Autophagy Deregulation in HIV-1-Infected Cells Increases Extracellular Vesicle Release and Contributes to TLR3 Activation.

Human immunodeficiency virus type 1 (HIV-1) infection can result in HIV-associated neurocognitive disorder (HAND), a spectrum of disorders characterized by neurological impairment and chronic inflammation. Combined antiretroviral therapy (cART) has elicited a marked reduction in the number of individuals diagnosed with HAND. However, there is continual, low-level viral transcription due to the lack of a transcription inhibitor in cART regimens, which results in the accumulation of viral products within infected cells. To alleviate stress, infected cells can release accumulated products, such as TAR RNA, in extracellular vesicles (EVs), which can contribute to pathogenesis in neighboring cells. Here, we demonstrate that cART can contribute to autophagy deregulation in infected cells and increased EV release. The impact of EVs released from HIV-1 infected myeloid cells was found to contribute to CNS pathogenesis, potentially through EV-mediated TLR3 (Toll-like receptor 3) activation, suggesting the need for therapeutics to target this mechanism. Three HIV-1 TAR-binding compounds, 103FA, 111FA, and Ral HCl, were identified that recognize TAR RNA and reduce TLR activation. These data indicate that packaging of viral products into EVs, potentially exacerbated by antiretroviral therapeutics, may induce chronic inflammation of the CNS observed in cART-treated patients, and novel therapeutic strategies may be exploited to mitigate morbidity.

Dihydroxyphenyl- and Heteroaromatic-Based Thienopyrimidinones to Tackle HIV-1 LEDGF/p75-Dependent IN Activity.

The spread of Human Immunodeficiency Virus (HIV) still represents a global public health issue of major concern, and would benefit from unveiling unique viral features as targets for drug design. In this respect, HIV-1 integrase (IN), due to the absence of homologs in human cells, is a popular target for the synthesis of novel selective compounds. Moreover, as drug-resistant viral strains are rapidly evolving, the development of novel allosteric inhibitors is acutely required. Recently, we have observed that Kuwanon-L, quinazolinones and thienopyrimidinones containing at least one polyphenol unit, effectively inhibited HIV-1 IN activity. Thus, in the present research, novel dihydroxyphenyl-based thienopyrimidinone derivatives were investigated for their LEDGF/p75-dependent IN inhibitory activity. Our findings indicated a close correlation between the position of the OH group on the phenyl moiety and IN inhibitory activity of these compounds. As catechol may be involved in cytotoxicity, its replacement by other aromatic scaffolds was also exploited. As a result, compounds 21-23, 25 and 26 with enhanced IN inhibitory activity provided good lead candidates, with 25 being the most selective for IN. Lastly, UV spectrometric experiments suggested a plausible allosteric mode of action, as none of the thienopirimidinones showed Mg2+ chelation properties otherwise typical of IN strand transfer inhibitors (INSTIs).

Discovery of Small Molecules Targeting the Frameshifting Element RNA in SARS-CoV-2 Viral Genome.

Targeting structured RNA elements in the SARS-CoV-2 viral genome with small molecules is an attractive strategy for pharmacological control over viral replication. In this work, we report the discovery of small molecules that target the frameshifting element (FSE) in the SARS-CoV-2 RNA genome using high-throughput small-molecule microarray (SMM) screening. A new class of aminoquinazoline ligands for the SARS-CoV-2 FSE are synthesized and characterized using multiple orthogonal biophysical assays and structure-activity relationship (SAR) studies. This work reveals compounds with mid-micromolar binding affinity (KD = 60 ± 6 µM) to the FSE RNA and supports a binding mode distinct from previously reported FSE binders MTDB and merafloxacin. In addition, compounds are active in in vitro dual-luciferase and in-cell dual-fluorescent-reporter frameshifting assays, highlighting the promise of targeting structured elements of RNAs with druglike compounds to alter expression of viral proteins.

HIV-1 Transcription Inhibition Using Small RNA-Binding Molecules.

