Journal of General Virology (2016), 97, 220–224
DOI 10.1099/jgv.0.000326
The CD8+ cell non-cytotoxic antiviral response affects RNA polymerase II-mediated human immunodeficiency virus transcription in infected CD4+ cells Dalibor Blazek,1,2 Fernando Teque,1 Carl Mackewicz,1,3 Matija Peterlin1 and Jay A. Levy1 Correspondence Jay A. Levy [email protected]
1
Department of Medicine, University of California San Francisco, 513 Parnassus Ave, Room S1280, San Francisco, CA 94143, USA
2
CEITEC-Masaryk University, 62500 Brno, Czech Republic
3
Amgen, Thousand Oaks, CA, USA
Received 13 August 2015 Accepted 21 October 2015
A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.
INTRODUCTION Long-term survival from human immunodeficiency virus (HIV) infection is associated with a CD8+ cell non-cytotoxic antiviral response (CNAR) (Walker et al., 1986). This innate cellular immune activity involves inhibition of viral transcription and is active against all HIV-1, HIV-2 and simian immunodeficiency virus isolates tested (Chen et al., 1993; Levy et al., 1996; Mackewicz et al., 1995; Tomaras et al., 2000; Walker et al., 1986). CNAR is mediated by the production of a soluble CD8+ cell antiviral factor (CAF) (Brinchmann et al., 1990; Levy, 2003; Walker & Levy, 1989). Healthy HIV-infected individuals, many of whom have lived for more than 10 years to over 35 years without treatment, show the presence of CNAR and the production of CAF by their CD8+ cells (Mackewicz et al., 1991; Barker et al., 1998; Go´mez et al., 1994). Thus, the characterization of CNAR/CAF and its specific mechanism of action is a major objective of our research. Previous work has indicated that several regions of the HIV-LTR promoter are not involved in the inhibition of transcription by CNAR/CAF (Bonneau et al., 2008; Shridhar et al., 2014). Deletion of these promoter sequences did One supplementary figure is available with the online Supplementary Material.
220
not lead to the production of viruses that are resistant to this CD8+ cell antiviral response. These findings have suggested that intracellular transcriptional factors may be targeted by CNAR/CAF and this regulatory action is responsible for the block in HIV transcription. In approaches to define the processes involved in the block in viral transcription, we have found that HIV-infected cells exposed to CAF or CD8+ cells expressing CNAR have a reduced amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral genetic regions. A role of positive transcriptional elongation factor b (PTEFb) is suggested, but a direct effect on P-TEFb complexes was not observed. The results suggest that a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.
RESULTS The effect of CAF on P-TEFb-mediated HIV-LTR transcription To determine the mechanism by which CNAR/CAF affects transcription, a transcriptional reporter system was used (Fig. 1). It consists of a plasmid reporter PG6LTR 000326 G 2015 The Authors
Printed in Great Britain
CNAR and RNA polymerase II-mediated HIV transcription
When we co-expressed the Gal-CycT1 chimera with pG6LTRCAT in the Jurkat cells, the levels of CAT activity increased 17-fold over basal levels (Fig. 1b, compare bar 2 to bar 1). To assess the effect on the transcriptional activity of the CAT reporter, we incubated transfected cells with CAF-containing culture fluids from CD8+ cells from three CNAR-positive subjects. Strikingly, incubation with CAF fluid decreased reporter activity substantially (Fig. 1b, compare bars 3, 4 and 5 to bar 2). The CD8+ cell fluids from CNAR-negative uninfected subjects did not affect CAT transcription levels (Fig. 1b, compare bars 6 and 7 to bar 2). This experiment indicated that CAF from CNAR-positive subjects can regulate the transcription of an HIV promoter-based reporter system.
(a) Plasmid constructs Gal-cyclinT1 Gal
CyclinT1
G6LTRCAT 6xGal binding sites
HIV-LTR
CAT
Reporter assay Gal-CycT1
RNAPII
6-Gal binding sites
CAT
HIV-LTR
(b) 40
Relative CAT activity
35 30
Effect of CAF on P-TEFb
25
It is well-established that P-TEFb is associated in two forms in cells: either in the catalytically inactive form in complex with other proteins including Hexim1 (so called the large complex of P-TEFb) or in a catalytically active free form (small complex) (Peterlin & Price, 2006). Because the above-described HIV-LTR based reporter system is dependent on Cdk9 activity, we incubated CD4+ cells with either CAF positive or negative fluids for 48 h and then separated the protein complexes from the CD4+ lysates by glycerol gradient centrifugation. Western blot analyses of the glycerol gradient fractions with Hexim1 and Cyclin T1 antibodies showed that the amounts of P-TEFb in the large and small complexes remained the same upon culture of the infected CD4+ cells with either CAF-positive or negative fluid (data not shown). Moreover, the total amounts of the CyclinT1, Cdk9 and Hexim1 proteins were the same in the CD4+ cells treated with both the CAF- positive and negative culture fluids (Fig. S1, available in the online Supplementary Material).
