Page 23 - O. Microbiology

Anti-viral Effects of HEXIM1 Derivative Chimeric Proteins on HIV-1 production
Hae-In Kim and Ji-Chang You

National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic
University of Korea, Seoul.

BACKGROUND
Viral trans-activator Tat can bring P-TEFb to the HIV promoter to induce HIV expression (1, 2). P-TEFb is comprised of cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9) (3, 4), and it is
required for transcription elongation for both HIV and host gene expression. 7SK small nuclear ribonucleoprotein complex (7SK snRNP) consisting of 7SK RNA and proteins HEXIM1, LARP7,
and MePCE regulate P-TEFb activity (5, 6). One of the 7SK snRNP components, HEXIM1, binds to the 7SK snRNA through RNA binding arginine-rich motif (ARM, residues 150–162), and to P-
TEFb through its CycT1 binding domain (TBD, residues 250–359) and its central inhibitory domain (ID, residues 200–211). The inhibitory domain which includes a PYNT motif (202Pro-203Tyr-
204Asn-205Thr) inhibits the cyclin-dependent kinase activity of P-TEFb by masking CDK9’s substrate-binding site (7, 8). TBD domain synergistically inhibits CDK9 activity with ID (9). The central
ARM region of the viral trans-activator Tat (residues 51–57) binds to 7SK RNA and escapes P-TEFb from inhibition by HEXIM1 (10). Tat's activation domain (AD) binds to CycT1, which can bind
to the bulge region of TAR (11). The Tat-TAR recognition motif of CycT1 binds to the central loop of TAR, allowing P-TEFb to be recruited into the RNAPII early elongation complex arrested at
the HIV transcription start site. CDK9 then stimulates transcriptional elongation to express viral genes from HIV promoters (12, 13). Recently, it has been shown that chimeric protein HEXIM1-Tat
(HT) which includes critical functional domains of Tat and HEXIM1 inhibits P-TEFb and binds RNA by combining the P-TEFb binding N-terminal domain of Tat to the acidic and/or central basic
domains of HEXIM1 (14). In this study, we combined HT with ACP which is a novel cell-penetrating peptide identified in our laboratory and verified whether anti-viral effects are enhanced. These
chimeric proteins could be proposed as a new candidate for anti-HIV-1 therapy.
METHODS
We fused ACP with HT and verified each anti-viral effect. pNL43-eGFP co-transfected with pACP-HT, pHT-ACP or
pHT into 293T cell. 24hrs after transfection, the cell lysate was used for Western blot (WB). The supernatant was
collected and used for ELISA, infection of MT4 cell and detection of GFP-positive infected cells by FACS analysis.
RESULTS

Figure 1. Structure of HEXIM1 derivative
chimeric proteins
The functional domains used from HEXIM1
and Tat include a HEXIM1 Arginine Rich
Motif (ARM, black box, residues 150–162)
that binds RNA, a HEXIM1 inhibitory
domain (ID, light grey box, residues 200–
211) that inhibits CDK9 through a PYNT
motif, and Tat transactivation domain (AD,
dark grey box, residues 2–48) that binds to
P-TEFb. The ACP (Blue box, residues 1-
55) which is a novel cell-penetrating
peptide identified in our laboratory inserted
in front or rear of HT.

Figure 2. Effect of HEXIM1 derivative chimeric proteins on virus
production in 293T cell.
293T cells co-transfected control plasmid or Myc-HEXIM1 or Myc-
tagged HEXIM1 derivative chimeric proteins or Flag-ACP with pNL4-
3eGFP and RFP. After 24hrs transfection, cells were lysed by RIPA
Figure 3. Chimeric protein ACP_HT with proteinase inhibitor cocktail and viral supernatant were
restrained infectivity of the progeny virus. harvested. (A) Western blot results of cell lysate. Upper panel:
MT4 cells were infected with viral supernatant Expression of ACP, HEXIM1 and its derivates. Lower panel:
as same p24 particles quantified by ELISA. (A) Expression level of viral proteins, RFP transfection control, and
Fluorescence microscope image. (B) Detection GAPDH house keeping gene. (B) Virus titer of viral supernatant
of GFP-positive infected cells by FACS analysis. quantified by p24 ELISA and normalized by RFP level. Statistical
Statistical significance was performed using significance was performed using one-way ANOVA. *P < 0.05, **P <
one-way ANOVA (*P < 0.05). 0.01, ***P < 0.0001 and n.s indicate non-significant.

