Analisis Faktor-Faktor yang Memengaruhi Tingkat Kepatuhan Wajib Pajak Orang Pribadi di Lingkungan Kantor Pelayanan Pajak Pratama, Tigaraksa Tangerang

Tax collection is not an easy matter. Active participation from the tax authorities also requires the willingness of the taxpayer. A public reaction can be seen from the taxpayer\u27s willingness to pay taxes. Willingness and awareness to pay taxes represent a value contributed by someone (which has been determined by regulation). Tax is used to finance public expenditures without any direct benefit. Taxpayer\u27s awareness about taxation functions as state funding is needed to improve tax compliance and to determine the level of tax compliance in implementing their tax obligations. Limitation of the scope of this study is the effect of the level of awareness of paying taxes, taxpayer\u27s understanding about tax benefits, tax penalties, and understanding of service quality to the tax authorities of individual taxpayer compliance in the fulfillment of tax obligations, as well as restricted to data obtained through questionnaires received and filled by the individual taxpayer of Tigaraksa Pratama Tax Office area. Data were obtained through questionnaire and processed and analyzed using parametric statistical tests and multiple linear regression with 4 independent variables and one dependent variable resulted in the conclusion that the factors that most influence taxpayer compliance in carrying out its tax liability is the use of sanctions against taxpayers who do not carry out its obligations under applicable legislation

Construction of lentiviral vectors.

<p>A) The HIV-1 provirus NL4.3 and the HIV-1-vector V^Hgenomic are shown. Large parts of the <i>gag, pol</i> and <i>env</i> genes are deleted in V^Hgenomic (see white bars in the deleted regions marked by shaded areas). The remaining <i>gag</i> sequence contains parts of the encapsidation signal (Psi, Ψ) and the <i>env</i> fragments contain splicing regulatory elements as well as the RRE. Due to deletions (shaded squares) and frameshift mutations (black asterisks in <i>gag</i> and <i>rev</i>) no viral genes are expressed from V^Hgenomic. Both vectors are drawn to scale. B) Schematic representation of the lentiviral vectors V^Hgenomic, V^Henv and V^Hnef. The intron between SD1 and SA5 or the introns between SD1 and SA5 and between SD4 and SA7 were deleted from V^Hgenomic in V^Henv or V^Hnef, respectively. Unspliced and spliced transcripts with splice sites (5′ splice sites in green and 3′ splice sites in blue) and <i>cis</i>-acting splicing regulatory elements (in orange) are shown. Please note that the unspliced Msd1-sa5 RNA of V^Henv is identical in sequence to the singly-spliced SD1-SA5 RNA of V^Hgenomic. Furthermore, the unspliced Msd1-sa5+Msd4-sa7 RNA of V^Hnef is identical to the fully-spliced SD1-SA5+SD4-SA7 RNA of V^Hgenomic and the singly-spliced Msd1-sa5+SD4-SA7 RNA of V^Henv. Arrowheads represent RT-PCR primers. C) and D) After cotransfection of lentiviral vectors with <i>tat</i> and <i>rev</i> expression plasmids into HEK293T cells cytoplasmic RNA was isolated and analyzed by RT-PCR with primer pairs depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048688#pone-0048688-g001" target="_blank">figure 1B</a>. Agarose gel electrophoretic analyses of PCR products are shown. The amplification products were sequenced to verify splicing between the indicated splice sites.</p

Rev-dependency of the infectious lentiviral vector titer.

<p>A) Cellular lysates and viral particles were harvested two days after transfection of HEK293T cells and were analyzed by an anti-CA Western Blot. The expression plasmid UTRgpRRE contains wild type <i>gag/gagpol</i> gene sequences combined with a part of the viral 5′UTR and the RRE. The Rev-independent <i>gag/gagpol</i> expression plasmid Hgp^syn encodes proteins with wild type amino acid sequences but the gene sequence is dramatically altered due to codon-optimization. B) HEK293 cells were infected with supernatants containing VSV-G pseudotyped lentiviral vectors produced in the presence or absence of Rev. Constant high Gag/GagPol protein levels were provided during vector production by cotransfection of the Rev-independent codon-optimized expression plasmid Hgp^syn. Two days later green fluorescent cells were counted to obtain the infectious titer as GFP forming units per ml of cell culture supernatant (GFU/ml). Titer of the negative control without VSV-G and Gag/GagPol was below 50 GFU/ml (data not shown). Mean values with SEM (standard error of mean) of log10 transformed results obtained in at least 4 independent experiments are shown. Statistical analysis was performed with an unpaired two-tailed t-test with 95% confidence interval. ***, p≤0.001; **, p≤0.01; *, p≤0.05; n.s., not statistically significant.</p

Cytoplasmic and virion-associated lentiviral vector RNA levels in the presence and absence of Rev.

