Life changing response to successive surgical interventions on cranial venous outflow: A case report on chronic fatigue syndrome.

Recognition of similarities between chronic fatigue syndrome and idiopathic intracranial hypertension (IIH) has raised suggestions that they might be connected, with chronic fatigue syndrome representing a mild version of IIH, sharing many of its symptoms, but without the signature features of elevated intracranial pressure that characterize the complete syndrome. A further development of this idea factors in the effects of a cerebrospinal fluid leak, a known complication of IIH, to explain cases where symptoms seem out of proportion to the apparent physiological disturbance. Cranial venous outflow obstruction has been proposed as the pathological substrate. We describe a patient with multiple symptoms, including headache and disabling fatigue, in which this model guided investigation and treatment. Specifically, CT and catheter venography identified focal narrowings of both jugular and the left brachiocephalic veins. Treatment of brachiocephalic obstruction was not feasible. However, in separate surgical procedures, relief of jugular venous obstruction produced incremental and significant clinical improvements which have proven durable over the length of follow-up. We suggest that investigating chronic fatigue syndrome under this model might not only bring benefit to individual patients but also will provide new insights into IIH and its relationship with spontaneous intracranial hypotension.

A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors.

Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5' leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5-gag interaction and a more stable structure with a U5-AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5' region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.

HIV-1 Packaging Visualised by In-Gel SHAPE.

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA-protein complex. Here, we apply the in-gel SHAPE (selective 2'OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag-dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.

Evaluating RNA Structural Flexibility: Viruses Lead the Way.

Our understanding of RNA structure has lagged behind that of proteins and most other biological polymers, largely because of its ability to adopt multiple, and often very different, functional conformations within a single molecule. Flexibility and multifunctionality appear to be its hallmarks. Conventional biochemical and biophysical techniques all have limitations in solving RNA structure and to address this in recent years we have seen the emergence of a wide diversity of techniques applied to RNA structural analysis and an accompanying appreciation of its ubiquity and versatility. Viral RNA is a particularly productive area to study in that this economy of function within a single molecule admirably suits the minimalist lifestyle of viruses. Here, we review the major techniques that are being used to elucidate RNA conformational flexibility and exemplify how the structure and function are, as in all biology, tightly linked.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS - CoV-2.

Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3' proximal genome body sequence to a 5' untranslated leader sequence. This leads to a fusion between the common 5' leader sequence and a 3' proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

The Interplay between ESCRT and Viral Factors in the Enveloped Virus Life Cycle.

Viruses are obligate parasites that rely on host cellular factors to replicate and spread. The endosomal sorting complexes required for transport (ESCRT) system, which is classically associated with sorting and downgrading surface proteins, is one of the host machineries hijacked by viruses across diverse families. Knowledge gained from research into ESCRT and viruses has, in turn, greatly advanced our understanding of many other cellular functions in which the ESCRT pathway is involved, e.g., cytokinesis. This review highlights the interplay between the ESCRT pathway and the viral factors of enveloped viruses with a special emphasis on retroviruses.

Development of a Novel Competitive qRT-PCR Assay to Measure Relative Lentiviral Packaging Efficiency.

Third-generation HIV-1-derived lentiviral vectors are successfully used as therapeutic agents in various clinical applications. To further promote their use, we attempted to enhance vector infectivity by targeting the dimerization and packaging properties of the RNA transfer vector based on the premise that these two processes are tightly linked. We rationally designed mutant vectors to favor the dimeric conformation, potentially enhancing genome packaging. Initial assessments using standard assays generated outputs of variable reproducibility, sometimes with conflicting results. Therefore, we developed a novel competitive qRT-PCR assay in a co-transfection setting to measure the relative packaging efficiencies of wild-type and mutant transfer vectors. Here we report the effect of the dimerization-stabilizing mutations on infectious and physical titers of lentiviral vectors together with their packaging efficiency, measured using our novel assay. Enhancing dimerization did not automatically lead to better vector RNA packaging, suggesting that, for vector functionality, sufficient flexibility of the RNA to adopt different conformations is more important than the dimerization capacity. Our novel competitive qPCR assay enables a more stringent analysis of RNA packaging efficiency, allowing a much more precise understanding of the links between RNA structure, packaging, and infectious titers that will be invaluable for future vector development.

Therapeutic applications of trans-splicing.

BACKGROUND: RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein. SOURCES OF DATA: Comprehensive literature search of published research papers and reviews using Pubmed. AREAS OF AGREEMENT: Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction. AREAS OF CONTROVERSY: Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge. GROWING POINTS: Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies. AREAS TIMELY FOR DEVELOPING RESEARCH: Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.

DDX5 potentiates HIV-1 transcription as a co-factor of Tat.

BACKGROUND: HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein-protein interactions and RNA and ATP binding. RESULTS: We demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable. CONCLUSION: DDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1.

