Reverse Transcription Process Research Articles

The Significance of the 2013 Nobel Prize in Chemistry and the Challenges Ahead

The Significance of the 2013 Nobel Prize in Chemistry and the Challenges Ahead

Single Molecule Study of HIV-1 Reverse Transcriptase Polymerization Activity in the Presence of NC

HIV-1 reverse transcriptase (RT) is a multifunctional polymerase, which synthesizes double-stranded proviral DNA from single-stranded viral RNA by catalyzing RNA- and DNA-dependent DNA polymerization and degrading RNA via its RNase H activity. Reverse transcription is an essential step in HIV-1 infection, and HIV-1 RT is the target of many anti-AIDs therapeutic drugs. HIV-1 nucleocapsid (NC) protein is a nucleic acid chaperone, which facilitates DNA duplex melting and re-annealing, and shows rapid protein-nucleic acid interaction kinetics. The effect of NC on the reverse transcription process is not fully understood. To gain insights into the polymerase activity of RT in the presence of NC protein, we use single molecule force spectroscopy to examine DNA polymerization activity of HIV-RT along long single-stranded DNA (ssDNA) templates with and without NC. Our preliminary observations show the polymerization activity of RT is dependent on the force on ssDNA templates; an increase in the force on ssDNA templates reduces the polymerization activity of HIV-RT. The observed exponential dependence of polymerization activity of RT with force on long ssDNA templates is consistent with previous single molecule studies, and NC appears to enhance the polymerization rate. Our studies will test the polymerization activity of RT in the presence of NC, allowing us to determine the biophysical mechanism by which NC enhances this activity.

RNA Structural Rearrangements during Reverse Transcription Initiation in HIV

Reverse transcription is the first step in the replication of the Human Immunodeficiency Virus (HIV) and is a target of multiple therapies. The initiation phase of reverse transcription in human immunodeficiency virus (HIV) is the slowest and least processive step of the reverse transcription process; however, premature initiation of reverse transcription leads to failed infection. During this phase, several structures between the viral genomic RNA and tRNA(Lys3) primer are formed and broken as reverse transcriptase transcribes viral genomic RNA into negative sense DNA. Using NMR spectroscopy, X-Ray crystallography, single molecule FRET spectroscopy, and biochemical methods, we are characterizing these RNA structural rearrangements in order to understand their role in regulating the initiation of reverse transcription in HIV.

Algorithms and Processing Pipeline For Error Correction and Detection Of Significant Mutations In The Kinase Domain Of BCR-ABL Analyzed By Next-Generation Sequencing: Implications For Clinical Practice Of Chronic Myeloid Leukemia

