Abortive Initiation Research Articles

Transcriptomic HIV-1 reservoir profiling reveals a role for mitochondrial functionality in HIV-1 latency.

Identifying cellular and molecular mechanisms maintaining HIV-1 latency in the viral reservoir is crucial for devising effective cure strategies. Here we developed an innovative flow cytometry-fluorescent in situ hybridization (flow-FISH) approach for direct ex vivo reservoir detection without the need for reactivation using a combination of probes detecting abortive and elongated HIV-1 transcripts. Our flow-FISH assay distinguished between HIV-1-infected CD4+ T cells expressing abortive or elongated HIV-1 transcripts in PBMC from untreated and ART-treated PWH from the Amsterdam Cohort Studies. This flow-FISH method was employed to isolate CD4+ T cells expressing abortive or elongated HIV-1 transcripts from five ART-naïve PWH for transcriptomic analysis by 3' RNA sequencing. Supervised cluster analysis identified several differentially expressed mitochondrial genes in infected CD4+ T cells with abortive HIV-1 transcripts compared to cells containing elongated HIV-1 transcripts. Notably, enhancing mitochondrial function induced HIV-1 transcription in PBMC from PWH. Our data strongly suggests that cellular metabolism is involved in maintaining HIV-1 latency and show that improving mitochondrial functions induces HIV-1 transcriptional activity in PWH. These findings underline the relevance of metabolic regulation in HIV-1 infection, and support the development of strategies modulating immunometabolism to target viral latency.

Products of abortive transcription can prime synthesis of chimeric oligonucleotides

The aim of the study was to search for signal RNAs potentially utilized by bacteria for intercellular interactions. This was not limited to a certain category of regulatory RNAs, which are contained in large numbers in all domains of life. The search was performed using RNA-seq data obtained for a wild-type strain of E. coli and its mutant derivative (Δdps) lacking the gene encoding nucleoid protein Dps. This protein can bind RNAs and is found in membrane structures, which indicates the possibility of its participation in their secretion. Since the spectra of intracellular and secreted RNAs differed, it was assumed that the secreted RNAs are somehow selected for secretion. Therefore, in the RNA sets with Dps-dependent secretion, we searched for and found reads with non-template nucleotides at the ends, point nucleotide substitutions, insertions and deletions, as well as regularly detected chimeras. One chimera with a 9-mer GCCAAGGCG at the 5'-end and a transcript from the fimA-fimI intergenic region at the 3'-end was chosen for detailed study. It was found that the 9-mer is a product of abortive transcription from the antisense promoter inside the ravA gene. It is efficiently secreted and forms chimeras with many RNAs, including their 5'-ends and fragments shortened from this terminus. Analysis of the 5'-ends of the 9-mer partners in different chimeras revealed their coding sites in the genome, a feature of which was the ability to form stable secondary structures and therefore cause transcription stops or pauses. The discovery of different chimeras with the 9-mer at the 5'-end witnesses that they are formed as a result of primed synthesis rather than random ligation. The possible biological role of transcription primed by abortive products, whose regulatory role has been established for the first time, is discussed.

Products of abortive transcription can prime synthesis of chimeric oligonucleotides

The aim of the study was to search for signal RNAs potentially utilized by bacteria for intercellular interactions. This was not limited to a certain category of regulatory RNAs, which are contained in large numbers in all domains of life. The search was performed using RNA-seq data obtained for a wild-type strain of E. coli and its mutant derivative (Δdps) lacking the gene encoding nucleoid protein Dps. This protein can bind RNAs and is found in membrane structures, which indicates the possibility of its participation in their secretion. Since the spectra of intracellular and secreted RNAs differed, it was assumed that the secreted RNAs are somehow selected for secretion. Therefore, in the RNA sets with Dps-dependent secretion, we searched for and found reads with non-template nucleotides at the ends, point nucleotide substitutions, insertions and deletions, as well as regularly detected chimeras. One chimera with a 9-mer GCCAAGGCG at the 5'-end and a transcript from the fimA-fimI intergenic region at the 3'-end was chosen for detailed study. It was found that the 9-mer is a product of abortive transcription from the antisense promoter inside the ravA gene. It is efficiently secreted and forms chimeras with many RNAs, including their 5'-ends and fragments shortened from this terminus. Analysis of the 5'-ends of the 9-mer partners in different chimeras revealed their coding sites in the genome, a feature of which was the ability to form stable secondary structures and therefore cause transcription stops or pauses. The discovery of different chimeras with the 9-mer at the 5'-end witnesses that they are formed as a result of primed synthesis rather than random ligation. The possible biological role of transcription primed by abortive products, whose regulatory role has been established for the first time, is discussed.

