Unspliced RNA Research Articles

Translation of HIV-1 unspliced RNA is regulated by 5' untranslated region structure.

HIV-1 must generate infectious virions to spread to new hosts and HIV-1 unspliced RNA (HIV-1 RNA) plays two central roles in this process. HIV-1 RNA serves as an mRNA that is translated to generate proteins essential for particle production and replication, and it is packaged into particles as the viral genome. HIV-1 uses several transcription start sites to generate multiple RNAs that differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. The virus relies on host machinery to translate its RNAs in a cap-dependent manner. Here, we demonstrate that the 5' context of HIV-1 RNA affects the efficiency of translation both in vitro and in cells. Although both RNAs are competent for translation, 3G RNA is translated more efficiently than 1G RNA. The 5' untranslated region (UTR) of 1G and 3G RNAs has previously been shown to fold into distinct structural ensembles. We show that HIV-1 mutants in which the 5' UTR of 1G and 3G RNAs fold into similar structures were translated at similar efficiencies. Thus, the host machinery translates two 99.9% identical HIV-1 RNAs with different efficiencies, and the translation efficiency is regulated by the 5' UTR structure.IMPORTANCEHIV-1 unspliced RNA contains all the viral genetic information and encodes virion structural proteins and enzymes. Thus, the unspliced RNA serves distinct roles as viral genome and translation template, both critical for viral replication. HIV-1 generates two major unspliced RNAs with a 2-nt difference at the 5' end (3G RNA and 1G RNA). The 1G transcript is known to be preferentially packaged over the 3G transcript. Here, we showed that 3G RNA is favorably translated over 1G RNA based on its 5' untranslated region (UTR) RNA structure. In HIV-1 mutants in which the two major transcripts have similar 5' UTR structures, 1G and 3G RNAs are translated similarly. Therefore, HIV-1 generates two 9-kb RNAs with a 2-nt difference, each serving a distinct role dictated by differential 5' UTR structures.

HIV-1 control in vivo is related to the number but not the fraction of infected cells with viral unspliced RNA.

In the absence of antiretroviral therapy (ART), a subset of individuals, termed HIV controllers, have levels of plasma viremia that are orders of magnitude lower than non-controllers (NC) who are at higher risk for HIV disease progression. In addition to having fewer infected cells resulting in fewer cells with HIV RNA, it is possible that lower levels of plasma viremia in controllers are due to a lower fraction of the infected cells having HIV-1 unspliced RNA (HIV usRNA) compared with NC. To directly test this possibility, we used sensitive and quantitative single-cell sequencing methods to compare the fraction of infected cells that contain one or more copies of HIV usRNA in peripheral blood mononuclear cells (PBMC) obtained from controllers and NC. The fraction of infected cells containing HIV usRNA did not differ between the two groups. Rather, the levels of viremia were strongly associated with the total number of infected cells that had HIV usRNA, as reported by others, with controllers having 34-fold fewer infected cells per million PBMC. These results reveal that viremic control is not associated with a lower fraction of proviruses expressing HIV usRNA, unlike what is reported for elite controllers, but is only related to having fewer infected cells overall, maybe reflecting greater immune clearance of infected cells. Our findings show that proviral silencing is not a key mechanism for viremic control and will help to refine strategies toward achieving HIV remission without ART.

Recombinant origin and interspecies transmission of a HERV-K(HML-2)-related primate retrovirus with a novel RNA transport element.

HERV-K(HML-2), the youngest clade of human endogenous retroviruses (HERVs), includes many intact or nearly intact proviruses, but no replication competent HML-2 proviruses have been identified in humans. HML-2-related proviruses are present in other primates, including rhesus macaques, but the extent and timing of HML-2 activity in macaques remains unclear. We have identified 145 HML-2-like proviruses in rhesus macaques, including a clade of young, rhesus-specific insertions. Age estimates, intact open reading frames, and insertional polymorphism of these insertions are consistent with recent or ongoing infectious activity in macaques. 106 of the proviruses form a clade characterized by an ~750 bp sequence between env and the 3' long terminal repeat (LTR), derived from an ancient recombination with a HERV-K(HML-8)-related virus. This clade is found in Old World monkeys (OWM), but not great apes, suggesting it originated after the ape/OWM split. We identified similar proviruses in white-cheeked gibbons; the gibbon insertions cluster within the OWM recombinant clade, suggesting interspecies transmission from OWM to gibbons. The LTRs of the youngest proviruses have deletions in U3, which disrupt the Rec Response Element (RcRE), required for nuclear export of unspliced viral RNA. We show that the HML-8-derived region functions as a Rec-independent constitutive transport element (CTE), indicating the ancestral Rec-RcRE export system was replaced by a CTE mechanism.

