A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 10⁷ CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 10⁶ cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays

A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 10⁷ CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 10⁶ cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays

Modeling and Simulation Based Analysis of Multi-Class Traffic with Look-Ahead Controlled Vehicles

Additional file 7: Fig. S7. TLR and PTEF-b inhibition impairs TLR-mediated HIV reactivation. a TLR ligands reactivate HIV in an NF-κB-dependent manner. Treatment of THP-1/HIV (HA3) cells with TNF-α (10 ng/mL) or TLR ligands (Pam3CSK4 at 0.1 µg/mL, HKLM at 108 cells/mL, poly (I:C) at 10 µg/mL, LPS at 1 µ/mL, flagellin at 1 µ/mL, FSL-1 at 1 µg/mL, imiquimod at 10 µg/mL, ssRNA40 at 5 µg/mL, and ODN2006 at 5 µM) for 16 h after a 2-h pre-incubation with either 100 µM of IKKγ NEMO binding domain inhibitory peptide (red bars; Inh Pep) or equivalent amount of the control peptide (blue bars; Imgenex) (X-axis). Y-axis represents % of GFP-expressing cells after FACS measurements and blue squares % of viable cells after PI exclusion quantification (right Y-axis). Error bars depict the standard deviation of three different experiments. b Partial inhibition of TNF-α-, IL-1β-, or TLR-mediated HIV reactivation by P-TEFb inhibitors. Human hµglia/HIV (HC01) and (HC69), and rat hT-CHME-5/HIV (HC03) and (HC14) microglial cells were untreated (black) or pre-treated with DRB (red; 10 µM) or flavopiridol (blue; 30 nM) for 30 min prior to treatment with TNF-α (30 ng/mL), IL-1β (10 pg/mL), LPS (1 µg/mL), or poly (I:C) (10 µg/mL), as shown in the X-axis, for 16 h prior to quantification of GFP (Y-axis)

Phosphorylation of CDK9 at Ser175 Enhances HIV Transcription and Is a Marker of Activated P-TEFb in CD4⁺ T Lymphocytes

<div><p>The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.</p></div

A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 10⁷ CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 10⁶ cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays

Post-translational modifications (PTMs) of CDK9 Isoform 2 (117 amino acid extension at N-Terminus) identified by tandem mass spectrometry analysis.

<p>Fold-changes in PTM levels after PMA or TCR activation are relative to the non-stimulated condition. Analyses of the CDK9 isoform 2 was performed using mass spectrometry data from FLAG-CDK9 affinity isolates.</p

Post-translational modifications (PTMs) of CycT1 identified by tandem mass spectrometry analysis.

<p>Fold-changes in PTM levels after PMA or TCR activation are relative to the non-stimulated condition. Analyses of CycT1 was performed using mass spectrometry data from FLAG-CDK9 affinity isolates.</p

Post-translational modifications (PTMs) of CDK9 Isoform 1 identified by tandem mass spectrometry analysis.

<p>Fold-changes in PTM levels after PMA or TCR activation are relative to the non-stimulated condition. Analyses of the CDK9 isoform 1 was performed using mass spectrometry data from FLAG-CDK9 affinity isolates.</p

Ser175 phosphorylation of CDK9 is rapidly induced by T-cell activation signals.

<p>(A) Affinity purification of FLAG-CDK9 complexes from Jurkat 2D10 cells and their identification by mass spectrometry. The percent values indicate the sequence coverage of the identified proteins. (B) Manually annotated MS/MS fragmentation spectra of the unmodified (upper) and phosphorylated (lower) CDK9 AFSLAK tryptic precursor peptides. (C) Ratio of phosphorylation of CDK9 at Ser175 and Thr186 in PMA stimulated (50 ng/ml) versus untreated cells with or without pretreatment with 20 µM U0126.</p

Signal-dependent phosphorylation of CDK9 at Ser175.

<p>(A) Detection of Ser175 phosphorylation by Western blotting after 1 h PMA (50 ng/mL) stimulation for wild type CDK9 and the T186A and T186D mutants. FLAG-CDK9 carrying the wild type sequence, or the S175A, S175D, T186A, or T186D mutations was stably expressed in latently infected Jurkat 2D120 cells using the MSCV retroviral expression system. Top panel: Whole cell extracts used for immunoprecipitation were immunoblotted for total CDK9. Note the slower migration of the ectopically expressed FLAG-CDK9 compared to the endogenous CDK9. Bottom three panels: Anti-FLAG-CDK9 immunoprecipitates were screened by immunoblotting for CDK9, pThr186, and pSer175 using a polyclonal antibody derived using a 19-residue peptide carrying a pSer175 epitope. (B) Validation of the epitope specificity of the pSer175 CDK9 antibody by peptide blocking. Purified antibody was pre-incubated overnight with pSer175 peptide epitope prior to immunoblotting anti-FLAG-CDK9 immunoprecipitates derived from control Jurkat T-cells, or 2D10 cells expressing FLAG-CDK9 before and after stimulation for 1 hr by PMA.</p