20 15 10 5 0
1 + G6LTRCAT Gal-emp.vect. + Gal-cyclinT1 CAF positive CAF negative
2 +
3 +
4 +
5 +
6 +
7 +
+
+ +
+ +
+ +
+
+
+
+
Fig. 1. CAF inhibits transcription from the HIV G6LTRCAT reporter gene. (a) Depiction of the HIV G6LTRCAT reporter assay. Six Galbinding sites (dark green rectangles) are bound by six molecules of Gal-CyclinT1 (Gal-CycT1; light green ovals). RNAPII (yellow oval) binds the HIV-LTR promoter (black rectangle). Active transcription results in CAT reporter gene (pink rectangle) expression. GalCycT1/Cdk9 phosphorylates the C-terminal domain of RNAPII (light green arrow) which results in the productive transcription of the CAT reporter gene (yellow arrow). (b) Jurkat T-cells described in (a) were transfected with either the Gal-empty vector or GalCycT1. The cells were also incubated for 48 h with CAF or control fluids from CNAR-positive and negative subjects, respectively. The grey bars correspond to relative CAT activity. The line bars represent mean and SD for two independent studies.
containing six Gal4 DNA-binding sites positioned upstream of the HIV-LTR, followed by the chloramphenicol acetyltransferase (CAT) reporter gene (Fig. 1a, see plasmid construct). Co-expression of the plasmid effector, coding for the chimeric Gal-CycT1 protein (Fig. 1a) leads to its recruitment to the PG6LTR promoter where the CycT1-associated kinase Cdk9 phosphorylates the C-terminal domain of RNAPII which activates transcription of the CAT gene (Taube et al., 2002) (Fig. 1a, see depiction of the reporter assay). http://jgv.microbiologyresearch.org
Chromatin immunoprecipitation (ChIP) studies To determine further the effect of CNAR/CAF on HIV transcription, we performed chromatin immunoprecipitation (ChIP) assays measuring the presence of RNAPII on the HIV genome in the infected CD4+ cells co-cultured with CD8+ cells from subjects that showed CNAR activity (Fig. 2). This experiment allowed us to measure the amount of RNAPII on the HIV provirus under the presence or absence of CNAR activity. We designed primers for the ChIP for the HIV promoter (LTR) and for two coding regions of HIV (gag and tat genes) (Fig. 2a). CD4+ cells were activated with phytohaemagglutinin (PHA) and IL-2 and infected with a single round HIV (pNL4-3 D Env GFP virus). The CD4+ cells were then incubated with CD8+ cells from a subject with CNAR or CD8+ cells from a normal uninfected subject that served as a control. After 2 days, the CD8+ cells were removed and the ChIP assay was performed with lysates from the HIV-infected CD4+ cells. 221
D. Blazek and others
(a) 337-457 5'LTR
5862-6011
1700-1875 vif
gag pol
1
tat vpu
tat
vpr rev
env
nef 3'LTR 9719
Location of primers on HIV genome
(b) Gag 120
100
100
80 CTRL CNAR
60 40 20 0
IgG
80 CTRL CNAR
60 40 20 0
RNAPII
IgG
RNAPII
Tat
120 Relative quantity
Relative quantity
Relative quantity
LTR 120
100 80 CTRL CNAR
60 40 20 0
IgG
RNAPII
Fig. 2. CD4+ cells infected with a single-round vesicular stomatitis virus (VSV)-HIV-GFP virus have less RNAPII on the HIV promoter, and the viral Gag and Tat coding regions, upon treatment with CD8+ cells from HIV-infected subjects with CNAR. (a) Location of primers used in ChIP assay on the HIV genome. Positions of the ChIP primers are indicated by red squares. The numbers above red squares represent the nucleotide positions of individual ChIP primers. Green rectangles represent individual HIV genes and numbers 1 and 9719 represent the first and final nucleotides of the HIV genome, respectively. (b) CD4+ cells in a CNAR assay result in lower recruitment of RNAPII to the HIV-LTR promoter and the HIV genome. ChIP analyses for the presence of RNAPII on the regions of the HIV genome (LTR, gag, tat) are shown. The amount of RNAPII on the LTR promoter and gag and tat genes in the control experiment was set at 100 %. The IgG corresponds to the empty bead control. The results of the study come from two independent experiments and quantitative PCR (qPCR) was performed in triplicate for each experiment. CNAR denotes results with CD8+ cells from CNAR-positive subjects; CTRL denotes results with CD8+ cells from normal uninfected subjects. The line bars represent mean and SD for two independent studies.
These ChIP studies with RNAPII antibody showed about a tenfold enrichment of RNAPII on the LTR promoter as well as on the tat and gag genes in comparison to the control ChIP with IgG empty beads (Fig. 2b, compare gray and black bars in IgG and RNAPII ChIP). Notably, incubation of the infected CD4+ cells with CNAR-positive CD8+ cells led to a three to fourfold decrease of RNAPII on the HIV-LTR promoter as well as on the gag and tat genes in comparison to the control cells (Fig. 2b, compare gray and black bars for RNAPII ChIP). These ChIP studies indicated that the levels of RNAPII on the HIV-LTR and on coding regions of the virus are diminished in the presence of CNAR-expressing CD8+ cells. The results provide an explanation for the decreased transcription in the CAT reporter assay following CAF treatment (Fig. 1b). They 222
support previous studies indicating a block in HIV transcription by CNAR/CAF (Chen et al., 1993; Mackewicz et al., 1995; Tomaras et al., 2000).
