- Open Access
The identification of unique serum proteins of HIV-1 latently infected long-term non-progressor patients
© Van Duyne et al; licensee BioMed Central Ltd. 2010
- Received: 15 March 2010
- Accepted: 6 July 2010
- Published: 6 July 2010
The search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. The comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patient's cellular homeostasis. In particular, the analysis of HIV-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. The utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to HIV-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum.
Here, the depletion of 12 of the most abundant serum proteins, followed by two-dimensional gel electrophoresis coupled with identification of these proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, has allowed for the identification of differentially expressed, low abundant serum proteins. We have analyzed and compared serum samples from HIV-1 infected subjects who are being treated using highly active antiretroviral therapy (HAART) to those who are latently infected but have not progressed to AIDS despite the absence of treatment, i.e. long term non-progressors (LTNPs). Here we have identified unique serum proteins that are differentially expressed in LTNP HIV-1 patients and may contribute to the ability of these patients to combat HIV-1 infection in the absence of HAART. We focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treatment of HIV-1 latently infected cell lines with p16INK4A decreases viral production despite it not being expressed endogenously in these cells.
Identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to HIV-1 infection in a given population.
- Jurkat Cell
- Cellular Viability
- Reverse Transcriptase Activity
- Abundance Fraction
- Serum Proteome
Human serum is derived from the liquid plasma component of the blood with the fibrinogens, or clotting factors, removed and is composed of small molecules such as salts, lipids, amino acids, sugars and approximately 60-80 mg of proteins/mL . Serum is a readily obtainable peripheral bodily fluid from which the protein profile directly reflects the normal or disease state of the organism [2–4]. Serum is a complex mixture of "classical" and "non-classical" proteins. Classical serum proteins are involved in a number of processes including proteolysis, inhibition, binding, transport, coagulation, and immune response and are often secreted from the liver, through the intestines, and into the bloodstream . "Non-classical" proteins are proteins that are not directly tied to any known function within the serum and often originate from cellular leakage or shedding, and may utilize the bloodstream for transportation . It is generally accepted that most of the significant changes in the serum will be found in these low abundant non-classical proteins, due to the hypothesis that the presence of these proteins should reflect changes in the diseased tissue. Indeed, serum commonly contains upwards of 10,000 different proteins at any given time that are being actively produced and secreted by all cells and tissues, therefore, the proteomic profile of serum can give insight into the systemic reaction to a disease state and can serve as a pool of differentially expressed proteins [2, 6–13]. Recently, the interest in characterizing the human serum proteome has increased due to the determination of disease biomarkers for early detection, diagnosis, and drug targeting; however, due to the extensive dynamic range of protein concentration within the serum, the identification of low abundance proteins suitable for biomarker determination is often masked. The 22 highly abundant proteins contained within serum constitute approximately 99% of the total serum proteins, including albumin, IgG, transferrin, haptoglobin, fibrinogen, etc. and interfere with the identification of low abundance proteins in the ng/mL concentration range. The presence of these highly abundant proteins necessitates the prefractionation of serum samples prior to analysis for low abundant proteins. Due to the dynamic insight the analysis of the serum proteome can relate to a disease state, of particular interest is the identification of low abundant proteins that change in expression or abundance in response to a disease state. These low abundant proteins could potentially arise as an early diagnostic for a disease state, or a therapeutic target.
Serum proteomics has emerged as an integral biomarker identification and diagnostic tool, especially for infectious diseases and oncology. Recently, novel serum biomarkers have been identified for liver fibrosis in hepatitis C virus (HCV) infected patients as well as unique protein signatures in SARS coronavirus infections, and infant hepatitis syndrome induced by human cytomegalovirus (HCMV) infection [14–16]. Characterization of the serum protein profile of these viral states helps provide insight into the expression changes associated with viral infection. In particular, HIV-1 infection, even at the acute phase, results in dramatic changes in both cellular and viral protein expression levels. As the HIV-1 viral tropism consists primarily of CD4+ T-cells, macrophages, and dendritic cells, the resulting protein changes can be seen systemically as infected cells travel throughout the body. Additionally, the nature of this viral infection supports the secretion of altered proteins into the blood and subsequently the serum due to the propensity of the virus to stimulate apoptosis of infected cells, therefore emptying cellular contents into the serum. These characteristics of HIV-1 infection suggest that the analysis of the serum of infected patients is an appropriate reflection of a patients' altered protein expression state.
