HIV-1 Reverse Transcriptase
(HIV-1 RT):
Structure of a Protein-DNA Complex


by Justin Miyamoto
© Justin Miyamoto 2005

Introduction:

The total number of people living with the human immunodeficiency virus (HIV) rose in 2004 to reach its highest level ever: an estimated 39.4 million. In 2004 alone, approximately 4.9 million people acquired HIV, while the global AIDS epidemic killed 3.1 million people in the past year. HIV has been identified as the etiologic agent causing AIDS, the world's most deadly infectious disease according to the World Health Organization. Since its discovery in 1981 by Michael Gottlieb, at the time a post-doctoral fellow in the UCLA School of Medicine, substantial amounts of research have been directed towards understanding HIV. HIV is a member of the retrovirus family. An essential element in the life cycle of this family of viruses is the requirement for integrating a copy of its genetic material (genome) into the human host cell genome before virus replication can occur. HIV RT is responsible for producing the DNA copy of the viral RNA genome that will be integrated into the human DNA. In this exhibition, HIV-1 RT is complexed to a double-stranded DNA.


Structure:

HIV-1 RT is composed of an extended, asymmetric heterodimer of two related chains, a 51-kD subunit (p51) of 440 amino acids and a 66-kD subunit (p66) of 560 amino acids. The p66 subunit shows an overall structural similarity to the polymerase domain of E. Coli DNA Pol I and like other polymerases, is analogous to a right hand. The p66 subunit folds into two domains, a polymerase and an RNase H domain. The polymerase domain is divided into four subdomains: the fingers, the palm, the thumb, and the connection.

Although the p51 subunit consists of the same domains as p66 (save RNase H), they are oriented differently. The large subunit adops an open, 'active' conformation that readily accomodates double-stranded DNA for catalysis. In contrast, the cataytically significant residues of the small subunit are largely inaccessible in p51. Mutagenesis studies have shown that the small subunit plays a primarily structural role. Additionally, it has been proposed that the p51 subunit forms part of the binding site for the initiating tRNA.

Retroviruses like HIV-RT replicate using three main activities:
1) 5'-->3' RNA directed DNA polymerization
(synthesizes DNA on the RNA template)
2) 5'-->3' DNA directed DNA polymerization
(synthesizes DNA on the DNA template)
3) exoribonuclease (degrades RNA in RNA:DNA hybrid)


Polymerase Site:

The first two of the aforementioned HIV-RT activities are accomplished by the polymerase site. This site resides mainly in two domains of the p66 subunit: the thumb and the palm. The interactions which stabilize HIV-RT to the ssRNA --activity #1-- and then the ssDNA template --activity #2-- (which is pictured here) are highly important for the processivity of the polymerase. Therefore, within these subunits there are a multitude of ammino acids which stabilize the DNA primer strand via van der Waals interactions and six particular amino acids which hydrogen bond: Asp185, Asp186, Met230, Lys263, Glu258, and Asn255. Lets look at these interactions in two sets. First focus on Asp185, Asp186, and Met230, three residues which stabilize the 3' end of the growing primer strand. Two hydrogen bonds are donated to the alpha phosphate while two are donated to the 3'OH. Additionally, six other van der Waals interactions occur between these residues and the DNA template. Second, examine the three residues Lys263, Glu258, and Asn255 Lys263, Gln 258, and Asn255 all donate a hydrogen bond to the phosphate backbone while both Lys263 and Gln258 donate one their respective ribose moieties. In addition, Gln258 donates an H-bond to the 5'alkyl group These three residues also provide four sets of van der Waals forces to stabilize the polymerase site.


RNAse H Domain:

Reload Protein
Again, remember that the goal of HIV-1 RT is to convert the single-stranded RNA gemone into double-stranded DNA. This task is accomplished through the collaboration of two enzymatic activites of RT: DNA polymerase and RNase H. The RNase H subunit is essential for the conversion of RNA into dsDNA as it degrades RNA present in the RNA-DNA duplex. This clears the path for polymerase to lay down a complementary DNA sequence onto the freshly cleared DNA template strand. Since both polymerase and RNAse H simultaneously contact the nucleic acid substrate and contribute to its binding, RNase H domain, plays a highly influential role in the efficiency of initiation in DNA synthesis as well.

