Nicole A. Haverland

Quantitative proteomics by SWATH-MS reveals altered expression of nucleic acid binding and regulatory proteins in HIV-1-infected macrophages

Human immunodeficiency virus type 1 (HIV-1) infection remains a worldwide epidemic, and innovative therapies to combat the virus are needed. Developing a host-oriented antiviral strategy capable of targeting the biomolecules that are directly or indirectly required for viral replication may provide advantages over traditional virus-centric approaches. We used quantitative proteomics by SWATH-MS in conjunction with bioinformatic analyses to identify host proteins, with an emphasis on nucleic acid binding and regulatory proteins, which could serve as candidates in the development of host-oriented antiretroviral strategies. Using SWATH-MS, we identified and quantified the expression of 3608 proteins in uninfected and HIV-1-infected monocyte-derived macrophages. Of these 3608 proteins, 420 were significantly altered upon HIV-1 infection. Bioinformatic analyses revealed functional enrichment for RNA binding and processing as well as transcription regulation. Our findings highlight a novel subset of proteins and processes that are involved in the host response to HIV-1 infection. In addition, we provide an original and transparent methodology for the analysis of label-free quantitative proteomics data generated by SWATH-MS that can be readily adapted to other biological systems.

Proteomic Analysis of Early HIV-1 Nucleoprotein Complexes

After entry into the cell, the early steps of the human immunodeficiency virus type 1 (HIV-1) replication cycle are mediated by two functionally distinct nucleoprotein complexes, the reverse transcription complex (RTC) and preintegration complex (PIC). These two unique viral complexes are responsible for the conversion of the single-stranded RNA genome into double-stranded DNA, transport of the DNA into the nucleus, and integration of the viral DNA into the host cell chromosome. Prior biochemical analyses suggest that these complexes are large and contain multiple undiscovered host cell factors. In this study, functional HIV-1 RTCs and PICs were partially purified by velocity gradient centrifugation and fractionation, concentrated, trypsin digested, and analyzed by LC-MS/MS. A total of seven parallel infected and control biological replicates were completed. Database searches were performed with Proteome Discoverer and a comparison of the HIV-1 samples to parallel uninfected control samples was used to identify unique cellular factors. The analysis produced a total data set of 11055 proteins. Several previously characterized HIV-1 factors were identified, including XRCC6, TFRC, and HSP70. The presence of XRCC6 was confirmed in infected fractions and shown to be associated with HIV-1 DNA by immunoprecipitation-PCR experiments. Overall, the analysis identified 94 proteins unique in the infected fractions and 121 proteins unique to the control fractions with ≥ 2 protein assignments. An additional 54 and 52 were classified as enriched in the infected and control samples, respectively, based on a 3-fold difference in total Proteome Discoverer probability score. The differential expression of several candidate proteins was validated by Western blot analysis. This study contributes additional novel candidate proteins to the growing published bioinformatic data sets of proteins that contribute to HIV-1 replication.

Mass spectrometric characterization of gelsolin isoforms

Gelsolin is the most widely expressed member of the actin capping and severing family of proteins. There are two isoforms of gelsolin: isoform 1, a secretory (plasma) protein that is 51 amino acids longer than isoform 2, a cytosolic protein, at the N-terminus; the first 27 amino acids is a signal sequence. Both isoforms are coded by a single gene and differ as a result of alternative initiation site/splicing. The level of gelsolin in the blood and cerebrospinal fluid (CSF) is altered in many diseases including amyloidoses and other neurodegenerative disorders. Although quantitative analysis of gelsolin has been reported, lack of suitable antibodies makes it impossible to differentiate these two isoforms by immunodetection techniques and no other technique is available. Therefore, ambiguity exists whether gelsolin present in circulation is isoform 1 or also isoform 2 released from lysed cells. We report in this communication a mass spectrometric approach to identify isoform 1 of gelsolin immunopurified from human plasma and CSF. Recombinant isoform 1 was used as reference.

