To investigate gender differences in the association between self-rated health (SRH) and impaired glucose tolerance (IGT) in subjects unaware of their glucose tolerance.A cross-sectional population-based study.The two municipalities of Vara and Skövde in south-western Sweden.A total of 2502 participants (1301 women and 1201 men), aged 30–75, were randomly selected from the population.IGT was regarded as the outcome measure and SRH as the main risk factor.The prevalence of IGT was significantly higher in women (11.9%) than in men (10.1%), (p = 0.029), as was the prevalence of low SRH . Both men and women with low SRH had a poorer risk factor profile than those with high SRH, and a statistically significant crude association between SRH and IGT was found in both men  and women . However, after controlling for several lifestyle factors and biomedical variables, the association was attenuated and remained statistically significant solely in men .The gender-specific associations found between SRH and IGT suggest that SRH may be a better indicator of IGT in men than in women. Future studies should evaluate the utility of SRH in comparison with objective health measures as a potential aid to health practitioners when deciding whether to screen for IGT and T2DM. People with previously unknown diabetes have lower self-rated health than those with normal glucose metabolism.The authors investigated whether previously unknown impaired glucose tolerance was associated with self-rated health, and whether the association differed by gender.Low self-rated health was independently associated with impaired glucose tolerance solely in men.Type 2 diabetes mellitus (T2DM) is a common chronic disease, which substantially increases the risk of cardiovascular disease (CVD). The prevalence of T2DM is estimated to continue to increase, thus posing a serious challenge at both an individual and a societal level . ImpaireSelf-rated health (SRH) is a subjective measure of health, which has been associated with increased mortality both in a general population ,5 and inIn 2002–2005, a health survey consisting of two visits to the local primary health care centre was conducted in the municipalities of Vara and Skövde, south- western Sweden, as part of the Skaraborg Project . ParticiAs described previously in greater detail , data onKnown diabetes was defined as a physician's diagnosis of diabetes. In participants without known diabetes, a standard oral glucose tolerance test (OGTT) was performed after a 10-hour overnight fast . Blood s2 (m2).The second visit, two weeks after the first, included a physical examination with anthropometric measurements. All participants were seen by one of the two specially trained nurses who conducted all study visits. Systolic and diastolic right brachial arterial blood pressures were recorded to the nearest 2 mm Hg, in a supine position after five minutes’ rest. Body weight was measured to the nearest 0.1 kg, standing height to the nearest centimetre, and body mass index (BMI) was calculated as weight (kg)/heightThe statistical analyses were performed using SPSS for Windows (version 19.0), and results are presented separately for men and women. Differences between genders and IGT (high/low), respectively, were examined by general linear model for continuous variables and by logistic regression analysis for proportions. IGT was considered to be the outcome measure and thus used as the dependent variable in all analyses, whereas either gender or SRH was used as the independent variable. Confounding by age, BMI, alcohol consumption, daily smoking, educational level, HOMA-ir, fasting glucose, hypertension, previous CVD, previous hyperlipidaemia, HDL cholesterol, lack of sleep, and LTPA was assessed by stratification and by multivariate analyses. The potential confounding factors were chosen based on a theoretical model of factors previously found to be associated with both SRH and IGT. All tests were two-sided and statistical significance was accepted at p < 0.05.The majority of participants gave a positive rating of their general health , althougWhen comparing participants with high versus low SRH, both men and women with low SRH were more often smokers, had significantly higher levels of HOMA-ir and BMI, and had significantly lower levels of HDL cholesterol . FurtherWhen the association between SRH and IGT was further explored in logistic regression analyses, a statistically significant association between SRH and IGT was revealed in both men and women in the crude model . In men,Both men and women with poor SRH had a more atherogenic risk-factor profile than participants with high SRH. In a crude model, a statistically significant inverse association between SRH and IGT was seen in both men and women. However, after adjustments for confounders the association remained solely in men.The large random, population-based sample and the high participation rates all strengthen the generalizability of the current results. The validity is also supported by the fact that two specially trained registered nurses performed all study visits. Furthermore, all participants without previously known diabetes were given an OGTT to systematically identify those with IGT, using current standard definitions . All queRegarding the association seen here between SRH and IGT, comparative studies are scarce. In an Australian cohort the association between SRH and IGT was explained by confounding in cross-sectional analyses , whereasAlthough the Australian studies ,9 includThe possibility of the current results reflecting an effect of IGT on SRH rather than the contrary must also be acknowledged. A qualitative study  within tIdentifying subjects with IGT and T2DM early on is important to facilitate lifestyle or medical interventions in order to treat manifest T2DM or prevent the development from IGT to T2DM. The independent association found here between SRH and IGT in men suggests that, at least in men, SRH might be a useful indicator to consider when health practitioners make decisions about screening for IGT and T2DM. Thus, future studies should evaluate the utility of SRH in comparison with objective health measures. Moreover, as no association between SRH and IGT was found in women in the present study, future studies should also further explore the relationships between gender-specific factors and IGT.


Phe from S. cerevisiae as well as hydrolytic activity of leadzyme. Pb(II) induced hydrolysis of the single phosphodiester bond in tRNAPhe is accompanied by release of 98 water molecules, while other molecule, leadzyme releases 86.The tertiary structure of nucleic acids results from an equilibrium between electrostatic interactions of phosphates, stacking interactions of bases, hydrogen bonds between polar atoms and water molecules. Water interactions with ribonucleic acid play a key role in its structure formation, stabilization and dynamics. We used high hydrostatic pressure and osmotic pressure to analyze changes in RNA hydration. We analyzed the lead catalyzed hydrolysis of tRNA Phe) with Pb(II). Yeast tRNAPhe can be very useful model for this study because its three-dimensional structure has been determined in many crystallographic studies  High hydrostatic pressure (HHP) is a convenient tool for study hydration because it allows to induce changes without addition external agents, e.g., salts. Both HHP as well as OP affect the structure of water , 11.Phe soaked with Pb(II) starts with abstraction of H+ from a ribose 2′-OH group by Pb(II) bound hydroxyl group. RNA hydrolysis is pH dependent and the optimum for Pb(II) is 7.5 while for Mg(II) is 8.5 ATP and 10 U T4 polynucleotide kinase and 1 × PNK buffer (Promega) was incubated at 37°C for 45 min and 99°C for 2 min. Than 0.8 nmol of tRNAPhe and 10 U of T4 RNA ligase (Boehringer) in of 20 μL of ligase buffer  were added and incubated 16 h at 4°C.6 μL of mixture containing 0.16 mM Cp, 20 μCi [γ-32P]ATP and 6 U T4 polynucleotide kinase (Promega) and kinase buffer (Promega) and incubated 30 min at 37°C. Reaction mixture was then separated in a 15% (tRNA) and 24% (L1) polyacrylamide gel with 7 M urea. The radioactive band was cut out, the RNA was eluted with buffer and precipitated with ethanol . C. Cobs veme Figs. b, 8b oneing step , 39. So ith FTIR . Howeverleadzyme , 20. Spoleadzyme . While iPhe caused by increasing pressure is connected with decrease of water volume while further effects arise from tRNA conformational changes.Our results show that cleavage of RNA with Pb(II) is pressure inhibited while spontaneous cut of RNA occurring in helical region is relatively less HHP dependent. It means that structural changes of RNA during in Pb(II) induced hydrolysis are bigger. Hydrolysis of single phosphodiester bond in the loop of RNA is accompanied by three times higher hydration changes comparing to cleavage in helix. The first step of volume change of tRNA


Covariation studies and phylogenetic analysis exploring the potential structure of RNA11 of rotaviruses suggested that, besides the previously predicted “modified panhandle” structure, the 5’ and 3’ termini of one of the isoforms of the bovine rotavirus UKtc strain may interact to form a tRNA-like structure (TRLS). Such TRLSs have been identified in RNAs of plant viruses, where they are important for enhancing replication and packaging. However, using tRNA mimicry assays (in vitro aminoacylation and 3’- adenylation), we found no biochemical evidence for tRNA-like functions of RNA11. Capping, synthetic 3’ adenylation and manipulation of divalent cation concentrations did not change this finding. NMR studies on a 5’- and 3’-deletion construct of RNA11 containing the putative intra-strand complementary sequences supported a predominant panhandle structure and did not conform to a cloverleaf fold despite the strong evidence for a predicted structure in this conserved region of the viral RNA. Additional viral or cellular factors may be needed to stabilise it into a form with tRNA-like properties.Rotaviruses are a major cause of acute gastroenteritis, which is often fatal in infants. The viral genome consists of 11 double-stranded RNA segments, but little is known about their  Reoviridae and are icosahedral triple-layered particles (TLPs). They contain a genome of 11 segments of double-stranded RNA encoding six structural viral proteins  and six non-structural viral proteins (NSP1-NSP6). After infection, enzymatic digestion of the outer particle layer in the cytoplasm produces double-layered particles (DLPs) that are transcriptionally active and extrude 11 ssRNA segments of positive polarity. These RNA molecules are either translated into viral proteins or function as templates for RNA replication, producing the double-stranded genome in progeny virions. Early viral morphogenesis and RNA replication occur in cytoplasmic inclusion bodies called viroplasms  ATP (Perkin Elmer) and a KinaseMax 5’- End Labelling Kit (Ambion) following manufacturer’s instructions. Radiolabelled RNA11 