Single-cell total-RNA profiling unveils regulatory hubs of transcription factors

Recent development of RNA velocity uses master equations to establish the kinetics of the life cycle of RNAs from unspliced RNA to spliced RNA (i.e., mature RNA) to degradation. To feed this kinetic analysis, simultaneous measurement of unspliced RNA and spliced RNA in single cells is greatly desired. However, the majority of single-cell RNA-seq chemistry primarily captures mature RNA species to measure gene expressions. Here, we develop a one-step total-RNA chemistry-based single-cell RNA-seq method: snapTotal-seq. We benchmark this method with multiple single-cell RNA-seq assays in their performance in kinetic analysis of cell cycle by RNA velocity. Next, with LASSO regression between transcription factors, we identify the critical regulatory hubs mediating the cell cycle dynamics. We also apply snapTotal-seq to profile the oncogene-induced senescence and identify the key regulatory hubs governing the entry of senescence. Furthermore, from the comparative analysis of unspliced RNA and spliced RNA, we identify a significant portion of genes whose expression changes occur in spliced RNA but not to the same degree in unspliced RNA, indicating these gene expression changes are mainly controlled by post-transcriptional regulation. Overall, we demonstrate that snapTotal-seq can provide enriched information about gene regulation, especially during the transition between cell states.

IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome

Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes. Here we systematically compared nuclear factors identified in published HIV-1 proteomic studies and performed our own mass spectrometry analysis using affinity-tagged HIV-1 and RSV Gag proteins mixed with nuclear extracts. We identified 57 nuclear proteins in common between HIV-1 and RSV Gag, and a set of nuclear proteins present in our analysis and ≥ 1 of the published HIV-1 datasets. Many proteins were associated with nuclear processes which could have functional consequences for viral replication, including transcription initiation/elongation/termination, RNA processing, splicing, and chromatin remodeling. Examples include facilitating chromatin remodeling to expose the integrated provirus, promoting expression of viral genes, repressing the transcription of antagonistic cellular genes, preventing splicing of viral RNA, altering splicing of cellular RNAs, or influencing viral or host RNA folding or RNA nuclear export. Many proteins in our pulldowns common to RSV and HIV-1 Gag are critical for transcription, including PolR2B, the second largest subunit of RNA polymerase II (RNAPII), and LEO1, a PAF1C complex member that regulates transcriptional elongation, supporting the possibility that Gag influences the host transcription profile to aid the virus. Through the interaction of RSV and HIV-1 Gag with splicing-related proteins CBLL1, HNRNPH3, TRA2B, PTBP1 and U2AF1, we speculate that Gag could enhance unspliced viral RNA production for translation and packaging. To validate one putative hit, we demonstrated an interaction of RSV Gag with Mediator complex member Med26, required for RNA polymerase II-mediated transcription. Although 57 host proteins interacted with both Gag proteins, unique host proteins belonging to each interactome dataset were identified. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.Graphical

Co-transcriptional splicing facilitates transcription of gigantic genes.

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.