<p>A) Cytoplasmic RNA was extracted two days after transfection and analyzed using quantitative RT-PCR protocols (please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048688#pone.0048688.s001" target="_blank">Materials and Methods S1</a> for experimental details). Transcript copy numbers per µg of cytoplasmic RNA are shown. B) Virion-associated RNA was isolated from cell culture supernatants of cells analyzed in A. Transcript copy numbers per ml of cellular supernatant were obtained after RT-qPCR analyses. Unspliced RNA levels of V^Hgenomic are shown in green. RNA levels of the singly-spliced SD1-SA5 RNA of V^Hgenomic and the unspliced Msd1-sa5 transcript of V^Henv are depicted in blue. These RNAs represent the class of singly-spliced transcripts. Shown in red are transcript levels of the multiply-spliced SD1-SA5+SD4-SA7 RNA of V^Hgenomic, the singly-spliced Msd1-sa5+SD4-SA7 RNA of V^Henv and the unspliced Msd1-sa5+Msd4-sa7 RNA of V^Hnef. These RNAs correspond to the class of fully-spliced transcripts. Mean values with SEM of log10 transformed RNA copy numbers obtained in 5 independent experiments are shown. Statistical analysis was performed with a one-way ANOVA combined with the Newman-Keuls post-test. ***, p≤0.001; **, p≤0.01; *, p≤0.05; n.s., not statistically significant.</p

Encapsidation efficiency in the presence and absence of Rev.

<p>The ratio of virion-associated and cytoplasmic RNA levels defines the encapsidation efficiency for all lentiviral vector transcripts detected. The log10 transformed ratios were calculated for each single data pair obtained in each single experiment for all the different RNA species examined. Mean values with SEM obtained are shown. Statistical analysis was performed with a one-way ANOVA combined with the Newman-Keuls post-test. ***, p≤0.001; **, p≤0.01; *, p≤0.05; n.s., not statistically significant.</p

Antibody response after VLP boost in DNA immunized mice.

<p>(A) Mice were immunized three times with DNA encoding different MPER display mutants and boosted with VLPs containing the different MPER display mutants eight to fifteen weeks after the last DNA immunization. (B) MPER-specific antibody levels in the sera of immunized mice three weeks after the last VLP immunization are presented as log₁₀ values of the relative light units (Log RLU) obtained in an MPER antibody ELISA. Mean and single values for each of the animals are shown.</p

Map of domains and amino acid sequences of the MPER display mutants.

<p>The open reading frames of the MPER display mutants contain a heterologous leader peptide (LP), an isoleucine zipper to promote trimerization, a common tag (Ollas-tag) for easy detection, a flexible linker region (G4S), various regions of gp41 (MPERmut) including an HIV-1 transmembrane region (TM), and the cytoplasmic domain of the VSV-G protein (CT). The amino acid sequence of the different domains is given in the one-letter code. The numbering backward as depicted indicates the number of MPER-derived amino acid residues expressed by each mutant. The target regions for 2F5 and 4E10 monoclonal antibodies are indicated by double arrow-lines.</p

Characterization of the MPER-specific antibody response.

<p>(A) Binding of antibodies in sera of mice immunized with MPER42 by the DNA-VLP regimen to MPER peptide in the presence of saturating amounts of 2F5 and 3D6 monoclonal antibodies. Significant competition of 2F5 with serum antibodies from five individual mice (MPER42-1 to MPER42-5) for binding to MPER peptide is marked by the bars. (B) Neutralizing activity of sera of two individual mice with the strongest antibody response after immunization with MPER42 DNA and VLP vaccines. Sera from a ΔEnv control mouse and from mice responding strongest to immunization with gp41 and gp160 by the DNA-VLP regimen were also analyzed. The 2F5 antibody at a concentration of 50 µg/ml was diluted as indicated and used as a positive control for neutralization.</p

Incorporation of MPER display mutants into VLPs.

<p>VLPs containing MPER display mutants were concentrated from the supernatant of transfected cells and analyzed by Western blot analysis under denaturing (A to C) and non-reducing conditions (D) using the antibodies 2F5 (A), 4E10 (B), anti-Ollas (C, D). Results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038068#pone-0038068-g003" target="_blank">Figures 3</a> A-D are representative of at least three WB analyses using different VLP preparations and different denaturing conditions. ^*MPER47 contains 5 amino acids residue of non-MPER origin at its N-terminus.</p

Influence of DNA priming on the MPER-specific antibody response after the VLP boost.

<p>(A) Mice (n = 5–6/group) were immunized with VLPs containing different MPER display mutant either with or without prior DNA immunization with plasmids encoding the same MPER display mutants. (B) MPER-specific antibody levels in the sera of immunized mice three weeks after the last VLP immunization are presented as Log values of the relative light units (Log RLU) obtained in an MPER antibody ELISA. Mean and single values for each of the animals are shown. Statistically significant differences between the groups treated with the different DNA and VLP vaccines were determined by one way analysis of variance followed by Bonferroni’s multiple comparison test and are indicated by horizontal bars with “a” indicating a p-value of <0.05. T-tests were performed to determine whether DNA priming enhances antibody levels for each of the different immunogens. Statistically significant differences are marked by dashed horizontal bars with “b” indicating a p-value of <0.05. Filled symbols are used for non-primed mice and opened symbols for DNA-primed mice. (C) Time course of MPER-specific antibodies in sera of individual mice (MPER42-1 to MPER42-5) immunized with MPER42 DNA and VLP vaccines.</p