GS-9822, a Preclinical LEDGIN Candidate, Displays a Block-and-Lock Phenotype in Cell Culture

ABSTRACT The ability of HIV to integrate into the host genome and establish latent reservoirs is the main hurdle preventing an HIV cure. LEDGINs are small-molecule integrase inhibitors that target the binding pocket of LEDGF/p75, a cellular cofactor that substantially contributes to HIV integration site selection. They are potent antivirals that inhibit HIV integration and maturation. In addition, they retarget residual integrants away from transcription units and toward a more repressive chromatin environment. As a result, treatment with the LEDGIN CX14442 yielded residual provirus that proved more latent and more refractory to reactivation, supporting the use of LEDGINs as research tools to study HIV latency and a functional cure strategy. In this study, we compared GS-9822, a potent, preclinical lead compound, with CX14442 with respect to antiviral potency, integration site selection, latency, and reactivation. GS-9822 was more potent than CX14442 in most assays. For the first time, the combined effects on viral replication, integrase-LEDGF/p75 interaction, integration sites, epigenetic landscape, immediate latency, and latency reversal were demonstrated at nanomolar concentrations achievable in the clinic. GS-9822 profiles as a preclinical candidate for future functional cure research.

Analysis of the intrinsic chromatin binding property of HIV-1 integrase and its regulation by LEDGF/p75 using human chromosomes spreads

ABSTRACTRetroviral integration requires the stable insertion of the viral genome into the host chromosomes. During this process, the functional integration complex must associate with cellular chromatin via the interaction between retroviral integrase and nucleosomes. The final association between the HIV-1 integration complex and the nucleosomal target DNA remains unclear and may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To date, there is no experimental system allowing the direct monitoring of this protein association with chromatin to depict the molecular mechanism of this process fully. To investigate this and understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase further, we used both biochemical approaches and an unedited chromosome-binding assays. Our study revealed that retroviral IN has an intrinsic ability to bind and recognize specific chromatin regions even in the absence of its cofactor. We also showed that this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromatin regions. Using these approaches, we also better determined the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. In addition to supporting a chromatin-binding function of the integrase protein acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate, our work precisely elucidates the mechanism of action of LEDGF/p75 in this crucial integration step.

The structure of APOBEC1 and insights into its RNA and DNA substrate selectivity

APOBEC1 (APO1), a member of AID/APOBEC nucleic acid cytosine deaminase family, can edit apolipoprotein B mRNA to regulate cholesterol metabolism. This APO1 RNA editing activity requires a cellular cofactor to achieve tight regulation. However, no cofactors are required for deamination on DNA by APO1 and other AID/APOBEC members, and aberrant deamination on genomic DNA by AID/APOBEC deaminases has been linked to cancer. Here, we present the crystal structure of APO1, which reveals a typical APOBEC deaminase core structure, plus a unique well-folded C-terminal domain that is highly hydrophobic. This APO1 C-terminal hydrophobic domain (A1HD) interacts to form a stable dimer mainly through hydrophobic interactions within the dimer interface to create a four-stranded β-sheet positively charged surface. Structure-guided mutagenesis within this and other regions of APO1 clarified the importance of the A1HD in directing RNA and cofactor interactions, providing insights into the structural basis of selectivity on DNA or RNA substrates.

HIV-1 Vpr mediates the depletion of the cellular repressor CTIP2 to counteract viral gene silencing

Mammals have evolved many antiviral factors impacting different steps of the viral life cycle. Associated with chromatin-modifying enzymes, the cellular cofactor CTIP2 contributes to HIV-1 gene silencing in latently infected reservoirs that constitute the major block toward an HIV cure. We report, for the first time, that the virus has developed a strategy to overcome this major transcriptional block. Productive HIV-1 infection results in a Vpr-mediated depletion of CTIP2 in microglial cells and CD4+ T cells, two of the major viral reservoirs. Associated to the Cul4A-DDB1-DCAF1 ubiquitin ligase complex, Vpr promotes CTIP2 degradation via the proteasome pathway in the nuclei of target cells and notably at the latent HIV-1 promoter. Importantly, Vpr targets CTIP2 associated with heterochromatin-promoting enzymes dedicated to HIV-1 gene silencing. Thereby, Vpr reactivates HIV-1 expression in a microglial model of HIV-1 latency. Altogether our results suggest that HIV-1 Vpr mediates the depletion of the cellular repressor CTIP2 to counteract viral gene silencing.

Metabolic cofactors act as initiating substrates for primase and affect replication primer processing

Recently a new, non-canonical type of 5’-RNA capping with cellular metabolic cofactors was discovered in bacteria and eukaryotes. This type of capping is performed by RNA polymerases, the main enzymes of transcription, which initiate RNA synthesis with cofactors. Here we show that primase, the enzyme of replication which primes synthesis of DNA by making short RNA primers, initiates synthesis of replication primers using the number of metabolic cofactors. Primase DnaG of E. coli starts synthesis of RNA with cofactors NAD+/NADH, FAD and DP-CoA in vitro. This activity does not affect primase specificity of initiation. ppGpp, the global starvation response regulator, strongly inhibits the non-canonical initiation by DnaG. Amino acid residues of a “basic ridge” define the binding determinant of cofactors to DnaG. Likewise, the human primase catalytic subunit P49 can use modified substrate m7GTP for synthesis initiation.For correct genome duplication, the RNA primer needs to be removed and Okazaki fragments ligated. We show that the efficiency of primer processing by DNA polymerase I is strongly affected by cofactors on the 5’-end of RNA. Overall our results suggest that cofactors at the 5’ position of the primer influence regulation of initiation and Okazaki fragments processing.Visual abstractA. Non-canonical capping of RNA by RNA polymerase. RNA polymerase uses cellular cofactor as initiating substrate for RNA synthesis, instead of NTP. Then RNA chain grows, while cofactor remains attached and serves as cap. B. Proposed mechanism of non-canonical initiation of RNA primer synthesis by DnaG primase during replication. DnaG primase initiates synthesis of the primer for DNA replication using cellular cofactor. Primer stays annealed with the DNA template. DNApolI encounters cofactor, which affects the removal of primer.

Quinoline-based Protein–protein Interaction Inhibitors of LEDGF/p75 and HIV Integrase: An In Silico Study

The failure of the Integrase Strand Transfer Inhibitors (INSTIs) due to the mutations occurring at the catalytic site of HIV integrase (IN) has led to the design of allosteric integrase inhibitors (ALLINIs). Lens epithelium derived growth factor (LEDGF/p75) is the host cellular cofactor which helps chaining IN to the chromatin. The protein-protein interactions (PPIs) were observed at the allosteric site (LEDGF/p75 binding domain) between LEDGF/p75 of the host cell and IN of virus. In recent years, many small molecules such as CX04328, CHIBA-3053 and CHI-104 have been reported as LEDGF/p75-IN interaction inhibitors (LEDGINs). LEDGINs have emerged as promising therapeutics to halt the PPIs by binding at the interface of both the proteins. In the present work, we correlated the docking scores for the reported LEDGINs containing quinoline scaffold with the in vitro biological data. The hierarchal clustering method was used to divide the compounds into test and training set. The robustness of the generated model was validated by q2 and r2 for the predicted set of compounds. The generated model between the docking score and biological data was assessed to predict the activity of the hits (quinoline scaffold) obtained from virtual screening of LEDGINs providing their structureactivity relationships to aim for the generation of potent agents.

CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses

ABSTRACTPestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCEOnly noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

Specific Inhibition of HIV Infection by the Action of Spironolactone in T Cells

ABSTRACTTat protein, the HIV transactivator, regulates transcription of the HIV genome by the host transcription machinery. Efficient inhibitors of HIV transcription that target Tat or the cellular cofactor NF-κB are well known. However, inhibition of HIV Tat-dependent transcription by targeting the general transcription and DNA repair factor II human (TFIIH) has not been reported. Here, we show that spironolactone (SP), an aldosterone antagonist approved for clinical use, inhibits HIV-1 and HIV-2 infection of permissive T cells by blocking viral Tat-dependent transcription from the long terminal repeat (LTR). We found that treatment of Jurkat and primary CD4+T cells with SP induces degradation of the XPB cellular helicase, a component of the TFIIH complex, without affecting cellular mRNA levels, T cell viability, or T cell proliferation. We further demonstrate that the effect of SP on HIV infection is independent of its aldosterone antagonist function, since the structural analogue, eplerenone, does not induce XPB degradation and does not inhibit HIV infection. Rescue experiments showed that the SP-induced block of HIV infection relies, at least partially, on XPB degradation. In addition, we demonstrate that SP specifically inhibits Tat-dependent transcription, since basal transcription from the LTR is not affected. Our results demonstrate that SP is a specific inhibitor of HIV Tat-dependent transcription in T cells, which additionally suggests that XPB is a cofactor required for HIV infection. Targeting a cellular cofactor of HIV transcription constitutes an alternative strategy to inhibit HIV infection, together with the existing antiretroviral therapy.IMPORTANCETranscription from the HIV promoter is regulated by the combined activities of the host transcription machinery and the viral transactivator Tat protein. Here, we report that the drug spironolactone—an antagonist of aldosterone—blocks viral Tat-dependent transcription, thereby inhibiting both HIV-1 and HIV-2 infection of permissive T cells. This inhibition relies on the degradation of the cellular helicase XPB, a component of the TFIIH transcription factor complex. Consequently, XPB appears to be a novel HIV cofactor. Our discovery of the HIV-inhibitory activity of spironolactone opens the way for the development of novel anti-HIV strategies targeting a cellular cofactor without the limitations of antiretroviral therapy of drug resistance and high cost.