DISCUSSION Previous studies have indicated that the CNAR suppresses HIV replication by blocking viral transcription (Chen et al., 1993; Mackewicz et al., 1995; Tomaras et al., 2000). This antiviral activity is associated with production of a soluble CAF (Levy, 2003). Specific regions of the HIV-LTR do not appear to be involved with this effect (Bonneau et al., 2008; Shridhar et al., 2014) so intracellular transcriptional factors seemed responsible. The results from the current Journal of General Virology 97
CNAR and RNA polymerase II-mediated HIV transcription
studies further demonstrate that CNAR/CAF activity is associated with transcriptional inhibition at the HIV-LTR. First, incubation of CAF-containing fluid from CNAR-positive subjects inhibited transcriptional activity of the integrated HIV-LTR transcriptional reporter while the fluid from the CNAR-negative subjects did not (Fig. 1). This anti-HIV activity of CD8+ cell fluids had no apparent effect on the formation or the amount of P-TEFb complexes (Fig. S1). Second, ChIP analyses of HIV-infected CD4+ cells revealed markedly diminished amounts of RNAPII on the HIV genome (both promoter and coding regions) upon co-culture with CD8+ cells from CNAR-positive subjects (Fig. 2). It is possible that the effects of CNAR/CAF on the virus genome result from a block in transcription of certain cellular proteins needed for HIV transcription. However, the levels of Cdk9 and Cyclin T1 were not substantially affected (Fig. S1). Moreover, previous work in our laboratory and others have shown that CNAR does not affect the level of maintenance genes expressed, such as glyceraldehyde-3phosphate dehydrogenase and the IL-2 receptor (Mackewicz et al., 2000) nor does this CD8+ cell anti-HIV response suppress reporter protein production in cells transfected with CMV (cytomegalovirus) or SV40 constructs (Shridhar et al., 2014) Therefore, the current results further suggest a previously unrecognized role of CNAR/CAF in the regulation of HIV-specific transcription. Importantly, HIV transcription is dependent on the kinase activity of P-TEFb (Peterlin & Price, 2006). Moreover, although P-TEFb regulates transcription of many human genes (Chao et al., 2000; Rahl et al., 2010) the HIV-LTR seems to be much more sensitive to P-TEFb activity for optimal HIV transcription (Ott et al., 2011; Peterlin & Price, 2006). Notably, in addition to the well-established role of P-TEFb in transcriptional elongation of HIV and human genes, P-TEFb can regulate the HIV-specific transcription complex assembly at the preinitiation step (Brady & Kashanchi, 2005; Raha et al., 2005). Importantly, in addition to the Hexim1-contaning LC, P-TEFb is also found in several other complexes including super elongation and Brd complexes (Peterlin & Price, 2006). These newly discovered P-TEFb complexes differ in their function and kinase activity (including their ability to activate HIV transcription). We can then speculate that one of those complexes might be important for HIV-specific transcription complex assembly at the preinitiation step and is sensitive to CNAR/CAF. However, given that CNAR/CAF leads to diminished levels of RNAPII on the HIV-LTR promoter and other viral genetic regions (Fig. 2), virtually any step during the preinitiation complex assembly could be sensitive to inhibition by this CD8+ cell non-cytotoxic anti-HIV activity.
METHODS CAF positive/CAF negative fluid. The HIV-infected subjects whose CD8+ cells produce CAF came from a cohort of healthy long-term survivors (LTS) who have been part of the Levy laboratory studies for many years (Barker et al., 1998; Levy, 1993). These individuals have
http://jgv.microbiologyresearch.org
been infected for more than 10 years, remain healthy with a CD4+ cell count above 500 cells ml21 and have a low viral load (v10 000 RNA copies ml21). The fluids used were selected at random from several LTS out of at least 20 who have been followed for many years. Healthy uninfected subjects, whose CD8+ cells did not show CNAR or CAF production, were blood donors to the Blood Centers of the Pacific, San Francisco. Using immunomagnetic (IM) beads (Life Technologies), CD8+ cells were obtained from PBMC from these HIV-infected subjects and stimulated for 3 days with anti-CD3 beads. Fluids were collected and assayed for anti-HIV activity by standard procedures (Mackewicz et al., 1994). Briefly, CD4+T-cells obtained by IM beads from the PBMC from normal donors were stimulated with phytohaemagglutinin (PHA) (3 mg ml21) for 3 days, washed and inoculated with the X4-tropic HIV isolate, HIV1SF2. This virus is chemokine-resistant and sensitive to CAF. After 1 h, the CD4+ cells were washed and CD8+ cell fluids were added to the culture. Fluids from the CD4+ cell cultures were changed on day 2 and day 4 post-infection and assayed for virus replication by the reverse transcriptase (RT) assay (Hoffman et al., 1985). Reduction of HIV replication by more than 50 % was considered positive. Negative fluids obtained from CD8+ cells from uninfected controls showed less than 15 % inhibition of HIV replication. Transient transfection and CAT assay. A chloramphenicol acetyl-
