Safety and Tolerability of COVID-19 Vaccine in Mast Cell Disorders Real-Life Data from a Single Centre in Italy.

Background In the past three years, COVID-19 has had a significant impact on the healthcare systems and people's safety worldwide. Mass vaccinations dramatically improved the health and economic damage caused by SARS-CoV-2. However, the safety of COVID-19 vaccines in patients at high risk of allergic reactions still has many unmet needs that should be clarified. Material and methods A retrospective, single-centre study was performed by collecting demographic and clinical data of patients with Mast Cell Disorders (MCDs) to evaluate the safety and tolerability of COVID-19 vaccinations. Moreover, any changes in the natural history of the underlying disease following the vaccine have been evaluated. Results This study included 66 patients affected with MCDs. Out of them, 52 (78.8%) received a COVID-19 vaccination and 41 (78.8%) completed the vaccination course. Premedication came first in 86.6% of our patients. A total of seven (4.5%) patients complained about an immediate reaction and two (1.3%) had a late reaction. Worsening of MCD history was observed in a single patient. Conclusions Despite the overall high risk of allergic reactions, our study did not reveal any increased risk for SARS-CoV-2 allergic reactions in MCD patients, thus supporting the recommendation in favour of the SARS-CoV-2 vaccination. However, due to the potentially increased rate of anaphylactic reactions, MCD patients should receive vaccine premedication and should be treated in a hospital setting after an allergological specialistic evaluation.

Subcutaneous infliximab CT-P13 without intravenous induction in psoriatic arthritis: A case report and pharmacokinetic considerations.

INTRODUCTION: The biosimilar CT-P13, the first and only subcutaneous (SC) infliximab formulation, is recommended for patients with psoriatic arthritis (PsA) and can be administered as a maintenance treatment, to be started 4 weeks after the induction treatment with 2 intravenous (IV) infliximab infusions. OBJECTIVE: To evaluate treatment with SC infliximab without prior IV infusion induction to meet patient needs. MATERIALS AND METHODS: After approval by the ethics review board and based on the schedule approved for rheumatoid arthritis, SC induction was performed with infliximab CT-P13 120 mg weekly for 4 weeks, followed by an injection of 120 mg every 2 weeks. RESULTS: After 4 months of therapy, joint symptoms were resolved, inflammation parameters were normalized (erythrocyte sedimentation rate) reduced from 42 to 16 mm/h, and C-reactive protein from 1.74 to 0.43 mg/dL), and clinical assessment parameters were improved. After 9 months of therapy, the clinical data remained stable, with no adverse events or local side effects. CONCLUSION: SC infliximab was successfully used without previous IV infusion induction. Although, to date, the induction of PsA treatment via the SC route is not foreseen, the known pharmacokinetic properties and the outcome improvements observed in our patient show that subcutaneous treatment induction, as is already done in the treatment of rheumatoid arthritis, is also possible.

MAURIVAX: A Vaccination Campaign Project in a Hospital Environment for Patients Affected by Autoimmune Diseases and Adult Primary Immunodeficiencies.

Background: Patients with autoimmune diseases (ADs) and primary immunodeficiencies (PIDs) are characterized by an increased risk of noninvasive and widespread infections as they are considered frail patients. In addition, many flares of the underlying disease are reported after routine vaccinations. To date, the vaccination rate in these two populations is suboptimal. According to the latest guidelines, targeted interventions are needed, such as strengthening the network of vaccination activities. Our project aimed to propose a pilot network for carrying out the recommended vaccinations in frail patients. Methods: The Allergy and Immunology Center of the Mauriziano Hospital in Turin, Italy started the "Maurivax" project, a facilitated pathway for frail patients to administer the recommended vaccinations in the setting of a dedicated structure where they could be properly followed up. Results: From June 2022 to February 2023, 49 patients underwent a vaccination consultation: 45 of them (91.8%) were subsequently vaccinated. Among these, 36 subjects (80%) were affected by an active AD and were already in treatment with immunosuppressive therapy or about to start it. Seven patients (15.5%) had a confirmed diagnosis of PID or showed a clinical presentation that was highly suggestive of that condition. Overall, twenty-seven patients (60%) showed a high-grade immunosuppression and six (13.3%) had a low-grade immunosuppression. No patients had a disease flare within 30 days from vaccination and no severe reactions after vaccination was observed. Conclusions: Adherence and vaccination safety at our immunology hospital vaccine clinic dedicated to patients with ADs and PIDs were high. We propose an effective model for managing vaccinations in frail patients in a specialist hospital setting.

New Onset of Giant Cell Arteritis following ChAdOx1-S (Vaxevria®) Vaccine Administration.

We report a 78-year-old man presenting with persistent headaches in vertex and temporo-parietal area; fatigue, worsening after walking; jaw claudication; scotomas; pharyngodynia; and dry cough after the second dose of the SARS-CoV-2 vaccine (ChAdOx1-S) administration. Laboratory findings showed an elevated C-reactive protein level and FDG-CT PET showed evidence of active large vessel vasculitis with diffuse abnormal artery uptake. Under suspicion of vasculitis, a temporal arteries biopsy was performed; the histopathologic findings demonstrated the transmural inflammatory infiltrate with giant cells, compatible with giant cell arteritis. Although the overall incidence of vaccine-triggered autoimmunity is low, rheumatologists worldwide should be aware of autoimmune diseases as a new potential adverse event of vaccines.

The Importance of Endoscopy with Biopsy: Real-World Evidence of Gastrointestinal Involvement in Primary Immunodeficiency in Two Main Northern Italian Centres.

INTRODUCTION: Inborn errors of immunity (IEI) represent a heterogeneous group of diseases in which the true prevalence of GI involvement is not well-known. This study evaluates the prevalence of lower GI manifestations in patients with common variable immunodeficiency (CVID), analysing the histologic findings in colonic samples and assessing any correlations with biochemical abnormalities. MATERIALS AND METHODS: A retrospective study was performed by collecting the data of IEI adult patients followed up at two main Northern Italian centres. Demographic and clinical data, and blood tests were collected. A colonoscopy with multiple biopsies in standard sites, in addition to a biopsy for any macroscopic lesion, was performed. The gastrointestinal Symptom Rating Scale for Irritable Bowel Syndrome (GSRS-IBS) and the short Inflammatory Bowel Disease Questionnaire (sIBDQ) were used to assess GI symptoms. RESULTS: 141 patients were included: 121 (86.5%) with CVID, 17 (12.1%) with IgG subclass deficiency, and 2 (1.4%) with X-linked agammaglobulinemia. Of the patients, 72 (51%) complained of GI symptoms. No differences were seen between patients receiving or not IgRT. GI infections were found in 9 patients (6.4%). No significant correlations were found between gut infections and symptoms or leukocyte infiltrates. Colonoscopy alterations were present in 79 patients (56%), and the most common were colon polyps (42%). Microscopical abnormalities were seen in 60 histologic samples (42.5%) and the most frequent was nodular lymphoid hyperplasia (40%). A leukocyte infiltrate was present in 67 samples (47.5%), and the most common was a lymphocyte infiltrate (33%). No correlation was found between GI symptoms and macroscopic alterations, whereas a positive correlation between symptoms and microscopic alterations was detected. CONCLUSIONS: GI symptoms and microscopic alterations in colon samples are closely related; hence, it is important to carry out serial colonic biopsies in every CVID patient, even in the absence of macroscopic lesions.

Disease activity at conception predicts lupus flare up to two years after birth: A multicentre long term follow-up study.

OBJECTIVE: To assess predicting factors that might influence systemic lupus erythematosus (SLE) disease activity in women in an extended follow-up period of two years after giving birth with clinical assessments every three months. METHODS: The study was design as an international retrospective study, enrolling 119 women with a first birth and with a two years follow-up. RESULTS: Joint involvement was present in 80% of patients, acute cutaneous in 64%, haematological in 54%, renal in 41% and 75% of patients were positive for anti-dsDNA. The mean SLE disease activity index 2000 (SLEDAI-2K) at diagnosis was 13.5±6.8 and at first birth was 2.8±4.4. At follow-up, 51.3% of patients had at least one flare after a mean time after birth of 9±6.3 months (mean flare per patient 0.94±1.1). The most frequent flare manifestations were joint involvement (48%), renal (33%), cutaneous (28%) and haematologic (20%). Patients with remission of disease (SLEDAI-2K=0; no clinical or laboratory manifestations of SLE) at conception had significantly lower rates of flares (18/49-37% vs. 43/70-61%; p=0.008). Patients who experienced a flare during pregnancy (17 patients) had higher rates of flares during follow-up (76% vs. 47%; p=0.019), lower time for first flare (4.4±2.3 months vs. 10.3±6.5; p<0.001), lower rate of remission of disease at conception (12% vs. 46%; p<0.001), lower rates of SLEDAI-2K at conception (5.9±5.6 vs. 2.3±4; p<0.001) and lower rates of exclusive breastfeeding (24% vs. 57%: p=0.009). Results were confirmed after performing multivariate analysis. CONCLUSION: Remission at conception can influence SLE disease positively, even at long-term. Planned pregnancy counseling is fundamental when managing SLE patients.

COPD, but Not Asthma, Is Associated with Worse Outcomes in COVID-19: Real-Life Data from Four Main Centers in Northwest Italy.