Due to innate genetic and phenotypic differences in the human population, significant variability exists in the susceptibility to HIV-1 infection. Amongst this diversity includes the well-studied CCR5Δ32 inherited mutation, which prevents the binding of R5-tropic HIV-1 strains to the CCR5 chemokine receptor on the surface of CD4+ T-cells, therefore preventing entry of the virus . Additionally, some individuals can be infected with HIV-1, however will not progress to AIDS even in the absence of therapy. These Long Term Non-Progressors (LTNPs) are often characterized as being infected with HIV-1 but are also disease free and sustain a normal CD4 T-cell count and a low viral load. Over the past 20 years, multiple studies have been aimed at determining the reason that these individuals are able to resist disease progression. There are studies that suggest that the virus infecting these cells could be deficient in some way, for example, Nef deficient viruses and Vpr R77Q mutations are associated with LTNPs [18–22]. A number of host factors have also been identified that may contribute to the observed resistance. LTNPs have a higher prevalence of the CCR5Δ32 allele [17, 23–25]. In addition, the presence of certain HLA genes including HLA-B27, HLA-B*5701, HLA-B*5401, and HLA-B*1507 have been linked to LTNP [26–28] however, the identified alterations do not account for all cases of LTNP. Therefore, the search for protective host factors is still an area of active investigation in hopes of obtaining information that could be of therapeutic value.
Here, we describe the detection of unique, low abundant serum proteins in latently infected HIV-1 LTNPs as compared to serum from patients undergoing HAART treatment, and those not infected with HIV-1. We attempted to characterize the underlying differences in LTNPs that contribute to the ability of these patients to combat HIV-1 infections. We have depleted 12 of the most highly abundant serum proteins from three sets of serum samples (uninfected, infected on HAART, LTNP) and identified differentially expressed proteins across the samples. In particular, we focus on the identified cellular protein p16INK4A which is found preferentially in LTNP patient serum samples, but is not present in patients undergoing HAART treatment. In vitro viral assays and viability studies confirm the loss of viral replication upon p16INK4A treatment in latently infected cell lines and the non-toxic effect of the same treatment in corresponding uninfected cell lines.
Depletion of the 12 highly abundant serum proteins allows for the identification of low abundant proteins
Patient samples obtained from the WIHS Interagency Cohort.
Protein Identification of Serum Depleted Samples from 1D SDS-PAGE
Human immunodeficiency virus type 1 enhancer binding protein 1
Ribonuclease III (Drosha)
ADAM metallopeptidase with thrombospondin type 1 motif, 18
Tax1-binding protein TXBP151
Heterochromatin protein 1, binding protein
Gga-Vhs domain & Beta-Secretase C-terminal phosphopeptide
MHC class I antigen
Coagulation factor V (Proaccelerin, labile factor)
Matrix metalloproteinase 2 preprotein
Ribosomal protein L27
Ribosomal protein L36a
2DGE and MALDI-TOF analysis of pooled, depleted serum samples identified unique low abundance proteins
Protein Identification of Serum Depleted Samples from 2D SDS-PAGE
Protein kinase 3
Cyclin D-dependent kinase 4 and 6-binding protein/p16
Serine/Threonine kinase 33
Kelch repeat domain containing protein 11
SNW1 protein/APAF1 interacting protein
Eukaryotic translation initiation factor 4B
FGFR1 oncogene partner
Anti-HIV-1 gp120 IgG 16c kappa light chain
PCTAIRE protein kinase 3
Pre-B-cell leukemia homeobox interacting protein 1
In addition to these low abundant protein identifications, the LTNP high abundant samples (gel "d") indicated the presence of the FGFR1 oncogenic partner and PCTAIRE protein kinase 3 as well as an anti-HIV-1 gp120 IgG 16 cκ light chain. FGFR1 oncogene partnered with the fibroblast growth factor receptor 1 (FGFR1) is thought to be associated with myeloproliferative disorders and as of yet is not associated with any HIV-1 protein interactions or associated disease phenotypes. PCTAIRE protein kinase 3, however, is a member of the serine/threonine family of protein kinases and more specifically, the cdc2/cdkx subfamily that plays a role in broad signal transduction pathways. This serine/threonine kinase family member has also been associated with the essential regulation of cell cycle progression, as well as transcription and DNA repair . This protein identification again demonstrates the role that HIV-1 infection plays in the dysregulation of cellular kinases and specifically, cell cycle progression.