molecules were purified using RNeasy spin columns (QIAGEN). To ensure structural uniformity, RNA was heated to 80°C for 10 minutes and allowed to cool to room temperature before partial cleavage by RNase T1 (Ambion), which cleaves unpaired guanine residues. Reactions were incubated at room temperature for 10 minutes in 10-μl reactions containing 1 μg carrier yeast tRNA (Ambion AM7119). The reactions were stopped by the addition of inactivation/precipitation buffer (supplied with RNase T1 by Ambion). Each reaction contained 2 μg of RNA11. Samples were analysed on 12 % polyacrylamide 7 M urea gels. The sequence ladder was generated from RV RNA11 by alkaline hydrolysis.Purified RNA11 molecules were radiolabelled at the 5’ terminus using [γ-32P] ATP into a yeast tRNA mixture or a crude wheat germ tRNA preparation . Approximately 4 % of the tRNA molecules in these crude tRNA preparations have incomplete 3’ CCA termini and therefore act as substrates for 3’ adenylation by CCA-NTase  ATP , and HeLa S100 fraction (1.0 μg). The latter is used as a source of tRNA-reactive enzymes  (Sigma-Aldrich).An RNA deletion construct (117 bases) of RV RNA11 was transcribed comprising the first 52 nt at the 5’ end and the last 61 nt at the 3’ end, joined by an artificial loop-forming sequence 5’-…UUCG…-3’. For this, two overlapping DNA templates were synthesized [(forward S1: 5’AGCTAGAACTGCAGAAAAAAGCTTAAAAAAAA3’), in a 5’-3’ direction, would produce a 32-nt overlapping sequence with the 3’ end of S1 , followed by the last 43 nt of the target construct ending in -CC. The last ‘C’ was the starting ‘C’ of a Pst I restriction site (underlined in the S2 reverse-complementary sequence). This is followed by the Hind III restriction site (shown in bold in the S2-reverse-complementary sequence), spaced between two stretches of spacer sequences.The forward template  contained an Eco RI restriction site in a 5’-3’ direction (underlined in the S1 sequence) and a T7 promoter sequence downstream (shown in bold in the S1 sequence), separated by four spacer nucleotides. The first 74 nt of the target 117-nt construct immediately followed the T7 promoter sequence. The reverse template  was designed such that reverse complementarities (S2-reverse-complementary: 5’acgagtcttcttcggaagatttgtaggtctgaCCTGAGAGGTCACTAGGGAGCTCCCCACTCCCGTTTTGTGACPfu DNA polymerase (Promega) with forward (SB1: 5’AGCTAGAAGAATTCATATTAATA 3’) and reverse  primers to produce a PCR product for cloning into the pUC19 vector (Invitrogen). The cycle conditions were as follows: initial melting at 99 °C for 1 min and one cycle of 99 °C for 30 s, 55 °C for 30 s and 72 °C for 10 min. This was followed by 30 cycles of 94 °C for 1 min, 55 °C for 30 s and 72°C for 2 min. The final extension cycle comprised incubation at 72 °C for 10 min. The resulting PCR product (175 bp) was cloned into pUC19 using restriction sites Eco R1 and Hind III. The pUC19 construct was then expanded by transforming and culturing E.coli DH-5α cells, followed by plasmid purification . The plasmid was then linearized by Pst I digestion, followed by removal of the 3’ overhang using T4 DNA polymerase (Promega) to obtain linear DNA terminating in the authentic rotavirus 3’ sequence of 5’…ACC-3’. This linearized plasmid was used as a template for obtaining the target 117-base RNA construct by in vitro transcription .The S1 and S2 DNA templates were used in a “hot-start PCR” reaction, using TAATACGACTCACTATAGGCAGCAGTGCAGTTATGACTC TCTGTAGTGACTTC3’ (T7 promoter sequence underlined) and 5’GCTCTCTA GAGAAGACAAGGCAACATCCAGACTGGACTCTCCAGTGACCGAAGTCACTACAGAGAGTCATAACTGC3’ (overlapping sequences shown in bold) [E.coli DH5α cells, which were used to produce milligram quantities of the recombinant plasmid. The plasmid was purified using a QIAfilter Plasmid Mega Kit (QIAGEN) and then linearised with BbsI before performing in vitro transcription using T7 RNA polymerase. The RNA transcripts were purified using weak anion-exchange FPLC [2O (v/v) and 0.25 mM d12-EDTA) at an RNA concentration of 0.5 mM. NMR data were acquired at 25°C on a Bruker Avance 800 spectrometer equipped with a 5-mm cryo-probe. Base pairing schemes were established from NOE patterns in 2D NOESY spectra recorded in 95 % H2O/5 % D2O (v/v) [A DNA template containing the T7 promoter, the desired RNA sequence, a two-nucleotide linker and a BbsI restriction site  flanked by HindIII and EcoRI restriction sites  was generated by PCR amplification using the following overlapping primers: 5’GAGCAAGCTin bold) . The temnge FPLC , and thein vivo conditions such as protein binding and physiological concentrations of divalent cations.Phylogenetic analysis of the sequences at the 5’ and 3’ termini of RNA11 of 94 group A RV strains identified two regions in the 3’ arm that showed both much higher levels of nucleotide variation than the surrounding sequences and also strong evidence of co-variation  is 5 bp long , while the T-loop equivalent is 8 bases long . Furthermore, the 5’…C28UCAG32…3’ sequence in the putative D-loop can potentially base-pair with the 3’…G630AGUC626…5’ sequence in the putative T-loop  in the presence of HeLa S100 (in comparison to reactions in which the cell extract was absent). The mean proportion of bound tRNA  expressed as a percentage of both bound and unbound tRNAs was 12.2 ± 1.9 % . Compared to this, the nonspecific binding of yeast tRNAs and 14C-amino acids in the absence of any aminoacylating enzyme was 2.2 ± 0.8 % (from 5 experiments). The addition of HeLa S100 increased the binding by more than fivefold compared to when the cell extract was not added to the tRNA and amino acid mixtures, and this difference was statistically significant (P<0.001). The radiolabelled amino acid mixture in the absence of any RNA or enzyme also produced similar nonspecific binding, i.e., 2.3 ± 0.8 % with the filter paper following washing (data from 4 experiments). Incubation of the HeLaS100 fraction only in the presence of radiolabelled amino acids produced nonspecific binding of 1.0 ± 0.4 % for 2 μl and 0.7 ± 0.04 % for 3 μl of the fraction.A eukaryotic yeast tRNA mixture (Ambion), used as a positive control, showed effective aminoacylation with P=0.36). Also, the percentage of bound RNA11 after attempts at aminoacylation was hardly higher than even the “no RNA and no enzyme” control. Increasing the concentration of viral RNA (from 100 ng to 2.7 μg) or increasing the amount of the amino acid mixture did not have any significant effect on the readings.The binding of boRV RNA11 with radiolabelled amino acids ranged from <1 to 2.2-fold compared to the negative control  , these RNAs became competent for valylation [In vitro incorporation of a 5’ cap into 3’-adenylated RNA11 did not have any effect on the outcome of aminoacylation assays (data not shown).The above aminoacylation assays were performed with original RNA11 transcripts assuming that crude HeLa S100 would initially adenylate the segments at their 3’ end (by CCA-NTase activity), followed by aminoacylation by aminoacyl synthetase activity. To facilitate the process, RNA11 was transcribed to have a 3’- CCA see "" and useThe presence of a tRNA-like conformation has been shown to be associated with RNA packaging in plant viruses . Rotavir2+ is believed to be required for optimal tRNA function, partially due to stabilisation of tertiary interactions between the loop regions [E. coli tRNAGlu has been shown to adopt an alternative conformation (similar to a panhandle) at low Mg2+ concentration [2+ in the reaction buffer did not augment in vitro 3’-adenylation of RNA11 (10.0-20.0 mM Mg2+) or aminoacylation (5.0-20.0 mM Mg2+) of RNA11 with 3’ A (data not shown).The presence of Mg regions , 32. Intntration . Howeverin vitro transcription [2, but only minor chemical shift changes around G-U wobble base pairs were observed, which can bind hexahydrated magnesium ions in the major groove. Most importantly, the NOE pattern in the middle helical segment was preserved (data not shown). These data led to the conclusion that the RNA11 deletion construct under these conditions adopts an extended structure consistent with a hairpin fold.Chemical and enzymatic probing data did not allow us to determine the overall secondary structure of RNA11 unambiguously . NMR specription . Size-exViral RNA often has to adopt a series of different structures to fulfil different functions during the viral life cycle . Often, in silico and in vitro studies supported the existence of long-range interactions between the 5’- and 3’-terminal regions of rotavirus RNA11 [Previous us RNA11 . The newin vivo packaging of BMV RNA3 is known to be dependent on a cis-acting 3’-TRLS [in vitro compared to the coat protein binding (CPB) conformer [Certain TRLSs have been reported to be involved in viral RNA packaging; e.g., the  3’-TRLS . Further 3’-TRLS . It has onformer .in vitro conditions used to make it a substrate for these modifying enzymes.In the light of the above, we tested biochemically whether the predicted tRNA-like structure of RV RNA11 also has properties comparable to other viral TRLSs. However, RV RNA11 could not be aminoacylated or adenylated like tRNA or TRLSs. Despite persuasive free-energy and covariation evidence for a tRNA-like structure, the resemblance must not be adequate under the in vitro as a panhandle structure rather than a cloverleaf conformation. The observed NMR data for the RNA11 construct did not conform to the NOE spectra typically observed for tRNAs [in vivo upon stabilization through interactions with other cellular or viral ligands or that the 5’- SL and/or the 3’- SL, but not the complete 4-helical junction, exist transiently in the folding intermediates to ensure the correct folding of RNA11. Given the strength of the phylogenetic data supported by structural predictions together with biochemical probing consistent with the existence of more than one possible terminal structure, the strong implication is that the termini of RNA11 do have alternative conformations, which may have distinct roles in the viral life cycle.The structure mapping data and NMR data from the 5’- and 3’-deletion construct suggest that RV RNA11 terminal sequences are more stable or tRNAs –40. It i


During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load.Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4 Human immunodeficiency virus type 1 (HIV-1) persists in infected persons despite advancements in treatment. Current treatment regimens, called highly active antiretroviral therapy (HAART), usually consist of combinations of three or more drugs from two or more classes of antiretroviral agents , resulting in non-productive infection The sources of persistent virus and residual viremia have been termed reservoirs, which have not been fully characterized. Several mechanisms could be contributing to viral persistence, including low-level viral replication or the persistence of long-lived cells that release virus slowly or episodically + T cell populations from HAART-treated people an abundance of MS viral mRNAs have been detected despite an absence of cellular activation Although the exact mechanisms of viral latency have not yet been elucidated, several transcription-based models have been proposed Non-human primate animal models of AIDS have been developed and are being used to address viral persistence during HAART gag RNA in tissues collected at necropsy from macaques treated with HAART Recently, our group analyzed the distribution of RT-SHIV DNA and + T cells gag RNA in the same tissues to determine whether productive viral infection or latency dominated in specific sites of viral persistence. Some of the macaques also received a regimen of valproic acid and prostratin as induction therapy designed to activate latent virus in the presence of HAART.In the present study, we addressed the possibility that MS transcripts might serve as indicators of viral persistence in tissues from RT-SHIV-infected macaques during HAART. Levels of MS transcripts might indicate newly infected cells because they are essential early in the viral replication cycle, before the temporal switch to production of full-length vRNA. MS mRNAs also accumulate in the nucleus of latently-infected resting memory CD4env mRNA is not spliced at this site. However, this codon is in frame and the observed C-to-T mutation would result in a stop codon (TAG) in the env reading frame.The plasmid TOPOtat was generated by cloning the MS transcript from RT-SHIV infected CEMx174 cells into a TOPO vector to serve as a DNA standard in the development of a TaqMan real-time PCR assay for the quantification of the MS transcript. Upon sequence analysis, plasmid clones containing PCR-amplified MS transcripts from RT-SHIV-infected CEMx174 cells demonstrated sequence variation at the coding exon-one and coding exon-two splice junction . The fir6 vRNA copies/mL) and subsequently cloned into a TOPO vector  and –3.43 for the +CAG template (r2 = 0.989), indicating amplification efficiencies of 92% and 96%, respectively. Because this single set of TaqMan primers and probe detected both viral sequence variants efficiently, it was used for all subsequent analysis in this study.TOPOtat was used for standardization of TaqMan real-time PCR in order to quantify the MS transcripts in tissues from RT-SHIV-infected macaques. Serial ten-fold dilutions of the plasmids TOPOtat-CAG and TOPOtat+CAG were used as templates in TaqMan PCR to generate standard curves. Results of TaqMan PCR using the SIVtat 284#2p probe (containing the CAG sequence) were linear over seven logs, whether the template contained the CAG nucleotides or not . Linear gag RNA and cellular GAPDH RNA were amplified from total cDNA isolated from RT-SHIV-infected CEMx174 cells. Samples were removed from the cycler every five cycles, through 25 cycles. TaqMan PCR for each target was used in single-plex reactions to quantify copy numbers of RNA. Linear regression analysis demonstrated that the preamplification step was linear through 25 cycles for all three targets with r2 values for MS, gag, and GAPDH RNA of 0.993, 0.980, and 0.995, respectively . To increase the sensitivity of the assay, a 25 cycle multiplex PCR preamplification step was developed. To test for linearity in the preamplification step, MS mRNA, ectively .GAPDH RNA in six different tissues from each of the animals was quantified to determine whether this housekeeping gene could be used to normalize viral RNA content. Multiplex preamplification PCR was performed on cDNA generated from 4 µg of total RNA isolated from tissues from all nine macaques. TaqMan PCR of the preamplified cDNA demonstrated that GAPDH RNA levels were similar in all tissues  for both MS mRNA and gag RNA for five of the tissues , with Pearson r values ranging from 0.9991 to 1.0000. Mmu 36544 had a plasma virus load at necropsy of 1.29×106 vRNA copies/mL .To confirm the observation that higher plasma viral loads were associated with higher levels of RNA in tissues, correlations of MS mRNA and termined . Correlaopies/mL . This plgag RNA, the ratios of MS mRNA levels to gag RNA levels were determined for samples that had detectable levels of both RNAs .To estimate whether tissues from animals treated with HAART displayed an abundance of MS mRNA relative to oth RNAs . This angag RNA in tissues from RT-SHIV-infected macaques treated with HAART. Due to the possibility that both low-level viral replication and long-lived cellular reservoirs might persist despite HAART, we elected to analyze total tissue lysates as opposed to populations enriched for resting memory CD4+ T lymphocytes in situ hybridization in tissues from SIV-infected macaques demonstrated that gag RNA was more prevalent than MS mRNA in the absence of HAART gag RNA was also present at higher levels than MS mRNA in tissues from HAART-treated, RT-SHIV-infected macaques.While it is apparent that HAART does not eradicate HIV-1 infection, reservoirs allowing for viral persistence during treatment are poorly understood. To help elucidate the mechanisms of viral persistence, we compared the distribution of viral MS mRNA and in vivo. Our results from tissues support the general pattern of splice acceptor usage previously described in the cell culture experiments, with 39% of the 28 clones that we sequenced utilizing the first splice acceptor and 61% utilizing the second acceptor  is used for coding exon-two of MS mRNA in HIV-1NL4-3-infected cells A similar pattern of three potential splice acceptors for coding exon-two of tat and rev mRNA is present in the genomes of HIV-1et al. previously reported on the presence of premature stop codons in the transmembrane domain of SIV envin vitro artifact due to SIV replication in unnatural human cells.The third variant that we observed in cDNA clones generated from RT-SHIV-infected CEMx174 cells was not observed in any of the clones generated from tissues. Kodama gag RNA in some. Because the gag RNA levels that were detected were at lower levels than in the other tissues, it is possible that viral replication or reactivation from latency is occurring in the thymus but at levels too low to detect the MS mRNA. However, the lack of MS mRNA suggested that the detected gag RNA in thymus might be virion-associated or trapped in cells such as FDCs TaqMan quantification of MS mRNA from the HAART-treated macaques indicated that no tissue had consistently detectable levels of MS mRNA. Interestingly, MS mRNA was not detected in any of the thymus samples despite detectable levels of gag RNA levels from well-suppressed animals (<50 vRNA copies/mL) were compared with the levels observed in animals with higher virus loads (>50 vRNA copies/mL). While none of the tissues contained an abundance of MS transcripts relative to gag RNA, the gastrointestinal samples from well-suppressed macaques had a significantly higher ratio of MS to gag RNA, which suggests a state of viral latency in these samples. Two recent studies by Yukl et al. analyzed biopsies taken from gastrointestinal compartments of HIV-1-infected patients during HAART gag RNA to vDNA, suggesting viral latency in these tissues despite high numbers of activated CD4+ T cells gag RNA content using a preamplification step may help to further elucidate the state of infection in these tissues.MS mRNA and gag RNA, which serves as genomic vRNA as well as mRNA for Gag and Pol proteins, was present at higher levels in these tissues. Importantly, while gag RNA is present in virions and can persist in FDCs in the absence of infection gag RNA in tissues correlate with plasma virus load.An accumulation of MS mRNA in several key lymphoid tissues was not observed despite the essential role of MS transcripts early in the infection cycle and evidence that MS mRNAs accumulate in the nucleus of latently-infected cells Macaca mulatta; Mmu) from the retrovirus-free colony of the California National Primate Research Center (CNPRC) were used. This study was approved by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) accredited University of California, Davis Institutional Care and Use Committee (IACUC). The UC Davis IACUC has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW). Animals were administered 10 mg/kg body weight ketamine-HCl  intramuscularly when necessary for immobilization. Additionally, analgesics were administered at the discretion of the CNPRC veterinary staff in an effort to minimize all animal pain and discomfort. Macaques were housed at the California National Primate Research Center, which is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). For housing, animals were maintained in cages with 4 square feet of floor space, or 6 square feet if over 10 kg, and fixed perch bars in a temperature-controlled BSL-2+ vivarium with continuous monitoring of temperature and humidity. Compatible animals were paired continuously or intermittently (separated at night) whenever possible. All animals had visual and auditory access to other macaques 24 hours per day. These animals were fed a balanced commercial macaque chow  twice daily and fresh produce twice weekly, with free access to water 24 hours per day. Supplemental food was provided when clinically indicated. Environmental enrichment was provided daily, included manipulanda  and novel foodstuffs.Nine juvenile, male Rhesus macaques . All macaques were humanely euthanized by overdose of sodium pentobarbital (60 mg/kg) administered by the intravenous route under ketamine sedation (10 mg/kg).5 50% tissue culture infectious doses of RT-SHIV as previously described For infection of animals, stocks of RT-SHIV were prepared from cell culture supernatants of CEMx174 cells transfected with the two viral half-clone plasmids as previously described RT-SHIV-infected rhesus macaques were treated with HAART as previously described After 26 weeks of HAART, six of the HAART-treated macaques were treated with prostratin and valproic acid for eight weeks in addition to continuing HAART. During the fist two weeks of the induction period, each animal received induction treatment five times. Over the final six weeks of the induction period, each animal received weekly treatment. Prostratin  was delivered to anesthetized animals at 0.1 mg/kg by infusion in saphenous or cephalic veins. Valproic acid  was delivered orally at 120 mg/kg in two doses, the first dose being delivered by orogastric intubation during anesthesia and the second being delivered in food later the same day. After the induction period, the animals were continued on HAART alone until euthanasia. Complete details of the induction treatment, including pharmacokinetics, will be published elsewhere .After 41 weeks of HAART, four of the animals  were sedated with ketamine-HCl and then humanely euthanized with a barbiturate overdose by veterinary pathologists and staff at the CNPRC. HAART was stopped in the remaining five animals  and plasma vRNA was monitored for viral rebound. After 16–20 weeks without HAART, these animals were subsequently euthanized.later RNA Stabilization Reagent  according to the