CCAT1 lncRNA is chromatin-retained and post-transcriptionally spliced

Super-enhancers are unique gene expression regulators widely involved in cancer development. Spread over large DNA segments, they tend to be found next to oncogenes. The super-enhancer c-MYC locus forms long-range chromatin looping with nearby genes, which brings the enhancer and the genes into proximity, to promote gene activation. The colon cancer-associated transcript 1 (CCAT1) gene, which is part of the MYC locus, transcribes a lncRNA that is overexpressed in colon cancer cells through activation by MYC. Comparing different types of cancer cell lines using RNA fluorescence in situ hybridization (RNA FISH), we detected very prominent CCAT1 expression in HeLa cells, observed as several large CCAT1 nuclear foci. We found that dozens of CCAT1 transcripts accumulate on the gene locus, in addition to active transcription occurring from the gene. The accumulating transcripts are released from the chromatin during cell division. Examination of CCAT1 lncRNA expression patterns on the single-RNA level showed that unspliced CCAT1 transcripts are released from the gene into the nucleoplasm. Most of these unspliced transcripts were observed in proximity to the active gene but were not associated with nuclear speckles in which unspliced RNAs usually accumulate. At larger distances from the gene, the CCAT1 transcripts appeared spliced, implying that most CCAT1 transcripts undergo post-transcriptional splicing in the zone of the active gene. Finally, we show that unspliced CCAT1 transcripts can be detected in the cytoplasm during splicing inhibition, which suggests that there are several CCAT1 variants, spliced and unspliced, that the cell can recognize as suitable for export.

Impact of influenza and pneumococcal vaccines on HIV persistence and immune dynamics during suppressive antiretroviral therapy.

We sought to determine if standard influenza and pneumococcal vaccines can be used to stimulate HIV reservoirs during antiretroviral therapy (ART). A prospective, randomized, double-blinded, placebo-controlled, crossover trial of two clinically recommended vaccines (influenza and pneumococcal). Persons with HIV on ART ( N = 54) were enrolled in the clinical trial. Blood was collected at baseline and days 2,4,7,14, and 30 postimmunizations. Levels of cellular HIV RNA and HIV DNA were measured by ddPCR. Expression of immunological markers on T cell subsets was measured by flow cytometry. Changes in unspliced cellular HIV RNA from baseline to day 7 postinjection between each vaccine and placebo was the primary outcome. Forty-seven participants completed at least one cycle and there were no serious adverse events related to the intervention. We observed no significant differences in the change in cellular HIV RNA after either vaccine compared with placebo at any timepoint. In secondary analyses, we observed a transient increase in total HIV DNA levels after influenza vaccine, as well as increased T cell activation and exhaustion on CD4 + T cells after pneumococcal vaccine. Clinically recommended vaccines were well tolerated but did not appear to stimulate the immune system strongly enough to elicit significantly noticeable HIV RNA transcription during ART.Clinicaltrials.gov identifier: NCT02707692.

HIV-1 RNA genome packaging: it's G-rated.

A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "G" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein Gag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired guanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different guanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize cis- and trans-acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.

Low unspliced cell-associated HIV RNA in early treated adolescents living with HIV on long suppressive ART.

Initiation of antiretroviral treatment (ART) in patients early after HIV-infection and long-term suppression leads to low or undetectable levels of HIV RNA and cell-associated (CA) HIV DNA and RNA. Both CA-DNA and CA-RNA, overestimate the size of the HIV reservoir but CA-RNA as well as p24/cell-free viral RNA can be indicators of residual viral replication. This study describes HIV RNA amounts and levels of cytokines/soluble markers in 40 well-suppressed adolescents who initiated ART early in life and investigated which viral markers may be informative as endpoints in cure clinical trials within this population. Forty adolescents perinatally infected with HIV on suppressive ART for >5 years were enrolled in the CARMA study. HIV DNA and total or unspliced CA-RNA in PBMCs were analyzed by qPCR/RT-qPCR and dPCR/RT-dPCR. Cell-free HIV was determined using an ultrasensitive viral load (US-VL) assay. Plasma markers and p24 were analyzed by digital ELISA and correlations between total and unspliced HIV RNA and clinical markers, including age at ART, Western Blot score, levels of cytokines/inflammation markers or HIV CA-DNA, were tested. CA-RNA was detected in two thirds of the participants and was comparable in RT-qPCR and RT-dPCR. Adolescents with undetectable CA-RNA showed significantly lower HIV DNA compared to individuals with detectable CA-RNA. Undetectable unspliced CA-RNA was positively associated with age at ART initiation and Western Blot score. We found that a higher concentration of TNF-α was predictive of higher CA-DNA and CA-RNA. Other clinical characteristics like US-VL, time to suppression, or percent CD4+ T-lymphocytes were not predictive of the CA-RNA in this cross-sectional study. Low CA-DNA after long-term suppressive ART is associated with lower CA-RNA, in concordance with other reports. Patients with low CA-RNA levels in combination with low CA-DNA and low Western Blot scores should be further investigated to characterize candidates for treatment interruption trials. Unspliced CA-RNA warrants further investigation as a marker that can be prioritized in paediatric clinical trials where the sample volume can be a significant limitation.