transferase (CAT) assay was performed as described previously (Taube et al., 2002). Briefly, Jurkat T-cells were transfected with 0.5 mg of the pG6TAR reporter plasmid and 2 mg of the Gal-CycT1 or Gal-empty vector plasmid using Mirus transIT transfection reagent. After 8 h, the medium was changed and cells were incubated with CAF or control CD8+ cell fluids for 48 h. Subsequently, the CD4+ cells were harvested and the CAT activity in the cell lysate was measured by a scintillation counter. The protein content of the whole-cell lysate for normalization was determined using a BCA protein determination kit (Pierce). In these cell culture studies, no cell toxicity or reduction in cell growth was observed. Plasmid constructs. Full-length pNL4-3-D E-EGFP was obtained
from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (catalogue number 11100; provided by Drs Haili Zhang, Yan Zhou and Robert Siliciano). The viral envelope pMD.G was obtained as a generous gift from the D. Trono laboratory (Lausanne). Transfection for virus recovery. Culture of 293T cells was per-
formed in Dulbecco’s modified Eagle’s medium (1|) (Life Technologies) supplemented with 10 % FBS, 2 mM L -glutamine and 1 mM sodium pyruvate. Cells were plated at 3.5|106 per 10 ml of medium in a 100 mM plate 1 day prior to transfection. Three hours before transfection, the cell culture medium was replaced with fresh growth medium. Transfections were performed using a ProFection Mammalian Transfection System-Calcium Phosphate kit (Promega Corporation). Briefly, 15 mg pNL 4-3-D E-EGFP and 5 mg pMD.G were used for each transfection in a 100 mm plate. Reagents were combined and incubated according to the protocol. At 16 h posttransfection, the medium was discarded, and fresh medium added to the culture. The culture fluid was then collected 48 and 72 h posttransfection. These fluids were spun at 1300 g for 15 min, and 500 ml samples taken from each collected time point and centrifuged at 12 000 g for 2 h. Supernatants were aspirated and samples were quantified for the amount of the vesicular stomatitis virus (VSV)HIV-GFP virus by the RT assay (Hoffman et al., 1985).
ChIP assay with HIV infected CD4 1 cells and Western blot analysis. Approximately 6|107 PHA-stimulated CD4+ cells were
infected with a single round of VSV-HIV-GFP virus derived as described above. After 1 h, 2.4|107 CD4+ cells were washed and 223
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incubated for 2 days either alone or with resting CD8+ cells (obtained from a CNAR-positive LTS) at a cell input ratio of 1 : 1. Then, the CD8+ cells were removed from the CD4+/CD8+ mixture with anti-CD8+ IM beads at a 6 : 1 (bead : cells) ratio (Life Technologies). Approximately 2|106 of the infected CD4+ cells were used for the ChIP procedure that was performed as described (Blazek et al., 2011; Taube et al., 2002). Immunoprecipitations were performed with 5 mg of RNAPII antibody (sc-899; Santa Cruz Biotechnology). Quantitative PCRs (qPCRs) were conducted with the following primers: LTR: forward (337–359), reverse (436–457); Gag: forward (1700–1722), reverse (1852–1875); Tat: forward (5862–5886), reverse (5987–6011). Glycerol gradient centrifugation was performed as described previously (Blazek et al., 2005). CD8+ cells from normal uninfected subjects served as controls. For these studies, equal numbers of cells were used and they grew at the same rate. Cell toxicity was assessed by trypan blue dye exclusion and no difference in the experimental versus control cultures was observed. Antibodies used for Western blot analyses were Cdk9 (Santa Cruz Biotechnology; sc484), CyclinT1 (Santa Cruz Biotechnology; sc8126) and Hexim1 (Everest Biotech; EB06964).
Chen, C. H., Weinhold, K. J., Bartlett, J. A., Bolognesi, D. P. & Greenberg, M. L. (1993). CD8+ T lymphocyte-mediated inhibition
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HIV replication by CD8+ T cells correlates with CD4 counts and clinical stage of disease. Clin Exp Immunol 97, 68–75. Hoffman, A. D., Banapour, B. & Levy, J. A. (1985). Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology 147, 326–335. Levy, J. A. (1993). HIV pathogenesis and long-term survival. AIDS 7,
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(CAF). Trends Immunol 24, 628–632.
Levy, J. A., Mackewicz, C. E. & Barker, E. (1996). Controlling HIV
pathogenesis: the role of the noncytotoxic anti-HIV response of CD8+ T cells. Immunol Today 17, 217–224. Mackewicz, C. E., Ortega, H. W. & Levy, J. A. (1991). CD8+ cell anti-
HIV activity correlates with the clinical state of the infected individual. J Clin Invest 87, 1462–1466. Mackewicz, C. E., Ortega, H. & Levy, J. A. (1994). Effect of cytokines
ACKNOWLEDGEMENTS
on HIV replication in CD4+ lymphocytes: lack of identity with the CD8+ cell antiviral factor. Cell Immunol 153, 329–343.
