Step-dose IL-7 treatment promotes systemic expansion of T cells and alters immune cell landscape in blood and lymph nodes.

We employed a dose-escalation regimen in rhesus macaques to deliver glycosylated IL-7, a cytokine critical for development and maintenance of T lymphocytes. IL-7 increased proliferation and survival of T cells and triggered several chemokines and cytokines. Induction of CXCL13 in lymph nodes (LNs) led to a remarkable increase of B cells in the LNs, proliferation of germinal center follicular T helper cells and elevated IL-21 levels suggesting an increase in follicle activity.

Dolutegravir Monotherapy of Simian Immunodeficiency Virus-Infected Macaques Selects for Several Patterns of Resistance Mutations with Variable Virological Outcomes.

Drug resistance remains a major concern for human immunodeficiency virus (HIV) treatment. To date, very few resistance mutations have emerged in first-line combination therapy that includes the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). , DTG selects for several primary mutations that induce low-level DTG resistance; secondary mutations, while increasing the level of resistance, however, further impair replication fitness, which raised the idea that DTG monotherapy may be feasible.

Correction: Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

[This corrects the article DOI: 10.1371/journal.ppat.

Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

Unlabelled: B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8+ T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (TFH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8+ T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8+ cells in LN.

T-cell responses to KSHV infection: a systematic approach.

Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs.

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

Unlabelled: HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations.

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized.

Characterization and favorable in vivo properties of heterodimeric soluble IL-15·IL-15Rα cytokine compared to IL-15 monomer.

Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium.

Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum.

IL-15 is an important cytokine for the function of the immune system, but the form(s) of IL-15 produced in the human body are not fully characterized. Coexpression of the single-chain IL-15 and the IL-15 receptor alpha (IL-15Rα) in the same cell allows for efficient production, surface display, and eventual cleavage and secretion of the bioactive IL-15/IL-15Rα heterodimer in vivo, whereas the single-chain IL-15 is poorly secreted and unstable. This observation led to the hypothesis that IL-15 is produced and secreted only as a heterodimer with IL-15Rα.

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture.

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines.

Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail.

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme.

HIV-1 inactivation by 4-vinylpyridine is enhanced by dissociating Zn(2+) from nucleocapsid protein.

Selective inactivation of critical cysteine residues in human immunodeficiency virus type one (HIV-1) was observed after treatment with 4-vinylpyridine (4-VP), with and without the membrane-permeable metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Chromatographic analysis showed that cysteines contained within nucleocapsid zinc fingers, in the context of whole virus or purified protein, were essentially unreactive, but became reactive when a chelator was included. Virus treated with 4-VP showed only a modest decrease in infectivity; after TPEN addition, nearly complete inactivation of HIV-1 occurred.

Mutational analysis of the C-terminal gag cleavage sites in human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6(Gag) proteins.

HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease.

HIV-1 virions are highly enriched in cholesterol relative to the cellular plasma membrane. We recently reported that a cholesterol-binding compound, amphotericin B methyl ester (AME), blocks HIV-1 entry and that single amino acid substitutions in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 confer resistance to AME. In this study, we defined the mechanism of resistance to AME.

Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.

Human immunodeficiency virus type 1 (HIV-1) infects CD4(+) T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells.

Recombinant production of anti-HIV protein, griffithsin, by auto-induction in a fermentor culture.

Griffithsin (GRFT) is a novel anti-HIV protein isolated from the red alga Griffithia sp. The potent anti-viral activity of GRFT against both laboratory and primary isolates of HIV at picomolar concentrations makes this protein an attractive candidate microbicide to prevent sexual transmission of HIV. Here, we describe the recombinant production and purification of a biologically active hexa-histidine-tagged GRFT (His-GRFT) from Escherichia coli.

Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp.

Griffithsin (GRFT), a novel anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp. The 121-amino acid sequence of GRFT has been determined, and biologically active GRFT was subsequently produced by expression of a corresponding DNA sequence in Escherichia coli. Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M- tropic HIV-1 with EC50 values ranging from 0.

Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant.

The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10(-6) relative to the wild type) and severely genomic RNA deficient.

A potent novel anti-HIV protein from the cultured cyanobacterium Scytonema varium.

A new anti-HIV protein, scytovirin, was isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein displayed potent anticytopathic activity against laboratory strains and primary isolates of HIV-1 with EC50 values ranging from 0.3 to 22 nM.

Retroviruses have differing requirements for proteasome function in the budding process.

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined.

Equine infectious anemia virus and the ubiquitin-proteasome system.

Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells.

Chemical cleavage at aspartyl residues for protein identification.

An alternative method to enzymatic digestion for protein identification by mass spectrometry has been developed that is based on chemical cleavage by formic acid. This method was tested on gel-purified apomyoglobin and BSA, as well as unknown proteins that cofractionate with Tyl-virus-like particles from Saccharomyces cerevisiae. Cleavage at aspartyl residues was found to be efficient and specific, and this specificity of cleavage lent itself easily to database searches.

A novel anti-HIV macrocyclic peptide from Palicourea condensata.

A 37 amino acid cyclic polypeptide has been isolated from the organic extract of the tropical tree Palicourea condensata. Palicourein (1) is the largest of a growing family of plant peptides that contain a cyclized amino acid backbone cross-linked via three internal disulfide bridges. Palicourein inhibits the in vitro cytopathic effects of HIV-1RF infection of CEM-SS cells with an EC50 value of 0.

Alteration of zinc-binding residues of simian immunodeficiency virus p8(NC) results in subtle differences in gag processing and virion maturation associated with degradative loss of mutant NC.

In all retroviruses analyzed to date (except for the spumaretroviruses), the Zn(2+)-coordinating residues of nucleocapsid (NC) perform or assist in crucial reactions necessary to complete the retrovirus life cycle. Six replication-defective mutations have been engineered in the two NC Zn(2+) fingers (ZFs) of simian immunodeficiency virus [SIV(Mne)] that change or delete specific Zn(2+)-interacting Cys residues and were studied by using electron microscopy, reversed-phase high-performance liquid chromatography, immunoblotting, and RNA quantification. We focused on phenotypes of produced particles, specifically morphology, Gag polyprotein processing, and genomic RNA packaging.

Role of the histidine residues of visna virus nucleocapsid protein in metal ion and DNA binding.

Zinc finger (ZF) domains in retroviral nucleocapsid proteins usually contain one histidine per metal ion coordination complex (Cys-X(2)-Cys-X(4)-His-X(4)-Cys). Visna virus nucleocapsid protein, p8, has two additional histidines (in the second of its two ZFs) that could potentially bind metal ions. Absorption spectra of cobalt-bound ZF2 peptides were altered by Cys alkylation and mutation, but not by mutation of the extra histidines.