The HAART responder patient samples (gels "b" and "e") also contained unique protein candidates: serine/threonine kinase 33, the Kelch repeat domain containing protein 11, and the SNW1 protein/APAF1 interacting protein in the low abundant fraction as well as the pre-B-cell leukemia homeobox interacting protein 1 in the high abundant fraction. Of functional interest is the general serine/threonine kinase 33 as we have already identified several cellular kinases of the same family. Additionally, the SNW1 protein is a transcriptional coactivator that induces the expression of vitamin D, retinoic acid, estrogen, and glucocorticoid associated genes. SNW1/SKIP interacts with HIV-1 Tat through the association with p-TEFb (cdk9/Cyclin T1) at the TAR RNA complex, stimulating HIV-1 transcription elongation . Interestingly, some of the protein spots identified the presence of serum albumin contamination (spots a2, d5, d7, and e2), which both served as an internal positive control for mass spectrometry and also indicated that the depletion columns are not completely efficient at removing contaminating high abundant proteins.
Validation of MS protein identifications by Western Blot
In order to address the concern that the protein signature of the pooled set of samples for each patient type may not be an accurate representation of the individual variability that could be present, we screened the low abundance fractions of the LTNPs for the presence of both p16INK4A and cdk4. Results in Figure 3B indicate the lack of detection of p16INK4A in any of the low abundant LTNP samples, especially as compared to the pooled sample "A" (lane 2). In contrast, p16INK4A was detectable in LTNP patients 1 and 2, as well as in the pooled sample "D" for the corresponding high abundance fractions (lanes 2, 3, 4). Due to the nature of the depletion step based on immuno-affinity, it is not surprising that p16INK4A is detectable in individual high abundant samples, as it is probably coupled to a larger, more abundant protein and was not efficiently depleted. Additionally, post-depletion, the low abundant fractions for each patient were very dilute and the protein levels were undetectable by traditional methods. Cdk4 was detectable in the LTNP low abundant individual samples with variable abundance, correlating with the data in Figure 3A. Interestingly, cdk4 was not detectable in the LTNP high abundant individual samples, indicating that this protein was effectively isolated away from the high abundant proteins. Although through a straight western blot, the levels of p16INK4A were undetectable, Figure 3C indicates that p16INK4A can indeed be immunoprecipitated out of the individual low abundant LTNP samples and subsequently detected by western blot. Lanes 1, 2, and 3 represent the pooled "A" sample and patient samples 2 and 3, respectively. The pooled "A" sample was incubated with α-IgG, and patient samples 2 and 3 were incubated with α-p16. These three immunoprecipitations were subjected to very stringent wash conditions of TNE600 + 0.1% NP-40, TNE300 + 0.1% NP-40, and TNE50 + 0.1% NP-40 in order to remove any non-specific proteins. As can be seen in Figure 3C, lanes 1-3 there is some non-specific p16 binding to the α-IgG negative control lane, however, the IPs from the two samples result in much higher percentage of p16INK4A present, especially in lane 3. As compared to lanes 4-10, where the salt washes were of less stringency, there are variable levels of p16INK4A IPed from each patient sample. Based on the background levels of p16INK4A in lane 1, we feel confident in concluding that the patients 2, 3, 4, and 6 have a detectable level of p16INK4A only after immunoprecipitation.