These research studies were supported by grants from the National Institutes of Health (RO1 A056992), the California HIV/AIDS Research Program (ID09-SF-058) and the Peter and Shelagh Godsoe Foundation. D.B. is supported by a project ‘‘CEITEC - Central European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068) and a GACR grant (14-09979S). The authors wish to thank Anne McGuire for assistance with the manuscript.
Mackewicz, C. E., Blackbourn, D. J. & Levy, J. A. (1995). CD8+T cells suppress human immunodeficiency virus replication by inhibiting viral transcription. Proc Natl Acad Sci U S A 92, 2308–2312. Mackewicz, C. E., Patterson, B. K., Lee, S. A. & Levy, J. A. (2000).
CD8+ cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration. J Gen Virol 81, 1261–1264. Ott, M., Geyer, M. & Zhou, Q. (2011). The control of HIV
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Journal of General Virology 97
DOI 10.1099/jgv.0.000326
The CD8+ cell non-cytotoxic antiviral response affects RNA polymerase II-mediated human immunodeficiency virus transcription in infected CD4+ cells Dalibor Blazek,1,2 Fernando Teque,1 Carl Mackewicz,1,3 Matija Peterlin1 and Jay A. Levy1 Correspondence Jay A. Levy [email protected]
1
Department of Medicine, University of California San Francisco, 513 Parnassus Ave, Room S1280, San Francisco, CA 94143, USA
2
CEITEC-Masaryk University, 62500 Brno, Czech Republic
3
Amgen, Thousand Oaks, CA, USA
Received 13 August 2015 Accepted 21 October 2015
A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.
INTRODUCTION Long-term survival from human immunodeficiency virus (HIV) infection is associated with a CD8+ cell non-cytotoxic antiviral response (CNAR) (Walker et al., 1986). This innate cellular immune activity involves inhibition of viral transcription and is active against all HIV-1, HIV-2 and simian immunodeficiency virus isolates tested (Chen et al., 1993; Levy et al., 1996; Mackewicz et al., 1995; Tomaras et al., 2000; Walker et al., 1986). CNAR is mediated by the production of a soluble CD8+ cell antiviral factor (CAF) (Brinchmann et al., 1990; Levy, 2003; Walker & Levy, 1989). Healthy HIV-infected individuals, many of whom have lived for more than 10 years to over 35 years without treatment, show the presence of CNAR and the production of CAF by their CD8+ cells (Mackewicz et al., 1991; Barker et al., 1998; Go´mez et al., 1994). Thus, the characterization of CNAR/CAF and its specific mechanism of action is a major objective of our research. Previous work has indicated that several regions of the HIV-LTR promoter are not involved in the inhibition of transcription by CNAR/CAF (Bonneau et al., 2008; Shridhar et al., 2014). Deletion of these promoter sequences did One supplementary figure is available with the online Supplementary Material.
220
not lead to the production of viruses that are resistant to this CD8+ cell antiviral response. These findings have suggested that intracellular transcriptional factors may be targeted by CNAR/CAF and this regulatory action is responsible for the block in HIV transcription. In approaches to define the processes involved in the block in viral transcription, we have found that HIV-infected cells exposed to CAF or CD8+ cells expressing CNAR have a reduced amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral genetic regions. A role of positive transcriptional elongation factor b (PTEFb) is suggested, but a direct effect on P-TEFb complexes was not observed. The results suggest that a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.
RESULTS The effect of CAF on P-TEFb-mediated HIV-LTR transcription To determine the mechanism by which CNAR/CAF affects transcription, a transcriptional reporter system was used (Fig. 1). It consists of a plasmid reporter PG6LTR 000326 G 2015 The Authors
Printed in Great Britain
CNAR and RNA polymerase II-mediated HIV transcription
When we co-expressed the Gal-CycT1 chimera with pG6LTRCAT in the Jurkat cells, the levels of CAT activity increased 17-fold over basal levels (Fig. 1b, compare bar 2 to bar 1). To assess the effect on the transcriptional activity of the CAT reporter, we incubated transfected cells with CAF-containing culture fluids from CD8+ cells from three CNAR-positive subjects. Strikingly, incubation with CAF fluid decreased reporter activity substantially (Fig. 1b, compare bars 3, 4 and 5 to bar 2). The CD8+ cell fluids from CNAR-negative uninfected subjects did not affect CAT transcription levels (Fig. 1b, compare bars 6 and 7 to bar 2). This experiment indicated that CAF from CNAR-positive subjects can regulate the transcription of an HIV promoter-based reporter system.
(a) Plasmid constructs Gal-cyclinT1 Gal
CyclinT1
G6LTRCAT 6xGal binding sites
HIV-LTR
CAT
Reporter assay Gal-CycT1
RNAPII
6-Gal binding sites
CAT
HIV-LTR
(b) 40
Relative CAT activity
35 30
Effect of CAF on P-TEFb
25
It is well-established that P-TEFb is associated in two forms in cells: either in the catalytically inactive form in complex with other proteins including Hexim1 (so called the large complex of P-TEFb) or in a catalytically active free form (small complex) (Peterlin & Price, 2006). Because the above-described HIV-LTR based reporter system is dependent on Cdk9 activity, we incubated CD4+ cells with either CAF positive or negative fluids for 48 h and then separated the protein complexes from the CD4+ lysates by glycerol gradient centrifugation. Western blot analyses of the glycerol gradient fractions with Hexim1 and Cyclin T1 antibodies showed that the amounts of P-TEFb in the large and small complexes remained the same upon culture of the infected CD4+ cells with either CAF-positive or negative fluid (data not shown). Moreover, the total amounts of the CyclinT1, Cdk9 and Hexim1 proteins were the same in the CD4+ cells treated with both the CAF- positive and negative culture fluids (Fig. S1, available in the online Supplementary Material).
