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HIV-1 reverse transcriptase complexed with FAB-28 monoclonal antibody fragments

Oliver Benes '03, Rebecca Burke '03,
Rebecca Palacios '03


I. Introduction

    The retrovirus HIV, and its subsequent progression to AIDS, is a rapidly growing worldwide epidemic.  HIV-1 reverse transcriptase is one of the key players in the mechanism of infection by this retrovirus.  The HIV-1 reverse transcriptase enzyme is responsible for copying a single-stranded viral RNA genome into double-stranded DNA (Sarafianos et al, 2001).  The newly created DNA can then be incorporated into the host genome; the host is mainly the human in the case of HIV.  The HIV-1 reverse transcriptase enzyme contains two main domains:  a DNA polymerase domain and a ribonuclease H (RNase H) domain.  The DNA polymerase is able to copy either an RNA or DNA template.  The function of the RNase H domain is to cleave and degrade the template RNA after DNA synthesis so that the newly made DNA can generate a second DNA strand.  The RNase H domain is also responsible for the integration of the duplex DNA into the host cell chromosome.
    Here we describe the crystal structure of HIV-1 reverse transcriptase complexed with two FAB-28 monoclonal antibody fragments and an DNA:RNA hybrid.  The FAB-28 heavy chain and FAB-28 light chain are not actual components of the reverse transcriptase enzyme.  The antibody fragments are complexed with the enzyme during the crystallization procedure in order to stabilize the enzyme structure.  This allows for a higher resolution crystallographic structure (Sarafianos et al, 2001).

II. General Structure

HIV-1 reverse transcriptase is a dimer composed of two distinct, but related chains.  The first of these two chains is a 66-kD subunit (p66) .  The other chain is a 51kD subunit (p51), which is related to p66.  However, the C-terminal RNase domain present in p66 is absent in p51 .  These two domains are responsible for the binding of the DNA:RNA substrate (Sarafianos et al, 2001; Kohlstaedt et al, 1992).

III. p66 and p51 subunits

The p66 subunit is the larger of the two subunits within HIV-1 reverse transcriptase .  This subunit contains the finger, palm, thumb, and connection subdomains as well as the RNase H subdomain .  The p51 subunit is the smaller of the two subunits in HIV-1 reverse transcriptase  .  This subunit also contains the finger, palm, thumb and connection subdomains  (Kohlstaedt et al, 1992).  The p51 subunit is a product of the same gene as the p66 subunit, however, the RNase H domain is absent in the p51 subunit as a result of proteolytic cleavage.  Click subunits to see a detailed schematic of the two subunits interacting with DNA (Flint, 2000). 

IV. DNA/RNA binding to Reverse Transcriptase

There are multiple interactions between the 2'-OH groups of the RNA template and the reverse transcriptase enzyme.  Residues serine 280 and arginine 284 of helix I in the p66 thumb are involved in the RNA-RT interactions .  In addition, residues glutamate 89 and glutamine 91 of the template grip in the p66 palm are involved in the RNA-RT interactions .  The p51 subunit also plays a role in the interactions between the RNA:DNA duplex and reverse transcriptase.  In particular residues lysine 395, glutamate 396, lysine 22, and lysine 390 of the p51 subunit interact with the DNA:RNA duplex   There are four regions in the nucleic acid that have characteristic geometrical conformations.  Structural analysis reveals that none of the four regions displays canonical A- or B-type geometry.  Region I assumes a conformation more closely related to that of A-form geometry.  .  Regions II, III, and IV exhibit a conformation that is intermediate between A-and B-form .  This conformation is known as the H-form, and is characterized by angles of the base pairs with respect to the helical axis, the dislocation of the base pairs from the helix axis, and the helical rise (Sarafianos et al, 2001).

V. The Polypurine Tract

The RNA polypurine tract (PPT) acts as the primer for (+) strand synthesis by resisting RNase H cleavage.  It is located at the 5' end of U3 in the RNA strand .  The left end of the upstream long terminal repeat (LTR) of the polypurine tract and the downstream LTR form the substrate on which the viral integrase enzyme acts.  The viral integrase enzyme inserts its linear viral DNA into the host genome at this site.  A key feature of the PPT are the A-tracts , which are stretches of four or more consecutive adenines.  Characteristics of the A-tracts include their extreme resistance to reconstitution around nucleosome cores, their straight chain conformation, and their narrow minor groove and  large propeller twist. During DNA binding, the integration host factor protein recognizes the A-tracts.  In particular, it recognizes the phosphates of the narrow minor groove.  In this way, the integration host factor recognizes the adenine regions based on conformation rather than on base-specific contacts (Sarafianos et al, 2001).

VI.  Unzipping of the PPT

In one section of the PPT, a departure from Watson-Crick base pairing is observed.  This particular region is referred to as the "unzipping of the PPT."  Features of this region include the melting of the first two base pairs of the 5'- end of the PPT, which results in an unpaired template base, Adenine 15 .  Following this unpaired nucleotide is a frame-shifted A-T base pair, Tempate A-16 and Primer T-15, and a GT mismatch, Template G-17 and Primer T-16 .  The nucleotide primer following this is the unpaired C-17, compensating for the unpaired nucleotide on the template strand .  This marks the return to Watson-Crick base pairing (Sarafianos et al, 2001).

VII. FAB-28 Monoclonal Antibody Fragments

The HIV-1 Reverse Transcriptase is complexed with two monoclonal antibody fragments, the FAB-28 light chain and the FAB-28 heavy chain .  The light chain contains 214 amino acids whereas the heavy chain is composed of 220 amino acids.   The heavy chain interacts with the p55 subunit of HIV-1 reverse transcriptase    but has no interations with the p66 subunit or the RNA-DNA hybrid.  These fragments do not play a role in the function of the HIV-1 reverse transcriptase and are merely present to stabilize the structure for crystallization.  A stablized structure allows for better resolution in the crystallization procedure.  It has been suggested that the fragments serve as a molecular clamp to constrain the conformational freedom of the enzyme (Sarafianos et al, 2001; Ding et al, 1998; Jacobo-Molina et al, 1991).

VIII. References

PDB from Molecules R Us file 1hys

Ding, Jianping, Kalyan Das, Yu Hsiou, Stefan G. Sarafianos, Arthur D. Clark, Jr., Alfredo Jacobo-Molina, Chris Tantillo, Stephen H. Hughes, and Edward Arnold. 1998. Strucutre and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody fab fragment at 2.8 A resolution. Implications of the Polymerase Active Site Region. 1095-1111.

Flint, S.J. Principles of Virology. 2000. ASM  Press.

Jacobo-Molina, Alfredo, Arthur D. Clark Jr., Roger L. Williams, Raymond G. Nann, Partick Clark, Andrea L. Ferris, Stephen H. Hughes, and Edward Arnold. 1991. Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffact x-rays to 3.5-A resolution. Proc. Natl. Acad. Sci. 88:10895-10899.

Kohlstaedt, L.A., J. Wang, J.M. Friedman, P.A. Rice, and T.A. Steitz. 1992. Crystal strucutre at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 256: 1783-1790.

Safarianos, Stefan G., Kaylan Das, Chris Tantillo, Arthur D. Clark, Jr., Jianping Ding, Jeanette M. Whitcomb, Paul L. Boyer, Stephen H. Hughes, and Edward Arnold. 2001. Crystal strucutre of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA. The EMBO Journal 20:1449-1461.