The HIV-1 transactivator protein Tat interacts with the transactivation response element (TAR) at the three-nucleotide UCU bulge to facilitate the recruitment of transcription elongation factor-b (P-TEFb) and induce the transcription of the integrated proviral genome. Therefore, the Tat-TAR interaction, unique to the virus, is a promising target for developing antiviral therapeutics. Currently, there are no FDA-approved drugs against HIV-1 transcription, suggesting the need to develop novel inhibitors that specifically target HIV-1 transcription. We have identified potential candidates that effectively inhibit viral transcription in myeloid and T cells without apparent toxicity. Among these candidates, two molecules showed inhibition of viral protein expression. A molecular docking and simulation approach was used to determine the binding dynamics of these small molecules on TAR RNA in the presence of the P-TEFb complex, which was further validated by a biotinylated RNA pulldown assay. Furthermore, we examined the effect of these molecules on transcription factors, including the SWI/SNF complex (BAF or PBAF), which plays an important role in chromatin remodeling near the transcription start site and hence regulates virus transcription. The top candidates showed significant viral transcription inhibition in primary cells infected with HIV-1 (98.6). Collectively, our study identified potential transcription inhibitors that can potentially complement existing cART drugs to address the current therapeutic gap in current regimens. Additionally, shifting of the TAR RNA loop towards Cyclin T1 upon molecule binding during molecular simulation studies suggested that targeting the TAR loop and Tat-binding UCU bulge together should be an essential feature of TAR-binding molecules/inhibitors to achieve complete viral transcription inhibition.

Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation.

Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets. An immobilised array of small molecular weight, drug-like compounds was panned against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1, known to be a major determinant in nucleocapsid formation. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly in vitro, which identified potent assembly inhibitors. Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5' labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T = 4 NCP formation) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation. The results imply that ∼50% of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.

Structural insights of the conserved "priming loop" of hepatitis B virus pre-genomic RNA.

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral polymerase with a cis-acting regulatory signal, designated epsilon (ε), located at the 5'-end of its pre-genomic RNA (pgRNA). Binding of polymerase to ε is also necessary for pgRNA encapsidation. While the mechanistic basis of this interaction remains elusive, mutagenesis studies suggest its internal 6-nt "priming loop" provides an important structural contribution. ε might therefore be considered a promising target for small molecule interventions to complement current nucleoside-analog based anti-HBV therapies. An ideal prerequisite to any RNA-directed small molecule strategy would be a detailed structural description of this important element. Herein, we present a solution NMR structure for HBV ε which, in combination with molecular dynamics and docking simulations, reports on a flexible ligand "pocket", reminiscent of those observed in proteins. We also demonstrate the binding of the selective estrogen receptor modulators (SERMs) Raloxifene, Bazedoxifene, and a de novo derivative to the priming loop.Communicated by Ramaswamy H. Sarma.

Dynamic bulge nucleotides in the KSHV PAN ENE triple helix provide a unique binding platform for small molecule ligands.

Cellular and virus-coded long non-coding (lnc) RNAs support multiple roles related to biological and pathological processes. Several lncRNAs sequester their 3' termini to evade cellular degradation machinery, thereby supporting disease progression. An intramolecular triplex involving the lncRNA 3' terminus, the element for nuclear expression (ENE), stabilizes RNA transcripts and promotes persistent function. Therefore, such ENE triplexes, as presented here in Kaposi's sarcoma-associated herpesvirus (KSHV) polyadenylated nuclear (PAN) lncRNA, represent targets for therapeutic development. Towards identifying novel ligands targeting the PAN ENE triplex, we screened a library of immobilized small molecules and identified several triplex-binding chemotypes, the tightest of which exhibits micromolar binding affinity. Combined biophysical, biochemical, and computational strategies localized ligand binding to a platform created near a dinucleotide bulge at the base of the triplex. Crystal structures of apo (3.3 Å) and ligand-soaked (2.5 Å) ENE triplexes, which include a stabilizing basal duplex, indicate significant local structural rearrangements within this dinucleotide bulge. MD simulations and a modified nucleoside analog interference technique corroborate the role of the bulge and the base of the triplex in ligand binding. Together with recently discovered small molecules that reduce nuclear MALAT1 lncRNA levels by engaging its ENE triplex, our data supports the potential of targeting RNA triplexes with small molecules.

