Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases

doi:10.1006/jmbi.1998.1739

Journal of Molecular Biology

Volume 278, Issue 4, 15 May 1998, Pages 757-765

https://doi.org/10.1006/jmbi.1998.1739 Get rights and content

Abstract

The reverse transcriptase (RT) of HIV-1 and feline immunodeficiency virus (FIV) consist of two subunits of 51 kDa (p51) and 66 kDa (p66). In order to elucidate the role of p51 in the heterodimer, chimeric HIV-1/FIV RT heterodimers were constructed and characterized. The FIV RT p51/HIV-1 RT p66 chimera showed a 2.5-fold higher RNase H activity than the natural HIV-1 RT, a 50% lower strand displacement DNA synthesis activity and resistance to the two RT inhibitors 3′-azido-3′-deoxythymidine triphosphate (AZTTP) and Nevirapine. The HIV-1 RT p51/FIV RT p66 chimera on the other hand had very similar properties to the natural FIV RT. The differences observed upon exchange of the p51 subunits suggest that the three-dimensional structure of the p51 subunit in the RT heterodimers is not completely conserved between the human and the feline lentiviruses. Finally, our data suggest an important role for the p51 subunit in maintaining the optimal structural integrity of the RT heterodimer. The different effects of the small subunits on the sensitivity to known RT inhibitors might be of importance in the development of novel drugs against HIV-1 RT.

Introduction

The human immunodeficiency virus type 1 (HIV-1) and the feline immunodeficiency virus (FIV) reverse transcriptases (RT) are responsible for replication of the lentiviral genomic single-stranded RNA to double-stranded DNA (for a recent review, see Hottiger & Hübscher, 1996). Both RTs consist of two polypeptides with common N termini, a 66 kDa subunit (p66), and a 51 kDa subunit (p51); both subunits are present in equimolar amounts and form a heterodimer. p51 is generated by cleavage of the RNase H domain (p15) at the C terminus of p66 by the virus-encoded protease. Sequence comparisons between HIV-1 RT and FIV RT revealed 48% identity and 67% similarity at the amino acid level.

The HIV-1 and FIV RT heterodimers have DNA polymerase and RNase H activities. Although both subunits are inactive as monomers, homodimers are able to perform RNA-dependent DNA synthesis Restle et al 1990, Hottiger et al 1994, Amacker et al 1995. In the RT heterodimer, the DNA polymerase activity resides exclusively in the p66 subunit Starnes et al 1988, Cheng et al 1991, Le Grice et al 1991, Boyer et al 1992. Very little is known about the function of p51 in the heterodimer. A stimulation of the strand displacement DNA synthesis activity of p66 by p51 was found for both HIV-1 (Hottiger et al., 1994) and FIV (Amacker et al., 1995) RT. Other studies suggested a role for p51 in increasing the processivity of p66 (Huang et al., 1992) and an involvement in tRNA binding (Jacques et al., 1994).

The structure of the HIV-1 RT heterodimer is highly asymmetric as the polymerase domains of the p66 and p51 subunits are arranged differently (Kohlstaedt et al., 1992). In order to gain further insight into the role of p51, stable and functionally active HIV-1/FIV RT heterodimers were reconstituted. They were characterized for RNA- and DNA-dependent DNA polymerase activity, strand-displacement DNA synthesis activity, RNase H activity and sensitivity to the RT inhibitors 3′-azido-3′-deoxythymidine triphosphate (AZTTP) and Nevirapine, and the data were compared with data obtained with the natural RT heterodimers. Here we demonstrate that striking differences between different chimeric HIV-1/FIV RTs exist. While the HIV-1 RT p51/FIV RT p66 (H51/F66 RT) had very similar properties to the FIV RT heterodimer (F51/F66 RT), the FIV RT p51/HIV-1 RT p66 (F51/H66 RT) behaved differently compared to the HIV-1 RT heterodimer (H51/H66 RT), suggesting that the function of the p51 subunit in the RT heterodimer is to preserve the optimal structural integrity in the RT heterodimer.

