HIV-1 Tat Protein

Salome Shubitidze '22 and Chris Goodall '22

Contents:

I. Introduction



The HIV-1 Tat protein is required to initiate transcription of the HIV genome. It is one of the first proteins expressed after an individual has been infected with HIV. Tat is a transactivator of transcription, meaning the protein activates RNA polymerase II—in this case by hijacking the host’s elongation machinery. Tat does this by binding to the HIV TAR element, which is a trans-activation response element that forms at the 5’ end of viral transcripts. This binding recruits an RNAP II general transcription factor called P-TEFb.



P-TEFb consists of subunits cyclin T1 and CDK9.



Once P-TEFb has been recruited, Tat makes contact with cyclin T1 and the T-loop of the CDK9 subunit in order to induce a conformational change in P-TEFb. This change results in phosphorylation of substrates in the RNAP II complex leading to the activation of elongation.

II. General Structure


Tat is a 14-Kda protein, encoded by two exons. Its transcriptional activation domain is made up of a cysteine rich region and a hydrophobic core motif. Tat engages with the cyclin T1 subunit of P-TEFb through zinc-mediated interactions, as well as van der waals and water-mediated interactions. The increased number of hydrogen bonds formed between Tat and cyclin T1 suggest that cyclin T1 is largely responsible for Tat’s structure, enabling the protein to complement the surface of the P-TEFb complex. Tat employs direct and indirect hydrogen bonds with the “T-loop” of the CDK9 subunit. Specifically a proline rich/acidic beta turn sequence section of Tat facilitates the interactions with CDK9.

III. TAT interactions with P-TEFb's cyclin T1 subunit

Of the 2 exons encoded in the viral message for the Tat protein, exon 1 is primarily involved with the binding of cyclin T1. Eighty-eight pecent of Tat’s interactions with the human p-TEFb involves the binding of Tat to the cyclin T1 subunit .

In addition to interacting with the 310 alpha helix, ZnF1 is also associated with the second of two alpha helices found in the complexed structure of Tat with P-TEFb. This interaction involves the binding of Zinc with Cys22, His33, Cys34, Cys37 in the Tat protein.

While not associated with a secondary alpha helix, Zn2 binds Cys261 of the Cyclin subunit along with Cys25, Cys27, Cys30 of the Tat protein.

Aside from zinc mediated interactions, HIV-1 Tat protein also engages cyclin T1 in a handful of hydrogen and Van der Waals bonding interactions. Tat forms 5 direct hydrogen bonds with cyclin: V54 Q97 P249 N250 interact with S16, W11, L43, A42, and L43 again respectively . And three indirect ones: Tat H13, P10, S46 interact with cyclin T1 L57, S55, Q40 via O286 O323 O324 respectively . The notably larger number of hydrogen bonds with cyclin T1 than intramolecular hydrogen bonding suggests that the Tat protein largely assumes its structure from its interactions with P-TEFb.

The Tat protein's Van der Waals bonding interactions with the cyclin T1 subunit involve two main tropes. First, a U-shaped a proline rich region near the carboxyl terminus of the tat protein interacts within a divot of the cyclin subunit found between cyclin repeats . Second, a cysteine rich region, central to the Tat protein, interacts with a secondary gap also found between cyclin repeats of the cyclin T1 subunit .

IV. CDK9 Subunit binding interactions

The remaining twelve percent of binding interactions mediated between the HIV-1 Tat protein and P-TEFb protein occur with the CDK9 subunit of the P-TEFb protein .

Although the HIV-1 Tat protein shares a functional similarity to that of other proteins complexed with the human P-TEFb protein, its interaction with the CDK9 subunit of the P-TEFb protein differs. Take for example the flavopiridol P-TEFb complex, a system critical in CDK9 regulation. Although both the flavopiridol and Tat complexes regulate CDK9 activity through phosphorylation, The HIV-1 Tat protein engages in a significantly reduced proportion of interactions with the CDK9 subunit of human P-TEFb.

Notably, the HIV-1 Tat protein’s binding interactions with the CDK9 subunit of human P-TEFb solely involve the “ ” of the CDK9 subunit.

A proline rich/acidic beta turn associated with the limited intramolecular hydrogen bonding interactions of the Tat protein facilitates connections between Tat and the CDK9 subunit. Notably, glutamate 9 of the Tat protein forms additional direct and indirect hydrogen bonds with the T-loop of the CDK9 subunit. Lys 144 of CDK9 forms two with this residue. Ser 175 Phe 174, Ala 173, and Lys 144 all form with this same glutamate.

VI. References

Bagashev, Asen, and Bassel E Sawaya. “Roles and functions of HIV-1 Tat protein in the CNS: an overview.” Virology journal vol. 10 358. 21 Dec. 2013, doi:10.1186/1743-422X-10-358

Gu J, Babayeva ND, Suwa Y, Baranovskiy AG, Price DH, Tahirov TH. Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4. Cell Cycle. 2014;13(11):1788-97. doi: 10.4161/cc.28756. Epub 2014 Apr 11. PMID: 24727379; PMCID: PMC4111725

Tahirov TH, Babayeva ND, Varzavand K, Cooper JJ, Sedore SC, Price DH. Crystal structure of HIV-1 Tat complexed with human P-TEFb. Nature. 2010 Jun 10;465(7299):747-51. doi: 10.1038/nature09131. PMID: 20535204; PMCID: PMC2885016.

Rice AP. The HIV-1 Tat Protein: Mechanism of Action and Target for HIV-1 Cure Strategies. Curr Pharm Des. 2017;23(28):4098-4102. doi: 10.2174/1381612823666170704130635. PMID: 28677507; PMCID: PMC5700838.

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