Allosteric Integrase Inhibitors Reveal a Role for Integrase During HIV-1 Maturation

Integration of the DNA copy of the HIV-1 genome is an essential step for virus replication and is mediated by a homotetrameric complex of the viral protein integrase (IN) in association with the ends of linear viral DNA (vDNA). HIV-1 integrates into actively transcribed genes, a trait mediated by cellular host cofactor LEDGF/p75. LEDGF/p75 engages IN in a pocket formed by dimerization of the IN catalytic core domain, a region that has been validated as a drug target for allosteric IN inhibitors (ALLINIs). Previous in vitro work suggested that ALLINIs function through disruption of two integration-associated functions: IN-vDNA complex formation and the IN-LEDGF/p75 interaction. We now demonstrate that ALLINI potency is accounted for during the late phase of HIV-1 replication where the inhibitors block the formation of the viral core, converting the normally electron-dense conical core to an eccentric phenotype where the electron-density exists as a condensate situated between a translucent core and the viral membrane. We have further elucidated the eccentric condensates to represent non-packaged viral ribonucleoprotein (vRNP) complexes and that either genetic or pharmacological inhibition of IN can impair vRNP encapsidation. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with the internal electron density. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose an active role for IN during HIV-1 maturation that involves initiating core morphogenesis and vRNP encapsidation.Medical Science

Allosteric Integrase Inhibitors Reveal a Role for Integrase During HIV-1 Maturation

Integration of the DNA copy of the HIV-1 genome is an essential step for virus replication and is mediated by a homotetrameric complex of the viral protein integrase (IN) in association with the ends of linear viral DNA (vDNA). HIV-1 integrates into actively transcribed genes, a trait mediated by cellular host cofactor LEDGF/p75. LEDGF/p75 engages IN in a pocket formed by dimerization of the IN catalytic core domain, a region that has been validated as a drug target for allosteric IN inhibitors (ALLINIs). Previous in vitro work suggested that ALLINIs function through disruption of two integration-associated functions: IN-vDNA complex formation and the IN-LEDGF/p75 interaction. We now demonstrate that ALLINI potency is accounted for during the late phase of HIV-1 replication where the inhibitors block the formation of the viral core, converting the normally electron-dense conical core to an eccentric phenotype where the electron-density exists as a condensate situated between a translucent core and the viral membrane. We have further elucidated the eccentric condensates to represent non-packaged viral ribonucleoprotein (vRNP) complexes and that either genetic or pharmacological inhibition of IN can impair vRNP encapsidation. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with the internal electron density. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose an active role for IN during HIV-1 maturation that involves initiating core morphogenesis and vRNP encapsidation.Medical Science

Engineered Hyperactive Integrase for Concerted HIV-1 DNA Integration

The DNA cutting and joining reactions of HIV-1 integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes

HRP2 Determines the Efficiency and Specificity of HIV-1 Integration in LEDGF/p75 Knockout Cells but Does Not Contribute to the Antiviral Activity of a Potent LEDGF/p75-Binding Site Integrase Inhibitor

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors

The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase

Background: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. Results: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. Conclusions: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0100-1) contains supplementary material, which is available to authorized users

Introductory programming: a systematic literature review

As computing becomes a mainstream discipline embedded in the school curriculum and acts as an enabler for an increasing range of academic disciplines in higher education, the literature on introductory programming is growing. Although there have been several reviews that focus on specific aspects of introductory programming, there has been no broad overview of the literature exploring recent trends across the breadth of introductory programming. This paper is the report of an ITiCSE working group that conducted a systematic review in order to gain an overview of the introductory programming literature. Partitioning the literature into papers addressing the student, teaching, the curriculum, and assessment, we explore trends, highlight advances in knowledge over the past 15 years, and indicate possible directions for future research

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID-19-associated vascular pathology in mice

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. In the present study, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID-19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells do not contribute significantly to the diverse vascular pathology associated with COVID-19

Serotonin Reduction in Post-acute Sequelae of Viral Infection

Post-acute sequelae of COVID-19 (PASC, Long COVID ) pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes

Neurotropic RNA Virus Modulation of Immune Responses within the Central Nervous System

The central nervous system (CNS) necessitates intricately coordinated immune responses to prevent neurological disease. However, the emergence of viruses capable of entering the CNS and infecting neurons threatens this delicate balance. Our CNS is protected from foreign invaders and excess solutes by a semipermeable barrier of endothelial cells called the blood–brain barrier. Thereby, viruses have implemented several strategies to bypass this protective layer and modulate immune responses within the CNS. In this review, we outline these immune regulatory mechanisms and provide perspectives on future questions in this rapidly expanding field

Neurotropic RNA Virus Modulation of Immune Responses within the Central Nervous System

The central nervous system (CNS) necessitates intricately coordinated immune responses to prevent neurological disease. However, the emergence of viruses capable of entering the CNS and infecting neurons threatens this delicate balance. Our CNS is protected from foreign invaders and excess solutes by a semipermeable barrier of endothelial cells called the blood–brain barrier. Thereby, viruses have implemented several strategies to bypass this protective layer and modulate immune responses within the CNS. In this review, we outline these immune regulatory mechanisms and provide perspectives on future questions in this rapidly expanding field