A model of HIV-1 pathogenesis that includes an intracellular delay

Mathematical modeling combined with experimental measurements have yielded important insights into HIV-1 pathogenesis. For example, data from experiments in which HIV-infected patients are given potent antiretroviral drugs that perturb the infection process have been used to estimate kinetic parameters underlying HIV infection. Many of the models used to analyze data have assumed drug treatments to be completely efficacious and that upon infection a cell instantly begins producing virus. We consider a model that allows for less then perfect drug effects and which includes a delay in the initiation of virus production. We present detailed analysis of this delay differential equation model and compare the results to a model without delay. Our analysis shows that when drug efficacy is less than 100%, as may be the case in vivo, the predicted rate of decline in plasma virus concentration depends on three factors: the death rate of virus producing cells, the efficacy of therapy, and the length of the delay. Thus, previous estimates of infected cell loss rates can be improved upon by considering more realistic models of viral infection.     

Small-molecule inactivation of HIV-1 NCp7 by repetitive intracellular acyl transfer

The zinc fingers of the HIV-1 nucleocapsid protein, NCp7, are prime targets for antiretroviral therapeutics. Here we show that S-acyl-2-mercaptobenzamide thioester (SAMT) chemotypes inhibit HIV by modifying the NCp7 region of Gag in infected cells, thereby blocking Gag processing and reducing infectivity. The thiol produced by SAMT reaction with NCp7 is acetylated by cellular enzymes to regenerate active SAMTs via a recycling mechanism unique among small-molecule inhibitors of HIV.     

Characterization of glycosylation profiles of HIV-1 transmitted/founder envelopes by mass spectrometry

The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workflow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan profiles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades.     

Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry

The pleomorphic nature of the immature and mature HIV-1 virions has made it difficult to characterize intersubunit interactions using traditional approaches. While the structures of isolated domains are known, the challenge is to identify intersubunit interactions and thereby pack these domains into supramolecular structures. Using high-resolution mass spectrometry, we have measured the amide hydrogen exchange protection factors for the soluble capsid protein (CA) and CA assembled in vitro. Comparison of the protection factors as well as chemical crosslinking experiments has led to a map of the subunit/subunit interfaces in the assembled tubes. This analysis provides direct biochemical evidence for the homotypic N domain and C domain interactions proposed from cryo-electron microscopy image reconstruction of CA tubes. Most significantly, we have identified a previously unrecognized intersubunit N domain-C domain interaction. The detection of this interaction reconciles previously discrepant biophysical and genetic data.     

Analysis of stereoisomers of chiral drug by mass spectrometry

Chiral molecules are of great importance in the life science since individual enantiomers may differ in biological activity, mechanism, and toxicity, making it necessary to explore efficient chiral analysis methods. Chromatography approaches are often used to differentiate enantiomers while mass spectrometry (MS) was thought to be blind in chiral analysis. With the development of MS technique, it began to play a more and more crucial part in chiral observation. In this review, we will give a detailed introduction of the analysis methods related to MS for chiral drugs, including its mechanism, applications, and future development.     

Accurate mass analysis of phosphoramidites by electrospray mass spectrometry

A method of accurate mass determination of phosphoramidites is described. The commonly used methanol/water/acid system was replaced by LiCl-containing acetonitrile and the concentrations of LiCl, poly(ethylene glycol), and phosphoramidite samples were optimized.     

HIV-1 neutralization coverage is improved by combining monoclonal antibodies that target independent epitopes

HIV-1 neutralizing monoclonal antibodies (MAbs) define key targets for vaccine development and are being considered for passive prevention of infection. We analyzed the interaction of MAbs to two independent epitopes on the viral envelope glycoprotein. Potently neutralizing MAbs to the CD4 binding site and V1V2 region displayed no in vitro cross-competition and displayed additive, though not synergistic, neutralization activity. Predicted neutralization coverage of a combination of two MAbs reached 97% on a 208-isolate panel.     

