HIV RNA splicing

Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods...

In vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5' end of the viral RNA...

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...

HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. Here, we characterize PCI domain-containing 2 (PCID2) protein, a subunit of the transcription and export complex 2 (TREX2) complex, to enforce transcriptional repression and post-transcriptional blocks to HIV-1 gene expression during latency. PCID2 bound the latent HIV-1 LTR (long terminal repeat) and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and latency reversal...

Retroviruses exploit a variety of host proteins to assemble and release virions from infected cells. To date, most studies that examined possible interacting partners of retroviral Gag proteins focused on host proteins that localize primarily to the cytoplasm or plasma membrane. Given the recent findings that several full-length Gag proteins localize to the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings that reveal previously unknown host processes. In this study, we systematically compared nuclear factors identified in published HIV-1 proteomic studies which had used a variety of experimental approaches...

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...

Epitranscriptomic RNA modifications play a crucial role in the posttranscriptional regulation of gene expression. N 6 -methyladenosine (m6 A) is the most prevalent internal modification of eukaryotic RNA and plays a pivotal role in RNA fate. RNA m6 A modification is regulated by a group of cellular proteins, methyltransferases (writers) and demethylases (erasers), which add and remove the methyl group from adenosine, respectively. m6 A modification is recognized by a group of cellular RNA-binding proteins (readers) that specifically bind to m6 A-modified RNA, mediating effects on RNA stability, splicing, transport, and translation...

Proliferation of HIV in astrocytes is limited when compared to CD4+ T lymphocytes, but the molecular mechanisms behind the disparity in cell-specific HIV propagations are unclear. In this study, we show that HIV-1 Tat determined nuclear export of Rev-viral RNA complex in CD4+ T lymphocytes by downregulating heterogenous nuclear ribonucleoprotein A2 (hnRNPA2)-mediated HIV-1 RNA splicing that increased the levels of unspliced/partially spliced viral RNA, thereby increasing virion production. Notably, hnRNPA2 was not downregulated in astrocytes due to limited Tat nuclear import...

Persistent HIV-1 reservoirs of infected CD4 T cells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained in vivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 T cells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells...

RNA processing plays a key role in gene expression, allowing for increased protein diversity and functional complexity. Consequently, modulating RNA processing can impact gene function. Given HIV-1's reliance on host RNA processing machinery for viral protein production/replication, modulators of this process could serve as novel anti-virals to complement and/or enhance existing therapies. In this study, screening of several serine-arginine-rich (SR) kinase inhibitors for their impact on HIV-1 gene expression identified harmine as an inhibitor of HIV-1 gene expression in several cell lines and primary CD4+ T cells/macrophages at low micromolar concentrations with limited cell toxicity...

The functional consequences of circular RNA (circRNA) expression on HIV-1 replication are largely unknown. Using a customized protocol involving direct RNA nanopore sequencing, here, we captured circRNAs from HIV-1-infected T cells and identified ciTRAN, a circRNA that modulates HIV-1 transcription. We found that HIV-1 infection induces ciTRAN expression in a Vpr-dependent manner and that ciTRAN interacts with SRSF1, a protein known to repress HIV-1 transcription. Our results suggest that HIV-1 hijacks ciTRAN to exclude serine/arginine-rich splicing factor 1 (SRSF1) from the viral transcriptional complex, thereby promoting efficient viral transcription...

We investigated in silico the secondary structure of the region encompassing DIS, SD and Psi hairpins in HIV-1 genomes of rare groups N, O and P, HIV-2 genomes and SIV genomes from chimpanzees, gorillas and monkeys. We found that the structure of this region in SIVcpzptt genomes of the 1st and the 2nd clusters is similar to that in HIV-1 genomes of groups M and N, respectively. Further, the structure of the region encompassing DIS, SD and Psi hairpins is similar in HIV-1 genomes of groups O and P and SIVgor genomes...

The complex between the Rev protein of HIV-1 and the Rev Recognition Element (RRE) within the virus RNA promotes nuclear export of unspliced or incompletely spliced viral transcripts and is required for virus transmission. Here, we have screened a virtual collection of compounds using a pharmacophore based on the chemical similarity of previously characterized inhibitors to identify new chemical scaffolds blocking the RRE-Rev interaction. The best molecules discovered with this strategy inhibited the complex by binding to the RRE and exhibited substantial antiretroviral activity (between 0...

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR...

We develop candidate HIV-1 vaccines, of which two components, ChAdOx1.tHIVconsv1 (C1) and ChAdOx1.HIVconsv62 (C62), are delivered by the simian adenovirus-derived vaccine vector ChAdOx1. Aberrant adenovirus RNA splicing involving transgene(s) coding for the SARS-CoV-2 spike was suggested as an aetiology of rare adverse events temporarily associated with the initial deployment of adenovirus-vectored vaccines during the COVID-19 pandemic. Here, to eliminate this theoretically plausible splicing phenomenon from the list of possible pathomechanisms for our HIV-1 vaccine candidates, we directly sequenced mRNAs in C1- and C62-infected nonpermissive MRC-5 and A549 and permissive HEK293 human cell lines...

Sin3 associated protein 18 (SAP18) is an evolutionary conserved protein, originally discovered in a complex with the transcriptional regulatory protein, Sin3. Subsequent investigations revealed SAP18 as an integral splicing component of the exon junction complex (EJC)-associated apoptosis-and splicing-associated protein (ASAP)/PNN-RNPS1-SAP18 (PSAP) complex. In association with Sin3, SAP18 contributes toward transcriptional repression of genes implicated in embryonic development, stress response, human immunodeficiency virus type 1 replication, and tumorigenesis...

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...

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol...

The transient activation of the cellular stress kinase, protein kinase RNA-activated (PKR), by double-helical RNA, especially by viral double-stranded RNA generated during replication, results in the inhibition of translation via the phosphorylation of eukaryotic initiation factor 2 α-chain (eIF2α). Exceptionally, short intragenic elements within primary transcripts of the human tumor necrosis factor ( TNF-α ) and globin genes, genes essential for survival, can form RNA structures that strongly activate PKR and thereby render the splicing of their mRNAs highly efficient...

RNA-binding protein with serine-rich domain 1 (RNPS1) gets deposited on the mRNA during the process of splicing and concomitantly associates with the exon junction complex (EJC). RNPS1 participates in post-transcriptional gene regulation, including constitutive and alternative splicing, transcriptional regulation and nonsense-mediated mRNA decay. In this study, we found that the tethering of RNPS1 or its isolated serine-rich domain (S domain) causes exon inclusion of an HIV-1 splicing substrate. In contrast, overexpressing the RRM domain of RNPS1 acts in a dominant negative manner and leads to the exon skipping of endogenous apoptotic pre-mRNAs (Bcl-X and MCL-1)...