations, whereas higher temperature or small amounts of urea increased them. The results reveal that intrinsically disordered protein regions transition between collapsed and extended conformations under native conditions.

Hairy cell leukemia: epidemiology, pharmacokinetics of cladribine, and long-term follow-up of subcutaneous therapy

Hairy cell leukemia is often reported as a disease of young males. The male predominance is strong, 4:1, but the median age in the Swedish national compulsory cancer registry is similar to that of follicular lymphoma, i.e. 62 years. The overall 6-year survival in the Swedish registry of patients diagnosed since 2000 is 80%, 93% of patients <60 years, and 68% of those >60 years. The yearly risk of secondary cancers is 1.75%. Cladribine is a prodrug which is selectively activated intracellularly. The intracellular initial half-life is 13 h and the terminal half-life is 30 h. Subcutaneous injection once daily is simple and effective due to 100% bioavailability and no local side effects from injection, and self-administration is easy. Long-term follow-up of Scandinavian patients treated with cladribine (mostly as subcutaneous injections) in the early 1990s shows a >80% 15-year survival from cladribine treatment in <60 years of age, but <50% in older patients. Survival from diagnosis of these patients was similar for those previously treated and untreated.

Joint Population Pharmacokinetic Analysis of Zidovudine, Lamivudine, and Their Active Intracellular Metabolites in HIV Patients

The population pharmacokinetic parameters of zidovudine (AZT), lamivudine (3TC), and their active intracellular metabolites in 75 naïve HIV-infected patients receiving an oral combination of AZT and 3TC twice daily as part of their multitherapy treatment in the COPHAR2-ANRS 111 trial are described. Four blood samples per patient were taken after 2 weeks of treatment to measure drug concentrations at steady state. Plasma AZT and 3TC concentrations were measured in 73 patients, and among those, 62 patients had measurable intracellular AZT-TP and 3TC-TP concentrations. For each drug, a joint population pharmacokinetic model was developed and we investigated the influence of different covariates. We then studied correlations between the mean plasma and intracellular concentrations of each drug. A one-compartment model with first-order absorption and elimination best described the plasma AZT concentration, with an additional compartment for intracellular AZT-TP. A similar model but with zero-order absorption was found to adequately described concentrations of 3TC and its metabolite 3TC-TP. The half-lives of AZT and 3TC were 0.81 h (94.8%) and 2.97 h (39.2%), respectively, whereas the intracellular half-lives of AZT-TP and 3TC-TP were 10.73 h (69%) and 21.16 h (44%), respectively. We found particularly a gender effect on the apparent bioavailability of AZT, as well as on the mean plasma and intracellular concentrations of AZT, which were significantly higher in females than in males. Relationships between mean plasma drug and intracellular metabolite concentrations were also highlighted both for AZT and for 3TC. Simulation with the model of plasma and intracellular concentrations for once- versus twice-daily regimens suggested that a daily dosing regimen with double doses could be appropriate.

On the Origin of Urinary Angiotensin II

To the Editor: With great interest we read the publication by Shao et al,1 who propose that angiotensin (Ang) II type 1 (AT1) receptor stimulation in the kidney results in urinary Ang II excretion. The authors have used an elegant approach, infusing rats with Val5-Ang II to allow a clear distinction from endogenous Ile5-Ang II, here denoted as Ang II. Val5-Ang II is believed to have the same properties as Ang II. We have followed a similar approach and infused 125I-labeled Ang II in pigs to distinguish it from endogenous Ang II.2 In these studies, 125I-Ang II accumulation in the kidney, largely, …

Lack of Minority K65R-Resistant Viral Populations Detected After Repeated Treatment Interruptions of Tenofovir/Zidovudine and Lamivudine in a Resource-Limited Setting

Lack of Minority K65R-Resistant Viral Populations Detected After Repeated Treatment Interruptions of Tenofovir/Zidovudine and Lamivudine in a Resource-Limited Setting

Emerging mutations at virological failure of HAART combinations containing tenofovir and lamivudine or emtricitabine