Exploring the Impact of In Vitro-Transcribed mRNA Impurities on Cellular Responses.

Advances in mRNA technology have enabled mRNA-based therapies to enter a new era of medicine. Such therapies benefit from a single, standardized in vitro transcription (IVT) manufacturing process applicable to a wide range of targets. This process includes several downstream purification steps, which aim to eliminate impurities that potentially affect safety and efficacy. However, it is not fully understood which impurities are the most critical; hence, some efforts are still needed to establish the correlation between RNA impurities and their role in limiting therapeutic efficacy. To study this relationship, we produced in vitro-transcribed mRNAs using several bacteriophage T7 RNA polymerases, including one wild-type and four engineered variants. Important attributes of the mRNA such as integrity, purity, and functional activity were then measured using advanced physicochemical and cellular assays. For impurities including abortive transcripts, mRNAs containing partial poly(A) tails, and double-stranded (ds)RNA byproducts, structure-function relationships have been established by tracking cellular responses (i.e., protein expression, reactogenicity) in multiple cell models. By varying the T7 RNA polymerase, different levels of sense-antisense dsRNA byproducts were measured by mass photometry, contributing directly to immunological reactogenicity in bone marrow-derived dendritic cells. T7 RNA polymerase differences with regard to short (<20 nucleotides) 3'-loopback dsRNA byproducts were also further investigated using native mass spectrometry by precisely resolving these impurities at the nucleotide level. Overall, this study highlights the importance of developing sensitive and advanced analytical methods to characterize IVT mRNA impurities and understand their interaction with cellular machinery in order to ensure quality control of RNA-based therapies.

Effective Synthesis of mRNA during In Vitro Transcription with Fewer Impurities Produced.

The remarkable efficacy of COVID-19 vaccines has established mRNA as a highly promising biomedical technology. However, the adequate application of mRNA therapeutics necessitates additional measures to mitigate the inherent immunogenicity, which is predominantly caused by dsRNA. As a byproduct of the in vitro transcription of mRNA, dsRNA was reported to be originated through several distinct mechanisms, including the extension of 3' loop-back hairpins, the extension of hybridized abortive transcripts, and promoter-independent transcription. The intricate mechanisms involved pose a dilemma as the reduction in dsRNA results in a concomitant decrease in other critical quality attributes of mRNA. Here, we demonstrate that the promoter binding motifs of T7 RNA polymerase directly impact the production of promoter-independent transcription-based dsRNA. Specifically, the G753A mutation significantly reduces the formation of dsRNA byproducts, which can further combine with modified nucleotides to enhance the effectiveness of dsRNA mitigation and with previously reported high-integrity mutation K389A to minimize side effects. Accordingly, the present study reports a cost-effective approach to synthesize high-purity, less immunostimulatory mRNA by using an engineered T7 RNA polymerase mutant.

Potential and application of abortive transcripts as a novel molecular marker of cancers.

Abortive transcript (AT) is a 2-19 nt long non-coding RNA that is produced in the abortive initiation stage. Abortive initiation was found to be closely related to RNA polymerase through in vitro experiments. Therefore, the distribution of AT length and the scale of abortive initiation are correlated to the promoter, discriminator, and transcription initiation sequence, and can be affected by transcription elongation factors. AT plays an important role in the occurrence and development of various diseases. Here we summarize the discovery of AT, the factors responsible for AT formation, the detection methods and biological functions of AT, to provide new clues for finding potential targets in the early diagnosis and treatment of cancers.