Introduction: Asthma, along with inhaled steroids, was initially considered a risk factor for worse clinical outcomes in COVID-19. This was related to the higher morbidity observed in asthma patients during previous viral outbreaks. This retrospective study aimed at evaluating the prevalence of asthma among patients admitted due to SARS-CoV-2 infection as well as the impact of inhaled therapies on their outcomes. Furthermore, a comparison between patients with asthma, COPD and the general population was made. Methods: All COVID-19 inpatients were recruited between February and July 2020 from four large hospitals in Northwest Italy. Data concerning medical history, the Charlson Comorbidity Index (CCI) and the hospital stay, including length, drugs and COVID-19 complications (respiratory failure, lung involvement, and the need for respiratory support) were collected, as well as the type of discharge. Results: patients with asthma required high-flow oxygen therapy (33.3 vs. 14.3%, p = 0.001) and invasive mechanical ventilation (17.9 vs. 9.5%, p = 0.048) more frequently when compared to the general population, but no other difference was observed. Moreover, asthma patients were generally younger than patients with COPD (59.2 vs. 76.8 years, p < 0.001), they showed both a lower mortality rate (15.4 vs. 39.4%, p < 0.001) and a lower CCI (3.4 vs. 6.2, p < 0.001). Patients with asthma in regular therapy with ICS at home had significantly shorter hospital stay compared to those with no treatments (25.2 vs. 11.3 days, p = 0.024). Discussion: Our study showed that asthma is not associated with worse outcomes of COVID-19, despite the higher need for respiratory support compared with the general population, while the use of ICS allowed for a shorter hospital stay. In addition, the comparison of asthma with COPD patients confirmed the greater frailty of the latter, according to their multiple comorbidities.

Insights Into Persistent HIV-1 Infection and Functional Cure: Novel Capabilities and Strategies.

Although HIV-1 replication can be efficiently suppressed to undetectable levels in peripheral blood by combination antiretroviral therapy (cART), lifelong medication is still required in people living with HIV (PLWH). Life expectancies have been extended by cART, but age-related comorbidities have increased which are associated with heavy physiological and economic burdens on PLWH. The obstacle to a functional HIV cure can be ascribed to the formation of latent reservoir establishment at the time of acute infection that persists during cART. Recent studies suggest that some HIV reservoirs are established in the early acute stages of HIV infection within multiple immune cells that are gradually shaped by various host and viral mechanisms and may undergo clonal expansion. Early cART initiation has been shown to reduce the reservoir size in HIV-infected individuals. Memory CD4+ T cell subsets are regarded as the predominant cellular compartment of the HIV reservoir, but monocytes and derivative macrophages or dendritic cells also play a role in the persistent virus infection. HIV latency is regulated at multiple molecular levels in transcriptional and post-transcriptional processes. Epigenetic regulation of the proviral promoter can profoundly regulate the viral transcription. In addition, transcriptional elongation, RNA splicing, and nuclear export pathways are also involved in maintaining HIV latency. Although most proviruses contain large internal deletions, some defective proviruses may induce immune activation by expressing viral proteins or producing replication-defective viral-like particles. In this review article, we discuss the state of the art on mechanisms of virus persistence in the periphery and tissue and summarize interdisciplinary approaches toward a functional HIV cure, including novel capabilities and strategies to measure and eliminate the infected reservoirs and induce immune control.

FRET-Based Detection and Quantification of HIV-1 Virion Maturation.

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

Visualizing Chromatin Modifications in Isolated Nuclei.

Modifications in chromatin structure are traditionally monitored by biochemical assays that provide average measurements of static events in a population of cells. Microscopy provides a method by which single cells or nuclei can be observed. Traditionally, microscopy has been used to image the nucleus by the application of immunostaining to chemically fixed samples or the use of exogenously expressed fluorescent proteins. This method represents an approach to observe changes in endogenous proteins relating to chromatin structure in real time. Here we describe a method for isolating transcriptionally and enzymatically active nuclei from live cells and visualizing events using fluorescently labeled antibodies. This method allows the observation of real time changes in chromatin architecture and can be used to observe the effects of drugs on nuclei while under microscopic observation.

Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes.

Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes (comprising 40-70% of all brain cells) likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/ß-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit ß-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to ß-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of ß-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of ß-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect ß-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development.

Benzodiazepines Drive Alteration of Chromatin at the Integrated HIV-1 LTR.

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.

Structural Determinants of the APOBEC3G N-Terminal Domain for HIV-1 RNA Association.