ESCRT-II functions by linking to ESCRT-I in human immunodeficiency virus-1 budding.

Human immunodeficiency virus (HIV) uses the ESCRT (endosomal sorting complexes required for transport) protein pathway to bud from infected cells. Despite the roles of ESCRT-I and -III in HIV budding being firmly established, participation of ESCRT-II in this process has been controversial. EAP45 is a critical component of ESCRT-II. Previously, we utilised a CRISPR-Cas9 EAP45 knockout cell line to assess the involvement of ESCRT-II in HIV replication. We demonstrated that the absence of ESCRT-II impairs HIV budding. Here, we show that virus spread is also defective in physiologically relevant CRISPR/Cas9 EAP45 knockout T cells. We further show reappearance of efficient budding by re-introduction of EAP45 expression into EAP45 knockout cells. Using expression of selected mutants of EAP45, we dissect the domain requirement responsible for this function. Our data show at the steady state that rescue of budding is only observed in the context of a Gag/Pol, but not a Gag expressor, indicating that the size of cargo determines the usage of ESCRT-II. EAP45 acts through the YPXL-ALIX pathway as partial rescue is achieved in a PTAP but not a YPXL mutant virus. Our study clarifies the role of ESCRT-II in the late stages of HIV replication and reinforces the notion that ESCRT-II plays an integral part during this process as it does in sorting ubiquitinated cargos and in cytokinesis.

A novel, sensitive dual-indicator cell line for detection and quantification of inducible, replication-competent latent HIV-1 from reservoir cells.

Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials.

A scale-free analysis of the HIV-1 genome demonstrates multiple conserved regions of structural and functional importance.

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 5' end of env.

HIV Silencing and Inducibility Are Heterogeneous and Are Affected by Factors Intrinsic to the Virus.

Transcriptionally silent HIV proviruses form the major obstacle to eradicating HIV. Many studies of HIV latency have focused on the cellular mechanisms that maintain silencing of proviral DNA. Here we show that viral sequence variation affecting replicative ability leads to variable rates of silencing and ability to reactivate. We studied naturally occurring and engineered polymorphisms in a recently identified exonic splice enhancer (ESEtat) that regulates tat mRNA splicing and constructed viruses with increased (strain M1), reduced (strain M2), or completely absent (strain ERK) binding of splicing factors essential for optimal production of tat mRNA resulting in a corresponding change in Tat activity. The mutations affected viral replication, with M1 having wild-type (WT) kinetics, M2 exhibiting reduced kinetics, and ERK showing completely abrogated replication. Using single-round infection with green fluorescent protein (GFP)-expressing viruses to study proviral gene expression, we observed progressively greater rates of silencing relating to the degree of ESEtat disruption, with the WT strain at 53%, strain M2 at 69%, and strain ERK at 94%. By stimulating infected cells with a latency reversal agent (phorbol myristate acetate [PMA], panobinostat, or JQ1), we observed that the dose required to achieve 50% of the maximum signal was lowest in the WT, intermediate in M2, and highest in ERK, indicating progressively higher thresholds for reactivation. These results suggest that the ability of silent proviruses to reactivate from latency is variable and that minor differences in the viral sequence can alter the proportion of silenced viruses as well as the threshold required to induce silenced viruses to reactivate and express.IMPORTANCE A reservoir of infected cells in which the HIV genome is transcriptionally silent is acknowledged to be the principal barrier to eradicating the virus from an infected person. A number of cellular processes are implicated in this silencing; however, the viral factors that may contribute remain underexplored. Here we examined mutations altering the correct splicing of HIV gene products as a model to study whether differences in viral sequence can affect either the proportion of viruses that are active or silent or their ability to reactivate. We found that some naturally occurring variations result in viruses that are silenced at a higher rate and require a proportionally increased stimulus for reactivation from latency. These data suggest that the silencing and reactivation behavior of HIV exists in a spectrum, influenced by factors intrinsic to the virus.

HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation.

A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual-who is known as the 'Berlin patient'-underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin's lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16 months after transplantation. HIV-1 remission has been maintained over a further 18 months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24 million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18 months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findings provide further support for the development of HIV-1 remission strategies based on preventing CCR5 expression.

Persistence of Imported Malaria Into the United Kingdom: An Epidemiological Review of Risk Factors and At-risk Groups.

