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The identification of unique serum proteins of HIV-1 latently infected long-term non-progressor patients.

Van Duyne R, Guendel I, Kehn-Hall K, Easley R, Klase Z, Liu C, Young M, Kashanchi F - AIDS Res Ther (2010)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: George Mason University, Department of Molecular and Microbiology, National Center for Biodefense & Infectious Diseases, Manassas, VA 20110, USA. bcmfxk@gwumc.edu.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

1D Demonstration of the depletion capabilities of the IgY-12 High Capacity SC Spin Column kit on patient serum. Depletion of patient serum was performed as indicated by manufacturer's instructions. Low and High abundant fractions were collected for each sample and run on a 1D 4-20% Tris-Gycine SDS-PAGE gel. A) Whole serum (lanes 2, 3) was incubated with the column containing antibodies against 12 of the high abundant serum proteins. Low abundant proteins (lanes 4, 5) were collected as the flowthrough, the column was washed (lanes 6, 7) and the high abundant proteins eluted (lanes 8, 9). Briefly the observed high abundant proteins were compared to the known sizes of the expected proteins as indicated. B) Equal volumes of serum from each of the six patients within each category (LTNP, HAART, and Negative) were pooled together to create a stock of each condition, independent of patient-to-patient variability. Twenty microliters of each stock was subjected to depletion and equal concentration of Low and High fraction were run on a 1D gel. Lanes 2, 3 and 4 are the low abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. Lanes 6, 8, and 10 are the high abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. The indicated arrows represent differentially expressed proteins that were excised, trypsinized, and identified using MALDI-TOF for preliminary protein screening.
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Figure 1: 1D Demonstration of the depletion capabilities of the IgY-12 High Capacity SC Spin Column kit on patient serum. Depletion of patient serum was performed as indicated by manufacturer's instructions. Low and High abundant fractions were collected for each sample and run on a 1D 4-20% Tris-Gycine SDS-PAGE gel. A) Whole serum (lanes 2, 3) was incubated with the column containing antibodies against 12 of the high abundant serum proteins. Low abundant proteins (lanes 4, 5) were collected as the flowthrough, the column was washed (lanes 6, 7) and the high abundant proteins eluted (lanes 8, 9). Briefly the observed high abundant proteins were compared to the known sizes of the expected proteins as indicated. B) Equal volumes of serum from each of the six patients within each category (LTNP, HAART, and Negative) were pooled together to create a stock of each condition, independent of patient-to-patient variability. Twenty microliters of each stock was subjected to depletion and equal concentration of Low and High fraction were run on a 1D gel. Lanes 2, 3 and 4 are the low abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. Lanes 6, 8, and 10 are the high abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. The indicated arrows represent differentially expressed proteins that were excised, trypsinized, and identified using MALDI-TOF for preliminary protein screening.