HIV-1 RNAs whose transcription initiates from the third deoxyguanosine of GGG tract in the 5' long terminal repeat serve as a dominant genome for efficient provirus DNA formation.

Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.

Single-Virion Analysis: A Method to Visualize HIV-1 Particle Content Using Fluorescence Microscopy.

HIV-1 virions incorporate viral RNA, cellular RNAs, and proteins during the assembly process. Some of these components, such as the viral RNA genome and viral proteins, are essential for viral replication, whereas others, such as host innate immune proteins, can inhibit virus replication. Therefore, analyzing the virion content is an integral part of studying HIV-1 replication. Traditionally, virion contents have been examined using biochemical assays, which can provide information on the presence or absence of the molecule of interest but not its distribution in the virion population. Here, we describe a method, single-virion analysis, that directly examines the presence of molecules of interest in individual viral particles using fluorescence microscopy. Thus, this method can detect both the presence and the distribution of molecules of interest in the virion population. Single-virion analysis was first developed to study HIV-1 RNA genome packaging. In this assay, HIV-1 unspliced RNA is labeled with a fluorescently tagged RNA-binding protein (protein A) and some of the Gag proteins are labeled with a different fluorescent protein (protein B). Using fluorescence microscopy, HIV-1 particles can be identified by the fluorescent protein B signal and the presence of unspliced HIV-1 RNA can be identified by the fluorescent protein A signal. Therefore, the proportions of particles that contain unspliced RNA can be determined by the fraction of Gag particles that also have a colocalized RNA signal. By tagging the molecule of interest with fluorescent proteins, single-virion analysis can be easily adapted to study the incorporation of other viral or host cell molecules into particles. Indeed, this method has been adapted to examine the proportion of HIV-1 particles that contain APOBEC3 proteins and the fraction of particles that contain a modified Gag protein. Therefore, single-virion analysis is a flexible method to study the nucleic acid and protein content of HIV-1 particles.

Validation of digital droplet PCR assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA for clinical studies in HIV-1 cure research

BackgroundCell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA (usRNA) are important virological parameters for monitoring HIV-1 persistence and activation of latent HIV-1. Assays fully validated by CLIA and/or GCLP standards are needed for future clinical trials that seek to evaluate treatments directed towards HIV-1 cure. ObjectivesTo determine performance characteristics of sensitive, moderate-throughput, digital droplet PCR (ddPCR) assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA that can detect a broad range of HIV-1 M-group subtypes. Study DesignTo evaluate linearity, limit of detection, precision, and accuracy of each assay, contrived specimens were analyzed in a background of uninfected PBMC. Detection breadth was evaluated by in silico analysis of primer and probes sets and analysis of material harvested from PBMC infected in vitro with various HIV-1 subtypes. A cohort of clinical specimens from viremic and virologically suppressed individuals was analyzed to demonstrate applicability to clinical research. ResultsThe empirically determined limit of detection of these assays was 29, 7, and 60 copies per million PBMC for HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA, respectively. The assays detect a broad range of HIV-1 M-group subtypes. Finally, analysis of clinical specimens demonstrate that these assays can detect low levels of cell-associated HIV-1 DNA, HIV-1 usRNA, and HIV-1 2-LTR circle and correlate with clinical histories and viral loads of untreated and antiretroviral treated individuals. ConclusionsWe report the clinical validation of three HIV reservoir assays with broad HIV-1 coverage for future cure studies.

A cohort-based study of host gene expression: tumor suppressor and innate immune/inflammatory pathways associated with the HIV reservoir size.

The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.

HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.

On a path toward a broad-spectrum anti-viral: inhibition of HIV-1 and coronavirus replication by SR kinase inhibitor harmine.

This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4+ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.

HIV Preintegration Transcription and Host Antagonism.

