Cellular kinases incorporated into HIV-1 particles: passive or active passengers?

Phosphorylation is one of the major mechanisms by which the activities of protein factors can be regulated. Such regulation impacts multiple key-functions of mammalian cells, including signal transduction, nucleo-cytoplasmic shuttling, macromolecular complexes assembly, DNA binding and regulation of enzymatic activities to name a few. To ensure their capacities to replicate and propagate efficiently in their hosts, viruses may rely on the phosphorylation of viral proteins to assist diverse steps of their life cycle. It has been known for several decades that particles from diverse virus families contain some protein kinase activity. While large DNA viruses generally encode for viral kinases, RNA viruses and more precisely retroviruses have acquired the capacity to hijack the signaling machinery of the host cell and to embark cellular kinases when budding. Such property was demonstrated for HIV-1 more than a decade ago. This review summarizes the knowledge acquired in the field of HIV-1-associated kinases and discusses their possible function in the retroviral life cycle

HIV-1-associated PKA acts as a cofactor for genome reverse transcription

BACKGROUND: Host cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle. RESULTS: NL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions. CONCLUSIONS: Altogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events

Turning high-throughput structural biology into predictive inhibitor design

A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilized advance is the increase in structural biology throughput, which has progressed from an artisanal endeavor to a monthly throughput of hundreds of different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high-throughput crystallography data into predictive models for ligand design. Here, we designed a simple machine learning approach that predicts protein–ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein–ligand complexes and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high-throughput crystallography campaign against the SARS-CoV-2 main protease (MPro), obtaining parallel measurements of over 200 protein–ligand complexes and their binding activities. This allows us to design one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a noncovalent and nonpeptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry

A Chemical Probe for Tudor Domain Protein Spindlin1 to Investigate Chromatin Function.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis

Involvement of cAMP dependent protein kinase (PKA) in the early steps of HIV-1 replication cycle

Les étapes précoces du cycle réplicatif du VIH-1 sont assistées par des cofacteurs cellulaires dont la nature et la fonction restent mal connues. Nos travaux ont caractérisé la contribution de la protéine kinase dépendante de l'AMP cyclique (PKA), dans la cellule cible ou incorporée dans la particule virale VIH-1, dans les étapes post-entrée du cycle réplicatif. Les virus dépourvus d'activité PKA se caractérisent par un défaut de la synthèse de l'ADN proviral. En absence de Nef, la perte d'activité PKA associée aux particules VIH-1 induit une baisse plus modérée du pouvoir infectieux. En outre, le contournement des voies d'entrée classiques, par l'utilisation de particules pseudotypées, rend l'infectiosité indépendante de l'activité PKA. L'action de PKA s'exercerait donc au sein de la particule VIH-1 assemblée et impliquerait à la fois la protéine Nef et les voies de transport des complexes de transcription inverse au cours des étapes post-entrée. De plus, l'inhibition de l'activité PKA dans les cellules cibles entraîne également un défaut de synthèse de l'ADN proviral. Nos résultats indiquent que PKA agit comme un cofacteur de la transcription inverse.The nature and function of cellular factors involved in post-entry steps of the HIV-1 life cycle are still poorly understood. We highlighted the role of cAMP-dependent protein kinase (PKA) in the early step of viral cycle, either as a host cellular protein in infected cells or as an incorporated protein into HIV-1 particles. PKA-deficient viruses failed to synthesize proviral DNA. In the absence of Nef, the loss of PKA activity associated to HIV-1 particles induces a minor diminution of infectiousness. Accordingly, VSV-G pseudotyped viruses, that use alternate entry pathway, exhibit full infectivity regardless of PKA deficiency. PKA action could therefore take place in the assembled HIV-1 particles, implying Nef protein and intracellular pathways of reverse transcription complexes. Moreover, the inhibition of PKA activity in target cells engenders a defective proviral DNA synthesis. Taken together, our data suggest that PKA may act as a cofactor required for HIV-1 reverse transcription

Rôle de la protéine kinase dépendante de l'AMPc (PKA) dans les étapes précoces du cycle réplicatif du VIH-1