Maternal Mortality and Morbidity Following Unsafe Abortion in a Tertiary Medical College Hospital

Background: Unsafe abortion refers to the termination of an undesired pregnancy by individuals lacking the requisite skills in an environment lacking minimal medical standards, or both. It stands as a prominent contributor to pregnancy-related mortality in Bangladesh. Notably, approximately one-third of all births in Bangladesh are unplanned and unwanted. The initiation of unsafe abortion may be attributed to the woman herself, an unqualified practitioner, or health workers operating in unhygienic conditions. Objectives: The primary aim of this study was to implement interventions aimed at mitigating maternal mortality and morbidity resulting from unsafe abortion. Materials and Methods: This cross-sectional study was conducted in the Department of Obstetrics and Gynecology at Khulna Medical College and Hospital, Khulna, spanning from July 2010 to June 2011. After applying exclusion criteria, which involved cases of medical termination of pregnancy and individuals with known medical conditions, a total of 145 abortion cases were admitted to two maternity units at Khulna Medical College Hospital. These cases exhibited a history of menstrual regulation (MR) and induced abortion performed by unskilled individuals, in unhygienic conditions, or both, throughout the study period. The primary outcome variables assessed in this study included age, marital status, education level, socio-economic status, parity, duration of amenorrhea, methods of induction, and maternal complications. Results: In this study, it has been seen that 588 (29.80%) of gynecological admissions are of abortion, and the incidence of unsafe abortion is 145 (24.65%) of all abortion patients. The majority are primarily educated and their socio-economic status is below average. Most of them are multipara. They had come to the hospital after the development of complications. Among the unsafe abortions, 9 (6.20%) expired and 136 (93.79%) improved after experiencing some sort of minor or major suffering. Conclusion: Maternal mortality is a key women’s health indicator. Reducing it is a global goal, but tracking progress is challenging, especially in developing countries with weak health data systems. Unsafe abortions are underreported, and many deny the truth even when in dire conditions. To address this, promoting modern contraceptives, training personnel for abortions, and emphasizing contraception’s importance is vital. Relaxing abortion laws may be necessary, as women may seek abortions regardless, risking their lives. Ensuring safe abortion access is essential for women’s rights and preventing harm to them and their families. Reducing unplanned pregnancies and providing safe abortion services are critical.

Detection of Naturally occurring abortive transcripts by Base-Stacking Hybridization Assisted Ligation and PCR amplification

Abortive transcripts (ATs) refer to nascent 2–10 nucleotides (nt) RNAs released by RNA polymerases before synthesizing productive RNAs. The quantitative detection of ATs is important for studying transcription initiation and the biological function of ATs; however, no method is available for the qualitative and quantitative assessment of such ultra-short oligonucleotides (typically shorter than 11 nt) in vivo at present, even with the LNA probes, the detection limit can only reach 11 nt. Here, we demonstrated the base stacking hybridization assisted ligation (BSHAL) technique, combined with TaqMan-MGB qPCR, can detect 4–10 nt ATs with a specificity of nucleotide resolution and a sensitivity of approximately 10 pM. By this technique, we detected endogenous ATs in cell lines, mice plasmas, and mice liver tissues, respectively, and proved that naturally occurring ATs do exist. We found that the 8 nt ATs of HMSB and Gapdh could be used as reference ATs for data normalization in Homo and mouse respectively, and 8 nt ATs of Afp and Gpc3 were suitable for use as plasma biomarkers of Hepatocellular carcinoma in mouse, indicate ATs are promising biomarkers. This study offers opportunities to study ATs and other ultra-short oligonucleotides in biological samples.

The RasGAP-associated endoribonuclease G3BP mediates stress granule assembly.

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149 whose dephosphorylation is induced by arsenite treatment. Critically, a non-phosphorylatable G3BP mutant (S149A) oligomerizes and assembles SG. These results suggest that G3BP is an effector of SG assembly and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.

Spontaneous HIV expression during suppressive ART is associated with the magnitude and function of HIV-specific CD4+ and CD8+ T cells

Spontaneous transcription and translation of HIV can persist during suppressive antiretroviral therapy (ART). The quantity, phenotype, and biological relevance of this spontaneously "active" reservoir remain unclear. Using multiplexed single-cell RNAflow-fluorescence in situ hybridization (FISH), we detect active HIV transcription in 14/18 people with HIV on suppressive ART, with a median of 28/million CD4+ Tcells. While these cells predominantly exhibit abortive transcription, p24-expressing cells are evident in 39% of participants. Phenotypically diverse, active reservoirs are enriched in central memory Tcells and CCR6- and activation-marker-expressing cells. The magnitude of the active reservoir positively correlates with total HIV-specific CD4+ and CD8+ Tcell responses and with multiple HIV-specific Tcell clusters identified by unsupervised analysis. These associations are particularly strong with p24-expressing active reservoir cells. Single-cell vDNA sequencing shows that active reservoirs are largely dominated by defective proviruses. Our data suggest that these reservoirs maintain HIV-specific CD4+ and CD8+ T responses during suppressive ART.

Processing and decay of 6S-1 and 6S-2 RNAs in Bacillus subtilis.

Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in Bacillus subtilis by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key-but mechanistically different-role of RNase J1. RNase J1 catalyzes 5'-end maturation of 6S-1 RNA, yet relatively inefficient and possibly via the enzyme's "sliding endonuclease" activity. 5'-end maturation has no detectable effect on 6S-1 RNA function, but rather regulates its decay: The generated 5'-monophosphate on matured 6S-1 RNA propels endonucleolytic cleavage in its apical loop region. The major 6S-2 RNA degradation pathway is initiated by endonucleolytic cleavage in the 5'-central bubble to trigger 5'-to-3'-exoribonucleolytic degradation of the downstream fragment by RNase J1. The four 3'-exonucleases of B. subtilis-RNase R, PNPase, YhaM, and particularly RNase PH-are involved in 3'-end trimming of both 6S RNAs, degradation of 6S-1 RNA fragments, and decay of abortive transcripts (so-called product RNAs, ∼14 nt in length) synthesized on 6S-1 RNA during outgrowth from stationary phase. In the case of the growth-retarded RNase Y deletion strain, we were unable to infer a specific role of RNase Y in 6S RNA decay. Yet, a participation of RNase Y in 6S RNA decay still remains possible, as evidence for such a function may have been obscured by overlapping substrate specificities of RNase Y, RNase J1, and RNase J2.

Social inequalities in utilization of a feminist telehealth abortion service in Brazil: A multilevel analysis.

The disruption caused by the COVID-19 pandemic on health services around the world boosted interest over telehealth models of care. In Brazil, where abortion is heavily restricted, abortion seekers have long relied on international telehealth services to access abortion pills. We conducted a cross-sectional multilevel study to assess the effect of individual and contextual social factors on utilization of one such service. For the individual-level, we analyzed data from the records of abortion seekers contacting this feminist international telehealth organization during 2019 (n = 25,920). Individual-level variables were age, race, education level and pregnancy length. Contextual-level units were states, for which we used data from the national Demographic Census and Household Surveys. Contextual-level variables were household income per capita, adjusted net school attendance rate, percentage of racialized women and income Gini Index. We fitted five multilevel Poisson Mixed-effects models with robust variance to estimate prevalence ratios (PR) of service utilization, which was defined as receiving abortion pills through the service. We found that only 8.2% of requesters got abortion pills through the service. Utilization was higher among women who were older, white, more educated and 5-8-weeks pregnant. Independently of this, service utilization was higher in states with higher income and education access, with lower proportions of racialized women, and located in the South, Southeast and Central-West regions. We concluded that while feminist telehealth abortion initiatives provide a life-saving service for some abortion seekers, they are not fully equipped to overcome entrenched social inequalities in their utilization, both at individual and contextual levels.

Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis

An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λPR promoter complexes. Strong position-dependences of overall rate constants (kcat/Km analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.

Mifepristone and Misoprostol for Undesired Pregnancy of Unknown Location.