High sequencing depths of NGS may cause false-positive variant calls of minor subclones (up to 10%). Errors inserted into the NGS pipeline during sample preparation and sequencing manifest by erroneous detections of mutations including point mutations. Thus, there is a need for algorithms for filtering data which occur by error from relevant biological information.We developed algorithms and a processing pipeline for error correction and detection of significant mutations after NGS of BCR-ABL kinase domain (KD). We validated our algorithms on the retrospective NGS analysis of 135 samples from 15 CML patients in chronic phase (median 8 samples per patient; range 5-19), who developed resistant mutations (confirmed by Sanger Sequencing, SS) during 2-4 lines of therapy.Amplicon libraries were prepared using reverse transcription and 2-step PCR. The second PCR was performed partly using fusion-primers designed within the IRON-II study research consortium (Roche Applied Science) which tested 4 overlapping amplicons and partly using alternative in-house set of fusion-primers that we have developed upfront and which utilized 3 overlapping amplicons covering the KD coding region. Key concept of our error control algorithm was to apply statistics used for bacterial mutation rate prediction, Lea-Coulson probability distribution (Lea and Coulson, J of Genet 1949), to distinguish sequencing pipeline errors from biologically relevant mutations. We postulated that spontaneous mutations in bacteria are similar phenomena as enzyme errors in vitro. In both processes there are new generations of bacteria or transcripts in which mutations or errors replicate exponentially. The error rate distributions based on analysis of c-ABL kinase domain of healthy donors (n=24) were fitted to Lea-Coulson distribution. From this analysis we derived, for each type of single nucleotide substitution, estimated thresholds based on which a particular mutation may be called significant by a self-developed statistical test. We cross-checked our results with results of standard Roche pipeline including GS Amplicon Variant Analyzer.Table 1 summarizes the estimated thresholds to be applied for transitions and transversions. Higher frequency of errors was found in case of using a 3-amplicon assay in comparison to a 4-amplicon assay. The PCR products in the 3-amplicon assay are 71 bp longer on average than in the 4-amplicon assay, thus the error frequency distribution may be dependent on the length of the sequence amplified. Using our algorithm we processed NGS data and reported significant mutations. Overall, no significant mutation that caused resistance during the treatment was detected at the time of diagnosis. During 1st line imatinib treatment 10 resistant mutations in 9 patients were detected as significant 2-5 months earlier than by SS. At the time of therapy switchover, in 3 patients the algorithm already detected minor populations of one of significant mutations F317L, T315I and M351T, while SS did not. These mutations manifested after the therapy switchover and caused treatment failure. After the therapy switch, baseline mutations were still significantly detectable by our algorithm in NGS data, but not by SS in 7 patients who achieved at the time of the analysis PCgR and MMR. In 5 patients, who subsequently failed therapy after switchover, resistant mutations were significantly detected by our algorithm in NGS data 2-9 months earlier than by SS. New minor mutations were revealed by NGS after the therapy switch in 8 patients.Table 1TRANSITIONS (%)TRANSVERSIONS (%)P valueA/GG/AT/CC/TA/C+C/A+T/A+A/T+T/G+G/T+C/G+G/C3-amplicon assay0.0112.24.5311.84.770.570.053.031.032.931.100.134-amplicon assay0.015.171.934.501.930.130.051.200.431.030.430.03Since enzymes create errors during reverse transcription, 2-step PCR and sequencing process, the error correction is an essential part of the bioinformatics pipeline for relevant interpretation of BCR-ABL KD mutations detected with the highly sensitive NGS assay. Our validated algorithm and processing pipeline for significant mutation evaluations from NGS data is helpful for future clinical practice as it filters errors and allows reporting only significant mutations. This avoids false-positive results and misleading interpretations which may negatively influence treatment management of CML patients.Supported by IGANT11555 and NT13899 Disclosures:Machova Polakova:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:NOVARTIS, BMS(Consultancy and speaker bureau), PFIZER, ARIAD ( Consultancy): Consultancy, Speakers Bureau. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Klamova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1

Reverse transcription is an important early step in retrovirus replication and is a key point targeted by evolutionarily conserved host restriction factors (e.g., APOBEC3G, SamHD1). Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a major target of antiretroviral drugs, and concerns regarding drug resistance and off-target effects have led to continued efforts for identifying novel approaches to targeting HIV-1 RT. Several observations, including those obtained from monocyte-derived macrophages, have argued that ribonucleotides and their analogs can, intriguingly, impact reverse transcription. For example, we have previously demonstrated that 5-azacytidine has its greatest antiviral potency during reverse transcription by enhancement of G-to-C transversion mutations. In the study described here, we investigated a panel of ribonucleoside analogs for their ability to affect HIV-1 replication during the reverse transcription process. We discovered five ribonucleosides-8-azaadenosine, formycin A, 3-deazauridine, 5-fluorocytidine, and 2'-C-methylcytidine-that possess anti-HIV-1 activity, and one of these (i.e., 3-deazauridine) has a primary antiviral mechanism that involves increased HIV-1 mutational loads, while quantitative PCR analysis determined that the others resulted in premature chain termination. Taken together, our findings provide the first demonstration of a series of ribonucleoside analogs that can target HIV-1 reverse transcription with primary antiretroviral mechanisms that include premature termination of viral DNA synthesis or enhanced viral mutagenesis.

Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10(26) unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 10(19) distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3'end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

Frequency of intron loss correlates with processed pseudogene abundance: a novel strategy to test the reverse transcriptase model of intron loss

BackgroundAlthough intron loss in evolution has been described, the mechanism involved is still unclear. Three models have been proposed, the reverse transcriptase (RT) model, genomic deletion model and double-strand-break repair model. The RT model, also termed mRNA-mediated intron loss, suggests that cDNA molecules reverse transcribed from spliced mRNA recombine with genomic DNA causing intron loss. Many studies have attempted to test this model based on its predictions, such as simultaneous loss of adjacent introns, 3'-side bias of intron loss, and germline expression of intron-lost genes. Evidence either supporting or opposing the model has been reported. The mechanism of intron loss proposed in the RT model shares the process of reverse transcription with the formation of processed pseudogenes. If the RT model is correct, genes that have produced more processed pseudogenes are more likely to undergo intron loss.ResultsIn the present study, we observed that the frequency of intron loss is correlated with processed pseudogene abundance by analyzing a new dataset of intron loss obtained in mice and rats. Furthermore, we found that mRNA molecules of intron-lost genes are mostly translated on free cytoplasmic ribosomes, a feature shared by mRNA molecules of the parental genes of processed pseudogenes and long interspersed elements. This feature is likely convenient for intron-lost gene mRNA molecules to be reverse transcribed. Analyses of adjacent intron loss, 3'-side bias of intron loss, and germline expression of intron-lost genes also support the RT model.ConclusionsCompared with previous evidence, the correlation between the abundance of processed pseudogenes and intron loss frequency more directly supports the RT model of intron loss. Exploring such a correlation is a new strategy to test the RT model in organisms with abundant processed pseudogenes.