Identification of an intracellular trafficking and assembly pathway for HIV-1 gag

Retroviral Gag proteins are membrane-bound polyproteins that are necessary and sufficient for virus-like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle production is determined. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to follow the trafficking of newly synthesized HIV-1 Gag. Gag first appears diffusely distributed in the cytosol, accumulates in perinuclear clusters, passes transiently through a multivesicular body (MVB)-like compartment, and then travels to the plasma membrane (PM). Sequential passage of Gag through these temporal intermediates was confirmed by live cell imaging. Induction of a transient rise in cytoplasmic calcium increased the amounts of Gag, Gag assembly intermediates and VLPs in MVBs, and resulted in a dramatic increase in VLP release. These results define an intracellular trafficking pathway for HIV-1 Gag that uses perinuclear compartments and the MVB as trafficking intermediates. We propose that the regulation of Gag association with MVB-like compartments regulates the site of HIV-1 budding and particle formation.     

Quantitative analysis of an N-oxide metabolite by fast atom bombardment tandem mass spectrometry

An assay for the N-oxide metabolite of a benzazepine drug by fast atom bombardment ionization with tandem mass spectrometric analysis on a triple quadrupole mass spectrometer has been developed and validated for urine and plasma samples. This methodology allows analysis of this metabolite directly in crude sample extracts, without the need for extensive chromatography or sample derivatization. Quantification was accomplished with the use of a stable isotope analog of the analyte as an internal standard, using the selected reaction monitoring mode of operation.     

Combined thin layer chromatography/mass spectrometry: an application of 252Cf plasma desorption mass spectrometry for drug monitoring

The pharmacokinetic analysis is performed in a three-step procedure: sample extraction, sample purification by thin layer chromatography (TLC) and quantitative sample detection by time-of-flight (TOF) mass spectrometry. 252Cf plasma desorption (PD) mass spectrometry utilizing the fission fragment-induced ionization and desorption of non-volatile compounds is suitable as a universal, non-destructive detector in TLC. Here TLC and mass spectrometry are operated in an off-line combination. As an example some pharmacokinetic data for etoposide (VP 16-213) together with calibration data are presented. The new experimental method is discussed in terms of sensitivity and detection limit.     

Early and prolonged antiretroviral therapy is associated with an HIV-1-specific T-cell profile comparable to that of long-term non-progressors

Background

Intervention with antiretroviral treatment (ART) and control of viral replication at the time of HIV-1 seroconversion may curtail cumulative immunological damage. We have therefore hypothesized that ART maintenance over a very prolonged period in HIV-1 seroconverters could induce an immuno-virological status similar to that of HIV-1 long-term non-progressors (LTNPs).

Methodology/Principal Findings

We have investigated a cohort of 20 HIV-1 seroconverters on long-term ART (LTTS) and compared it to one of 15 LTNPs. Residual viral replication and reservoirs in peripheral blood, as measured by cell-associated HIV-1 RNA and DNA, respectively, were demonstrated to be similarly low in both cohorts. These two virologically matched cohorts were then comprehensively analysed by polychromatic flow cytometry for HIV-1-specific CD4⁺ and CD8⁺ T-cell functional profile in terms of cytokine production and cytotoxic capacity using IFN-γ, IL-2, TNF-α production and perforin expression, respectively. Comparable levels of highly polyfunctional HIV-1-specific CD4⁺ and CD8⁺ T-cells were found in LTTS and LTNPs, with low perforin expression on HIV-1-specific CD8⁺ T-cells, consistent with a polyfunctional/non-cytotoxic profile in a context of low viral burden.

Conclusions

Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs, strengthening the recent emphasis on the positive impact of early treatment initiation and paving the way for further interventions to promote virological control after treatment interruption.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

The Siva protein is a novel intracellular ligand of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells

In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4⁺ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4⁺ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56^Lck, indicating that Siva-1 participates in a pathway independent of the p56^Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF

Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS.