Difference between revisions of "Part:BBa K2748000"

Latest revision as of 02:29, 18 October 2018

U24 protein from human herpesvirus 6A

U24 protein from human herpesvirus 6A is a C-terminal membrane anchored protein that can downregulate T cell receptors via endosomal recycling inhibition.

Usage and Biology

U24 is a small (87aa) tail-anchored protein (Sullivan and Coscoy, 2010) that can downregulate TCR/CD3 complex from the cell surface by exclusion of CD3 from Rab11-containing recycling endosomes and thus inhibiting TCR complex recycling back to the surface (Sullivan and Coscoy, 2008). While it was demonstrated later that U24 also downregulate transferrin (Sullivan and Coscoy, 2010), its action is relatively specific, without affecting the surface level of ICAM-1, MHC class I, ULBP1, ULBP2, CD4 and CD8 (Sullivan and Coscoy, 2008). Since U24 does not colocalized with CD3, it is believed that the downregulation does not rely on the interaction of U24 with CD3 but instead results from interaction of U24 and the endosomal recycling machinery (Sullivan and Coscoy, 2010). In addition, unlike proteins from other herpesviruses that downregulate TCRs or B cell receptors (BCRs), U24 does not activate lymphocyte signaling pathways (Sullivan and Coscoy, 2008). Furthermore, it was demonstrated that U24 can impair T cell activation by antigen presenting cells (Sullivan and Coscoy, 2008).

Design Considerations

The nucleotide sequence of U24 coding sequence was retrieved from NCBI nucleotide database and synthesized by IGE Biotechnology LTD. Biobrick prefix and suffix was added by PCR using the following primers,

U24-prefix: 5’ cggaattcgcggccgcttctagATGGATCCCCCTCGGACGC 3’

U24-suffix: 5’ AActgcagcggccgctactagtaTCATCGCCTTTGACGATTCACAT 3’

and ligated onto the pSB1C3 plasmid backbone obtained from digestion of pSB1C3-lacI-RFP(Part:BBa_J04450). The sequence was comfirmed by Sanger sequencing by IGE Biotechnology LTD using VF2 as the forward primer.

Note that this part comes with an stop codon at the end of the sequence.

Also note that when performing western blots, bands are detected at around 20kDa instead of 10kDa.

A proline 7-9 alanine substitution mutant can be used as negative control for it is non-functional (Sullivan and Coscoy, 2010)

Characterization

For more details, please check out our [http://2018.igem.org/Team:SYSU-CHINA#/Demonstrate result page]!

In iGEM 2018, SYSU-CHINA attempted to develop a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of Human Herpesvirus 6. Since it is the major part of our project, we conducted extensive research on U24 and obtained valuable results..

In order to determine the optimal concentration of dox for induction, we collected and analyzed fluorescence data and performed western blot analysis.

Interestingly, although the predicted molecular weight of U24 protein is approximately 10kDa, two bands were detected at 20kDa, consistent with previous research (Sullivan and Coscoy, 2010). This suggested that U24 undergoes extensive post-translational modifications. However, the types and functions of these modifications remain unknown.

Figure 2: Fluorescence and western blot analysis of U24 expression under different concentration of doxycycline

In order to determine the expression time course after adding dox, equal amount of HEK293T cells were seeded in a 6-well plate and transfected with ptetON-GFP-T2A-U24. Dox was added at the same time in all wells and equal number of cells in each well were harvested at the indicated time for western blot analysis. The result was analyzed with ImageJ and the amount of protein of interest was normalized using β-actin as internal controls.

Figure 3: Time course of U24 expression

In order to determine the degradation rate of U24, equal amount of HEK293T cells were seeded in a 6-well plate and transfected with ptetON-GFP-T2A-U24. Dox was added at the same time in all wells and cycloheximide (CHX), a protein synthesis inhibitor, was added to each well 24h post transfection. Equal number of cells in each well were harvested at indicated time pointe for western blot analysis. The result was analyzed with ImageJ and the amount of protein of interest was normalized using β-actin as internal controls.

Figure 4: Degradation curve of U24

To verify the ability of U24 to downregulate TCRs, and its potential use in TCR T therapy, we transduced Jurkat cells with lentiviral vector ptetON-GFP-T2A-U24 and ptetON-GFP-T2A-U24(P7-9A) as control. Dox was added 48h prior to flow cytometry. The cells were stained with PE-conjugated anti-CD3 monoclonal antibody (UCHT-1, LifeTechnologies, Thermo Scientific) and subject to flow cytometry. The results indicated U24 can downregulate surface CD3.

Figure 5: U24 downregulates surface CD3 level in Jurkat cells

To determine effect of U24 on CD3 is reversible,we transduced Jurkat cells with lentiviral vector ptetON-GFP-T2A-U24 and ptetON-GFP-T2A-U24(P7-9A) as control. Dox was added 48h prior to flow cytometry. Dox was removed from 1 well of ptetON-GFP-T2A-U24 transduced cells and continued in the other well. The cells were stained with PE-conjugated anti-CD3 monoclonal antibodyand subject to flow cytometry.The results indicated that the effect of U24 on surface CD3 is reversible.

Figure 6: Reversible effect of U24 on surface CD3 level in Jurkat cells

We cotransduced Jurkat cells with lentiviral vector pEF1a-CAR and lentiviral vector ptetON-GFP-T2A-U24 or ptetON-GFP-T2A-U24(P7-9A) as control. Dox was added 48h prior to flow cytometry. The cells were stained with biotin-protein L and PE-streptavidin and subject to flow cytometry. However, only modest, if any, effect on surface CAR level was observed.

Figure 7: Modest effect of U24 on surface chimeric antigen receptor level in Jurkat cells

To test whether U24 has cytotoxic effect, we transfected HEK293T cells with ptetON-GFP-T2A-U24 and Dox was added after transfection. The viable cell count and dead cell count were determined using typhan blue staining. As a control, we transfected HEK293T cells with ptetON-GFP-T2A-U24(P7-9A) or Lipofectamine 3000 only with Dox added after transfection, to rule out toxicity induced by transfection, Dox or the expression vector itself. Three parallel experiments were conducted.

Growth curves were drawn for cells transfected with each vector. No significant different was seen in the viable cells count and dead cells count at each time point (Student’s T test, double tailed, P>0.05), indicating that U24 has little, if any, toxic effect on cells

Figure 8: U24 has little cytotoxicity, determined by growth curve

References

Sullivan, B.M., and Coscoy, L. (2008). Downregulation of the T-cell receptor complex and impairment of T-cell activation by human herpesvirus 6 u24 protein. Journal of virology 82, 602-608.

Sullivan, B.M., and Coscoy, L. (2010). The U24 protein from human herpesvirus 6 and 7 affects endocytic recycling. Journal of virology 84, 1265-1275.

Sequence and Features