To compare the emergence of drug-resistant HIV variants at failure of lamivudine (3TC)/tenofovir (TDF)-containing or emtricitabine (FTC)/TDF-containing HAART as a consequence of the different 3TC and FTC intracellular half-lives. Retrospective evaluation of 859 patients selected from an Italian HIV resistance database (Antiretroviral Resistance Cohort Analysis). Patients were selected for analysis if treated with a HAART whose nucleoside/nucleotide reverse transcriptase inhibitor backbone was either 3TC/TDF or FTC/TDF; if they experienced a virological failure after at least 6 months of plasma HIV-RNA undetectability; and if HIV genotypes before treatment and at failure were available. Univariate and multivariate logistic regression analyses were done to detect predictors of resistance mutations emerging at failure. Of 714 patients failing with 3TC/TDF and 145 with FTC/TDF, 35.8 and 21.1% were in Centers for Disease Control and Prevention stage C, and 8.8 and 15.2% were on first-line HAART, respectively. At multivariate analysis, the emergence of K70R (P = 0.002), M184V (P = 0.031), T215F (P = 0.020) and Y181C (P = 0.005) was significantly more common in 3TC-treated than in FTC-treated patients, with an odds ratio of 4, 1.56, 1.89 and 3.84, respectively. Despite their close structural similarity, 3TC and FTC are associated with a significantly different rate of drug resistance at treatment failure when combined with TDF in HAART regimens independently of the third drug used.

Low- and high-risk human papillomavirus E7 proteins regulate p130 differently

The E7 protein of high-risk human papillomaviruses (HR HPVs) targets pRb family members (pRb, p107 and p130) for degradation; low-risk (LR) HPV E7 only targets p130 for degradation. The effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. These results suggest that there may be divergent mechanisms by which LR and HR HPV E7 target p130 for degradation.

An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages

Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half-life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis-database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine-rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin-related modifier (SUMO) chain (SUMO-2 K42, SUMO-3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.

Lamivudine for the treatment of HIV

Lamivudine is a nucleoside reverse transcriptase inhibitor that is widely used for the treatment of HIV-1 infection in combination with other antiretrovirals. It is a highly effective agent that can be dosed once or twice daily due to its long intracellular half-life. It also has one of the best tolerability and long-term safety profiles among all antiretroviral agents and continues to be preferred as part of initial or subsequent combination therapy in HIV-infected patients.

Cell-penetrating d-Isomer Peptides of p53 C-terminus: Long-term Inhibitory Effect on the Growth of Bladder Cancer

To investigate whether a single application of the membrane-permeable D-isomer of the p53 C-terminus connected with a retro-inverso version of the NH(2)-terminal 20-amino acid peptide of the influenza virus hemagglutinin-2 protein (riHA2) inhibited the growth of bladder cancer cells. The transduction of p53 using poly-arginine is useful for targeting and suppressing the growth of bladder cancer cells. However, the protein's intracellular half-life is short, and repeated application is necessary to achieve an anti-tumor effect. The p53 carboxyl-terminal peptides covalently coupled with cell-penetrating peptides were synthesized with D- or L-amino acids. Moreover, the peptides were connected with riHA2 by a disulfide bridge. Human bladder cancer cell lines were incubated with each peptide and cell viability was assessed with the WST assay. Apoptotic cells were confirmed by Hoechst and active capase-3 staining. The p53 peptides were injected into severe combined immunodeficiency disease mice transplanted with J82 cells to investigate their anti-tumor effect on bladder tumors. A survival curve was plotted using the Kaplan-Meier method. A single application of cell-penetrating D-isomer peptides of the p53 C-terminus connected with riHA2 (d11R-p53C'-riHA2 and dFHV-p53C'-riHA2) inhibited the growth and induced the apoptosis of bladder cancer cells. The tumor-bearing mice treated only with vehicle had a mean survival time of 12 days, whereas treatment with d11R-p53C'-riHA2 resulted in a long-term survival rate of 50%. Peptide transduction therapy using the D-isomer p53 C-terminal peptide with riHA2 may be an innovative method for the treatment of bladder cancer.

Alu-repeat-induced deletions within theNCF2gene causing p67-phox-deficient chronic granulomatous disease (CGD)

Mutations that impair expression or function of the components of the phagocyte NADPH oxidase complex cause chronic granulomatous disease (CGD), which is associated with life-threatening infections and dysregulated granulomatous inflammation. In five CGD patients from four consanguineous families of two different ethnic backgrounds, we found similar genomic homozygous deletions of 1,380 bp comprising exon 5 of NCF2, which could be traced to Alu-mediated recombination events. cDNA sequencing showed in-frame deletions of phase zero exon 5, which encodes one of the tandem repeat motifs in the tetratricopeptide (TPR4) domain of p67-phox. The resulting shortened protein (p67Delta5) had a 10-fold reduced intracellular half-life and was unable to form a functional NADPH oxidase complex. No dominant negative inhibition of oxidase activity by p67Delta5 was observed. We conclude that Alu-induced deletion of the TPR4 domain of p67-phox leads to loss of function and accelerated degradation of the protein, and thus represents a new mechanism causing p67-phox-deficient CGD.

A Model for Protein Turnover: the Work of Robert T. Sauer

The Structural Stability of a Protein Is an Important Determinant of Its Proteolytic Stability in Escherichia coli(Parsell, D. A., and Sauer, R. T. (1989) J. Biol. Chem. 264, 7590–7595)Identification of C-terminal Extensions That Protect Proteins from Intracellular Proteolysis(Bowie, J. U., and Sauer, R. T. (1989) J. Biol. Chem. 264, 7596–7602) The Structural Stability of a Protein Is an Important Determinant of Its Proteolytic Stability in Escherichia coli (Parsell, D. A., and Sauer, R. T. (1989) J. Biol. Chem. 264, 7590–7595) Identification of C-terminal Extensions That Protect Proteins from Intracellular Proteolysis (Bowie, J. U., and Sauer, R. T. (1989) J. Biol. Chem. 264, 7596–7602) Robert T. Sauer grew up in the Hudson River Valley in New York. He attended Amherst College and received his B.A. in biophysics in 1972. Before and after graduating from Amherst, he worked as a research technician at Massachusetts General Hospital in Boston. Eventually, Sauer enrolled in graduate school at Harvard University. His Ph.D. research focused on how the modular structure of phage λ repressor allowed it to act as a regulator of gene expression. A year before receiving his Ph.D. in 1979, Sauer joined the biology department at Massachusetts Institute of Technology (MIT). He worked his way up through the academic ranks and eventually became Salvador E. Luria Professor of Biology, a title that he continues to hold today. When he started his lab at MIT, Sauer continued his research on the regulation of gene expression, studying the mechanisms that allow transcription factors to recognize their specific binding sites in chromosomal DNA. Over the years, his interests shifted to protein folding and how amino acid sequence dictates the three-dimensional structure, stability, and susceptibility of proteins to degradation. Some of his findings are detailed in the two papers reprinted here that were published back to back in the Journal of Biological Chemistry (JBC). At the time the papers were published, it was believed that the proteolytic susceptibility of a protein was related to the thermodynamic stability of its native structure. However, it was difficult to prove this hypothesis because experiments inevitably ended up altering more than one property of the protein being studied. For example, a side chain modification meant to alter protein stability could also affect degradation if it acted as a recognition signal. As detailed in the first Classic, Sauer and his graduate student Dawn Parsell came up with a system in which they could alter protein stability without changing other variables. They constructed a set of variants of the N-terminal DNA-binding domain of λ repressor with a wide range of melting temperatures. Pulse-chase experiments showed that the most thermally stable proteins had the longest intracellular half-lives. The pair also showed that second-site mutations that increased the thermodynamic stability of the native N-terminal domain also suppressed intracellular degradation. This paper was followed by one in which Sauer and his graduate student James Bowie showed that intracellular degradation can also be affected by mechanisms independent of a protein's thermodynamic stability. The scientists isolated revertants of defective mutants in the Arc repressor of bacteriophage P22 and found that five of the six reverting mutations were frameshifts resulting in C-terminal extensions. The mutations prolonged the half-lives of the proteins but did not alter their thermodynamic stability, structure, oligomeric form, or DNA-binding properties. Furthermore, fusion of one of these tails to the C-terminal end of a mutant form of the N-terminal domain of λ repressor prevented proteolysis of the protein. Sauer hypothesized that the C-terminal sequences prevented degradation by blocking the proteins' recognition by the cell's proteolytic machinery. From these experiments, Sauer proposed a bipartite model for protein turnover in which the thermodynamic stability of a protein determines the fraction of the protein that will be in an unfolded and proteolytically susceptible state, whereas sequence determinants within the unfolded protein chain determine how effectively it will act as a substrate for proteolytic enzymes. Most recently, Sauer's research has focused on the structure and function of the molecular machines that disassemble macromolecular complexes and unfold native proteins. Sauer has received many honors and awards in recognition of his contributions to science and teaching. These include the MIT School of Science Prize for Excellence in Graduate Teaching (1998), the Protein Society's Amgen Award (2001) and Hans Neurath Award (2007), and election to the American Academy of Arts and Sciences (1993), the National Academy of Sciences (1996), and the American Academy of Microbiology (1996). He also served as president of the Protein Society (1997–1999) and head of the MIT Department of Biology (1999–2004).

Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β- l -2′,3′-Didehydro-2′,3′-Dideoxy-N 4 -Hydroxycytidine: Relevance for Activation in HepG2 Cells

Beta-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED(50)] = 0.03 microM; 50% cytotoxic dose [CD(50)] = 2,500 microM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[(3)H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5'-mono-, 5'-di-, and 5'-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ss-d-N(4)-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5'-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 microM l-[(3)H]Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 +/- 2.7 pmol/10(6) cells. The predominant peak belonged to 5-diphosphate, with 43.5 +/- 4.3 pmol/10(6) cells. The intracellular half-life of the 5'-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5'-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5'-diphosphate during a drug-free incubation. The high 5'-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.

Luciferase protection against proteolytic degradation: A key for improving signal in nano-system biology

Luciferase is most widely used bioluminescence protein in biotechnological processes, but the enzyme is susceptible to proteolytic degradation, thereby its intracellular half-life decreased. Osmolytes are known to enhance the stability of proteins and protect them in a native folded and functional state. The effects of osmolytes, including sucrose, glycine and DMSO on the stability of luciferase were investigated. To different extents, all osmolytes protected the luciferase towards proteolytic degradation in a concentration-dependent manner. The results showed that 1.5 M sucrose, 1.5 M glycine and 15% DMSO are the best. The ability of these osmolytes to protect luciferase against proteolysis decreased from sucrose, glycine, and finally DMSO. Enzymatic kinetic data showed that the luciferase activity is significantly kept in the presence of sucrose and glycine compared to DMSO, particularly at high temperatures. Bioluminescence intensity, circular dichroism (CD), intrinsic and ANS fluorescence experiments showed change in secondary and tertiary luciferase structure. These results suggest that osmolytes exert an important effect on stabilization of luciferase conformation; decreasing the unfolding rate, preventing adaptation and binding of luciferase at the active site of proteases, thereby the proteolytic digestion reduced and its active conformation was kept.

Tenofovir disoproxil fumarate for the treatment of hepatitis B infection.

More than 350 million individuals worldwide are chronically infected with hepatitis B virus (HBV). Individuals with chronic hepatitis B infection carry a significantly increased risk of life-threatening liver sequelae including cirrhosis, hepatic decompensation and hepatocellular carcinoma. Currently, antiviral therapy options for chronic HBV consist of immunomodulators, nucleoside analogues and nucleotide analogues. Tenofovir disoproxil fumarate, an oral prodrug of the phosphonate nucleotide tenofovir, was recently approved in 2008 for the treatment of chronic hepatitis B in the European Union and the United States. Tenofovir disoproxil fumarate is safe, well tolerated and has long plasma and intracellular half-lives, thus allowing for once-daily administration. Since its approval, tenofovir disoproxil fumarate has become recognized as a first-line treatment option for the management of chronic hepatitis B infection in both treatment-naive and treatment-experienced patients.

Ubiquitin-fusion as a strategy to modulate protein half-life: A3G antiviral activity revisited

The human APOBEC3G (A3G) is a potent inhibitor of HIV-1 replication and its activity is suppressed by HIV-1 virion infectivity factor (Vif). Vif neutralizes A3G mainly by inducing its degradation in the proteasome and blocking its incorporation into HIV-1 virions. Assessing the time needed for A3G incorporation into virions is, therefore, important to determine how quickly Vif must act to induce its degradation. We show that modelling the intracellular half-life of A3G can induce its Vif-independent targeting to the ubiquitin-proteasome system. By using various amino acids (X) in a cleavable ubiquitin-X-A3G fusion, we demonstrate that the half-life (t1/2) of X-A3G can be manipulated. We show that A3G molecules with a half-life of 13 min are incorporated into virions, whereas those with a half-life shorter than 5 min were not. The amount of X-A3G incorporated into virions increases from 13 min (Phe-A3G) to 85 min (Asn-A3G) and remains constant after this time period. Interestingly, despite the presence of similar levels of Arg-A3G (t1/2=28 min) and Asp-A3G (t1/2=65 min) into HIV-1 Deltavif virions, inhibition of viral infectivity was only evident in the presence of A3G proteins with a longer half-life (t1/2 > or = 65 min).

Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis

Firefly luciferase (EC.1.13.12.7) from Photinus pyralis is a single polypeptide chain (62 kDa), responsible for emission of yellow-green (557 nm) light, known to be most efficient bioluminescence system that make it an excellent tool for reporter in nano-system biology. However, it is very sensitive to proteolytic degradation, which reduces its intracellular half-life, leads to loss in sensitivity and precision in analytic applications. In order to generate more stable luciferases against protease digestion, we substituted two tryptic sites: R(213), R(337) and also next residue to it (Q(338)) with another amino acids. Overall, all mutations brought about structural changes that indicated more compact structure upon mutation, which revealed by enhancement of tryptophan fluorescence, decreases flexibility and less surface hydrophobic pockets. In general, structural changes associated with a clear improvement in thermostability and resistance against trypsin hydrolysis. In particular, R337Q mutant shows higher light stability in mammalian cell culture, which makes it as a suitable reporter for imaging.