manufacturer’s protocol. Briefly, sections of tissue no greater than 0.5 cm per dimension were cut from tissues using sterile scalpels. Tissues were then stored in RNAlater overnight at 4°C. The following day they were transferred to 1.5 ml centrifuge tubes and stored at −80°C until nucleic acid extraction.In order to protect RNA in tissues from degradation at necropsy, sections of tissue were treated with Qiagen RNATissues were disrupted for isolation of RNA with QIAzol Lysis Reagent (Qiagen) using a Qiagen TissueRuptor (a rotor-stator homogenizer). Frozen samples were weighed and 700 µL of QIAzol per 50 mg of tissue was added. The samples were chopped with sterile scalpels and then disrupted using the TissueRuptor with autoclaved, disposable probes to prevent carry-over contamination. The lysates were separated into 700 µL aliquots before the addition of 150 µL of chloroform , followed by 15 sec of vigorous shaking. After three minutes at room temperature, the samples were centrifuged at 12,000×g at 4°C. The aqueous phase was removed from each sample for RNA isolation and the remainder of each lysate was saved for DNA isolation (see below).For RNA extraction, 1.5 volumes of molecular grade absolute ethanol (Sigma) was added to the aqueous phase and the samples were applied to Qiagen miRNeasy mini columns according to manufacturer’s protocol. Two sequential DNase digestions using the RNase-Free DNase Set (Qiagen) were performed on each sample according to the on-column treatment procedure. RNA was eluted from each column using 52 µL of molecular grade water. The optical density of each sample was measured using a spectrophotometer at 260 nm and the samples were diluted to 10 µg of RNA per 50 µL of molecular grade water. An additional DNase treatment was performed using TURBO DNase  according to the manufacturer’s protocol. The RNA was then stored in aliquots at −80°C.Viral RNA was also isolated from 140 µL of plasma using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The RNA was eluted in molecular grade water and aliquots were stored at −80°C.DNA was isolated from the fraction of QIAzol lysates not used for total RNA isolation. Purification of DNA was accomplished by precipitation of genomic DNA from the organic phenol/chloroform phase of each lysate by adding 0.3 volumes of molecular grade absolute ethanol (Sigma). Samples were centrifuged at 2000×g for five minutes at 4°C to pellet the DNA, and the supernatant was discarded. The DNA was washed twice with 700 µL of 0.1 M sodium citrate containing 10% ethanol with a centrifugation step after each wash as described above. Next, the DNA was washed with 75% ethanol, followed by another centrifugation step. The DNA was then dissolved in 500–1,400 µL of molecular grade water. The DNA solution was sheared using a Qiagen QIAshredder according to manufacturer’s instructions in order to reduce viscosity.Escherichia coli, purified using a Qiagen Plasmid Maxi prep kit, and then the sequence was verified.A portion of the RT-SHIV MS transcript was cloned into a vector to be used as a standard for the development of a real-time TaqMan PCR assay. As a source for the MS transcripts, CEMx174 were infected with RT-SHIV at a multiplicity of infection of 0.01. Total RNA was isolated from the infected cells 48 hrs post-infection using Qiagen RNeasy columns according to the manufacturer’s protocol. MS mRNA was amplified from the total RNA preparations in a one-step RT-PCR using Invitrogen SuperScript III Reverse Transcriptase (RT) and Platinum Taq DNA polymerase (Invitrogen) with 45 cycles of amplification and primers SIV tatF6616 and SIV tatR9157  at 200 nE. coli, and sequenced.Due to sequence variation in TOPOtat at the splice site of the MS transcript, viral cDNAs from various sources were cloned and sequenced to determine whether the variation was splicing variation or viral mutation. Preamplified cDNA from macaque tissues (prepared as described below) were amplified by nested PCR in order to generate amplicons suitable for TOPO-TA cloning and subsequent sequencing. The preamplified cDNA PCR product was diluted ten-fold and 1 µL was used as a template for second round PCR with Advantage 2 DNA Polymerase  using primers SIV MS TranF6814 and SIV MS TranR9117  at 250 n6 cell equivalents of genomic DNA or 20 µL of cDNA. First round reaction conditions were: 95°C for 2 min, followed by 25 cycles of 95°C for 20 s, 55°C for 20 s, 72°C for 1 min and a final extension step at 72°C for 10 min. Second round PCR was performed with primers SIVmac239 8510 and SIV MS TranR9117 and 1 µL of a ten-fold dilution of the first round PCR product. Second round reaction conditions were identical to those of the first round, except that a 30 second extension time was used and products were amplified for 30 cycles. PCR products were cloned into pCR2.1-TOPO (Invitrogen), propagated in E. coli, and submitted for sequence analysis at the UC Davis sequencing facility.Nested PCR was used to amplify a portion of unspliced full-length virus from plasma vRNA, RNA from infected PBM cells, and DNA from PBM cells and mesenteric lymph node samples from infected macaques. The region amplified for sequence analysis included a portion of the Tat/Rev encoding region and the intron, corresponding to bases 7980 through 8871 of SIVmac239 genome (GenBank accession number M33262) tat and rev mRNA. To avoid detection of full-length unspliced vRNA or viral DNA, the probe was designed to span the coding exon-one and coding exon-two splice junction of the MS transcripts. For standardization, plasmid DNA was quantified spectrophotometrically at 260 nm and copy numbers were determined. Ten-fold serial dilutions of this stock were used as templates in a 12 µL TaqMan PCR assay using Applied Biosystems (ABI) Universal Master Mix  on an ABI 7900 cycler. The primers used were SIV MS TranF6814 and SIV MS TranR9117 at 400 nM and the TaqMan probe SIVtat284#2p was used at 80 nM (E) was determined as previously described TOPOtat was used as a standard for the development of a TaqMan PCR assay for the detection of the MS transcripts. The primers were designed to target both at 80 nM . Amplifigag RNA, and cellular GAPDH mRNA in total RNA from RT-SHIV-infected CEMx174 cells and in tissues collected from macaques at necropsy. cDNA was generated from total RNA using SuperScript III RT, random hexamers, and RNase inhibitor (Invitrogen) as previously described gag and GAPDH as previously described t) obtained from the TaqMan PCR analysis were used to determine the levels of MS mRNA using the standard equation obtained from the TOPOtat standard curve. Viral gag and cellular GAPDH RNA levels were also determined based on standard curves as previously described gag RNA levels were normalized by the preamplified GAPDH RNA levels for standardization purposes.TaqMan PCR was used to quantify MS mRNA,


When appended to 5′-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5′-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic potential of targeted RIG-I signaling activation.Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs) in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I through the presence of a 5′-triphosphate (5′-ppp) on the viral RNA in association with a 3′ poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine “core” of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5′-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein  in vitro and hepatic innate immune responses in vivo. Our studies identify long poly-uridine sequences with interspersed ribocytosines as an HCV PAMP motif that drives optimal RIG-I signaling.Pathogen recognition receptors (PRRs) are critical components of the innate immune response to viral pathogens, and function in the host to recognize pathogen-associated molecular patterns (PAMPs) in viral proteins or nucleic acids. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic PRR that senses viral RNA inside an infected cell. RIG-I recognizes hepatitis C virus (HCV) RNA as non-self through the presence of both a 5′-triphosphate (5′-ppp) and a 3′ poly-U/UC tract within the viral RNA. Here we examined the RNA structure-function attributes that define the HCV poly-U/UC tract as non-self to RIG-I, including nucleotide composition. We found that the 34 nucleotide poly-uridine “core” (U-core) within the HCV poly-U/UC tract RNA was required for non-self recognition by RIG-I, and interspersed ribocytosine nucleotides were also important to induce optimal RIG-I signaling. RIG-I/RNA binding studies revealed that RIG-I formed weaker interactions with HCV RNAs lacking poly-U sequences, and RNA interaction with multiple domains of RIG-I was required to activate signaling. Finally, RIG-I recognition of the U-core within the poly-U/UC tract activated anti-HCV responses  Mammalian cells respond to acute virus infection through the actions of host pathogen recognition receptors (PRRs) that recognize viral pathogen-associated molecular patterns (PAMPs). The RIG-I-like receptors (RLRs) are cytoplasmic RNA helicases that function as PRRs for the recognition of RNA virus infection. The RLRs include RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Whereas RIG-I and MDA5 encode tandem amino-terminal caspase activation and recruitment domains (CARDs), LGP2 lacks CARDs and is thought to play a regulatory role in signaling initiated by RIG-I or MDA5 The process of RIG-I signaling activation has been revealed through structure-function studies. In addition to the N-terminal CARDs, RIG-I possesses a central DExD/H box RNA helicase/ATPase domain and a C-terminal repressor domain (RD) RIG-I is essential for host cell recognition of a variety of RNA viruses in vitro anti-HCV responses and hepatic innate immune responses in vivo. Thus, RIG-I recognition of the U-core within the poly-U/UC tract of the 5′-ppp HCV RNA is the trigger of innate antiviral immunity to HCV infection. Poly-uridine sequences could thereby offer a novel application for innate immune stimulation in vaccine vectors and antiviral therapeutic strategies for controlling RNA virus infection.In this study, we evaluate the properties of RIG-I recognition of HCV RNA by conducting a detailed structure-function analysis of RIG-I and poly-U/UC RNA interactions and innate immune signaling. Our results show that the poly-uridine core (U-core) within the HCV poly-U/UC tract was essential for recognition by RIG-I, indicating that RIG-I recognizes long poly-uridine regions as non-self motifs within 5′-ppp RNA. In addition, we found that the affinity of RIG-I/RNA binding interactions, and critical contacts between the PAMP RNA and RIG-I helicase domain, both defined an HCV RNA recognition sequence in which long poly-uridine sequences (>U17) with interspersed ribocytosines induced in vitro from a DNA template using the T7 RNA polymerase, which resulted in the RNA products having a 5′-ppp and three guanine nt at the 5′ end To determine the RNA sequence elements in the HCV poly-U/UC tract required for non-self