RT activity of HIV-1 infected cells decreases in vitro in the presence of exogenous p16INK4A
Treatment of uninfected cells with p16INK4A does not affect cellular viability
To detect whether p16INK4A had an effect on normal or uninfected cells, we performed an MTT assay to screen for the percentage of cells viable after p16INK4A treatment. Figure 4B depicts CEM, Jurkat, and H9 uninfected T-cell lines, as well as the uninfected monocytic U937 cell line treated with GST as well as GST-p16INK4A. CEM, H9, and U937 control cells showed no appreciable decrease in cellular viability upon 48 hours of treatment with any of the four conditions. Interestingly, in the presence of 0.5 ug of GST-p16INK4A, Jurkat cells exhibit an almost 40% decrease in cellular viability. We next performed western blots on whole cell extracts from all four of these uninfected cell lines and observed only Jurkat cells exhibiting an endogenous expression of p16INK4A (Figure 4D, lane 2). This suggests that the decrease in cellular viability seen in Jurkat cells (Figure 4B) treated with p16 INK4A can be correlated with the expression of exogenous p16INK4A in these cells, resulting in an increase in cdk4,6/Cyclin D inhibition and an increase in apoptosis. Interestingly, no endogenous levels of p16INK4A are detected in the infected J1.1 cells.
Cellular Rb levels decrease as a result of the exogenous addition of p16INK4A to Jurkat T cells
P16INK4A is a critical member of the Rb tumor-suppressor pathway which acts to arrest the cell-cycle at G1/S by inhibiting the binding of cdk4/6 to Cyclin D1 and subsequently inhibiting the phosphorylation of Rb. In Figure 4C, we investigate the levels of Rb present in Jurkat cells alone (lane 1) compared to Jurkat cells treated with an excess of GST or GST-p16INK4A (2.5 μg, lanes 2 and 3). Interestingly, upon treatment of exogenous GST-p16INK4A, we observed a decrease in cellular levels of Rb; indeed there is also a decrease in Rb with GST treatment alone The Rb antibody used detects total Rb levels in the cell, therefore we could not assume a loss of phosphorylation due to the inhibitory effect of p16INK4A on cdk4/6. A recent paper has addressed the literature-wide discrepancies of RB dephosphorylation vs. degradation in response to drug treatment or cell senescence in various cell types . It is possible that the increased amount of p16INK4A present in these cells has induced a proteasomal degradation of Rb that has not otherwise been characterized in T cells. The cell line panel in Figure 4D was also screened for the presence of endogenous levels of Rb in these cell lines, and interestingly there is a high degree of variability. The T cell lines CEM, Jurkat, and the HIV-1 infected J1.1 have the highest endogenous levels of Rb. Interestingly, the monocytic cell lines U937 and the HIV-1 infected U1 have the lowest amount of Rb present, with almost completely undetectable levels in HIV-1 infected U1 cells. The variability supports the discrepancies seen in the literature about hypo-, hyper-phosphorylation of Rb, as well as depletion or degradation of Rb during cell cycle or cellular responses.
Purified GST-p16 is found intracellularly in Jurkat, J1.1, U937, and U1 after treatment
In order to confirm that the effects seen by GST and GST-p16INK4A treatment in Figure 4A, B, and C, we checked to ensure that the purified proteins are actually entering the cell. Jurkat, J1.1, U937, and U1 cell lines were treated with an excess (2.5 μg) of GST or GST-p16 (Figure 4E). At 48 hours post treatment, the cells were harvested, washed extensively, lysed, and incubated with Glutathione-Sepharose beads overnight. The Glutathione-Sepharose beads were washed extensively to remove any non-specific proteins with buffers containing salts and detergents. The bound proteins were subjected to Western blot for the presence of p16INK4A as shown in Figure 4E. Jurkat whole cell extract served as the positive control (lanes1 in both blots) and a higher molecular weight band corresponding to GST-p16INK4a was observed in the GST-p16INK4A pulldown lanes for each of the cell lines (lanes 4 and 7 in both blots). The lack of detection in the untreated cell lysate incubated with beads alone indicates that the protein detected in lanes 5 and 8 are specifically the GST-bound proteins. These studies confirm that the GST proteins are indeed entering the cells when incubated in the extracellular environment.