AN INSIGHT OF COTTON LEAF CURL VIRUS: A DEVASTATING PLANT PATHOGENIC BEGOMOVIRUS

Genome of plant viruses consists of either RNA or DNA. DNA viruses can be categorized into two types, (1) circular double-stranded DNA (dsDNA), these viruses are able to replicate through the process of reverse transcription from RNA (the caulimoviruses and badnaviruses), (2) viruses that have circular single-stranded DNA (ssDNA), which replicate through a dsDNA intermediate (the Geminivirus and nanoviruses). Begomoviruses are whiteflies transmitted geminiviruses and infect many economically important dicotyledonous crops including cotton, potato, tomato, cassava and chili. Begomoviruses cause leaf curl disease in cotton. In this review article, the Cotton Leaf Curl Virus (CLCuV) has been introduced systematically.

DYNAMICAL MODEL OF IN-HOST HIV INFECTION: WITH DRUG THERAPY AND MULTI VIRAL STRAINS

In this paper, a mathematical model for the effect of drug therapy on the in-host dynamics of HIV is considered and analyzed. As the process of reverse transcription is highly error prone, it causes mutation of virus which results in the emergence of drug resistant virus. This is also accounted in the model and corresponding model with both drug resistant and drug sensitive viral strains is studied. We found that, if reproductive ratios for both the strains are less than one, the virus population goes to extinction. If the reproductive ratio of either strain is greater than one and the reproductive ratio of drug resistant virus is smaller than that of drug sensitive virus then both the virus strains persist and infection is not cleared. However if reproductive ratio of drug resistant virus is greater than that of drug sensitive virus then the drug resistant virus out-competes the drug sensitive virus and only drug resistant virus survives. Hence the ratio of two reproduction ratios works as invading capacity threshold value for drug resistant strain. We also noted that by increasing the effective efficacy of the drug, virus may be cleared. Numerical simulations are performed to support and elaborate the analytical findings.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18-24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development, apoptosis, and cell cycle regulation. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes by binding to their target messenger RNAs (mRNAs). Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets. Methods used to investigate miRNA expression include SYBR green I dye-based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis. Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

Differential Effects of Vpr on Single-cycle and Spreading HIV-1 Infections in CD4+ T-cells and Dendritic Cells

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) contributes to viral replication in non-dividing cells, specifically those of the myeloid lineage. However, the effects of Vpr in enhancing HIV-1 infection in dendritic cells have not been extensively investigated. Here, we evaluated the role of Vpr during infection of highly permissive peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells and compared it to that of monocyte-derived dendritic cells (MDDCs), which are less susceptible to HIV-1 infection. Infections of dividing PBMCs and non-dividing MDDCs were carried out with single-cycle and replication-competent HIV-1 encoding intact Vpr or Vpr-defective mutants. In contrast to previous findings, we observed that single-cycle HIV-1 infection of both PBMCs and MDDCs was significantly enhanced in the presence of Vpr when the viral stocks were carefully characterized and titrated. HIV-1 DNA quantification revealed that Vpr only enhanced the reverse transcription and nuclear import processes in single-cycle HIV-1 infected MDDCs, but not in CD4+ T-cells. However, a significant enhancement in HIV-1 gag mRNA expression was observed in both CD4+ T-cells and MDDCs in the presence of Vpr. Furthermore, Vpr complementation into HIV-1 virions did not affect single-cycle viral infection of MDDCs, suggesting that newly synthesized Vpr plays a significant role to facilitate single-cycle HIV-1 infection. Over the course of a spreading infection, Vpr significantly enhanced replication-competent HIV-1 infection in MDDCs, while it modestly promoted viral infection in activated PBMCs. Quantification of viral DNA in replication-competent HIV-1 infected PBMCs and MDDCs revealed similar levels of reverse transcription products, but increased nuclear import in the presence of Vpr independent of the cell types. Taken together, our results suggest that Vpr has differential effects on single-cycle and spreading HIV-1 infections, which are dependent on the permissiveness of the target cell.

The Removal of RNA Primers from DNA Synthesized by the Reverse Transcriptase of the Retrotransposon Tf 1 Is Stimulated by Tf 1 Integrase

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process.

HIV-1 Polymerase Inhibition by Nucleoside Analogs: Cellular- and Kinetic Parameters of Efficacy, Susceptibility and Resistance Selection

Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.

TRNA Annealing onto the HIV-1 Genome Studied by FRET Spectroscopy and Microscopy

In the HIV life cycle, reverse transcription allows conversion of a single stranded genomic RNA into double stranded DNA, capable of integration. A prerequisite for the reverse transcription process is the formation of the initiation complex between the RNA genome and a host-cell tRNA that is used as a primer by reverse transcriptase. The nucleocapsid protein (NC) is involved in this process, which requires hybridization of the tRNA to the complementary primer binding site (PBS) sequence on the RNA genome. Besides the tRNA-PBS interaction, other interactions were proposed to play a role such as an 8-nucleotide motif in the U5 region of the untranslated leader RNA, named the primer activation signal (PAS). We investigate the tRNA annealing process and the presence of a tRNA-PAS interaction using fluorescence resonance energy transfer (FRET) spectroscopy and single molecule FRET microscopy. In our assay, fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the region of the HIV-1 genome in which the PBS sequence is located. We observe better folding of the RNA molecules in the presence of NC than with heat annealing and a large change in conformation of the RNA molecule upon tRNA annealing. Our results give further evidence that tRNA also interacts with the viral RNA PAS motif.

Recruitment of the Nuclear Form of Uracil DNA Glycosylase into Virus Particles Participates in the Full Infectivity of HIV-1

The HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the accuracy of reverse transcription. This role of Vpr was related to the recruitment of the nuclear form of the uracil DNA glycosylase (UNG2) enzyme into virus particles, but several conflicting findings have been reported regarding the role of UNG2 encapsidation on viral infectivity. Here, we report that the catalytic activity of UNG2 was not required for influencing HIV-1 mutation, and this function of UNG2 was mapped within a 60-amino-acid domain located in the N-terminal region of the protein required for direct interaction with the p32 subunit of the replication protein A (RPA) complex. Importantly, enforced recruitment of overexpressed UNG2 into virions resulted in a net increase of virus infectivity, and this positive effect on infectivity was also independent of the UNG2 enzymatic activity. In contrast, virus infectivity and replication, as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of either endogenous UNG2 or RPA p32. Taken together, these results demonstrate that incorporation of UNG2 into virions has a positive impact on HIV-1 infectivity and replication and positively influences the reverse transcription process through a nonenzymatic mechanism involving the p32 subunit of the RPA complex.

E17A mutation in HIV-1 Vpr confers resistance to didanosine in association with thymidine analog mutations

BackgroundHIV-1 accessory Vpr protein is involved in the reverse transcription process and has been shown to modulate the virus mutation rate. This process may play a role in the kinetics of appearance of drug resistance mutations under antiretroviral treatment. MethodsVpr sequences were analyzed from plasma viruses derived from 97 HIV-1-infected individuals failing antiretroviral treatment and 63 antiretroviral-naïve patients. Vpr genetic variability was analyzed for association with specific drug treatment and drug resistance mutations. Biological and virological experiments were employed to characterize a mutation in Vpr found to be associated with virological failure. ResultsE17A mutation located in the first α-helix of Vpr was more prevalent in HAART-treated individuals compared to untreated individuals. E17A was associated with thymidine analog mutations (TAMs) in reverse transcriptase M41L, L210W and T215Y and with the use of didanosine in the patients’ treatment histories. E17A had no impact on the biochemical and functional properties of Vpr, and did not affect kinetics of replication of wild-type or TAMs-containing viruses. However, its association with TAMs and the use of didanosine was consistent with phenotypic susceptibility assays showing a significant 3-fold decrease in didanosine susceptibility of viruses harboring Vpr E17A combined with TAMs compared to viruses harboring TAMs alone. ConclusionThese findings highlight a novel role of Vpr in HIV-1 drug resistance. Vpr E17A confers resistance to didanosine when associated with TAMs. Whether Vpr E17A facilitates excision of didanosine is still to be determined.