Characterizing and circumventing sequence restrictions for synthesis of circular RNA in vitro.

Just as eukaryotic circular RNA (circRNA) is a product of intracellular backsplicing, custom circRNA can be synthesized in vitro using a transcription template in which transposed halves of a split group I intron flank the sequence of the RNA to be circularized. Such permuted intron-exon (PIE) constructs have been used to produce circRNA versions of ribozymes, mimics of viral RNA motifs, a streptavidin aptamer, and protein expression vectors for genetic engineering and vaccine development. One limitation of this approach is the obligatory incorporation of small RNA segments (E1 and E2) into nascent circRNA at the site of end-joining. This restriction may preclude synthesis of small circRNA therapeutics and RNA nanoparticles that are sensitive to extraneous sequence, as well as larger circRNA mimics whose sequences must precisely match those of the native species on which they are modelled. In this work, we used serial mutagenesis and in vitro selection to determine how varying E1 and E2 sequences in a thymidylate synthase (td) group I intron PIE transcription template construct affects circRNA synthesis yield. Based on our collective findings, we present guidelines for the design of custom-tailored PIE transcription templates from which synthetic circRNAs of almost any sequence may be efficiently synthesized.

Scaffold hopping and optimisation of 3',4'-dihydroxyphenyl- containing thienopyrimidinones: synthesis of quinazolinone derivatives as novel allosteric inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H.

Bioisosteric replacement and scaffold hopping are powerful strategies in drug design useful for rationally modifying a hit compound towards novel lead therapeutic agents. Recently, we reported a series of thienopyrimidinones that compromise dynamics at the p66/p51 HIV-1 reverse transcriptase (RT)-associated Ribonuclease H (RNase H) dimer interface, thereby allosterically interrupting catalysis by altering the active site geometry. Although they exhibited good submicromolar activity, the isosteric replacement of the thiophene ring, a potential toxicophore, is warranted. Thus, in this article, the most active 2-(3,4-dihydroxyphenyl)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one 1 was selected as the hit scaffold and several isosteric substitutions of the thiophene ring were performed. A novel series of highly active RNase H allosteric quinazolinone inhibitors was thus obtained. To determine their target selectivity, they were tested against RT-associated RNA-dependent DNA polymerase (RDDP) and integrase (IN). Interestingly, none of the compounds were particularly active on (RDDP) but many displayed micromolar to submicromolar activity against IN.

3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis.

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a cis-acting regulatory signal located at the 5' terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs).

Adeno-Associated Viral Vector Mediated Expression of Broadly- Neutralizing Antibodies Against HIV-Hitting a Fast-Moving Target.

The vast genetic variability of HIV has impeded efforts towards a cure for HIV. Lifelong administration of combined antiretroviral therapy (cART) is highly effective against HIV and has markedly increased the life expectancy of HIV infected individuals. However, the long-term usage of cART is associated with co-morbidities and the emergence of multidrug-resistant escape mutants necessitating the development of alternative approaches to combat HIV/AIDS. In the past decade, the development of single-cell antibody cloning methods has facilitated the characterization of a diverse array of highly potent neutralizing antibodies against a broad range of HIV strains. Although the passive transfer of these broadly neutralizing antibodies (bnAbs) in both animal models and humans has been shown to elicit significant antiviral effects, long term virologic suppression requires repeated administration of these antibodies. Adeno-associated virus (AAV) mediated antibody gene transfer provides a long-term expression of these antibodies from a single administration of the recombinant vector. Therefore, this vectored approach holds promises in the treatment and prevention of a chronic disease like HIV infection. Here, we provide an overview of HIV genetic diversity, AAV vectorology, and anti-HIV bnAbs and summarize the promises and challenges of the application of AAV in the delivery of bnAbs for HIV prevention and therapy.