Fascaplysin treatment mimics the exogenous p16INK4A treatment
Fascaplysin treatment increases apoptosis in Jurkat cells and arrests latently infected J1.1 cells at G1/S in vitro
The global proteomic analysis of serum proteins is not without its challenges; however the presence or absence of proteins in such bodily fluids of patients is often the most accurate reflection of cellular leakage or secretion of proteins in response to a disease state. Unfortunately, the classical serum proteome contains a large concentration of high abundance proteins that mask the individual proteins that are unique to a particular phenotype. The identification of such low abundance serum proteins is attractive as a method of early detection of a disease state or a response to infection. Here, we demonstrate that the detection of unique proteins in the serum of HIV-1 infected long-term non-progressors may be indicative of a natural "immunity" to the progression of HIV-1 infection. Specifically, we have identified p16INK4A, a cdk4/6 inhibitor, as preferentially present in pooled serum of HIV-1 LTNP patients, as opposed to HIV-1 infected individuals responding to HAART treatment.
P16INK4A is a member of the INK4/ARF family of endogenous cdki's that serve to regulate cell cycle progression through the inhibition of specific cdk/Cyclin interactions. P16INK4A is a critical member of the Rb/p16 tumor-suppressor pathway which inhibits the activation of cdk4/6, preventing the progression through the cell cycle. This tumor-suppressive pathway is mutated in close to 100% of human cancers and specific loss-of-function mutations are found within the Rb gene or the CDKN2A gene encoding p16INK4A. In addition to the direct inactivation of these two proteins, cancer cells also override this regulatory pathway by overexpressing cdk/Cyclins as well as inducing the endogenous loss of expression of other cdk inhibitors. The loss of p16INK4A results in the constitutive activation of cdk4/6 as well as pRb hyperphosphorylation, therefore bypassing the anti-oncogenic senescence induced by this cdk inhibitor. In addition to necessitating an oncogenic state, the regulation and manipulation of cellular cdks and Cyclins is also critical for an active HIV-1 viral infection and occurs mainly through the HIV-1 transactivator Tat. Tat interacts with the cdk2/Cyclin E complex to phosphorylate cdk7 and assist in the phosphorylation of the C-terminal Domain (CTD) of RNA Pol II at the viral LTR (long-terminal repeat) to promote viral transcription [55–57]. Cdk2 is also involved in the direct phosphorylation of Tat at both serine 16 and 46, which are critical for efficient Tat activity [56, 57]. HIV-1 transactivation is also heavily dependent on the recruitment of the cdk9/Cyclin T1 complex to the viral TAR element where it also assists in the phosphorylation of the CTD of RNA Pol II as well as an autophosphorylation event which is important for the localization to the nucleus . Taking into consideration the recruitment of cellular kinases for HIV-1 transcription, it is not surprising that pharmacological cdk inhibitors have been developed (analogous to endogenous cdki's) as a template to specifically inhibit these kinases. Therefore, the presence of the cdk inhibitor p16INK4A in LTNP serum is suggestive of an existing defense mechanism in these patients that imparts a predisposition to a lack of HIV-1 disease progression.