recognition by RIG-I, we developed multiple poly-U/UC RNA constructs encoding changes in distinct regions termed the 5′arm, U-core, and 3′arm, and based on the HCV genotype 1b consensus (Con1) poly-U/UC sequence . We alsoWe further evaluated Huh7 and Huh7.5 cell responses to HCV poly-U/UC tract RNA construct derivatives . NeitherAnalysis of specific poly-U/UC tract constructs also revealed that interspersed ribocytosine nucleotides between the poly-U sequences in the RNA were necessary to achieve optimal RIG-I signal induction. We noticed that substitution of the last 26 nucleotides in the 3′arm of the poly-U/UC tract to ribocytosines (pU/UC 3′C26) resulted in a significant decrease in IFN-β-promoter induction compared to the wild-type Con1 pU/UC sequence . Removal10), 50% (EC50), or 90% (EC90) of each RNA as determined by EMSA  with purified recombinant RIG-I protein and 10 pmol of each poly-U/UC RNA construct . Compreh by EMSA . The EC  by EMSA . The rem10 values for the various poly-U/UC RNA constructs ranged from 0.65–3.04 pmol RIG-I, and EC50 values ranged from 2.28–7.31 pmol RIG-I. Noting that all RNAs included a 5′-ppp, we found that the X-region RNA did not bind to RIG-I in the range of 0–10 pmol of protein, while the X-region-U34 RNA had an EC10 value of 1.42 pmol RIG-I and >50% of the RNA bound to 8 pmol of RIG-I, demonstrating that the inserted U34 sequence promoted stable binding interactions between RIG-I and the X-region-U34 RNA. In addition, we found that EC10 values were significantly larger  for RNAs that did not signal  versus RNAs that demonstrated PAMP activity and signaled to the IFN-β-promoter . The non-signaling Δcore and U8core RNAs both contain large deletions in the U-core region, while the RNAs that induced RIG-I signaling all contained poly-U sequences >17 nt in length. These data reveal that RIG-I forms only weak interactions with 5′-ppp RNAs lacking poly-U sequences, and demonstrate that the 34 nt poly-U core of the HCV pU/UC tract is required to confer stable RIG-I/RNA binding interactions. There was not a significant difference in the EC50 or EC90 values between the signaling and non-signaling RNAs, indicating that differences in RNA affinity may play an important role in determining RIG-I signaling activation at lower, more physiologically relevant concentrations of RIG-I.As shown in 10  and EC50 (P = 0.004) values measured for no/low signaling RNAs compared to high signaling RNAs, thus demonstrating that the Δcore and U8core RNAs (lacking a poly-U core) form significantly weaker interactions with RIG-I than those RNAs with stronger PAMP activity. A significant difference was also detected when comparing the EC10 values measured for no/low signaling compared to medium signaling RNAs (P = 0.0263). In general, the EC90 for all RNA constructs was >10 pmol RIG-I regardless of whether or not the RNA could drive RIG-I activation. However, there was still a significant difference in the EC90 values between RNAs that did not signal  and the RNAs that induced high signaling to the IFN-β-promoter . Thus, our data link HCV RNA sequences containing poly-U motifs with stronger RIG-I binding and enhanced signaling for an overall potent PAMP activity. Taken together, our data indicate that the strength of RIG-I/RNA binding interactions defines an HCV RNA recognition sequence in which long poly-uridine sequences (>U17) with interspersed ribocytosines drive optimal RIG-I binding and signaling to induce the innate immune response.We also examined RIG-I/RNA affinity among RNAs denoted as non-signaling/low signaling , medium signaling , and high signaling  based on their ability to induce different levels of signaling to the IFN-β promoter. There was a statistically significant increase in the ECRIG-I is maintained in an auto-repressed conformation in uninfected cells where the CARDs interact with either the C-terminal repressor domain (RD), the helicase insertion domain (Hel2i), or both domains We found that all RNA constructs were able to bind and protect the RIG-I RD, albeit variably, from trypsin proteolysis in a dose-dependent manner . While wTo determine how the poly-U core sequence imposes PAMP activity that initiates RIG-I signaling of innate immunity, we measured HCV production in Huh7 cells that were first transfected with poly-U/UC RNA constructs to stimulate RIG-I signaling . These ain vivo, we examined the ability of the constructs to trigger hepatic innate immune responses using an intraperitoneal injection model in mice. This model recapitulates RIG-I-dependent PAMP signaling of hepatic innate immunity triggered by HCV RNA in vivo RNA transfection reagent and injected into wild-type C57BL/6 mice, and livers were collected 8 hrs later for assessment of the hepatic innate immune response. Expression of IFN-β mRNA and several interferon-stimulated genes (ISGs) were measured using real-time quantitative PCR  mRNA expression was significantly higher in mice transfected with Con1 pU/UC RNA compared to either X-region or Δcore RNA, indicating that the U34-core of the pU/UC tract is required for PAMP activity that induces the expression of IFN-β and other ISGs. Expression of hepatic Ifit2 mRNA was also higher in mice transfected with X-region-U34 RNA compared to X-region RNA, although the difference in gene expression did not reach statistical significance, implicating the requirement of poly-U RNA sequences for activation of hepatic innate immune responses in vivo. Expression of Ifit2 also correlated with ISG54 protein expression as shown by immunohistochemical staining of mouse liver sections  composition see . In termin vivo. Together, our studies identify long poly-uridine sequences (>U17) with interspersed ribocytosines as an HCV PAMP motif that drives optimal RIG-I signaling.We have demonstrated that the 34 nucleotide poly-uridine core of the HCV poly-U/UC tract is required for non-self recognition by RIG-I. Interspersed ribocytosine nucleotides between the poly-U sequences in the RNA were also important for optimal RIG-I signaling to the IFN-β promoter. Our RIG-I/RNA binding studies found that RIG-I formed weaker interactions with HCV RNAs lacking poly-U sequences, while the 34 nt poly-U core of the poly-U/UC tract was required to stimulate stronger RIG-I/RNA binding interactions. Additionally, limited-trypsin proteolysis studies revealed that while the RIG-I RD interacts with the 5′-ppp terminus of HCV RNA, interactions between the helicase domain and poly-uridine HCV RNA sequences are required to activate RIG-I signaling. Finally, we found that poly-U/UC RNA variants with high RIG-I signaling activity induced significant anti-HCV responses in cultured cells and also induced hepatic innate immune responses Our previous studies also demonstrated the importance of the 5′-ppp for non-self recognition by RIG-I, wherein 5′-ppp was necessary but not sufficient for PAMP activity conferred by HCV RNA RIG-I is maintained in an auto-repressed conformation in uninfected cells Our results imply a model of RIG-I interaction with the HCV PAMP RNA in which the RIG-I RD interacts with the 5′-ppp terminus of HCV ligand RNA. While kissing loop interactions between structures in the 5′ and 3′ NTR of the HCV genomic strand RNA All HCV genomes contain a poly-U sequence in the 3′ NTR; therefore, certain restrictions must exist which prevent viral evolution of this genomic region to mitigate RIG-I recognition. Indeed, previous studies have reported that the poly-U/UC tract is essential for HCV RNA replication HCV encompasses 6 major genotypes and multiple subtypes, and this increased viral diversity results in highly variable treatment outcomes in vitro and hepatic innate immune responses in vivo, thus providing a potential target for restricting HCV infection. Similar poly-uridine molecules could be used to induce antiviral immune responses in conjunction with IFN-based and viral protease-targeted therapies to improve HCV clearance in chronically-infected subjects. In addition, vaccine strategies that activate RIG-I signaling pathways may be required to induce appropriate immune responses to RNA viruses, including HCV, which are highly sensitive to IFN and other innate antiviral ISGs in the absence of viral antagonism. Given that RIG-I can activate innate antiviral immune responses upon recognition of poly-uridine sequence motifs, incorporation of poly-U sequences into vaccine vectors could act as an adjuvant to mimic the natural early immune response following virus infection.HCV evades the host immune response using multiple mechanisms, while host PRRs target critical regions of viral RNA or protein to suppress HCV replication C57BL/6 mice were housed under pathogen-free conditions in the animal facility at the University of Washington. Experiments and procedures were performed with approval from the University of Washington Institutional Animal Care and Use Committee . Methods for mice use and care were performed in accordance with the guidelines of the University of Washington Institutional Animal Care and Use Committee, and also follow the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.Huh7 cells and Huh7.5 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml of penicillin and streptomycin. Huh7.5 cells encode a mutant RIG-I protein that cannot signal Renilla-luc The plasmids pIFN-β-luc, pCMV-5′-TAATACGACTCACTATAGGTGGCTCCATCTTAGCCCTA-3′) and X-regionR (5′-ACTTGATCTGCAGAGAGGCCAGTATCA-3′). The amplified PCR product was purified by agarose gel extraction using the QIAquick gel extraction kit (Qiagen) as per the manufacturer's protocol. Full-length HCV RNA was produced from the pJFH-1 plasmid (genotype 2a) as previously described et al.All in vitro transcribed RNAs contain a 5′-triphosphate (5′-ppp) and three guanine nucleotides at the 5′ end to enhance T7 polymerase transcription. HCV X-region 5′-ppp RNA was synthesized from a T7 promoter-linked PCR product generated from the pX-region-c4 plasmid using the primers X-regionF (Renilla-luc (Renilla luciferase) plasmids using the FuGENE 6 transfection reagent and protocol (Roche). Transfected Huh7 or Huh7.5 cells were incubated at 37°C for 18 hours, then split into 48-well plates and incubated for an additional 12 hours prior to RNA transfection. RNA transfection was conducted in a 48-well plate format using the TransIT-mRNA Transfection kit (Mirus) as per the manufacturer's instructions. RNA transfection was conducted using either equal numbers of moles of each RNA or 350 ng RNA, depending on the experiment. Following RNA transfection, cells were incubated an additional 18 hours and luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega). All conditions and experiments were conducted in triplicate.Huh7 or Huh7.5 cells were plated on 10 cm dishes, and 24 hours later cells were transfected with 5.76 µg pIFN-β-luc (firefly luciferase) and 0.24 µg pCMV-2; 1.5 mM DTT). Reactions were incubated at 37°C for 15 minutes, then 4× native sample buffer  was added to samples. Products were separated on a 