Characterizing the Latent HIV-1 Reservoir in Patients with Viremia Suppressed on cART: Progress, Challenges, and Opportunities.

Modern combination antiretroviral therapy (cART) can bring HIV-1 in blood plasma to level undetectable by standard tests, prevent the onset of acquired immune deficiency syndrome (AIDS), and allow a near-normal life expectancy for HIV-infected individuals. Unfortunately, cART is not curative, as within a few weeks of treatment cessation, HIV viremia in most patients rebounds to pre-cART levels. The primary source of this rebound, and the principal barrier to a cure, is the highly stable reservoir of latent yet replication-competent HIV-1 proviruses integrated into the genomic DNA of resting memory CD4+ T cells. In this review, prevailing models for how the latent reservoir is established and maintained, residual viremia and viremic rebound upon withdrawal of cART, and the types and characteristics of cells harboring latent HIV-1 will be discussed. Selected technologies currently being used to advance our understanding of HIV latency will also be presented, as will a perspective on which areas of advancement are most essential for producing the next generation of HIV-1 therapeutics.

Molecular recognition of HIV-1 RNAs with branched peptides.

Targeting RNA offers the potential in many diseases of a therapeutic treatment. Due to its large surface area and ability to adopt different conformations, targeting RNA has proven challenging. Medium-sized branched peptides are of the size to competitively bind RNA while remaining cell permeable, stable in vivo, and non-toxic. Additionally, the ease in generating a large library followed by high-throughput screening provides a way to suggest a scaffold with high diversity that is capable of targeting the structure and sequence of RNA. The ability to select various types of amino acid modifications in the branched peptide allows for variable structures and interactions of the branched peptide but can result in too large a task if not approached properly. In this chapter, we discuss a strategy to selectively recognize RNAs of interest through high throughput screening of branched peptides, validation of hits and biophysical characterization, leading by example with our experience in targeting HIV-1 RNAs with branched peptides.

Interplay of primary sequence, position and secondary RNA structure determines alternative splicing of LMNA in a pre-mature aging syndrome.

Aberrant splicing in exon 11 of the LMNA gene causes the premature aging disorder Hutchinson-Gilford Progeria Syndrome. A de novo C1824T mutation activates an internal alternative 5' splice site, resulting in formation of the disease-causing progerin protein. The underlying mechanism for this 5' splice site selection is unknown. Here, we have applied a combination of targeted mutational analysis in a cell-based system and structural mapping by SHAPE-MaP to comprehensively probe the contributions of primary sequence, secondary RNA structure and linear splice site position in determining in vivo mechanisms of splice site choice in LMNA. While splice site choice is in part defined by sequence complementarity to U1 snRNA, we identify RNA secondary structural elements near the alternative 5' splice sites and show that splice site choice is significantly influenced by the structural context of the available splice sites. Furthermore, relative positioning of the competing sites within the primary sequence of the pre-mRNA is a predictor of 5' splice site usage, with the distal position favored over the proximal, regardless of sequence composition. Together, these results demonstrate that 5' splice site selection in LMNA is determined by an intricate interplay among RNA sequence, secondary structure and splice site position.

Evolution of the HIV-1 Rev Response Element during Natural Infection Reveals Nucleotide Changes That Correlate with Altered Structure and Increased Activity over Time.

The HIV-1 Rev response element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promote the nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly, and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The early RRE was less functionally active than the late RRE, despite differing in sequence by only 4 nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over 6 years of untreated infection. RRE sequence diversity varied over the course of infection, with evidence of selection pressure that led to sequence convergence as disease progressed being found. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding.IMPORTANCE HIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev response element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account.

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA.

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a Kd value of 410 nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.

Selective Small-Molecule Targeting of a Triple Helix Encoded by the Long Noncoding RNA, MALAT1.

Metastasis-associated lung adenocarcinoma transcript 1 ( Malat1/ MALAT1, mouse/human), a highly conserved long noncoding (lnc) RNA, has been linked with several physiological processes, including the alternative splicing, nuclear organization, and epigenetic modulation of gene expression. MALAT1 has also been implicated in metastasis and tumor proliferation in multiple cancer types. The 3' terminal stability element for nuclear expression (ENE) assumes a triple-helical configuration that promotes its nuclear accumulation and persistent function. Utilizing a novel small molecule microarray strategy, we identified multiple Malat1 ENE triplex-binding chemotypes, among which compounds 5 and 16 reduced Malat1 RNA levels and branching morphogenesis in a mammary tumor organoid model. Computational modeling and Förster resonance energy transfer experiments demonstrate distinct binding modes for each chemotype, conferring opposing structural changes to the triplex. Compound 5 modulates Malat1 downstream genes without affecting Neat1, a nuclear lncRNA encoded in the same chromosomal region as Malat1 with a structurally similar ENE triplex. Supporting this observation, the specificity of compound 5 for Malat1 over Neat1 and a virus-coded ENE was demonstrated by nuclear magnetic resonance spectroscopy. Small molecules specifically targeting the MALAT1 ENE triplex lay the foundation for new classes of anticancer therapeutics and molecular probes for the treatment and investigation of MALAT1-driven cancers.

Polymorphisms in KSHV-encoded microRNA sequences affect levels of mature viral microRNA in Kaposi Sarcoma lesions.

Background: We previously reported Kaposi sarcoma-associated herpesvirus (KSHV) microRNA sequence variants in clinical samples correlated with increased risk of multicentric Castleman's disease (MCD). We then demonstrated that microRNAs with variant sequence have different maturation and mature microRNA expression in vitro. Here, we illustrate the association between microRNA sequence and changes in mature microRNA levels within Kaposi sarcoma (KS) lesions. Methods: KSHV microRNA sequences were determined from 20 KS lesions and 4 control skin biopsies from individuals evaluated for KS. Levels of mature KSHV microRNAs were measured with 21 custom small RNA qRT-PCR assays using RNA RNU6B as endogenous control. Results: The levels of 13 KSHV-encoded microRNAs were elevated in KS lesions compared to control biopsies. MicroRNA 9-5p was strongly down regulated in South African vs. US biopsies. Low levels of K12-9-5p were associated with single nucleotide polymorphisms (SNPs) in miR-K12-9-5p, 4-5p, 5-3p, 7-3p and pri-miR-K12-3. One SNP in pri-miR-K12-3 resulted in down regulation of miR-K12-6-3p, 8-3p, 10-3p, 12-5p and the upregulation of 5-5p, illustrating sequence variants outside pre-microRNAs were also associated with changes in mature microRNA levels. Conclusions: The levels of mature KSHV-encoded microRNAs in KS lesions correlate with sequence variation reflecting changes in secondary and tertiary RNA structure.

APOL1 risk allele RNA contributes to renal toxicity by activating protein kinase R.

APOL1 risk alleles associate with chronic kidney disease in African Americans, but the mechanisms remain to be fully understood. We show that APOL1 risk alleles activate protein kinase R (PKR) in cultured cells and transgenic mice. This effect is preserved when a premature stop codon is introduced to APOL1 risk alleles, suggesting that APOL1 RNA but not protein is required for the effect. Podocyte expression of APOL1 risk allele RNA, but not protein, in transgenic mice induces glomerular injury and proteinuria. Structural analysis of the APOL1 RNA shows that the risk variants possess secondary structure serving as a scaffold for tandem PKR binding and activation. These findings provide a mechanism by which APOL1 variants damage podocytes and suggest novel therapeutic strategies.