BACKGROUND: The United Kingdom documented a decline of >30% in imported cases of malaria annually between 1996 and 2003; however, there are still approximately 1700 cases and 5-10 deaths each year. Prophylaxis health messages focus on families returning to their country of origin. METHODS: We reviewed 225 records of patients seen in Cambridge University Hospital Foundation Trust [CUHFT], a tertiary referral center in Cambridge, England. All records of patients seen in CUHFT between 2002-2016 were analyzed in the context of national figures from Public Health England. RESULTS: Between 2004-2016, there was no decrease in imported cases of malaria locally or nationally. Plasmodium falciparum remains responsible for most imported infections (66.7%); Plasmodium vivax contributed 15.1%, Plasmodium malariae 4%, and Plasmodium ovale 6.7%; 7.5% (17/225) of patients had an incomplete record. Most cases were reported in people coming from West Africa. Sierra Leone and the Ivory Coast had the highest proportions of travelers being infected at 8 and 7 per 1000, respectively. Visiting family in the country of origin (27.8%) was the commonest reason for travel. However, this was exceeded by the combined numbers traveling for business and holidays (22.5% and 20.1%, respectively). Sixty percent of patients took no prophylaxis. Of those who did, none of the patients finished their chemoprophylaxis regimen. CONCLUSIONS: Significant numbers of travelers to malarious countries still take no chemoprophylaxis. Health advice about prophylaxis before travel should be targeted not only at those visiting family in their country of origin but also to those traveling for holiday and work.

DDX17 Specifically, and Independently of DDX5, Controls Use of the HIV A4/5 Splice Acceptor Cluster and Is Essential for Efficient Replication of HIV.

HIV splicing involves five splice donor and eight splice acceptor sequences which, together with cryptic splice sites, generate over 100 mRNA species. Ninety percent of both partially spliced and fully spliced transcripts utilize the intrinsically weak A4/A5 3' splice site cluster. We show that DDX17, but not its close paralog DDX5, specifically controls the usage of this splice acceptor group. In its absence, production of the viral envelope protein and other regulatory and accessory proteins is grossly reduced, while Vif, which uses the A1 splice acceptor, is unaffected. This is associated with a profound decrease in viral export from the cell. Loss of Vpu expression causing upregulation of cellular Tetherin compounds the phenotype. DDX17 utilizes distinct RNA binding motifs for its role in efficient HIV replication, and we identify RNA binding motifs essential for its role, while the Walker A, Walker B (DEAD), Q motif and the glycine doublet motif are all dispensable. We show that DDX17 interacts with SRSF1/SF2 and the heterodimeric auxiliary factor U2AF65/35, which are essential splicing factors in the generation of Rev and Env/Vpu transcripts.

A new method for detecting signal regions in ordered sequences of real numbers, and application to viral genomic data.

We present a fast, robust and parsimonious approach to detecting signals in an ordered sequence of numbers. Our motivation is in seeking a suitable method to take a sequence of scores corresponding to properties of positions in virus genomes, and find outlying regions of low scores. Suitable statistical methods without using complex models or making many assumptions are surprisingly lacking. We resolve this by developing a method that detects regions of low score within sequences of real numbers. The method makes no assumptions a priori about the length of such a region; it gives the explicit location of the region and scores it statistically. It does not use detailed mechanistic models so the method is fast and will be useful in a wide range of applications. We present our approach in detail, and test it on simulated sequences. We show that it is robust to a wide range of signal morphologies, and that it is able to capture multiple signals in the same sequence. Finally we apply it to viral genomic data to identify regions of evolutionary conservation within influenza and rotavirus.

An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

BACKGROUND: NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. RESULTS: Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. CONCLUSIONS: NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.

No evidence of ongoing evolution in replication competent latent HIV-1 in a patient followed up for two years.

The persistence of infected T cells harbouring intact HIV proviruses is the barrier to the eradication of HIV. This reservoir is stable over long periods of time despite antiretroviral therapy. There has been controversy on whether low level viral replication is occurring at sanctuary sites periodically reseeding infected cells into the latent reservoir to account its durability. To study viral evolution in a physiologically relevant population of latent viruses, we repeatedly performed virus outgrowth assays on a stably treated HIV positive patient over two years and sequenced the reactivated latent viruses. We sought evidence of increasing sequence pairwise distances with time as evidence of ongoing viral replication. 64 reactivatable latent viral sequences were obtained over 103 weeks. We did not observe an increase in genetic distance of the sequences with the time elapsed between sampling. No evolution could be discerned in these reactivatable latent viruses. Thus, in this patient, the contribution of low-level replication to the maintenance of the latent reservoir detectable in the blood compartment is limited.

Innovations in the quantitative virus outgrowth assay and its use in clinical trials.

A robust measure of the size of the latent HIV reservoir is essential to quantifying the effect of interventions designed to deplete the pool of reactivatable, replication competent proviruses. In addition to the ability to measure a biologically relevant parameter, any assay designed to be used in a clinical trial needs to be reproducible and scalable. The need to quantify the number of resting CD4+ T cells capable of releasing infectious virus has led to the development of the quantitative viral outgrowth assay (VOA). The assay as originally described has a number of features that limit its scalability for use in clinical trials; however recent developments reducing the time and manpower requirements of the assay, while importantly improving reproducibility mean that it is becoming much more practical for it to enter into more widespread use. This review describes the background to VOA development and the practical issues that they present in utilising them in clinical trials. It describes the innovations that have made their usage more practical and the limitations that still exist.