APOBEC3G (A3G) is a cellular protein that inhibits HIV-1 infection through virion incorporation. The interaction of the A3G N-terminal domain (NTD) with RNA is essential for A3G incorporation in the HIV-1 virion. The interaction between A3G-NTD and RNA is not completely understood. The A3G-NTD is also recognized by HIV-1 Viral infectivity factor (Vif) and A3G-Vif binding leads to A3G degradation. Therefore, the A3G-Vif interaction is a target for the development of antiviral therapies that block HIV-1 replication. However, targeting the A3G-Vif interactions could disrupt the A3G-RNA interactions that are required for A3G's antiviral activity. To better understand A3G-RNA binding, we generated in silico docking models to simulate the RNA-binding propensity of A3G-NTD. We simulated the A3G-NTD residues with high RNA-binding propensity, experimentally validated our prediction by testing A3G-NTD mutations, and identified structural determinants of A3G-RNA binding. In addition, we found a novel amino acid residue, I26 responsible for RNA interaction. The new structural insights provided here will facilitate the design of pharmaceuticals that inhibit A3G-Vif interactions without negatively impacting A3G-RNA interactions.

Real-time visualization of chromatin modification in isolated nuclei.

Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented. This article has an associated First Person interview with the first author of the paper.

Long-Term Outcomes and Discard Rate of Kidneys by Decade of Extended Criteria Donor Age.

BACKGROUND AND OBJECTIVES: Extended criteria donors represent nowadays a main resource for kidney transplantation, and recovery criteria are becoming increasingly inclusive. However, the limits of this approach are not clear as well as the effects of extreme donor ages on long-term kidney transplantation outcomes. To address these issues, we performed a retrospective study on extended criteria donor kidney transplantation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In total, 647 consecutive extended criteria donor kidney transplantations performed over 11 years (2003-2013) were included. Donor, recipient, and procedural variables were classified according to donor age decades (group A, 50-59 years old [n=91]; group B, 60-69 years old [n=264]; group C, 70-79 years old [n=265]; and group D, ≥80 years old [n=27]). Organs were allocated in single- or dual-kidney transplantation after a multistep evaluation including clinical and histologic criteria. Long-term outcomes and main adverse events were analyzed among age groups and in either single- or dual-kidney transplantation. Kidney discard rate incidence and causes were evaluated. RESULTS: Median follow-up was 4.9 years (25th; 75th percentiles: 2.7; 7.6 years); patient and graft survival were comparable among age groups (5-year patient survival: group A, 87.8%; group B, 88.1%; group C, 88.0%; and group D, 90.1%; P=0.77; graft survival: group A, 74.0%; group B, 74.2%; group C, 75.2%; and group D, 65.9%; P=0.62) and between dual-kidney transplantation and single-kidney transplantation except for group D, with a better survival for dual-kidney transplantation (P=0.04). No difference was found analyzing complications incidence or graft function over time. Kidney discard rate was similar in groups A, B, and C (15.4%, 17.7%, and 20.1%, respectively) and increased in group D (48.2%; odds ratio, 5.1 with A as the reference group; 95% confidence interval, 2.96 to 8.79). CONCLUSIONS: Discard rate and long-term outcomes are similar among extended criteria donor kidney transplantation from donors ages 50-79 years old. Conversely, discard rate was strikingly higher among kidneys from octogenarian donors, but appropriate selection provides comparable long-term outcomes, with better graft survival for dual-kidney transplantation.

The inhibition of microRNAs by HIV-1 Tat suppresses beta catenin activity in astrocytes.

BACKGROUND: Long term infection with HIV-1, even in the context of therapy, leads to chronic health problems including an array of neurocognitive dysfunctions. The viral Tat protein has previously been implicated in neuropathogenesis through its effect on astrocytes. Tat has also been shown to inhibit the biogenesis of miRNAs by inhibiting the activity of the cellular Dicer protein in an RNA dependent fashion. Whether there is a mechanistic connection between the ability of HIV-1 Tat to alter miRNAs and its observed effects on cells of the central nervous system has not been well examined. RESULTS: Here, we examined the ability of HIV-1 Tat to bind to and inhibit the production of over 300 cellular miRNAs. We found that the Tat protein only binds to and inhibits a fraction of the total cellular miRNAs. By mapping the downstream targets of these miRNAs we have determined a possible role for Tat alterations of miRNAs in the development of neuropathogenesis. Specifically, this work points to suppression of miRNAs function as the mechanism for Tat suppression of ß-catenin activity. CONCLUSIONS: The discovery that HIV-1 Tat inhibits only a fraction of miRNAs opens new areas of research regarding changes in cellular pathways through suppression of RNA interference. Our initial analysis strongly suggests that these pathways may contribute to HIV-1 disruption of the central nervous system.

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein.

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.

Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

UNLABELLED: To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. IMPORTANCE: Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNAs stayed at the plasma membrane for 15 to 60 min in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately 1/10 to 1/3 of the RNAs that reached the plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained temporal information on RNA-Gag interactions that lead to RNA encapsidation.