The development of pharmacological cdki's and peptide mimetics is a commonly used approach for both cancer and viral therapeutics. For instance, p16INK4A peptides have previously been developed, which, when conjugated to localization proteins/peptides, were able to block cell cycle progression in breast cancer and colon cancer cell lines in vitro as well as reduce tumor size in an in vivo mouse model of pancreatic cancer [59–61]. These peptides have also been used for intracellular delivery to leukemia and lymphoma derived cells [59, 62]. These studies have demonstrated the ability of cdki-derived peptides to be used for both direct cell cycle arrest and the treatment of metastatic cancers. This type of therapy is an appropriate approach to cancerous states where the oncogenic mutation results in a loss of function or expression of the p16INK4A protein. Here, we show that p16INK4A is only endogenously expressed in the Jurkat T-cell line and additional treatment of Jurkat cells with exogenous p16INK4A results in a decrease in cellular viability. All additional uninfected and infected cell lines used in this study did not express endogenous levels of p16INK4A, suggesting that a loss of function or expression may be present in these particular cell types. More importantly, the treatment of stably infected HIV-1 cell lines, J1.1 and U1, with exogenous p16INK4A resulted in a decrease in viral replication with a minimal decrease in cellular viability, suggesting that the presence of p16INK4A in the context of an in vitro HIV-1 infection may mimic an in vivo infection in a LTNP patient. The same dosage of p16 INK4A needed for a decrease in viral replication in infected cells is not toxic to uninfected cells, with the exception of Jurkat cells, indicating that there may be a threshold for which the effect of this cdk inhibitor begins to harm the cell and potentially induce cell cycle arrest and apoptosis.
In this study we also chose to reinforce the p16INK4A-induced cdki effects seen on cellular viability and viral replication with a known cdk4/6/Cyclin D inhibitor, Fascaplysin. We show that treatment of Jurkat cells with this inhibitor also results in a decrease in cellular viability, consistent with the effect seen by treatment with endogenous p16INK4A. Additionally, the treatment of both stably infected HIV-1 cell lines, J1.1 and U1, with Fascaplysin resulted in a decrease in viral replication with a minimal decrease in cellular viability. The correlation of this pharmacological cdki inhibitor data with the functional-cdki p16INK4A data suggests that the preferential inhibition of the cdk4/6/Cyclin D interaction by LTNPs may contribute to this phenotype.
The high and low abundance fractions analyzed for each of the three patient types were a pooled representation of six individual patient samples that almost certainly have inherent variability. Indeed, when assaying for p16INK4A in high and low abundance serum fractions for each individual LTNP patient, p16INK4A was present in only a subset of the patients analyzed. This indicates that although p16INK4A may serve as a unique serum protein indicating a LTNP HIV-1 infected status, individual person-to-person changes will alter the serum proteome profile and needs to be taken into consideration. Differences in expression of p16INK4A can be due to either a host cellular response to the infection or from an influence of the infection itself. Two different scenarios need to be considered when examining the potential importance of this protein in defining a viral disease state such as a LTNP patient; the effect on the host and the effect on the virus.
Finally, many complicating factors can contribute to the altered state of proteins present in the serum, not the least of which is the length of time in which the patient has been infected, age, gender, coinfections, and other preexisting conditions such as cancer or metabolic diseases. Therefore, it is important to take into consideration some of these factors when applying global proteomic analyses to HIV-1 patient derived samples.
Cell Culture and Protein Reagents
293T endothelial kidney cell line was harvested for whole cell extract and used as a positive control. 293Ts were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 1% L-glutamine, and 1% streptomycin/penicillin (Quality Biological). Latently infected HIV-1 cell lines J1.1 (T-cells) and U1 (monocytes) were used for RT assays and whole cell extracts were obtained for Western blots. Uninfected CEM, Jurkat, and H9 cell lines (T-cells) and uninfected U937 cell line (monocytes) were used for viability assays and whole cell extracts were obtained for Western blots. Uninfected cells were grown in RPMI-1640 media containing 10% FBS, 1% L-glutamine, and 1% streptomycin/penicillin (Quality Biological). All cells were incubated at 37°C and 5% CO2. GST-p16INK4A was a generous gift from Dr. Ming-Daw Tsai, Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan
Serum Samples and Serum Depletion
Eighteen subject serum samples (6 LTNP, 6 HIV infected subjects receiving HAART therapy, and 6 uninfected individuals) were obtained through Washington DC site of the Women's Interagency HIV Study (WIHS) (Table 1). WIHS is an NIH multicenter study of the natural history of HIV-1 infection in women . LTNPs were defined by WIHS as being HIV Infected, but disease free for at least five years, a CD4 count of greater than 500 at all visits, and no history of anti-retroviral therapy. Serum samples were subjected to depletion of the 12 most abundant serum proteins using the ProteomeLab IgY-12 High Capacity Spin Column Proteome Partitioning kit from Phenomenex (Torrance, CA). This spin column consists of anti-human serum albumin, anti-IgG, anti-fibrinogen, anti-transferrin, anti-IgA, anti-IgM, anti-HDL (anti-apo A-I and anti-apo A-II), anti-haptoglobin, anti-α1-antitrypsin, anti-α1-acid glycoprotein and anti-α2-macroglobulin conjugated to polymeric microbeads. Twenty microliters of each serum sample was diluted 1:25 in dilution buffer and ran over the spin column. Low abundant proteins were collected in the flowthrough and subsequent high abundant, bound proteins were retained and eluted with a low pH stripping buffer. Protein concentrations of both low and high abundant fractions were calculated and pooled as indicated.
