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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 22, 2003 as doi:10.1096/fj.02-0512fje. |
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Laboratory of Immunology, Interdisciplinary Research Center of Autoimmune Diseases, Department of Medical Science, University of Eastern Piedmont, Novara, Italy;
* Department of Genetics, Biology and Biochemistry, University of Turin, Turin, Italy;
Department of Physiological Chemistry, Graduate School of Pharmaceutical Science, University of Tokyo, Hongo, Tokyo, Japan; and
Division of Retrovirology, Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy
2Correspondence: Laboratory of Immunology, Department of Medical Science, "A. Avogadro" University of Eastern Piedmont, Via Solaroli 17, I-28100 Novara, Italy. E-mail: asavarino{at}medscape.com
SPECIFIC AIMS
We have investigated the molecular mechanisms by which CD38 inhibits fusion of human immunodeficiency viruses (HIV) to target cells, since they may be open for exploitation in new anti-HIV strategies. CD38 is a leukocyte surface molecule expressed by naive and activated lymphocytes, and its expression level on activated T cells is an indicator of AIDS progression. We have previously shown that CD38 displays lateral association with CD4 on the T cell surface and that the viral envelope glycoprotein gp120 potentiates this association. We showed that expression of CD38 down-modulated HIV-1 entry into the human MT-4 T cell line (CD4+CXCR-4+) and gp120 attachment to mouse cells expressing human CD4.
The aims of this study were to 1) map the CD38 domains involved in the anti-HIV-1 effects and 2) investigate the mechanisms and specificity of these effects.
PRINCIPAL FINDINGS
1. CD38 down-modulates HIV-1 fusion and CD4-mediated attachment of gp120 to cells
The first set of experiments assessed the specificity of anti-HIV effect of CD38. By stable transfection on the MT-2 human T cell line (CD4+CXCR-4+CD38-), we produced a clone expressing high levels of CD38 (MT-2.CD38) and a mock-transfected clone (MT-2.M). De novo infection with HIV-1IIIB showed that p24 production was significantly lower in the MT-2.CD38 cells. Syncytium assays showed that CD38 expression inhibited fusion of uninfected MT-2 cells with chronically HIV-1IIIB-infected H9 cells (H9IIIB). Flow cytometry showed that staining with FITC-conjugated gp120IIIB (FITC-gp120) was weaker in MT-2.CD38 than in MT-2.M cells. The specificity of the inhibition of FITC-gp120 binding to CD4 was assessed on stable transfectants of the SR.hCD4 T cell line expressing human CD4 and either human CD38 or other human control molecules, i.e., CD95, CD59, or CD31. Staining with FITC-gp120 showed that expression of CD38 alone inhibited gp120 attachment. This is thus specific and CD38 may be assumed to specifically interfere with gp120 binding to CD4.
2. CD38’s anti-HIV effect depends on the extracellular membrane-proximal region
We evaluated the role of the intracellular (IC), transmembrane (TM), and extracellular (EC) portions. Five truncated forms of CD38 were used: one lacking the IC domain (CD3824–300, spanning amino acids 24–300), a soluble form lacking the IC and TM domains (sCD3845–300), and three forms lacking different portions of the EC COOH terminus (CD381–285, CD381–289, and CD381–191). Since some of these forms lacked the epitopes recognized by the available mAbs, we added a myc tag at their NH2 terminus to detect their expression by an anti-myc mAb. De novo infection with HIV-1IIIB of MT-2 cells stably transfected with the truncated forms showed they all inhibited viral replication to an extent similar to wild-type CD38 (Fig. 1
). Since all constructs shared the EC membrane-proximal sequence (aa 45–191), the next step was to map the anti-HIV activity within this region.
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3. The extracellular membrane-proximal region of CD38 displays significant homology with the V3 loop of gp120
The sequence spanning the amino acids 45–74 attracted our attention since it displayed similarities with a large portion of the V3 loop of gp120, including identity of the conserved GPG triplet at the tip of the V3 loop, a partially conserved region that influences HIV-1 attachment/fusion. Multiple alignments with the DIALIGN algorithm showed that most of these amino acids were in significant alignment with the V3 loop consensus sequences for the main HIV-1 subgroupings, SIV cpz (phylogenetically close to the common ancestor of the HIV-1 groups), and HIV-2 (P<0.01).
4. Peptides containing the amino acid 52–57 sequence of CD38 reproduce the anti-HIV effects of the full-length molecule
We tested whether a soluble synthetic peptide (sCD3851–74) spanning a large portion of the V3-like region of CD38 displayed anti-HIV effects. This peptide dose-dependently inhibited p24 production in CD38- MT-4 cells de novo infected with HIV-1IIIB (Fig. 2
A), syncytium formation in H9IIIB/MT-2 cocultures (Fig. 2B
) and FITC-gp120 attachment to MT-4 cells (Fig. 2C
). It also inhibited replication of phylogenetically unrelated R5, X4, R5/X4 HIV-1, and HIV-2 primary isolates in both cell lines and peripheral blood mononuclear cells (PBMC), but did not inhibit a HIV-1 vector (pRRL.sin.hPGK.GFP) pseudotyped with the vesicular stomatitis virus envelope protein (Fig. 2D
). This indicates that the inhibition requires interaction of gp120 with target cells. Furthermore, sCD3851–74 inhibited gp120 binding to recombinant CD4 (Fig. 2E
).
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The inhibitory concentrations were strikingly decreased when a multiple branched peptide (MBP) construct (sCD3851–74 MBP) was used. This inhibited infection of MT-4 cells by HIV-1IIIB and infection of PBMC by primary isolates in the nanomolar range.
5. Involvement of a GPGTTK motif in the anti-HIV-1 effect of CD38
We then evaluated the role of the CD3852–57 sequence (GPGTTK) corresponding to the gp120 V3 loop tip in our alignments. We found that an MBP GPGTTK peptide (sCD3852–57-MBP) dose-dependently inhibited infection of MT-4 cells, whereas no anti-HIV effect was displayed by a modified sCD3851–74 peptide in which amino acids 51–57 were replaced by a random sequence (TSHALSA) that maintained the same overall charge (sCD3858–74).
Last, we evaluated the role of GPGTTK by transfecting MT-2 cells with a CD38 construct lacking this sequence (CD38
52–57). De novo infection with HIVIIIB showed that CD3852–57 did not display anti-HIV-1 activity. Fluorescence resonance energy transfer experiments also showed that it did not display lateral interaction with CD4 in intact cells.
CONCLUSIONS
This study shows that CD38 inhibits HIV replication at an early step and its 52–57 sequence plays a crucial role. Data in the acellular model show that CD38-derived peptides containing this sequence directly interfere with gp120 binding to CD4, although contemporary interactions with other cell surface molecules involved in HIV entry cannot be ruled out. One explanation may be that this sequence (homologous to the V3 loop tip in our alignments) competes for CD4 binding with the gp120 V3 loop, which may establish a secondary interaction between gp120 and CD4. An alternative explanation might be that CD38-derived peptides destabilize the gp120/CD4 complex by altering its electrical charge or energetics.
The effects of CD38 on HIV replication may help clarify some aspects of virus/host interactions. This study supports our previously published model that HIV-1 preferentially infects CD38- cells and that the high levels of HIV-1 expression displayed by activated (CD38+) T cells are due to post-entry events. Peptides from the membrane-proximal region of CD38 inhibit primary HIV-1 and HIV-2 isolates from different subtypes and with different coreceptor usage, with no apparent cell toxicity at the concentrations inhibiting HIV. These effects encourage further studies aimed at determining their potential therapeutic use or application as "topic microbicides" for limiting sexual transmission of HIV.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0512fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0512fje 
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) was sometimes added at the NH2 terminus to detect expression with an anti-myc mAb. B) Inhibition of p24 production on day 5 after infection in de novo HIV-1IIIB-infected transfectants expressing full-length CD38 or truncated forms including a soluble (s) form consisting solely of the extracellular domain (sCD3845–300). Values are representative of 3 experiments. Data using MT-2.M cells are shown as a negative control. Values using a full-length tagCD38 show that the tag per se does not affect the inhibitory effects. C) Staining for surface CD38, CD4, and CXCR-4 in MT-2-cell transfectants. The panel shows the median fluorescence intensity (MeFI) mathematically converted to linear fluorescence intensity and subtracted of the control antibody staining. The inhibitory effects on HIV-1 replication cannot be attributed to discrepancies in expression of CD4 and of CXCR-4, since this was similar in all cell lines.