ACP-HT or HT-ACP inhibited viral protein expression more effectively than HT. ACP-HT and HT-ACP depleted the p24 level of
the supernatant up to 95.7% and 81.3% respectively, while HT reduced p24 level only up to 52.2%. Also, the expression of ACP-
HT or HT-ACP induced 65.3% and 41.8% decrease of GFP positive cells respectively. The results suggested that ACP-HT or
HT-ACP could inhibit virus production and restrain the infectivity of the progeny virus.

CONCLUSION REFERENCES

Both fusion proteins ACP-HT and HT- 1. Cary DC, Fujinaga K, Peterlin BM. Molecular mechanisms of HIV latency. The Journal of clinical investigation. 2016
2. Kao SY, Calman AF, Luciw PA, Peterlin BM. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature. 1987
ACP outperform the anti-viral effect of 3. Garber ME, Wei P, KewalRamani VN, Mayall TP, Herrmann CH, Rice AP, et al. The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue
that is not conserved in the murine CycT1 protein. Genes & development. 1998
HT. These novel chimeric proteins 4. Wei P, Garber ME, Fang SM, Fischer WH, Jones KA. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to
TAR RNA. Cell. 1998
efficiently inhibit HIV-1 replication and 5. Fujinaga K, Luo Z, Peterlin BM. Genetic analysis of the structure and function of 7SK small nuclear ribonucleoprotein (snRNP) in cells. The Journal of biological chemistry. 2014
denote new candidates for anti-HIV-1 6. Peterlin BM, Brogie JE, Price DH. 7SK snRNA: a noncoding RNA that plays a major role in regulating eukaryotic transcription. Wiley interdisciplinary reviews RNA. 2012
7. Yik JH, Chen R, Pezda AC, Samford CS, Zhou Q. A human immunodeficiency virus type 1 Tat-like arginine-rich RNA-binding domain is essential for HEXIM1 to inhibit RNA
therapy. polymerase II transcription through 7SK snRNA-mediated inactivation of P-TEFb. Mol Cell Biol. 2004
8. Kobbi L, Demey-Thomas E, Braye F, Proux F, Kolesnikova O, Vinh J, et al. An evolutionary conservedHexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb. Proc Natl
Acad Sci U S A. 2016
9. Czudnochowski N, Bosken CA, Geyer M. Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition. Nat Commun. 2012
10. Barboric M, Yik JH, Czudnochowski N, Yang Z, Chen R, Contreras X, et al. Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription. Nucleic Acids
Contact information Res. 2007
11. Fujinaga K, Taube R, Wimmer J, Cujec TP, Peterlin BM. Interactions between human cyclin T, Tat, and the transactivation response element (TAR) are disrupted by a cysteine to
tyrosine substitution found in mouse cyclin T. Proceedings of the National Academy of Sciences of the United States of America. 1999
12. Mousseau G, Mediouni S, Valente ST. Targeting HIV transcription: the quest for a functional cure. Current topics in microbiology and immunology. 2015
haein2809@catholic.ac.kr 13. D’Orso I, Frankel AD. RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation. Nature structural & molecular biology. 2010
14. Leoz M, Kukanja P, Luo Z, Huang F, Cary DC, Peterlin BM, Fujinaga K. HEXIM1-Tat chimera inhibits HIV-1 replication. PLoS pathogens. 2018