2D-Gel Electrophoresis (2DGE) and MALDI-TOF MS
Five hundred micrograms of pooled LTNP, HAART, and Negative patient samples for both Low and High abundance fractions were subjected to isoelectric focusing on an IPG strip, pH 3.0-10.0 and further subjected to SDS-PAGE on a 4-20% Criterion Tris-Glycine gel. The gels were stained with Coomassie Blue and protein spots of interest were excised. Spots were vortexed, washed, and equilibrated in 25 mM NH4HCO3, broken into smaller pieces, and vortexed and washed 3X with 50% ACN/25 mM NH4HCO3 to remove the Coomassie from the gel. Gel pieces were vortexed and washed with 100% ACN to dehydrate the gel pieces. Gel pieces were re-swelled with up to 200 ng of trypsin (in enough volume to cover the gel) and incubated on ice for 30 min. Residual trypsin was removed, 20 μl of 25 mM NH4HCO3 or enough to cover the gel was added to the gel pieces and the reactions were incubated overnight at 37°C. Peptides were extracted with 1X dH2O wash with brief vortexing and sonication, followed by 3X washes with 60% ACN/5% TFA. Extracted peptides were pooled together and a SpeedVac was utilized to reduce the volume to approximately 10 μl. Twenty microliters of 0.1% TFA was added to each tube and peptides were desalted using C18 ZipTips (Millipore) according to manufacturer's instructions. Peptides were spotted on MALDI sample plate 1:1 with α-cyano-4-hydroxy cinnamic acid (CHCA) matrix solution: 10 mg CHCA, 500 μl 100% ACN, 500 μl 0.1% TFA. Positive control calibration peptide solution of Bradykinin, Angiotensin II, P14R, and ACTH was spotted along with negative control empty gel slice. Mass peaks obtained were entered into Mascot http://www.matrixscience.com/ and ProFound http://prowl.rockefeller.edu/prowl-cgi/profound.exe databases for peptide mass fingerprinting analysis.
Fifty microliters of pooled low abundance LTNP serum sample "A" and individual low abundance LTNP serum samples (1-6) were incubated with either α-IgG or α-p16INK4A as indicated, volume was brought up to 500 μl with TNE50 + 0.1% NP-40, rotating overnight at 4°C. Extracts were incubated with 50 μl of a 30% slurry of Protein A + G Agarose beads (Calbiochem #IP05) for 2 hrs, rotating, at 4°C. Beads were washed with indicated salt washes (TNE600 + 0.1% NP-40, TNE300 + 0.1% NP-40, TNE150 + 0.1% NP-40, or TNE50 + 0.1% NP-40), bound proteins were removed from the beads with Laemmli buffer and subjected to Western blots against p16INK4A.