20 15 10 5 0
1 + G6LTRCAT Gal-emp.vect. + Gal-cyclinT1 CAF positive CAF negative
2 +
3 +
4 +
5 +
6 +
7 +
+
+ +
+ +
+ +
+
+
+
+
Fig. 1. CAF inhibits transcription from the HIV G6LTRCAT reporter gene. (a) Depiction of the HIV G6LTRCAT reporter assay. Six Galbinding sites (dark green rectangles) are bound by six molecules of Gal-CyclinT1 (Gal-CycT1; light green ovals). RNAPII (yellow oval) binds the HIV-LTR promoter (black rectangle). Active transcription results in CAT reporter gene (pink rectangle) expression. GalCycT1/Cdk9 phosphorylates the C-terminal domain of RNAPII (light green arrow) which results in the productive transcription of the CAT reporter gene (yellow arrow). (b) Jurkat T-cells described in (a) were transfected with either the Gal-empty vector or GalCycT1. The cells were also incubated for 48 h with CAF or control fluids from CNAR-positive and negative subjects, respectively. The grey bars correspond to relative CAT activity. The line bars represent mean and SD for two independent studies.
containing six Gal4 DNA-binding sites positioned upstream of the HIV-LTR, followed by the chloramphenicol acetyltransferase (CAT) reporter gene (Fig. 1a, see plasmid construct). Co-expression of the plasmid effector, coding for the chimeric Gal-CycT1 protein (Fig. 1a) leads to its recruitment to the PG6LTR promoter where the CycT1-associated kinase Cdk9 phosphorylates the C-terminal domain of RNAPII which activates transcription of the CAT gene (Taube et al., 2002) (Fig. 1a, see depiction of the reporter assay). http://jgv.microbiologyresearch.org
Chromatin immunoprecipitation (ChIP) studies To determine further the effect of CNAR/CAF on HIV transcription, we performed chromatin immunoprecipitation (ChIP) assays measuring the presence of RNAPII on the HIV genome in the infected CD4+ cells co-cultured with CD8+ cells from subjects that showed CNAR activity (Fig. 2). This experiment allowed us to measure the amount of RNAPII on the HIV provirus under the presence or absence of CNAR activity. We designed primers for the ChIP for the HIV promoter (LTR) and for two coding regions of HIV (gag and tat genes) (Fig. 2a). CD4+ cells were activated with phytohaemagglutinin (PHA) and IL-2 and infected with a single round HIV (pNL4-3 D Env GFP virus). The CD4+ cells were then incubated with CD8+ cells from a subject with CNAR or CD8+ cells from a normal uninfected subject that served as a control. After 2 days, the CD8+ cells were removed and the ChIP assay was performed with lysates from the HIV-infected CD4+ cells. 221
D. Blazek and others
(a) 337-457 5'LTR
5862-6011
1700-1875 vif
gag pol
1
tat vpu
tat
vpr rev
env
nef 3'LTR 9719
Location of primers on HIV genome
(b) Gag 120
100
100
80 CTRL CNAR
60 40 20 0
IgG
80 CTRL CNAR
60 40 20 0
RNAPII
IgG
RNAPII
Tat
120 Relative quantity
Relative quantity
Relative quantity
LTR 120
100 80 CTRL CNAR
60 40 20 0
IgG
RNAPII
Fig. 2. CD4+ cells infected with a single-round vesicular stomatitis virus (VSV)-HIV-GFP virus have less RNAPII on the HIV promoter, and the viral Gag and Tat coding regions, upon treatment with CD8+ cells from HIV-infected subjects with CNAR. (a) Location of primers used in ChIP assay on the HIV genome. Positions of the ChIP primers are indicated by red squares. The numbers above red squares represent the nucleotide positions of individual ChIP primers. Green rectangles represent individual HIV genes and numbers 1 and 9719 represent the first and final nucleotides of the HIV genome, respectively. (b) CD4+ cells in a CNAR assay result in lower recruitment of RNAPII to the HIV-LTR promoter and the HIV genome. ChIP analyses for the presence of RNAPII on the regions of the HIV genome (LTR, gag, tat) are shown. The amount of RNAPII on the LTR promoter and gag and tat genes in the control experiment was set at 100 %. The IgG corresponds to the empty bead control. The results of the study come from two independent experiments and quantitative PCR (qPCR) was performed in triplicate for each experiment. CNAR denotes results with CD8+ cells from CNAR-positive subjects; CTRL denotes results with CD8+ cells from normal uninfected subjects. The line bars represent mean and SD for two independent studies.
These ChIP studies with RNAPII antibody showed about a tenfold enrichment of RNAPII on the LTR promoter as well as on the tat and gag genes in comparison to the control ChIP with IgG empty beads (Fig. 2b, compare gray and black bars in IgG and RNAPII ChIP). Notably, incubation of the infected CD4+ cells with CNAR-positive CD8+ cells led to a three to fourfold decrease of RNAPII on the HIV-LTR promoter as well as on the gag and tat genes in comparison to the control cells (Fig. 2b, compare gray and black bars for RNAPII ChIP). These ChIP studies indicated that the levels of RNAPII on the HIV-LTR and on coding regions of the virus are diminished in the presence of CNAR-expressing CD8+ cells. The results provide an explanation for the decreased transcription in the CAT reporter assay following CAF treatment (Fig. 1b). They 222
support previous studies indicating a block in HIV transcription by CNAR/CAF (Chen et al., 1993; Mackewicz et al., 1995; Tomaras et al., 2000).
DISCUSSION Previous studies have indicated that the CNAR suppresses HIV replication by blocking viral transcription (Chen et al., 1993; Mackewicz et al., 1995; Tomaras et al., 2000). This antiviral activity is associated with production of a soluble CAF (Levy, 2003). Specific regions of the HIV-LTR do not appear to be involved with this effect (Bonneau et al., 2008; Shridhar et al., 2014) so intracellular transcriptional factors seemed responsible. The results from the current Journal of General Virology 97
CNAR and RNA polymerase II-mediated HIV transcription
studies further demonstrate that CNAR/CAF activity is associated with transcriptional inhibition at the HIV-LTR. First, incubation of CAF-containing fluid from CNAR-positive subjects inhibited transcriptional activity of the integrated HIV-LTR transcriptional reporter while the fluid from the CNAR-negative subjects did not (Fig. 1). This anti-HIV activity of CD8+ cell fluids had no apparent effect on the formation or the amount of P-TEFb complexes (Fig. S1). Second, ChIP analyses of HIV-infected CD4+ cells revealed markedly diminished amounts of RNAPII on the HIV genome (both promoter and coding regions) upon co-culture with CD8+ cells from CNAR-positive subjects (Fig. 2). It is possible that the effects of CNAR/CAF on the virus genome result from a block in transcription of certain cellular proteins needed for HIV transcription. However, the levels of Cdk9 and Cyclin T1 were not substantially affected (Fig. S1). Moreover, previous work in our laboratory and others have shown that CNAR does not affect the level of maintenance genes expressed, such as glyceraldehyde-3phosphate dehydrogenase and the IL-2 receptor (Mackewicz et al., 2000) nor does this CD8+ cell anti-HIV response suppress reporter protein production in cells transfected with CMV (cytomegalovirus) or SV40 constructs (Shridhar et al., 2014) Therefore, the current results further suggest a previously unrecognized role of CNAR/CAF in the regulation of HIV-specific transcription. Importantly, HIV transcription is dependent on the kinase activity of P-TEFb (Peterlin & Price, 2006). Moreover, although P-TEFb regulates transcription of many human genes (Chao et al., 2000; Rahl et al., 2010) the HIV-LTR seems to be much more sensitive to P-TEFb activity for optimal HIV transcription (Ott et al., 2011; Peterlin & Price, 2006). Notably, in addition to the well-established role of P-TEFb in transcriptional elongation of HIV and human genes, P-TEFb can regulate the HIV-specific transcription complex assembly at the preinitiation step (Brady & Kashanchi, 2005; Raha et al., 2005). Importantly, in addition to the Hexim1-contaning LC, P-TEFb is also found in several other complexes including super elongation and Brd complexes (Peterlin & Price, 2006). These newly discovered P-TEFb complexes differ in their function and kinase activity (including their ability to activate HIV transcription). We can then speculate that one of those complexes might be important for HIV-specific transcription complex assembly at the preinitiation step and is sensitive to CNAR/CAF. However, given that CNAR/CAF leads to diminished levels of RNAPII on the HIV-LTR promoter and other viral genetic regions (Fig. 2), virtually any step during the preinitiation complex assembly could be sensitive to inhibition by this CD8+ cell non-cytotoxic anti-HIV activity.
