1 Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea
2 Center for AIDS Research, National Institute of Health, Seoul 122-701, Korea
3 TaKaRa-Bio Inc., Seta 3-4-1, Otsu, Shiga, Japan
Correspondence
Sunyoung Kim
sunyoung{at}plaza.snu.ac.kr
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ABSTRACT |
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INTRODUCTION |
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Several regions of the Nef protein have been reported to be involved in down-regulation of CD4 and MHC class I proteins (Geyer et al., 2001; Geyer & Peterlin, 2001
; Piguet et al., 1999
). A myristoylation signal (Geyer et al., 1999
), amino acid 5758 region (Grzesiek et al., 1996
), the dileucine (LL) motif (Bresnahan et al., 1998
; Craig et al., 1998
; Greenberg et al., 1998a
) and the EE155 diacidic sequence (Benichou et al., 1994
; Janvier et al., 2001
; Piguet et al., 1999
) were reported to be necessary for CD4 down-regulation, while for MHC class I the proline rich (PxxP) motif (Greenberg et al., 1998b
; Mangasarian et al., 1999
) and an acidic amino acid stretch around the amino acid 65 region (Piguet et al., 2000
) were identified as key motifs. However, the roles of some regions are controversial. For example, the EE155 region has been reported to be involved in CD4 down-regulation by interacting with
-COP protein, but contradictory observations have also been made (Janvier et al., 2001
; Piguet et al., 1999
). Most of this work on the biochemical and genetic characterization of Nef protein have employed nef sequences isolated from standard laboratory strains such as HIV-1 NL4-3 and SF2. These strains have been maintained for a long period in transformed cell lines. However, it is now clear that the nef gene is not even necessary for virus replication in the in vitro cell culture system and thus such nef sequences might not represent the nef sequence present in vivo. Based on this rationale, our group and others have been isolating and characterizing nef sequences directly from infected individuals at various clinical stages. It was quite clear from these studies that variation in Nef amino acid sequences is significantly high among primary isolates, ranging up to 30 % even within the same subtype, forcing us to revisit some of the previous work done on the laboratory nef sequences.
We previously reported that two primary nef sequences, KS2 (subtype B) and K306 (subtype D), significantly differ in their pattern of CD4 down-regulation from the NL4-3 nef sequence (Yoon et al., 2001). For example, KS2 Nef could efficiently down-regulate MHC class I, but not CD4. It was reasoned that such differential effects could be used to localize the regions or motifs important for the biochemical effects of Nef. In this report, we continued the analysis of KS2 Nef and found the EE region around amino acid position 155 to be crucial for CD4 down-regulation. During this analysis, it was also determined that the effects of Nef are more prominent during the early stage of nef expression and also in primary T cells.
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METHODS |
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Transfections and transductions.
Retroviral constructs were transfected to 293T cells by a three-plasmid transfection method (Soneoka et al., 1995) using the calcium phosphate method. Cell culture supernatant was taken 2 days after transfection and filtered through a 0·45 µm-pore size filter. 5x105 human T lymphoid lines, Jurkat and CEM-SS cells, were incubated with virus supernatant in the presence of 8 µg polybrene ml1 for 48 h in a 37 °C incubator. The virus supernatants were then replaced with fresh medium containing 1 mg G418 ml1. Following selection in G418, Nef was detected by Western blot analysis. All transductions were tested and found positive for Nef expression. For cells transiently expressing Nef, the constructs were transfected to 293T cells by a three-plasmid transfection method using FuGene6 (Roche). Culture supernatants were taken 2 days post-transfection and transduced three times at 12 h intervals by centrifugation. The virus supernatant was concentrated 100-fold by centrifugation at 29 000 r.p.m. in a Beckman SW-40 rotor for 2 h at 20 °C.
Purification of CD4+ T cells from PBMCs.
CD4+ T cells were purified from the peripheral blood mononuclear cells (PBMCs) of healthy individuals. PBMCs were separated by using a FicollPaque Plus (Pharmacia Biotech) density gradient and then the CD4+ T cells were purified by panning, as described previously (Wysocki & Sato, 1978). Cells were stimulated with 13 µg phytohaemagglutinin ml1 (Sigma) and cultured with 25 ng IL-2 ml1 (Endogen) in RPMI complete medium for 8 days. The percentage of CD4+ T cells in the purified cells was determined by flow cytometry.
Antibodies.
Rabbit anti-Nef serum was obtained from the NIH AIDS Research and Reference Reagent Program. Polyclonal GFP antiserum was purchased from Invitrogen. Mouse monoclonal anti-FLAG M2 antibody, anti-rabbit IgGperoxidase conjugate, anti-mouse IgGR-phycoerythrin-conjugated antibody and anti-mouse IgG1 antibody were purchased from Sigma. Anti-mouse IgGhorseradish peroxidase conjugate was purchased from Pierce and R-phycoerythrin-conjugated mouse anti-human monoclonal CD4 antibody or FITC-conjugated anti-human MHC class I antibody from BD PharMingen.
Western blot analysis.