Western blots were performed to validate proteins identified from depleted serum samples. Whole cell extracts were obtained from cell culture pellets washed twice with 25 mL of phosphate buffered saline (PBS) with Ca2+ and Mg2+ (Quality Biological) and centrifuged once more. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na3VO4, 1 mM DTT, one complete protease cocktail tablet/50 mL) and incubated on ice for 20 min, with a gently vortexing every 5 min. Cell lysates were transferred to eppendorf tubes and were centrifuged at 10,000 rpm for 10 min. Supernatants were transferred to a fresh tube where protein concentrations were determined using Bio-Rad protein assay (Bio-Rad, Hercules, CA). Antibodies against cdk4 (sc-749), p16 (sc-467), PCTAIRE (sc-174), Rb (sc-50), and Actin (sc-1615) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against HP1α and HP1γ were purchased from Cell Signaling (Danvers, MA).
J1.1 and U1 cells (2 × 106) were treated with GST or GST-p16 (0.1 or 0.5 μg) or Fascaplysin (100 nM, 500 nM, or 1 μM) and supernatants collected to test for the presence of virus 48 hours post treatment. Jurkats were treated with GST or GST-p16 (2.5 μg) for 48 hours prior to HIV-1 infection with the dual tropic strain 89.6. Supernatants were collected at various time points post infection to test for the presence of virus post treatment. Viral supernatants (10 μl) were incubated in a 96-well plate with reverse transcriptase (RT) reaction mixture containing 1X RT buffer (50 mM Tris-HCl, 1 mM DTT, 5 mM MgCl2, 20 mM KCl), 0.1% Triton, poly(A) (1U/ml), pd(T) (1U/ml), and [3H]TTP. The mixture was incubated overnight at 37°C, and 5 μl of the reaction mix was spotted on a DEAE Filtermat paper, washed four times with 5% Na2HPO4, three times with water, and then dried completely. RT activity was measured in a Betaplate counter (Wallac, Gaithersburg, MD).
Five thousand cells were plated per well in a 96-well plate and the next day cells were treated with GST or GST-p16 (0.1 or 0.5 μg) or Fascaplysin (100 nM, 500 nM, or 1 μM). Forty-eight hours later, 10 μl MTT reagent (5 mg/ml) was added to each well and plates incubated at 37°C for 3 hours. Next, 100 μl of DMSO was added to each well to solubilize the violet crystals. The assay was read at 570 nM.
Cell cycle analysis
Cells were washed with PBS and fixed with 70% ethanol. Following rehydration in PBS, cells were stained in PBS containing 25 ug/ml propidium iodide (Sigma), 10 ug/ml RNase A (Sigma) and 0.1% NP-40. Cells were analyzed on a BD FacsCalibur flow cytometer. Cell cycle analysis and measurement of apoptosis was performed using CellQuest software. Aggregates and debris were excluded by gating on the FL2W and FL2A parameters. Apoptosis was considered to be the population of cells that were sub-G1.
We would like to thank the members of the Kashanchi lab for experiments and assistance with the manuscript. We thank Dr. Ming-Daw Tsai (Institute of Biological Chemistry, Academia Sinica) for the GST-p16INK4A plasmid constructs. Most of the data on the current manuscript was generated using funds from NIH grants AI078859, AI074410 and AI043894. Data in this manuscript were collected by the Women's Interagency HIV Study (WIHS) Collaborative Study Group with centers (Principal Investigators) at New York City/Bronx Consortium (Kathryn Anastos); Brooklyn, NY (Howard Minkoff); Washington, DC Metropolitan Consortium (Mary Young); The Connie Wofsy Study Consortium of Northern California (Ruth Greenblatt); Los Angeles County/Southern California Consortium (Alexandra Levine); Chicago Consortium (Mardge Cohen); Data Coordinating Center (Stephen Gange). The WIHS is funded by the National Institute of Allergy and Infectious Diseases (UO1-AI-35004, UO1-AI-31834, UO1-AI-34994, UO1-AI-34989, UO1-AI-34993, and UO1-AI-42590) and by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (UO1-HD-32632). The study is co-funded by the National Cancer Institute, the National Institute on Drug Abuse, and the National Institute on Deafness and Other Communication Disorders. Funding is also provided by the National Center for Research Resources (UCSF-CTSI Grant Number UL1 RR024131). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. Rachel Van Duyne is a predoctoral student in the Microbiology and Immunology Program of the Institute for Biomedical Sciences at The George Washington University.
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