HIV-1 Replication in Monocyte-derived Dendritic Cells is Stimulated by Melarsoprol, One of the Main Drugs Against Human African Trypanosomiasis

Human African trypanosomiasis (HAT) is a disease caused by the protozoan parasite Trypanosoma brucei, the causative agent of sleeping sickness that is still endemic in well defined regions of sub-Saharan Africa. Co-infections with this human pathogen and human immunodeficiency virus (HIV) are not uncommon, but their potential interaction has been little studied. The organo-arsenical drug melarsoprol has been widely used for the treatment of late stage trypanosomiasis since the early 1950s and is still widely used despite very serious adverse effects. Because arsenic trioxide, another trivalent arsenical structurally related to melarsoprol, has been shown to markedly increase HIV replication in dendritic cells (DCs), we tested the effect of melarsoprol on virus replication in various primary human immune cell types, including DCs. We show here that this medicinal drug stimulates the replication of several strains of HIV-1, specifically in monocyte-derived DCs, and also renders such cells susceptible to HIV-2 infection. The drug acts mainly through an increase in the efficacy of the reverse transcription process, and this effect is mediated, at least partly, by an inhibition of expression of the cellular restriction factor APOBEC3G. These observations raise concerns about the harmful effect that melarsoprol might exert on the natural history of HIV in co-infected patients and on virus transmission.

Development of a Method to Control the RNA Extraction and Reverse Transcription Steps for the Detection of Dengue Virus Present in Human Blood Samples

Molecular biology techniques based on the detection of genomic sequences by reverse transcription combined with polymerase chain reaction (PCR) have enabled the detection of different RNA viruses in serum or plasma samples. Since the dengue epidemic outbreak declared in Argentina in 2009, numerous patients' samples were analyzed for the acute phase of infection. One of the main methodological drawbacks is the lack of internal control to measure the effectiveness of the viral extraction and reverse transcription process. In this article, we propose to standardize a molecular method to detect beta actin (β-Act) and glucose 6 phosphate dehydrogenase (G6PDH) complementary DNAs (cDNAs) present in patient's plasma/serum, as a control process. RNA extraction, reverse transcription, and PCRs for human G6PDH, β-Act, and the dengue virus genome were performed. cDNA fragments for β-Act and G6PDH were amplified for all samples, regardless of the presence or absence of viral RNA. Amplification of β-Act and G6PDH cDNAs can be used as a control for the extraction and reverse transcription processes during dengue virus detection. This could also be a useful method for controlling the above steps when infections caused by other RNA viruses are studied, even if another methodology is employed, such as real-time PCR.

Retroviral Restriction Factors TRIM5α: Therapeutic Strategy to Inhibit HIV-1 Replication

Tripartite motif protein 5-alpha (TRIM5α) is a cytoplasmic protein that efficiently recognizes the incoming capsid (CA) protein of retroviruses and potently inhibits virus infection in a species-specific manner. Through directly recognizing and interacting with HIV CA, TRIM5α is capable of disrupting the ordered process of viral uncoating, eventually interfering with HIV-1 reverse transcription and virus replication. TRIM5α protein contains four domains: RING domain, B-box 2 domain, coiled-coil domain, and B30.2 domain (SPRY) domain. All of the domains are necessary for efficient retrovirus restriction and the B30.2 domain has been shown to be the determinant of the specificity of restriction. Species-specific innate resistance against viral infections offers novel avenues for antiviral therapeutics. Various mutants of TRIM5α have been described which differently affect the HIV-1 reverse transcription process. This makes the establishment of new and improved models for HIV replication and AIDS pathogenesis by monitoring endogenous TRIM5α an attractive approach. TRIM5α-mediated restriction is modulated by the host protein Cyclophilin A (Cyp A) which could effectively interact with the CA of HIV-1. Here we will review the structure and roles of TRIM5α protein, the interaction between Cyp A and TRIM5α, as well as gene therapy strategies associated with TRIM5α to inhibit HIV-1 infection.

Initiation of HIV Reverse Transcription: Is Enzyme Flipping Required?

Liu and colleagues have recently studied dynamic changes in the orientation of HIV reverse transcriptase (RT) on its nucleic acid substrate during initiation of DNA synthesis. The authors employed a single molecule FRET assay and revealed the existence of an equilibrium between polymerase-competent and “flipped” polymerase-incompetent orientations. RT flipping correlates with enzyme pausing during initiation, while the transition to the processive elongation phase correlates with increases in the population of polymerase-competent complexes. The potential biological significance of these findings is discussed in this commentary in lieu of the entire process of reverse transcription.