METHODS CAF positive/CAF negative fluid. The HIV-infected subjects whose CD8+ cells produce CAF came from a cohort of healthy long-term survivors (LTS) who have been part of the Levy laboratory studies for many years (Barker et al., 1998; Levy, 1993). These individuals have
http://jgv.microbiologyresearch.org
been infected for more than 10 years, remain healthy with a CD4+ cell count above 500 cells ml21 and have a low viral load (v10 000 RNA copies ml21). The fluids used were selected at random from several LTS out of at least 20 who have been followed for many years. Healthy uninfected subjects, whose CD8+ cells did not show CNAR or CAF production, were blood donors to the Blood Centers of the Pacific, San Francisco. Using immunomagnetic (IM) beads (Life Technologies), CD8+ cells were obtained from PBMC from these HIV-infected subjects and stimulated for 3 days with anti-CD3 beads. Fluids were collected and assayed for anti-HIV activity by standard procedures (Mackewicz et al., 1994). Briefly, CD4+T-cells obtained by IM beads from the PBMC from normal donors were stimulated with phytohaemagglutinin (PHA) (3 mg ml21) for 3 days, washed and inoculated with the X4-tropic HIV isolate, HIV1SF2. This virus is chemokine-resistant and sensitive to CAF. After 1 h, the CD4+ cells were washed and CD8+ cell fluids were added to the culture. Fluids from the CD4+ cell cultures were changed on day 2 and day 4 post-infection and assayed for virus replication by the reverse transcriptase (RT) assay (Hoffman et al., 1985). Reduction of HIV replication by more than 50 % was considered positive. Negative fluids obtained from CD8+ cells from uninfected controls showed less than 15 % inhibition of HIV replication. Transient transfection and CAT assay. A chloramphenicol acetyl-
transferase (CAT) assay was performed as described previously (Taube et al., 2002). Briefly, Jurkat T-cells were transfected with 0.5 mg of the pG6TAR reporter plasmid and 2 mg of the Gal-CycT1 or Gal-empty vector plasmid using Mirus transIT transfection reagent. After 8 h, the medium was changed and cells were incubated with CAF or control CD8+ cell fluids for 48 h. Subsequently, the CD4+ cells were harvested and the CAT activity in the cell lysate was measured by a scintillation counter. The protein content of the whole-cell lysate for normalization was determined using a BCA protein determination kit (Pierce). In these cell culture studies, no cell toxicity or reduction in cell growth was observed. Plasmid constructs. Full-length pNL4-3-D E-EGFP was obtained
from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (catalogue number 11100; provided by Drs Haili Zhang, Yan Zhou and Robert Siliciano). The viral envelope pMD.G was obtained as a generous gift from the D. Trono laboratory (Lausanne). Transfection for virus recovery. Culture of 293T cells was per-
formed in Dulbecco’s modified Eagle’s medium (1|) (Life Technologies) supplemented with 10 % FBS, 2 mM L -glutamine and 1 mM sodium pyruvate. Cells were plated at 3.5|106 per 10 ml of medium in a 100 mM plate 1 day prior to transfection. Three hours before transfection, the cell culture medium was replaced with fresh growth medium. Transfections were performed using a ProFection Mammalian Transfection System-Calcium Phosphate kit (Promega Corporation). Briefly, 15 mg pNL 4-3-D E-EGFP and 5 mg pMD.G were used for each transfection in a 100 mm plate. Reagents were combined and incubated according to the protocol. At 16 h posttransfection, the medium was discarded, and fresh medium added to the culture. The culture fluid was then collected 48 and 72 h posttransfection. These fluids were spun at 1300 g for 15 min, and 500 ml samples taken from each collected time point and centrifuged at 12 000 g for 2 h. Supernatants were aspirated and samples were quantified for the amount of the vesicular stomatitis virus (VSV)HIV-GFP virus by the RT assay (Hoffman et al., 1985).
ChIP assay with HIV infected CD4 1 cells and Western blot analysis. Approximately 6|107 PHA-stimulated CD4+ cells were
infected with a single round of VSV-HIV-GFP virus derived as described above. After 1 h, 2.4|107 CD4+ cells were washed and 223
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incubated for 2 days either alone or with resting CD8+ cells (obtained from a CNAR-positive LTS) at a cell input ratio of 1 : 1. Then, the CD8+ cells were removed from the CD4+/CD8+ mixture with anti-CD8+ IM beads at a 6 : 1 (bead : cells) ratio (Life Technologies). Approximately 2|106 of the infected CD4+ cells were used for the ChIP procedure that was performed as described (Blazek et al., 2011; Taube et al., 2002). Immunoprecipitations were performed with 5 mg of RNAPII antibody (sc-899; Santa Cruz Biotechnology). Quantitative PCRs (qPCRs) were conducted with the following primers: LTR: forward (337–359), reverse (436–457); Gag: forward (1700–1722), reverse (1852–1875); Tat: forward (5862–5886), reverse (5987–6011). Glycerol gradient centrifugation was performed as described previously (Blazek et al., 2005). CD8+ cells from normal uninfected subjects served as controls. For these studies, equal numbers of cells were used and they grew at the same rate. Cell toxicity was assessed by trypan blue dye exclusion and no difference in the experimental versus control cultures was observed. Antibodies used for Western blot analyses were Cdk9 (Santa Cruz Biotechnology; sc484), CyclinT1 (Santa Cruz Biotechnology; sc8126) and Hexim1 (Everest Biotech; EB06964).
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ACKNOWLEDGEMENTS
on HIV replication in CD4+ lymphocytes: lack of identity with the CD8+ cell antiviral factor. Cell Immunol 153, 329–343.
These research studies were supported by grants from the National Institutes of Health (RO1 A056992), the California HIV/AIDS Research Program (ID09-SF-058) and the Peter and Shelagh Godsoe Foundation. D.B. is supported by a project ‘‘CEITEC - Central European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068) and a GACR grant (14-09979S). The authors wish to thank Anne McGuire for assistance with the manuscript.
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Journal of General Virology 97