Transduced cells were washed with PBS and lysed with lysis buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA, 0·5 % Triton X-100, 1 mM PMSF, 0·5 mM DTT) by freezing and thawing; lysates were clarified by centrifugation. Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane (Amersham). After blocking with 5 % skimmed milk in Tris-buffered saline (10 mM Tris pH7·5, 0·15 M NaCl), blots were probed with appropriate primary antibody and incubated with horseradish peroxidase-conjugated secondary antibody. Filters were washed and visualized by enhanced chemiluminescence (Pierce). The total levels of GFP, FLAG and Nef in MIGnef were assessed by Western blot analysis with the corresponding antibodies. The blot was probed with an antibody against FLAG and then stripped and reprobed with an antibody specific for GFP. Equivalent amounts of proteins as used in previous blots were electrophoresed and then Western blotting was performed with Nef (SF2). For reprobing, blots were treated with Restore stripping solution (Pierce).
Flow cytometry analysis.
Flow cytometric analysis of CD4 or MHC class I expression in cells transduced with retrovirus constructs expressing various Nef was performed on a Becton Dickinson FACSort, using CellQuest (BD) data acquisition and analysis software. Briefly, 1x106 cells were incubated with saturating amounts of phycoerythrin-conjugated human anti-CD4 or anti-MHC class I monoclonal antibodies. Cells were then washed three times with PBS and resuspended in 500 µl PBS. CD4 or MHC class I expression levels were measured by FACS.
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RESULTS AND DISCUSSION |
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We also measured levels of MHC class I in these transduced T cell lines to test whether the EE155 region was also involved in MHC class I down-regulation. For NL4-3 Nef, the relative MHC class I down-regulation in Jurkat cells was approximately 36·4±13·07 % and 23±6·43 % for Nefs containing and lacking the EE155 region, respectively (Fig. 5B, C), while it was 30±1·16 % and 23±4·69 % for KS2 Nef. Similar results were obtained in CEM-SS. These data indicated that the EE155 region did not exert any significant influence on MHC class I down-regulation, consistent with the previous result showing that KS2 Nef down-regulates MHC class I as efficiently as NL4-3 (Yoon et al., 2001
).
Analysis of clinical samples from KS2 and other pathologically defined individuals
Because KS2 is defined as an LTNP, we compared the nef sequence and basic clinical data of KS2 with those of eight other individuals whose pathological status has been followed by the National Institute of Health in Korea, and whose nef sequences were determined also (Table 1). One of the noticeable observations made from KS2 was that this individual maintained an unusually high number of CD8+ T cells in peripheral blood over the 6-year period, ranging from 1252 to 2000 cells mm3. Because CD4+ T cells were maintained at around 540 cells, the individual showed a relatively lower ratio of CD4/CD8, ranging from 0·24 to 0·47. This is in contrast to other LTNPs whose CD4/CD8 ratio was generally higher than 0·5 at almost all time-points, but similar to that of the three RPs (rapid progressors) compared in this study. Since the last observation made in March 2003, KS2 showed no sign of any significant change in these numbers, and KS2 is still not undergoing any drug treatment. The significance of this finding remains unclear at this time.
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Analysis of nef sequences available from the public database
We checked the 481 nef sequences available from the Los Alamos HIV Sequence Database. Seven nef sequences (approx. 1·5 % of the total) lack the EE155 region, while one of the two Es was replaced with other amino acids in 88 nef sequences (approx. 18·3 %). Among 34 nef sequences isolated from 21 LTNPs available from the PubMed database, only one sequence did not have the EE155 region, while no replacement was found in other sequences. Five nef sequences from five RPs were also available; one had the replaced EE155 region, while all the others contained the two glutamic acids.
We found the EE155 region crucial for CD4 down-regulation by Nef protein. Thus far two regions, the LL165 motif and the EE155 region, have been reported to play a role(s) in CD4 down-regulation. However, the involvement of the latter region has been controversial (Janvier et al., 2001; Piguet et al., 1999
). Our data are more consistent with the findings of Piguet et al. showing that the EE155 region is needed for CD4 down-regulation. The actual mechanism of how the EE155 interacts with CD4 molecules remains to be elucidated, and various biochemical experiments are in progress.
There have been a few reports showing correlation between the presence of particular Nef regions and disease progression (Mariani & Skowronski, 1993; Asamitsu et al., 1999
). However, data on the possible relationship between the EE155 region in particular, CD4 down-regulation and AIDS pathogenesis is extremely limited. One report discusses the possible role of the EE155 region in CD4 down-regulation (Mariani & Skowronski, 1993
), while the other report indicates no role (Asamitsu et al., 1999
). However, the number of individuals used in these studies was only one or two, numbers too low to derive any conclusion. In our study, we analysed eight individuals, whose status of disease progression was identified, as well as 34 Nef sequences from 21 LTNPs available from the database. Although the profile of CD4+ and CD8+ T cells in the individual KS2 is intriguing, we did not discover any significant correlation between the progress of disease and presence of two glutamic acids. Based on the bulk of previous reports, it is likely that there are multiple factors involved in Nef-driven CD4 down-regulation, and thus it may not be possible to come to any acceptable conclusion about AIDS pathogenesis and the EE155 region alone. However, the EE155 region was very well conserved and present in the majority of nef sequences, indicating a crucial role in Nef protein function. Biological and clinical data are interesting enough to warrant further studies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 November 2003;
accepted 6 January 2004.
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