2% agarose gel  and RNA was visualized using SYBR Gold nucleic acid stain (Invitrogen). Gel-shift images were analyzed using ImageJ software , and RNA-protein binding curves were graphed using Prism 5 software (GraphPad).Various amounts of purified recombinant full-length RIG-I protein (0–30 pmol) were mixed with 10 pmol RNA and 10 µl ATPase reaction buffer , 0.67 µl AMP-PNP (10 mg/ml), and nuclease-free water up to 10 µl total volume. Reactions were incubated for 5 minutes at room temperature. Sequencing grade TPCK-treated modified trypsin (Promega) was added to the RIG-I/RNA mixtures at a protease∶protein ratio of 1∶20 (w/w), and the reactions were incubated at 37°C for 15 minutes. Proteolysis was stopped by adding 0.5 µl TLCK (1 mg/ml) and incubating reactions for 5 minutes at room temperature. SDS-PAGE Laemmli sample buffer (Bio-Rad) was then added to the samples and reaction products were analyzed by SDS-PAGE followed by silver stain using the SilverQuest silver staining kit (Invitrogen) as per the manufacturer's instructions.Various amounts of RNA (0–10 pmol) were mixed with 30 pmol purified RIG-I protein, 2 µl of 5× reaction buffer . Reactions were incubated at 37°C for 15 minutes, ATP (Sigma) was added to the reaction mixture at a final concentration of 1 mM, and the reactions were incubated at 37°C for 15 minutes. Free-phosphate concentration was determined using BIOMOL Green reagent (Enzo Life Sciences) in a microplate format and absorbance was measured at OD630 nm.Various amounts of RNA (0–1 pmol) were mixed with 5 pmol purified RIG-I protein in a total of 25 µl ATPase reaction buffer  as per the manufacturer's instructions and incubated at 37°C for 12 hours. The transfection media was removed and the cells were washed gently with complete DMEM. Transfected cells were then infected with cell culture adapted JFH-1 HCV (MOI = 0.1) in 100 µl total media volume and incubated at 37°C for 3 hours. The virus inoculum was then removed and 300 µl complete DMEM was added, and the cells were incubated at 37°C for 48 hours. HCV-infected cell supernatants were collected and titered on Huh7.5 cells. For the HCV titer assay, Huh7.5 cells were plated on 48-well plates and incubated for 12–24 hours at 37°C, media was removed, and 100 µl of infectious supernatants were added to cells using the following dilutions . Cells were incubated with supernatants at 37°C for 3 hours, the virus inoculum was removed and complete DMEM (300 µl) was added, and cells were incubated at 37°C for 48 hours. Media was then removed and the cells were washed 2 times with phosphate buffered saline (PBS). Huh7.5 cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature. Cell monolayers were permeablized with a solution of PBS/0.2% Triton X-100 for 15 minutes at room temperature, washed with PBS, and then incubated with 10% fetal bovine serum in PBS for 10 minutes. After rinsing with PBS, cells were incubated with a human antiserum specific for HCV  for 1 hour, washed three times with PBS, then incubated for 1 hour with donkey anti-human-HRP secondary antibody. Cells were washed 3 times with PBS, then immunoreactive cells were visualized using the Vector VIP substrate kit for peroxidase (Vector Laboratories) following the manufacturer's instructions. Cells were allowed to dry and focus forming units were counted to determine HCV titers in the cell supernatants. All conditions were conducted in triplicate.Huh7 cells were plated on 48-well plates and incubated for 12–24 hours at 37°C. Cells were transfected with 350 ng RNA using the in vivo RNA transfection reagent (Altogen Biosystems), and were euthanized 8 hours later for comparative measurement of mRNA and protein expression. Following systemic PBS perfusion to remove contaminating blood cells, mouse livers were recovered and fixed in 4% formalin solution for 24 hours and immunohistochemistry staining for mouse ISG54  was performed as described Mouse experiments and procedures were performed with approval from the University of Washington Institutional Animal Care and Use Committee. C57BL/6 mice were transfected via intraperitoneal injection with 200 µg RNA using a lipid-based IFN-β, CCL5, Ifit2, ISG15, and GAPDH were purchased from SABiosciences. Results were normalized to the expression of mouse GAPDH mRNA.Mouse liver sections were collected following systemic PBS perfusion and soaked in RNAlater reagent (Ambion). Liver sections were homogenized and RNA was extracted using the RNeasy kit (Qiagen). Synthesis of cDNA was conducted using the iScript select cDNA synthesis kit (Bio-Rad) with both oligo(dT) and random primers following the manufacturer's instructions. One-step real-time quantitative PCR was performed with SYBR Green master mix (Applied Biosystems) using an ABI PRISM 7300 Real-Time PCR System. Gene specific primers for mouse Figure S1RNA gel images. A) In vitro transcribed RNAs were visualized on denaturing 8 M urea polyacrylamide gels. RNA was stained using SYBR Gold nucleic acid stain. B) Full-length HCV JFH1 RNA was visualized on a denaturing formaldehyde agarose gel. RNA was stained using ethidium bromide.(TIF)Click here for additional data file.


C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function.The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom.Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins. C. elegans unc-76. The 376 and 385 amino acid containing isoforms of the UNC-76 protein arise by alternative splicing of the same gene and showed no strong similarity to any previously characterized proteins, but similar human sequences were identified in the expressed sequence tag database, dbEST FEZ1 and FEZ2.The FEZ (fasciculation and elongation protein zeta) family designation was first purposed by Bloom and colleagues in 1997 by genetic analysis of unc-76 function is necessary for normal fascicle structure and is required specifically for axon-axon interactions in C. elegans. The human FEZ1 gene was able to restore partial locomotion and axonal fasciculation in the C. elegans unc-76 mutants in germ-line transformation experiments, indicating that both the function and the structure of the FEZ proteins have been conserved in evolution Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of phenotypes observed in kinesin mutants and thereby suggesting that UNC-76 is required for kinesin-dependent axonal transport The C. elegans UNC-76, 45 interactions for human FEZ1 and 17 interactions for human FEZ2 according to BIND, HPRD, BioGRID data and previous research published by our group The homologous proteins UNC-76, FEZ1 and FEZ2, share a conserved predicted coiled-coil region at their C-terminal regions unc-76 to FEZ1 and FEZ2 is predicted to have occurred during the two rounds of whole-genome duplication in the chordate ancestral line, after the divergence of cephalochordates but before the splitting of the teleosts and tetrapods taxa.Here, we report that nucleotide sequences belonging to the FEZ family can be found in many eukaryotic species, but that most of the putative homologs are not only functionally uncharacterized but even remain unrecognized as FEZ family members in the databases. Our phylogenetic analysis of the FEZ family of proteins indicates that the ancestral gene of the FEZ family is found only in the animal kingdom. The gene duplication of C. elegans UNC-76 (248–372), human FEZ1 (221–392) and human FEZ2 (207–353) as baits and human prey clones from cDNA libraries demonstrated that the pattern of protein-protein interactions (PPIs) is highly conserved between UNC-76 and FEZ1. These orthologues did however not interact with 19 proteins that interacted specifically with FEZ2, although FEZ2 interacted with all 40 proteins that interact with FEZ1/UNC-76. In summary, we found largely overlapping PPI patterns for FEZ1 and UNC-76 and extended PPIs for FEZ2. Interestingly, our data provide an explanation for the ability of FEZ1 to rescue the defects caused by unc-76 mutations in nematodes and to the lack of strong defects in FEZ1 deficient mice, since the continuous presence of the ubiquitous expressed paralogue FEZ2 may be able to compensate lack of FEZ1 because its PPI pattern contains all the interactions found for FEZ1.The most conserved region in FEZ proteins is its C-terminus, and we and others showed previously that this region is involved in the association with other proteins C. elegans UNC-76 sequence were performed at the NCBI Web site http://www.ncbi.nlm.nih.gov/BLAST/ using the non-redundant protein sequence database available at August 16, 2009. After six rounds of iteration, all UNC-76, FEZ1 and FEZ2 orthologs with an E-value of 0.005 or below were selected and all redundant sequences were excluded (except to B. floridae). All sequences were collected in FASTA format for further analysis as shown in http://www.ebi.ac.uk/Tools/clustalw2/index.html at default settings. The alignments were then shaded using the multiple sequence alignment editor GENEDOC http://www.nrbsc.org/gfx/genedoc/index.html. The identity and similarity were calculated using default settings by NPS@: Network Protein Sequence Analysis trough the alignments of human proteins FEZ1 and FEZ2 and C. elegans UNC-76 protein  Protein Psi-Blast http://mobyle.pasteur.fr/cgi-bin/portal.py?jobs=http://mobyle.pasteur.fr/data/jobs/protpars. Parsimony analyses were performed using the protein alignment as input. Bootstrap values were obtained by using SEQBOOT and creating 1000 Bootstrap data sets. Analysis was then performed using PROTPARS with standard parameters. The “M” option was invoked for the analysis of the multiple data sets generated by SEQBOOT.PHYLIP version 3.5c C. elegans UNC-76 (NM_074311.3) was PCR-amplified using a specific primer set  as well as the template plasmid pSU001, that had been generously provided by Dr. Hengartner EcoRI and SalI restriction sites into the vector pBTM116. The orientation, frame, and correctness of sequence of each insert DNA was confirmed by restriction endonuclease analysis and automated DNA sequencing. These are no new cell lines but only cDNA clones obtained by in vitro experiments as described.The plasmid constructions of pBMT116-FEZ1 (221–392) (NM_005103.3) and pBTM-FEZ2 (207–353) (NM_005102.2) have been described previously Using FEZ1 as a bait in a screening, Assmann et al. (2006) C. elegans UNC-76 (248–372) as additional baits fused to LexA in vector pBTM116, as described above and previously Yeast two-hybrid assays were performed with human FEZ1 (221–392) and +, Trp+, and His+) were transferred onto nylon membranes, permeabilized in liquid nitrogen, and placed on Whatman 3 MM paper previously soaked in Z buffer  containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside . After incubation at 37°C for 1 h, the yeast cells forming dark blue colonies were taken from replica plates for further analysis (primary screen).beta-Galactosidase activity in yeast cells was determined by the filter assay method. Yeast transformants  and at the C-terminus (unc-76 gene duplication (see details below), suggesting that new nuclear functions may have been acquired in FEZ1 afterwards. Although there is no striking NLS in FEZ2 sequences, they contain a conserved polybasic region (e.g. KKKKK) with conserved amino acid substitutions in the FEZ1 related NLS. The NLS is less well conserved in all UNC-76 sequences.A database of sequences judged to be members of the FEZ protein family was compiled  section.terminus . The latFEZ1 and FEZ2 genes originated from the ancestral unc-76 by gene or whole genome duplications before the radiation of the Actinopterygii but after the divergence of the amphioxus branch and hence concomitant origin of the chordata. In fact, according to an amphioxus-human synteny analysis by Putnam unc-76 gene seems to have duplicated in the chordate stemline into FEZ1 and FEZ2 genes.A phylogenetic tree  was geneC. elegans protein UNC-76, which may have preserved ancient features.So far, only interactions for C-terminal of FEZ proteins are described, as by our screenings using C termini of FEZ proteins , as well as by other publications describing FEZ proteins recovered as prey in other screens  HIS3 and LacZ reporters were sequenced. 