The mixed lineage kinase-3 inhibitor URMC-099 improves therapeutic outcomes for long-acting antiretroviral therapy

During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the mixed lineage kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2Rγc-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance. From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the mixed lineage kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future.

Granulocyte-Macrophage Colony Stimulating Factor Exerts Protective and Immunomodulatory Effects in Cortical Trauma

Neurodegeneration after traumatic brain injury is facilitated by innate and adaptive immunity and can be harnessed to affect brain repair. In mice subjected to controlled cortical impact (CCI), we show that treatment with granulocyte macrophage colony stimulating factor (GM-CSF) affects regulatory T cell numbers in the cervical lymph nodes coincident with decreased lesion volumes and increased cortical tissue sparing. This paralleled increases in neurofilament and diminished reactive microglial staining. Transcriptomic analysis showed that GM-CSF induces robust immune neuroprotective responses seven days following CCI. Together, these results support the therapeutic potential of GM-CSF for TBI.

Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation

BackgroundProteomic-based discovery of biomarkers for disease has recently come under scrutiny for a variety of issues; one prominent issue is the lack of orthogonal validation for biomarkers following discovery. Validation by ELISA or Western blot requires the use of antibodies, which for many potential biomarkers are under-characterized and may lead to misleading or inconclusive results. Gelsolin is one such biomarker candidate in HIV-associated neurocognitive disorders.MethodsSamples from human (plasma and CSF), monkey (plasma), monocyte-derived macrophage (supernatants), and commercial gelsolin (recombinant and purified) were quantitated using Western blot assay and a variety of anti-gelsolin antibodies. Plasma and CSF was used for immunoaffinity purification of gelsolin which was identified in eight bands by tandem mass spectrometry.ResultsImmunoreactivity of gelsolin within samples and between antibodies varied greatly. In several instances, multiple bands were identified (corresponding to different gelsolin forms) by one antibody, but not identified by another. Moreover, in some instances immunoreactivity depended on the source of gelsolin, e.g. plasma or CSF. Additionally, some smaller forms of gelsolin were identified by mass spectrometry but not by any antibody. Recombinant gelsolin was used as reference sample.ConclusionsOrthogonal validation using specific monoclonal or polyclonal antibodies may reject biomarker candidates from further studies based on misleading or even false quantitation of those proteins, which circulate in various forms in body fluids.

Investigation of the HIV-1 matrix interactome during virus replication

Like all viruses, human immunodeficiency virus type 1 (HIV‐1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell‐based inhibitors.

Alterations in the nuclear proteome of HIV-1 infected T-cells

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein-protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection.

The Proteomic Characterization of Plasma or Serum from HIV-Infected Patients.

Proteomics holds great promise for uncovering disease-related markers and mechanisms in human disorders. Recent advances have led to efficient, sensitive, and reproducible methods to quantitate the proteome in biological samples. Here we describe the techniques for processing, running, and analyzing samples from HIV-infected plasma or serum through quantitative mass spectroscopy.

Proteomic profiling of HIV-infected T-cells by SWATH mass spectrometry

Viral pathogenesis results from changes in host cells due to virus usurpation of the host cell and the innate cellular responses to thwart infection. We measured global changes in protein expression and localization in HIV-1 infected T-cells using subcellular fractionation and the Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) proteomic platform. Eight biological replicates were performed in two independent experimental series. In silico merging of both experiments identified 287 proteins with altered expression (p < .05) between control and infected cells- 172 in the cytoplasm, 84 in the membrane, and 31 in nuclei. 170 of the proteins are components of the NIH HIV interaction database. Multiple Reaction Monitoring and traditional immunoblotting validated the altered expression of several factors during infection. Numerous factors were found to affect HIV infection in gain- and loss-of-expression infection assays, including the intermediate filament vimentin which was found to be required for efficient infection.