Les étapes précoces du cycle réplicatif du VIH-1 sont assistées par des cofacteurs cellulaires dont la nature et la fonction restent mal connues. Nos travaux ont caractérisé la contribution de la protéine kinase dépendante de l'AMP cyclique (PKA), dans la cellule cible ou incorporée dans la particule virale VIH-1, dans les étapes post-entrée du cycle réplicatif. Les virus dépourvus d'activité PKA se caractérisent par un défaut de la synthèse de l'ADN proviral. En absence de Nef, la perte d'activité PKA associée aux particules VIH-1 induit une baisse plus modérée du pouvoir infectieux. En outre, le contournement des voies d'entrée classiques, par l'utilisation de particules pseudotypées, rend l'infectiosité indépendante de l'activité PKA. L'action de PKA s'exercerait donc au sein de la particule VIH-1 assemblée et impliquerait à la fois la protéine Nef et les voies de transport des complexes de transcription inverse au cours des étapes post-entrée. De plus, l'inhibition de l'activité PKA dans les cellules cibles entraîne également un défaut de synthèse de l'ADN proviral. Nos résultats indiquent que PKA agit comme un cofacteur de la transcription inverse.The nature and function of cellular factors involved in post-entry steps of the HIV-1 life cycle are still poorly understood. We highlighted the role of cAMP-dependent protein kinase (PKA) in the early step of viral cycle, either as a host cellular protein in infected cells or as an incorporated protein into HIV-1 particles. PKA-deficient viruses failed to synthesize proviral DNA. In the absence of Nef, the loss of PKA activity associated to HIV-1 particles induces a minor diminution of infectiousness. Accordingly, VSV-G pseudotyped viruses, that use alternate entry pathway, exhibit full infectivity regardless of PKA deficiency. PKA action could therefore take place in the assembled HIV-1 particles, implying Nef protein and intracellular pathways of reverse transcription complexes. Moreover, the inhibition of PKA activity in target cells engenders a defective proviral DNA synthesis. Taken together, our data suggest that PKA may act as a cofactor required for HIV-1 reverse transcription.MONTPELLIER-BU Médecine UPM (341722108) / SudocSudocFranceF

Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors

HIV-1 entry and fusion with target cells is an important target for antiviral therapy. However, a few currently approved treatments are not effective as monotherapy due to the emergence of drug resistance. This consideration has fueled efforts to develop new bioavailable inhibitors targeting different steps of the HIV-1 entry process. Here, a high-throughput screen was performed of a large library of 100,000 small molecules for HIV-1 entry/fusion inhibitors, using a direct virus–cell fusion assay in a 384 half-well format. Positive hits were validated using a panel of functional assays, including HIV-1 specificity, cytotoxicity, and single-cycle infectivity assays. One compound—4-(2,5-dimethyl-pyrrol-1-yl)-2-hydroxy-benzoic acid (DPHB)—that selectively inhibited HIV-1 fusion was further characterized. Functional experiments revealed that DPHB caused irreversible inactivation of HIV-1 Env on cell-free virions and that this effect was related to binding to the third variable loop (V3) of the gp120 subunit of HIV-1 Env. Moreover, DPHB selectively inhibited HIV-1 strains that use CXCR4 or both CXCR4 and CCR5 co-receptors for entry, but not strains exclusively using CCR5. This selectivity was mapped to the gp120 V3 loop using chimeric Env glycoproteins. However, it was found that pure DPHB was not active against HIV-1 and that its degradation products (most likely polyanions) were responsible for inhibition of viral fusion. These findings highlight the importance of post-screening validation of positive hits and are in line with previous reports of the broad antiviral activity of polyanions

Discovery of a selective inhibitor for the YEATS domains of ENL/AF9

Eleven-nineteen leukemia (ENL) contains an epigenetic reader domain (YEATS domain) that recognizes lysine acylation on histone 3 and facilitates transcription initiation and elongation through its interactions with the super elongation complex (SEC) and the histone methyl transferase DOT1L. Although it has been known for its role as a fusion protein in mixed lineage leukemia (MLL), overexpression of native ENL, and thus dysregulation of downstream genes in acute myeloid leukemia (AML), has recently been implicated as a driver of disease that is reliant on the epigenetic reader activity of the YEATS domain. We developed a peptide displacement assay (histone 3 tail with acylated lysine) and screened a small-molecule library totaling more than 24,000 compounds for their propensity to disrupt the YEATS domain-histone peptide binding. Among these, we identified a first-in-class dual inhibitor of ENL ( Kd = 745 ± 45 nM) and its paralog AF9 ( Kd = 523 ± 53 nM) and performed "SAR by catalog" with the aim of starting the development of a chemical probe for ENL