59 different proteins were identified using FEZ2 (207–353) as a bait  in a direct one-on-one analysis against all 59 FEZ2 interacting proteins , Table 1C. elegans UNC-76 seemed to be mostly conserved since the divergence of the deuterostomes (represented by the human FEZ1) from the “protostomes” (represented by C. elegans UNC-76), which occurred around 650 million years ago.To our great surprise, the pattern of interaction of UNC-76, in relation to the proteins identified in the screening assay of two-hybrid and FEZ2 as bait, was almost identical to that observed for FEZ1 , Fig. 3.In this context it is worthwhile to differentiate between nuclear and cytoplasmic proteins that interact with the FEZ family proteins. A significant proportion of the interacting proteins are nuclear or predicted to be nuclear . Among these 18 nuclear proteins identified in the screen with FEZ2, a high proportion of 15 (ca. 83%) also interacted with FEZ1 . This maIn 2000, Walhout and collaborators Three different fates have been proposed after the duplication of a genes In this context it is also interesting to analyze the origin of the proteins fished with the FEZ2 bait from either bone marrow or fetal brain cDNA libraries and to correlate this with the supposed expression patterns  and function  . Of the C. elegans UNC-76 interacted also with a large number of proteins. Genome-wide studies have shown that deletions of hub protein encoding genes are three times more likely to be lethal than deletions of non-hubs, a phenomenon known as the centrality-lethality rule C. elegans mutations in the unc-76 gene, which although not lethal, cause deleterious abnormalities in the elongation of neuronal axons along other axonal surfaces  during the animals development, which results in a “paralyzed” phenotype, where the worm has severly reduced body movements (therefore the designation unc for uncoordinated movements). These abnormalities can be complemented by the expression of human FEZ1 The large number of proteins that were identified to interact FEZ1, led to its classification as a hub protein C. elegans genome, which contains only unc-76. The duplication of the unc-76 gene occurred most likely in the stemline of the chordates . The reasons why both are maintained may be manifold, including dosage effects or a promoter specific regulation that must be maintained in the neurons (FEZ1). Data from the literature suggest that FEZ1 expression is increased upon NGF treatment, a neuronal tissue specific factor Many of the proteins identified by our screen with FEZ2 interact with proteins that also interact with each other, increasing therefore the likelihood that these are true interactions of biological relevance. We like to highlight here the interactions of pleiotrophin (PTN), protein tyrosine phosphatase, receptor type S (PTPRS) (Retinoic acid receptors (RARs) are transcriptional activators which are activated by binding of their ligands and act in concert with a combination retinoid X type receptors . Even in the absence of ligand, RAR-RXR heterodimers bind to DNA sequences known as response elements for RA, but in this state they recruit co-repressors. These co-repressors mediate the negative modulation of transcription by recruitment of the histone deacetylase complex and the transfer of methyl radicals to the DNA bound histones, thereby stabilizing the nucleosome. The binding of the ligand retinoic acid causes however conformational changes in the binding domain of RAR, which in return promote the release of the co-repressors and the recruitment of co-activators. While some co-activators interact with factors of the basal transcription machinery, others induce chromatin remodeling and specific transcriptional activation. Data from our previous two-hybrid and pull-down assays confirmed the interaction of FEZ1 and FEZ2 proteins related to activation of transcription (BAF60a) and the repression of transcription (SAP30L) unc-76 mutations in nematodes, since the PPI pattern of FEZ1 and UNC-76 are highly similar. Furthermore, no strong defects are caused in mice lacking the FEZ1, probably due to the compensatory presence of its paralogue FEZ2, which interacted with all FEZ1 interacting proteins, and additional new interactors that are FEZ2 specific. Our new data will hopefully stimulate and facilitate further studies on the functional role of proteins of the FEZ family in the development and function of both neuronal and non-neuronal cells. Furthermore, studies such as ours may contribute to our ever growing understanding of how protein networks function and specially how they came about and were modified and adapted during evolution. In the future we may devise new and more complete ways to visualize and comprehend how these networks were shaped throughout time.In summary, we have described here the molecular expansion of the FEZ protein family in the chordate stemline, most likely after two rounds of whole-genome duplication. We have identified many new members of the FEZ family from different species and a highly conserved C-terminal domain, which is responsible to mediate the majority of the protein-protein interactions. Our analyses indicate that the ancestral UNC-76 protein function and protein interaction profile is surprisingly conserved in animals, evolutionary distinct as vertebrates and nematodes. The analysis of the phylogeny and the detailed protein interaction profiles uncovered a likely functional divergence between FEZ1 and FEZ2, since FEZ2 acquired in respect to FEZ1/UNC-76 additional new interactors and is ubiquitously expressed and not restricted to neuronal cells, as is FEZ1/UNC-76. Our data provide an explanation for the ability of human FEZ1 to rescue the defects caused by Figure S1Amino acid sequence alignment of the members of the FEZ family. Names of the sequences are given in the (PDF)Click here for additional data file.Figure S2Identities and similarities between human proteins FEZ1 and FEZ2 and C. elegans UNC-76. A general scheme of the FEZ family proteins is shown at the top. The identity and similarity comparisons were made of two-by-two proteins both for the complete protein alignment as well as for local alignment of FEZ fragments by NPS@ . I = identity, S = strongly similar, W = weakly similar, D = different.(TIF)Click here for additional data file.Table S1FEZ family proteins used for amino acid sequence alignment.(DOC)Click here for additional data file.


The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium. Electron microscopy of packaged HIV-1 genomic RNAs revealed that the two RNA molecules are strongly associated with each other through their 5′ ends, which are termed as dimer linkage structure et. al. proposed the existence of various stem loop motifs and the genomic RNA associated with infected cells in the dimerization and packaging competent BMH form, and they argued against the existence of the LDI conformer gag translation initiation region. The U5-AUG and SL1 regions appear to play an important role in dimerization Secondary structure of 5′-UTR of HIV-1 genomic RNA lead a riboswitch model in which extensive structural rearrangements of leader sequence could influence the presentation of stem-loop 1 (SL1), polyA hairpin, other RNA signals and thus dimerization, and template switching efficiency The retroviruses can use template switching to repair a break or damage in the RNA templates, a process known as forced copy-choice recombination et.al. found that most of the U5/L stem-loop is not required, only 10 bases within the lower SL1 stem-loop is required for complete RNA dimerization et al. suggested that transfer efficiency was severely compromised upon deletion of the DIS hairpin region from the UTR sequence and was enhanced when this element was inserted within a non-UTR sequence in vivo and that a region far from the DIS/DLS affected dimer formation in the retrovirus genome. Thus, there are many discrepancies between the in vitro and in vivo data on HIV-1 RNA dimerization Dimerization of retroviral RNA is a prerequisite to viral encapsidation/packaging Antisense and sense oligonucleotide have been previously been shown to inhibit HIV-1 RNA dimerization In the present study for the first PNAs were designed and synthesized in order to elucidate the importance of SL1, U5-AUG and PolyA Hairpin in dimerization and template-switching processes and the effect of these PNAs on the equilibrium between two alternative conformations. Our data reveal the importance of SL1 and AUG region as a potential target for antisense PNA as we observed strong inhibitory effect on dimerization and template-switching processes, whereas PNAs targeted against U5 region and PolyA hairpin signal showed little or no inhibition. Based on the results of our study, we suggest that the interference of HIV-1 leader RNA dimerization and template switching processes can be augmented by the use of antisense PNAs.All antisense PNA oligomers listed in The concentrations of PNAs were calculated on the basis of absorbance from the molar extinction coefficient of the nucleobases. The antiparallel PNA: RNA and PNA: DNA duplexes were prepared in 10 mM sodium phosphate buffer, pH 7 containing NaCl (100 mM) and EDTA (0.1 mM) and annealed by keeping the samples at 95°C for 5 min and followed by slow cooling to room temperature . Absorbance versus temperature profiles were obtained by monitoring at 260 nm with a UV-vis spectrophotometer scanning from 25 to 90°C temperature at a ramp rate of 0.2°C/minute. The measurements were performed in a 1-cm path length quartz cuvette containing PNA: RNA duplex at 2.5 µm concentration. Similar experiments were performed with PNA: DNA duplex to compare affinity of PNA with DNA. The melting studies were done on Cary 300 UV-Visible Spectrophotometer.A TCC TAA TAC GAC TCA CTA TAG GGA GGT