Mentions: To begin the identification of unique serum proteins, we obtained 18 subject serum samples: six LTNP, six HIV-1 infected subjects receiving HAART therapy (HAART) and six HIV-uninfected individuals through the Washington DC site of the Women's Interagency HIV Study (WIHS) Georgetown site (Table 1). WIHS is an NIH multicenter study of the natural history of HIV-1 infection in women [29]. LTNPs are defined by WIHS as being HIV-1 infected, but disease free for at least five years, having a CD4 count of greater than 500 at all visits and having no history of anti-retroviral therapy. The difficulty associated with analyzing serum is the presence of a high abundance of proteins which mask potential low abundance biomarkers. To overcome this obstacle, we utilized the ProteomeLab IgY serum depletion kit which removes 12 of the most abundant proteins in serum: albumin, IgG, transferrin, fibrinogen, IgA, α2-macroglobulin, IgM, α1-antitrypsin, haptoglobin, α1-acid glycoprotein, apolipoprotein A-I, and apolipoprotein A-II. As can be observed in Figure 1A, whole serum (lanes 2, 3) contains many proteins and is too complex to allow for confident identification of specific proteins. However, when the high abundant proteins (Figure 1, lanes 8, 9) are removed, lower abundant proteins that were originally masked (Figure 1, lanes 4, 5) are able to be analyzed. Along these lines, we found the ProteomeLab IgY serum depletion kit to be the most appropriate and reproducible manner in which to fractionate our serum samples into high and low abundant fractions. We applied this depletion strategy to pooled patient samples, combining equal volumes of whole serum from each of the six patients per sample set (LTNP, HAART, and Negative), which were subsequently depleted into low and high abundance fractions. We began the analysis with pooled samples to assist in the identification of HIV-1 infection specific protein identification as opposed to identifying individual patient and serum variability. These pooled samples were separated based on 1D SDS-PAGE (Figure 1B) and comparisons between LTNP, HAART, and Negative low abundant samples were carried out via in-gel trypsin digestion, peptide elution and desalting, followed by MALDI-TOF mass spectrometry as indicated by numbered arrows marking excised bands. The subsequent protein identifications served as a preliminary indication of differentially expressed proteins between the three patient types. These observations, as summarized in Table 2, provide an insight into the relevance of proteins identified in the context of the state of HIV-1 infection. Of particular interest in Table 2 is the identification of HIV-1 enhancer binding protein 1, (HIVEP1), Ribonuclease III, and heterochromatin protein 1 binding protein in the low abundance LTNP fraction. HIVEP1 is a member of the ZAS family of proteins which bind the promoter and enhancer regions of both cellular genes and infectious viruses, including HIV-1. Also known as PRDII-BF1 or MBP-1, this transcription factor binds to both the NF-κB and the TAR transactivation response DNA elements on the HIV-1 LTR in both the presence and absence of HIV-1 Tat [30,31]. It is not surprising that a transcription factor such as HIVEP1 would be present during HIV-1 infection; however, the identification of this protein is not necessarily a marker for a LTNP phenotype. Ribonuclease III, or Drosha, is a cellular enzyme found in the nucleus which serves to cleave double-stranded RNA hairpin transcripts as a key step in the production of miRNAs in the RNA interference pathway. Interestingly, heterochromatin protein 1, or HP1 is a member of the chromatin remodeling family of proteins, which can bind histones at methylated lysine residues and can interact with many chromatin-associated nonhistone proteins. The HP1 family of proteins has been associated with promoting a heterochromatic cellular state, where latently HIV-1 infected cells can persist as a transcriptionally silent provirus [32,33]. It may be of interest that an HP1 binding protein would be present in the serum of an HIV-1 infected patient as HP1, including its subtypes α, β, and γ, could be involved in the control of various stages of infection. It is possible that the association of this HP1 binding protein with varying subtype of HP1 could explain the differences in patient phenotypes, especially those that result in an altered susceptibility to viral infection.


The identification of unique serum proteins of HIV-1 latently infected long-term non-progressor patients.

Van Duyne R, Guendel I, Kehn-Hall K, Easley R, Klase Z, Liu C, Young M, Kashanchi F - AIDS Res Ther (2010)

1D Demonstration of the depletion capabilities of the IgY-12 High Capacity SC Spin Column kit on patient serum. Depletion of patient serum was performed as indicated by manufacturer's instructions. Low and High abundant fractions were collected for each sample and run on a 1D 4-20% Tris-Gycine SDS-PAGE gel. A) Whole serum (lanes 2, 3) was incubated with the column containing antibodies against 12 of the high abundant serum proteins. Low abundant proteins (lanes 4, 5) were collected as the flowthrough, the column was washed (lanes 6, 7) and the high abundant proteins eluted (lanes 8, 9). Briefly the observed high abundant proteins were compared to the known sizes of the expected proteins as indicated. B) Equal volumes of serum from each of the six patients within each category (LTNP, HAART, and Negative) were pooled together to create a stock of each condition, independent of patient-to-patient variability. Twenty microliters of each stock was subjected to depletion and equal concentration of Low and High fraction were run on a 1D gel. Lanes 2, 3 and 4 are the low abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. Lanes 6, 8, and 10 are the high abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. The indicated arrows represent differentially expressed proteins that were excised, trypsinized, and identified using MALDI-TOF for preliminary protein screening.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908552&req=5