Retrovirus integration is an obligatory step for the viral life cycle, but large amounts of unintegrated DNA (uDNA) accumulate during retroviral infection. For simple retroviruses, in the absence of integration, viral genomes are epigenetically silenced in host cells. For complex retroviruses such as HIV, preintegration transcription has been found to occur at low levels from a large population of uDNA even in the presence of host epigenetic silencing mechanisms. HIV preintegration transcription has been suggested to be a normal early process of HIV infection that leads to the syntheses of all three classes of viral transcripts: multiply-spliced, singly-spliced, and unspliced genomic RNA; only viral early proteins such as Nef are selectively translated at low levels in blood CD4 T cells and macrophages, the primary targets of HIV. The initiation and persistence of HIV preintegration transcription have been suggested to rely on viral accessory proteins, particularly virion Vpr and de novo Tat generated from uDNA; both proteins have been shown to antagonize host epigenetic silencing of uDNA. In addition, stimulation of latently infected resting T cells and macrophages with cytokines, PKC activator, or histone deacetylase inhibitors has been found to greatly upregulate preintegration transcription, leading to low-level viral production or even replication from uDNA. Functionally, Nef synthesized from preintegration transcription is biologically active in modulating host immune functions, lowering the threshold of T cell activation, and downregulating surface CD4, CXCR4/CCR5, and HMC receptors. The early Tat activity from preintegration transcription antagonizes repressive minichromatin assembled onto uDNA. The study of HIV preintegration transcription is important to understanding virus-host interaction and antagonism, viral persistence, and the mechanism of integrase drug resistance. The application of unintegrated lentiviral vectors for gene therapy also offers a safety advantage for minimizing retroviral vector-mediated insertional mutagenesis.

Plant immunity suppressor SKRP encodes a novel RNA-binding protein that targets exon 3' end of unspliced RNA.

The regulatory roles of RNA splicing in plant immunity are emerging but still largely obscure. We reported previously that Phytophthora pathogen effector Avr3c targets a soybean protein SKRP (serine/lysine/arginine-rich protein) to impair soybean basal immunity by regulating host pre-mRNA alternative splicing, while the biochemical nature of SKRP remains unknown. Here, by using Arabidopsis as a model, we studied the mechanism of SKRP in regulating pre-mRNA splicing and plant immunity. AtSKRP confers impaired plant immunity against Phytophthora capsici and associates with spliceosome component PRP8 and splicing factor SR45, which positively and negatively regulate plant immunity, respectively. Enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq) showed AtSKRP is a novel RNA-binding protein that targets exon 3' end of unspliced RNA. Such position-specific binding of SKRP is associated with its activity in suppressing intron retention, including at positive immune regulatory genes UBP25 and RAR1. In addition, we found AtSKRP self-interact and forms oligomer, and these properties are associated with its function in plant immunity. Overall, our findings reveal that the immune repressor SKRP is a spliceosome-associated protein that targets exon 3' end to regulate pre-mRNA splicing in Arabidopsis.

The transcriptomic landscape of normal and ineffective erythropoiesis at single-cell resolution.

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic and always reflect ineffective erythropoiesis yet result from diverse genetic mutations. To delineate shared mechanisms that lead to cell death, we studied the fate of single erythroid marrow cells from individuals with DBA or MDS-5q. We defined an unhealthy (vs healthy) differentiation trajectory using transcriptional pseudotime and cell surface proteins. The pseudotime trajectories diverge immediately after cells upregulate transferrin receptor (CD71), import iron, and initiate heme synthesis, although cell death occurs much later. Cells destined to die express high levels of heme-responsive genes, including ribosomal protein and globin genes, whereas surviving cells downregulate heme synthesis and upregulate DNA damage response, hypoxia, and HIF1 pathways. Surprisingly, 24%± 12% of cells from control subjects follow the unhealthy trajectory, implying that heme might serve as a rheostat directing cells to live or die. When heme synthesis was inhibited with succinylacetone, more DBA cells followed the healthy trajectory and survived. We also noted high numbers of messages with retained introns that increased as erythroid cells matured, confirmed the rapid cycling of colony forming unit-erythroid, and demonstrated that cell cycle timing is an invariant property of differentiation stage. Including unspliced RNA in pseudotime determinations allowed us to reliably align independent data sets and accurately query stage-specific transcriptomic changes. MDS-5q (unlike DBA) results from somatic mutation, so many normal (unmutated) erythroid cells persist. By independently tracking erythroid differentiation of cells with and without chromosome 5q deletions, we gained insight into why 5q+ cells cannot expand to prevent anemia.