To compare immediate initiation with delayed initiation of medication abortion among patients with an undesired pregnancy of unknown location. This retrospective cohort study used electronic medical record data from the Planned Parenthood League of Massachusetts (2014-2019) for patients who requested medication abortion with a last menstrual period (LMP) of 42 days or less and pregnancy of unknown location (no gestational sac) on initial ultrasonogram. Clinicians could initiate medication abortion with mifepristone followed by misoprostol while simultaneously excluding ectopic pregnancy with serial serum human chorionic gonadotropin (hCG) testing (same-day-start group) or establish a diagnosis with serial hCG tests and repeat ultrasonogram before initiating treatment (delay-for-diagnosis group). We compared primary safety outcomes (time to diagnosis of pregnancy location [rule out ectopic], emergency department visits, adverse events, and nonadherence with follow-up) between groups. We also reported secondary efficacy outcomes: time to complete abortion, successful medication abortion (no uterine aspiration), and ongoing pregnancy. Of 5,619 medication abortion visits for patients with an LMP of 42 days or less, 452 patients had pregnancy of unknown location (8.0%). Three patients underwent immediate uterine aspiration, 55 had same-day start, and 394 had delay for diagnosis. Thirty-one patients (7.9%), all in the delay-for-diagnosis group, were treated for ectopic pregnancy, including four that were ruptured. Among patients with no major ectopic pregnancy risk factors (n=432), same-day start had shorter time to diagnosis (median 5.0 days vs 9.0 days; P=.005), with no significant difference in emergency department visits (adjusted odds ratio [aOR] 0.90, 95% CI 0.43-1.88) or nonadherence with follow-up (aOR 0.92, 95% CI 0.39-2.15). Among patients who proceeded with abortion (n=270), same-day start had shorter time to complete abortion (median 5.0 days vs 19.0 days; P<.001). Of those who had medication abortion with known outcome (n=170), the rate of successful medication abortion was lower (85.4% vs 96.7%; P=.013) and the rate of ongoing pregnancy was higher (10.4% vs 2.5%; P=.041) among patients in the same-day-start group. In patients with undesired pregnancy of unknown location, immediate initiation of medication abortion is associated with more rapid exclusion of ectopic pregnancy and pregnancy termination but lower abortion efficacy.

Crosstalk between R848 and abortive HIV-1 RNA-induced signaling enhances antiviral immunity.

Pathogens trigger multiple pattern recognition receptors (PRRs) that together dictate innate and adaptive immune responses. Understanding the crosstalk between PRRs is important to enhance vaccine efficacy. Abortive HIV‐1 RNA transcripts are produced during acute and chronic HIV‐1 infection and are known ligands for different PRRs, leading to antiviral and proinflammatory responses. Here, we have investigated the crosstalk between responses induced by these 58 nucleotide‐long HIV‐1 RNA transcripts and different TLR ligands. Costimulation of dendritic cells (DCs) with abortive HIV‐1 RNA and TLR7/8 agonist R848, but not other TLR agonists, resulted in enhanced antiviral type I IFN responses as well as adaptive immune responses via the induction of DC‐mediated T helper 1 (TH1) responses and IFNγ+CD8+ T cells. Our data underscore the importance of crosstalk between abortive HIV‐1 RNA and R848‐induced signaling for the induction of effective antiviral immunity.

Illusion of Transcriptional Regulations by Rapid DNA Binding of a Regulator and Essential Requirement of Abortive Initiation for Regulations

Illusion of Transcriptional Regulations by Rapid DNA Binding of a Regulator and Essential Requirement of Abortive Initiation for Regulations

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes.

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase

Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome’s very 3’-end. The synthesis of short abortive leader transcripts (leaderRNAs) has been linked to transcription initiation for some NNS viruses. Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60–80. LeaderRNA synthesis requires hexamer phasing in the 3’-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3’-leader promoter. We further found different genomic 3’-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3’-leader promoter, i.e., at the second nucleotide of the genome 3’-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus.

Rapid preparation of 6S RNA-free B. subtilis σA-RNA polymerase and σA

The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σA-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σA-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also report expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.

Persistent HIV-1 transcription in CD4+ T cells from ART-suppressed individuals can originate from biologically competent proviruses

HIV-1 is able to persist in the face of potent antiretroviral therapy (ART). A number of strategies are being explored to allow ART-free viral remission or viral eradication. In order to gauge the progress of these strategies, assays with which to measure viral reservoir size and activity are needed. In a large percentage of aviremic individuals on suppressive ART, viral transcripts can be detected in peripheral blood CD4+ T cells. While this cell-associated RNA has been considered as a marker of viral reservoir activity, it is unclear whether cell-associated viral transcripts in aviremic individuals originate from biologically competent proviruses as opposed to being a product of abortive transcription from defective proviruses. We assessed whether cell-associated viral RNA in peripheral blood CD4+ T cells from aviremic individuals on ART originated from biologically competent proviruses. We demonstrate that cell-associated viral RNA transcripts were highly related to viral sequences obtained by ex vivo outgrowth. This relationship was also observed when viral transcription in the outgrowth cultures was limited to donor CD4+ T cells. Our study indicates that cell-associated viral RNA warrants further consideration as a viral reservoir surrogate in individuals on suppressive ART.