Figure 1: 1D Demonstration of the depletion capabilities of the IgY-12 High Capacity SC Spin Column kit on patient serum. Depletion of patient serum was performed as indicated by manufacturer's instructions. Low and High abundant fractions were collected for each sample and run on a 1D 4-20% Tris-Gycine SDS-PAGE gel. A) Whole serum (lanes 2, 3) was incubated with the column containing antibodies against 12 of the high abundant serum proteins. Low abundant proteins (lanes 4, 5) were collected as the flowthrough, the column was washed (lanes 6, 7) and the high abundant proteins eluted (lanes 8, 9). Briefly the observed high abundant proteins were compared to the known sizes of the expected proteins as indicated. B) Equal volumes of serum from each of the six patients within each category (LTNP, HAART, and Negative) were pooled together to create a stock of each condition, independent of patient-to-patient variability. Twenty microliters of each stock was subjected to depletion and equal concentration of Low and High fraction were run on a 1D gel. Lanes 2, 3 and 4 are the low abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. Lanes 6, 8, and 10 are the high abundance fractions of the pooled LTNP, HAART, and Negative patients, respectively. The indicated arrows represent differentially expressed proteins that were excised, trypsinized, and identified using MALDI-TOF for preliminary protein screening.
Mentions: To begin the identification of unique serum proteins, we obtained 18 subject serum samples: six LTNP, six HIV-1 infected subjects receiving HAART therapy (HAART) and six HIV-uninfected individuals through the Washington DC site of the Women's Interagency HIV Study (WIHS) Georgetown site (Table 1). WIHS is an NIH multicenter study of the natural history of HIV-1 infection in women [29]. LTNPs are defined by WIHS as being HIV-1 infected, but disease free for at least five years, having a CD4 count of greater than 500 at all visits and having no history of anti-retroviral therapy. The difficulty associated with analyzing serum is the presence of a high abundance of proteins which mask potential low abundance biomarkers. To overcome this obstacle, we utilized the ProteomeLab IgY serum depletion kit which removes 12 of the most abundant proteins in serum: albumin, IgG, transferrin, fibrinogen, IgA, α2-macroglobulin, IgM, α1-antitrypsin, haptoglobin, α1-acid glycoprotein, apolipoprotein A-I, and apolipoprotein A-II. As can be observed in Figure 1A, whole serum (lanes 2, 3) contains many proteins and is too complex to allow for confident identification of specific proteins. However, when the high abundant proteins (Figure 1, lanes 8, 9) are removed, lower abundant proteins that were originally masked (Figure 1, lanes 4, 5) are able to be analyzed. Along these lines, we found the ProteomeLab IgY serum depletion kit to be the most appropriate and reproducible manner in which to fractionate our serum samples into high and low abundant fractions. We applied this depletion strategy to pooled patient samples, combining equal volumes of whole serum from each of the six patients per sample set (LTNP, HAART, and Negative), which were subsequently depleted into low and high abundance fractions. We began the analysis with pooled samples to assist in the identification of HIV-1 infection specific protein identification as opposed to identifying individual patient and serum variability. These pooled samples were separated based on 1D SDS-PAGE (Figure 1B) and comparisons between LTNP, HAART, and Negative low abundant samples were carried out via in-gel trypsin digestion, peptide elution and desalting, followed by MALDI-TOF mass spectrometry as indicated by numbered arrows marking excised bands. The subsequent protein identifications served as a preliminary indication of differentially expressed proteins between the three patient types. These observations, as summarized in Table 2, provide an insight into the relevance of proteins identified in the context of the state of HIV-1 infection. Of particular interest in Table 2 is the identification of HIV-1 enhancer binding protein 1, (HIVEP1), Ribonuclease III, and heterochromatin protein 1 binding protein in the low abundance LTNP fraction. HIVEP1 is a member of the ZAS family of proteins which bind the promoter and enhancer regions of both cellular genes and infectious viruses, including HIV-1. Also known as PRDII-BF1 or MBP-1, this transcription factor binds to both the NF-κB and the TAR transactivation response DNA elements on the HIV-1 LTR in both the presence and absence of HIV-1 Tat [30,31]. It is not surprising that a transcription factor such as HIVEP1 would be present during HIV-1 infection; however, the identification of this protein is not necessarily a marker for a LTNP phenotype. Ribonuclease III, or Drosha, is a cellular enzyme found in the nucleus which serves to cleave double-stranded RNA hairpin transcripts as a key step in the production of miRNAs in the RNA interference pathway. Interestingly, heterochromatin protein 1, or HP1 is a member of the chromatin remodeling family of proteins, which can bind histones at methylated lysine residues and can interact with many chromatin-associated nonhistone proteins. The HP1 family of proteins has been associated with promoting a heterochromatic cellular state, where latently HIV-1 infected cells can persist as a transcriptionally silent provirus [32,33]. It may be of interest that an HP1 binding protein would be present in the serum of an HIV-1 infected patient as HP1, including its subtypes α, β, and γ, could be involved in the control of various stages of infection. It is possible that the association of this HP1 binding protein with varying subtype of HP1 could explain the differences in patient phenotypes, especially those that result in an altered susceptibility to viral infection.

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: George Mason University, Department of Molecular and Microbiology, National Center for Biodefense & Infectious Diseases, Manassas, VA 20110, USA. bcmfxk@gwumc.edu.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus