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

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

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

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

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

Sso7d-IN is functional in virions.

<p>The assay is based on the ability of IN expressed as a Vpr fusion protein to transcomplement N/N virus lacking a functional integrase. A, HIV-1 infectivity normalized to the level obtained with Vpr-IN complementation. The Vpr fusions used for complementation and the infections that were conducted in the presence of RAL are indicated. Sso7d(mut) contains the mutations W24A and R43E which abrogate DNA binding. Graphed are averages with standard deviation for n = 3 (infections with RAL or Vpr-IN-D64A) or n = 6 independent experiments. B, Western blot of IN deletion mutant virus produced with indicated Vpr fusions probed for IN (left panel) and p24 (right panel). All Vpr-IN constructs yielded similar levels of packaged IN protein. The anti-IN antibody 8E5 recognizes the C-terminus (262–271) of IN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105078#pone.0105078-Nilsen1" target="_blank">[18]</a> while the anti-p24 was from Abcam.</p

Optimization of reaction conditions with Sso7d-IN and oligonucleotide DNA substrates.

<p>Concerted integration bands are indicated with arrows. A, Effect of donor DNA length. The reactions were carried with 1 µM Sso7d-IN (Gly-11) and 0.5 µM viral DNA substrate containing a “GC rich” motif in 20 mM HEPES pH 7.5, 10 mM DTT, 5 mM MgCl₂, 4 µM ZnCl₂, 100 mM NaCl, and 300 ng pGEM-9zf. B, Reactions were carried with 1 µM Sso7d-IN differing in the length of the glycine linker. C, Concerted integration under optimized conditions. The ratio of Sso7d-IN (Gly-11) to donor DNA (U5-25) was kept constant at 2∶1. Sso7d-IN concentrations are 0.4 µM (lane 1), 1.0 µM (lane 2), 2.0 µM (lane 3), 4.0 µM (lane 4) and 8.0 µM (lane 5). 25% glycerol was included in the reaction buffer. The DNA smear (S) below the linear concerted integration product results from multiple integrations into the same target DNA (depicted in D).</p

EMSA of intasomes assembled with Sso7d-IN (Gly-11) and a 25 bp DNA substrate (FAM labeled U5-25).

<p>To prevent non-specific DNA binding, 10 µg/ml of heparin was added to the reaction mixture after intasome assembly as well as into 3% agarose gels. A, Intasomes assemble with Sso7d-IN (lane 3), but not with wild-type HIV-1 IN (lane 1) or the Sso7d domain alone (lane 2). B, Sso7d-IN specifically assembles intasomes on LTR-U5 sequence (lane 1), but not on “CA/GT mut” (lane 2) or “3 bp mismatch” (lane 3) DNAs. In the “CA/GT mut” DNA, the conserved “CA” dinucleotide is replaced by “GT” (highlighted in the sequence). “3 bp mismatch” was prepared by replacing of “ACT” with “TGA” at the 5′ end of the non-joining strand.</p

Sso7d-IN is a hyperactive IN.

<p>A, Schematic of the IN fusion proteins. NED, N-terminal extension domain NTD, N-terminal domain; CCD, catalytic core domain; CTD, C-terminal domain. B, Comparison of the solubilities of wild-type HIV-1 IN and Sso7d-IN. Proteins were incubated at the indicated NaCl concentrations in 20 mM HEPES pH 7.5, 10% glycerol, 5 mM DTT and 1 mM EDTA, centrifuged and the supernatants and pellets were analyzed by SDS PAGE. C, Schematic of the <i>in vitro</i> integration reaction with a double stranded oligonucelotide mimicking viral LTR-U5 and a circular target DNA. D, Strand transfer reaction carried with either wild-type HIV-1 IN or Sso7d-IN with an 11 amino acid linker and a fluorescently labeled viral DNA substrate (U5-25) in 20 mM HEPES pH 7.5, 10 mM DTT, 5 mM MgCl₂, 4 µM ZnCl₂, 100 mM NaCl, 300 ng pGEM-9zf and 0.5 µM viral DNA substrate. The position of concerted and half-site integration products is indicated. The same gel was visualized by either ethidium bromide staining (left panel) or a Typhoon 8600 fluorescence scanner (right panel).</p