It is important then, to look at the residues which ensure proper positioning of the DNA-RNA template within HIV-1 RT. Many of the RT contacts with the nucleic acid involve the sugar phosphate backbone, consistent with the fact that RT can copy a wide variety of different templates. Five of the amino acids in the p66 subunit which compose the RNase H Primer Grip, hydrogen bond directly with the oxygens of the alpha phosphate within the DNA template backbone. These are: Ala360, His361, Tyr501, Lys476, and Thr473. Additionally, Gly359 and Ile505 add van der Waals interactions to stabilize the phosphate backbone while Glu 475 and Thr473 hydrogen bond with the ribose moiety of a dNTP.


dNTP Binding Site:

1.Reload Protein (click twice)
2.Center and Color Protein

After HIV-RT has secured onto the DNA (or RNA) template, it faces the task of adding base specific nucleotides (dNTPs) to produce a new daughter strand. In addition to the stabilizing features of the polymerase site, there are also RT-template or RT-metal interactions which stabilize the dNTP binding site and promote the polymerization reaction. A majority of these stabilizing interactions are found within the palm domain, contacting the minor groove. First, distinguish the Major Groove from the Minor Groove. Follow the minor groove towards HIV-RT and visualize the palm domain of HIV-1 RT contacting the backside of the DNA template. Examine residues Ile 94, Pro 157, and Ile184 making van der Waals contacts with the DNA minor groove. Take note of Tyr 183 which hydrogen bonds to the minor groove.

Within the palm domain there are also two conserved Mg ions which assist polymerization of the incoming dNTP. To explain their functions, we will arbitrarily distinguish them as Metal A and Metal B. The unesterfied oxygens of the beta and gamma phosphhates, as well as the esterified oxygen bridging the alpha and beta phosphates are coordinated by Metal A By stabilizing the negative charge on the pyrophosphate group, Metal A makes it a better leaving group. Metal B also stabilizes a negative charge, this on the 3' oxygen of the growing daughter strand. Since this lowers the pKa of the 3' OH, Metal B makes the oxygen a better nucleophile. During polymerization, both Metal A and Metal B bind tightly and stabilize the pentavalent transition state on the alpha phosphate. Together, this proposed 2-Metal Ion Mechanism provides cooperative stabilization of the incoming dNTP and is the basis of the polymerization reaction.

In addition to the 2-Metal Ion Mechanism, there stands a multitude of amino acids which coordinate the triphosphate moiety. For instance, the sidechain of Arg 72, which lies flat against the dNTP base, donates hydrogen bonds to the alpha-phosphate and esterified oygen bridging the alpha and beta phosphates. The NH group of Lys 65 also donates hydrogen bonds to the gamma phosphate at both the esterified, and non-esterified oxygens. The main-chains of Asp 113 and Ala 114, donate hydrogen bonds to the beta and gamma phophates, respectively, while the main chain-NH of Tyr 115 donates a hydrogen bond to the 3'OH of the incoming dNTP. Thought here we have focused on a sampling of the amino acids which comprise the dNTP binding pocket, each play a siginificant role in undertsanding structure-activity relations.


Conclusions:

As a member of the retrovirus family, HIV-1 RT displays two significant activities: polymerization and exoribonuclease. Each of these activities is accomplished thanks to the high binding specificity of HIV-1 RT to its template strand. Note that a majority of the protein-template interactions are coordinated with either the phosphate backbone or sugar moiety. Thus, HIV-1 RT is specific to (DNA/RNA) shape, but not specific base sequence--implying how RT can use both DNA and RNA as a template. Additionally, like most other polymerase enzymes, HIV-1 RT utilizes a two metal mechanism to catalyze the polymerase reaction.

For more information on milestones in the fight against HIV/AIDS see FDA approved tests and treatments. Learn more about HIV-1 RT structure from the National Cancer Institute's HIV Drug Resistance Program


References:

  1. Guiterrez-Rivas, M. et al. Mutational Analysis of Phe160 within the "Palm" Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcripatse. Journal of Molecular Bio. 290. 615-625 (1999).
  2. Jaeger, J. et al. The structure of HIV-1 reverse transcripatse complexed with an RNA pseudoknot inhibitor. The European Molecular Biology Organization Journal. 17. 4535-4542 (1998).
  3. Fleming, P. et al. HIV prevalence in the United States, 2000 [Abstract 11]. Presented at the Ninth Conference on Retroviruses and Opportunistic Infections. Seattle, WA. February 24-28, 2002.
  4. Huang, H. et al. Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistance. Science. 282. 1669-1675 (1998).
  5. Julias, J. et al. Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo. Proceedings of the National Academy of Science. 99. 9515-9520 (2002).
  • PDB Code = 1J5O
  • PDB Code = 1RTD