CTC TCT GGT TAG ACC A 3′) and downstream primer GR (5′ GCC CAT ACT ATA TGT TTT AAT 3′), containing a T7 promoter directly upstream of the +1 position of the wild type HIV-1 transcript and ends to the 5′ end of the gag gene (position +1 to 447 relative to the transcription start site +1). PCR reaction was separated on 1% agarose gel and purified using the QIAEX II Gel Extraction Kit (Qiagen). In vitro transcription was carried out using MAXI script T7 transcription kit (Ambion). The radiolabeled transcripts were synthesized by using (α-32P) UTP (1µl) (BARC India) and were purified on 4% denaturing TBE polyacrylamide gels. The RNA bands were excised and quantified by either UV-absorbance measurements or scintillation counting.The DNA template (474 bp) for transcription was made by PCR amplification of the plasmid pYU-2 (5′) using upstream primer T7F (5′ GGTAA TAC GAC TCA CTA TAG GGA GGT CTC TCT GGT TAG ACC AA TCC  3′) and antisense primer T7R (5′ CTC TCT CCT TCT AGC CTC CGC 3′) in Taq Buffer (10 mM Tris-HCl (pH 9), 1.5 mM MgCl2, 50 mM KCl and 0.01% gelatin) under the following temperature conditions – (94°C - 4 min)×1, ×40, (72°C for 10 min)×1 and finally at 4°C for ∞. Donor construct was prepared by PCR amplification of the +90−447 sub-region  of 5′UTR including gag gene of HIV-1using the primer set BR (5′ GTG CTT TAA GTA GTG TGT GCC 3′) and GR (5′ GCC CAT ACT ATA TGT TTT AAT 3′)  of 5′UTR of HIV-1 using sense primer T7F (5′ GG AAT 3′)  in Taq b2) with varying concentration of antisense PNA oligomers and the samples were then analyzed on native gels containing 0.25X TBM buffer (25 mM Tris-HCl pH 8.9 and 22.5 mM borate with 5 mM MgCl2) and run at 150 V at 4°C. All quantifications were performed using a phosphor imager. The composition of monomer buffer is 50 mM Na-cacodylate pH 7.5, 250 mM KCl.Dimerization assays were performed using a radiolabeled RNA template concentration of ∼2pmol. The RNA template was first incubated at 65°C for 10 min and then at 37°C for 30 min in 10 µl of dimerization buffer , formamide buffer , dimerization buffer 50 mM Na-cacodylate  and then analyzed on a 4% non-denaturing polyacrylamide 0.25X TBE gels and run at 120 V at 4°C. For conformer assays, the radiolabeled HIV-1 RNA transcript (∼2pmol) was incubated at above mentioned conditions in 10 µl of TEN buffer with increasing concentration of PNA oligomers and then analyzed in 4% non-denaturing polyacrylamide 0.25X TBE gels.Approximately 2 pmol of radiolabeled RNA template was heated at 65°C for 10 min, slowly cooled to 37°C and kept for further 30 min at this temperature in 10 µl of Tris buffer , TN buffer , TN buffer with 0.1 mM, 1 mM or 5 mM MgClGCC CAT ACT ATA TGT TTT AAT 3′) was labeled at 5′ end by incubating at 37°C for 30 min with T4 polynucleotide kinase and (γ-32P) ATP in the reaction buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM DDT, 1 mM spermidine and 0.1 mM EDTA). The radiolabeled primer (GR) was heated with donor and acceptor transcripts at a molar ratio of 1∶2:10 at 85°C for 2 min and then snap-cooled on ice. RNA (100 nM) with increasing concentration of anti-sense PNA oligomers (1 to 20 pM) were added to 20 µl dimerization buffer and incubated at 65°C for 10 min and then cooled to 37°C and maintained for further 30 min. Reverse transcription was carried out by incubating above reaction mixture at 10 nM of RNA concentration, at 37°C for 30 min in a mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 100 µM concentrations of each dNTP, and 0.2 U of HIV-1 RT/µl. The reaction was terminated by the addition of 10 mM EDTA. The samples were analyzed by running at 150 V on a 6% denaturing polyacrylamide TBE gels. TBE gel bands were quantified and the template-switching efficiency of each PNAs was calculated.The efficiency of heterodimerization between the donor and acceptor RNA templates was performed by incubating radiolabeled donor transcript with increasing molar ratio of unlabeled acceptor transcript in dimerization buffer and analyzed on 4% native TBM gel. For template-switching assay, Primer GR  and 80.5°C (Δ Tm 16.9°C) with RNA. Similarly the other PNAs namely, 98, 102, 251 and 332 also showed thermal stability with corresponding RNA i.e.; 83.9°C, 82.8°C, 84.8°C, 81.4°C respectively, than the corresponding PNA: DNA duplexes . PNA 73 and PNA 110 also have stabilizing effect with corresponding PNA than the DNA  in melting temperature of duplexes (data not shown).The thermal melting studies were done to show the binding affinity of synthetic PNA to RNA and DNA. The T the DNA . The sam2 at 65°C with varying concentration of PNA oligomers  transcript, RNA was incubated in different buffers then analyzed on TBM . Based oligomers  & Fig. 6ligomers . In contligomers . PNA251 ligomers . PNA237&in vitro . PNA332ein vitro , whereasin vitro .The schematic representation of both donor and acceptor RNA templates is shown in The combined dimerization and template-switching assay showed varying degrees of PNA inhibition of dimerization; PNA237, 251 and 271 exhibited an inhibitory effect on template-switching efficiency .Maximum inhibition was observed with PNA251, followed by 271 and 237 respectively . PNA332 2. These results indicate that all the PNA oligomers (Table1), except PNA332 denature the LDI conformation and shift the equilibrium towards the BMH conformation .Although we cannot correlate the melting temperature of oligomers to their effect on conformational switching leading to inhibition of dimerization but, thermal stability measurements of duplexes demonstrate the target selectivity of PNA to RNA over DNA, showcasing PNA an efficient nucleic acids based therapeutic agent.The antisense oligonucleotide offers an attractive concept, based on the complementarity to a unique sequence on the RNA target, leading to the interference with a given function by “hybridization arrest” PNA73 & 98, target the exposed loop and lower stem of poly A hairpin respectively, destabilized LDI conformation at higher concentration (up to 20 molar excess) . An intein vitro conditions in vitro dimerization of HIV-1 RNA by these three PNAs was observed to be in the following order: PNA251>PNA271>PNA237  and 331 -344 (AUG) duplex in infected cells in vivo, which clearly support our results (14). Nucleotide resolution SHAPE data also did not confirm the in vitro models of RNA and suggested that dimerization in mature viruses occur through formation of intermolecular duplexes (28), neither through TAR stem nor through intramolecular base pairing in DIS stem. Sakuragi et. al. proposed a novel HIV-1 dimerization linkage sequence (DLS) structural model, showing a thermodynamically stable pseudoknot like structure having a unique shape with new stem like duplex (GCGUC –GACGC) and alongwith it U5 : AUG duplex sunken in this structure The coupled dimerization and template-switching assay demonstrated that PNA237, 251 and 271, targeted against the DIS hairpin, reduced template-switching and dimerization with similar potencies. Inhibition is presumably due to annealing of these PNAs to their complementary sequences within the DIS hairpin region and blockage of initiation and maturation of loop-loop kissing complex, which was necessary for the formation of a stable dimer complex between two RNA molecules and consequently inhibit the strand transfer during reverse transcription. Therefore, our results confirmed a direct relationship between the efficiency of template-switching and the stability of DIS-mediated kissing-loop complex in vitro . It was et al. and Palliart et al. did not observe long distance interacting conformers in the RNA isolated from the virion but they also did not rule out possibility of a preform which melt to produce dimerization competent form. The disappearance of long distance interacting conformers may be attributed to the more pronounced BMH form which is dimeric and packaging competent, obtained in in vivo conditions, though the former form cannot be neglected in the minor population or for short period of time. The ex vivo structure of 5′ UTR is not thermodynamically stable which shows that LDI conformer is kinetically unfavorable for reverse transcription of RNA (14). We also demonstrated that SL1 or DIS is necessary for initiation and maturation or stabilization of loop-loop kissing complex during the dimerization process and hence template switching process. We may conclude that PNAs can be used as a tool to unravel and correlate the various conformationally controlled viral processes, thereby providing a potent antisense molecule for efficient attenuation of emergence of virulent HIV strains.Based on all above mentioned results, we may conclude that HIV-1 leader RNA adopt two alternative conformations i.e.; LDI and BMH and the switch between these two conformations regulates some of the leader- encoded functions such as RNA dimerization Figure S1Panel A. A1: UV absorbance of PNA 73 with antiparallel RNA, A2 corresponding first derivative, A3 UV absorbance of PNA 73 with antiparallel DNA, A4 corresponding first derivative. Panel B. B1: UV absorbance of PNA 98 with antiparallel RNA, B2 corresponding first derivative, B3 UV absorbance of PNA 98 with with antiparallel DNA, B4 corresponding first derivative. Panel C. C1: UV absorbance of PNA 102 with antiparallel RNA, C2 corresponding first derivative, C3 UV absorbance of PNA 102 with antiparallel DNA, C4 corresponding first derivative. Panel D. D1: UV absorbance of PNA 110 with antiparallel RNA, D2 corresponding first derivative, D3 UV absorbance of PNA 110 with antiparallel DNA, D4 corresponding first derivative.Melting profiles of PNA with corresponding RNA and DNA duplexes. (TIF)Click here for additional data file.Figure S2Melting profiles of PNA with corresponding RNA and DNA duplexes. Panel A. A1: UV absorbance of PNA 237 with antiparallel RNA, A2 corresponding first derivative, A3 UV absorbance of PNA 237 with antiparallel DNA, A4 corresponding first derivative. Panel B. B1: UV absorbance of PNA 251 with antiparallel RNA, B2 corresponding first derivative, B3 UV absorbance of PNA 251with antiparallel DNA, B4 corresponding first derivative. Panel C. C1: UV absorbance of PNA 271 with antiparallel RNA, C2 corresponding first derivative, C3 UV absorbance of PNA 271 with antiparallel DNA, C4 corresponding first derivative. Panel D. D1: UV absorbance of PNA 332 with antiparallel RNA, D2 corresponding first derivative, D3 UV absorbance of PNA 332 with antiparallel DNA, D4 corresponding first derivative.(TIF)Click here for additional data file.Figure S3Gel electrophoresis to evaluate the effect of PNA oligomers on the template-switching efficiency between the acceptor and donor HIV-1 transcripts: Acceptor, donor transcripts and radiolabeled GR primer  were incubated in the dimerization buffer with increasing molar excess of respective PNA oligomer, as indicated at the top of each gel. This reaction mixture was used for the next template-switching efficiency assay, for details see (TIF)Click here for additional data file.