Explore the vast range of titles available.

Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme that produces IP6 by phosphorylation of inositol pentakisphosphate (IP5), were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but until recently, it was not known if IP5 could also function in promoting assembly in vivo. Here we addressed whether there is an absolute requirement for IP6 or IP5 in the production of infectious HIV-1 particles. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KO but not wildtype cells. Several attempts to make IP6/IP5 deficient stable cells were not successful, but transient expression of the enzyme multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in near ablation of IP6 and IP5. Under these conditions, we found that HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction) demonstrating an IP6/IP5 requirement. However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found to show a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus, which displayed similar sensitivity as HIV-1. We were not able to determine if producer cell IP6/IP5 is required at additional steps beyond assembly because viral particles devoid of both molecules could not be generated. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissivity to HIV-1 infection.

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir. To monitor the presence of novel SARS-CoV-2 variants in New York City, Smyth et al. perform deep-sequencing of the receptor binding domain of S protein in wastewater samples and find novel cryptic lineages containing mutations affecting ACE2-tropism and showing decreased neutralization by antibodies.

A short, 14-amino-acid segment called SP1, located in the Gag structural protein 1 , has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane 2 , 3 . Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors 4 , 5 . Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP 6 ) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP 6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP 6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP 6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1. Inositol hexakisphosphate, which is found in all mammalian cells, binds to two separate sites to promote the assembly and maturation of HIV-1 virus particles.

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based ‘gain of function’ assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.

The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein βTrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the βTrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both βTrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/βTrCP ubiquitin ligase.

Suicide genes have been widely investigated for their utility as therapeutic agents and as tools for in vitro negative selection strategies. Several methods for delivery of suicide genes have been explored. Two important considerations for delivery are the quantity of delivered cargo and the ability to target the cargo to specific cells. Delivery using a lentiviral vector is particularly attractive due to the ability to encode the gene within the viral genome, as well as the ability to limit off-target effects by using cell type-specific glycoproteins. Here, we present the design and validation of a diphtheria toxin A (DTA)-encoding lentiviral vector expressing DTA under the control of a constituitive promoter to allow for expression of DTA in a variety of cell types, with specificity provided via selection of glycoproteins for pseudotyping of the lentiviral particles. DTA exerts its toxic activity through inhibition of eukaryotic translation elongation factor 2 (eEF2) via adenosine diphosphate (ADP)-ribosylation of a modified histidine residue, diphthamide, at His715, which blocks protein translation and leads to cell death. Thus, we also detail development of DTA-resistant cell lines, engineered through CRISPR/Cas9-mediated knockout of the diphthamide 1 (DPH1) gene, which enable both robust virus production by transfection and evaluation of DTA-expressing virus infectivity.

In this Letter, the Protein Data Bank (PDB) accessions were incorrectly listed as '6BH5, 6BHT and 6BHS' instead of '6BHR, 6BHT and 6BHS'; this has been corrected online.In this Letter, the Protein Data Bank (PDB) accessions were incorrectly listed as '6BH5, 6BHT and 6BHS' instead of '6BHR, 6BHT and 6BHS'; this has been corrected online.