Section snippets

Reconstitution, purification and identification of HIV-1/FIV RT heterodimers

To purify and reconstitute the individual subunits of HIV-1 and FIV RT, the p51 and the p66 subunits were separately cloned and expressed as histidine-tagged proteins (see Materials and Methods). Each of the four RT subunits was separately purified over a Co²⁺ affinity column. Afterwards, the following heterodimers were constructed: HIV-1 RT p51/p66 (H51/H66), FIV RT p51/p66 (F51/F66), HIV-1 RT p51/FIV RT p66 (H51/F66) and FIV RT p51/HIV-1 RT p66 (F51/H66). Reconstitution was started by mixing

Discussion

We have examined the role of the p51 subunit in the RT heterodimer by reconstituting chimeric HIV-1/FIV RTs (H51/F66 RT and F51/H66 RT), which could be purified as stable heterodimers, as shown by gel filtration analysis. As controls for activity tests, reconstituted HIV-1 and FIV RT heterodimers were used, and no differences were found from RT expressed in a p66/p51 or p66/protease double-expression system, respectively. This is also confirmed by identical K_i values for several RT inhibitors

Chemicals

Radioactively labelled nucleotides and 3′-azido-3′-deoxythymidine triphosphate (AZTTP) were purchased from Amersham. All other reagents were of analytical grade and purchased from Merck (Darmstadt, Germany) or Fluka (Buchs, Switzerland). Nevriapine (BI-RG-587) was kindly provided by P. Ganong (Boehringer Ingelheim Pharmaceuticals, Ridgefield, Conn).

Buffers

The following buffers were used. Buffer A: 10 mM sodium phosphate, 10 mM imidazole (pH 7.2), 0.5 M NaCl, 0.4 mM phenylmethylsulfonyl fluoride

Acknowledgements

We thank Drs Stuart Le Grice, Giovanni Maga and Robert Keller for critical reading of the manuscript, Zophonı́as Jónsson for structure comparisons and Helen Greutert for technical assistance with the HPLC. This work was supported by the Swiss National Science Foundation (AIDS program 3139-047297-96) and by the Kanton of Zürich.

References (28)

W.A Beard et al.
Site-directed mutagenesis of HIV reverse transcriptase to probe enzyme processivity and drug binding
Curr. Opin. Biotech.
(1994)
N Cheng et al.
Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase
Biochem. Biophys. Res. Commun.
(1991)
M Hottiger et al.
Strand displacement activity of the human immunodeficiency virus type 1 reverse transcriptase heterodimer and its individual subunits
J. Biol. Chem.
(1994)
S.C Huang et al.
Contribution of the p51 subunit of HIV-1 reverse transcriptase to enzyme processivity
Biochem. Biophys. Res. Commun.
(1992)
P.S Jacques et al.
Modulation of HIV-1 reverse transcriptase function in “selectively deleted” p66/p51 heterodimers
J. Biol. Chem.
(1994)
W.B Parker et al.
Mechanism of inhibition of human immunodeficiency virus type 1 reverse transcriptase and human DNA polymerases alpha, beta, and gamma by the 5′-triphosphates of carbovir, 3′-azido-3′-deoxythymidine, 2′,3′-dideoxyguanosine and 3′-deoxythymidine
J. Biol. Chem.
(1991)
J.E Reardon et al.
Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5′-triphosphate and 3′-azido-3′-deoxythymidine 5′-triphosphate
J. Biol. Chem.
(1990)
T Restle et al.
Dimerization of human immunodeficiency virus type 1 reverse transcriptase. A target for chemotherapeutic intervention
J. Biol. Chem.
(1990)
M.C Starnes et al.
Enzyme activity gel analysis of human immunodeficiency virus reverse transcriptase
J. Biol. Chem.
(1988)
C Tantillo et al.
Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance
J. Mol. Biol.
(1994)

R.L Thimmig et al.

Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase

J. Biol. Chem.

(1993)

H Zhang et al.

Enzymatic properties and sensitivity to inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with Glu-138 → Arg and Tyr-188 → His mutations

Antivir. Res.

(1994)

M Amacker et al.

Feline immunodeficiency virus reverse transcriptaseexpression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits

J. Virol.

(1995)

M Amacker et al.

HIV-1 nucleocapsid protein and replication protein A influence the strand displacement DNA synthesis of lentiviral reverse transcriptases

AIDS

(1997)

Cited by (30)

Human immunodeficiency virus reverse transcriptase: 25 years of research, drug discovery, and promise
2012, Journal of Biological Chemistry
Citation Excerpt :
Despite identity in primary sequence, the p66 and p51 subdomains adopt significantly different folds, i.e. although the p66 DNA polymerase domain exhibits an open extended structure with a large active-site cleft, the equivalent region of p51 forms a closed compact structure incapable of participating in catalysis (19, 21). Both the DNA polymerase and RNase H activities are catalyzed by the p66 subunit, whereas the proposed roles for p51 include providing structural support to p66 (19, 20, 22, 23), facilitating p66 loading onto a template-primer (24), and stabilizing the appropriate p66 conformation during tRNA-primed initiation of reverse transcription (25, 26). In contrast to p66, which undergoes large-scale motions (especially the fingers and thumb subdomains), the p51 subunit is essentially rigid (27).
Synthesis of integration-competent, double-stranded DNA from the (+)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA- and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (−)- and (+)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here.
Purification of untagged HIV-1 reverse transcriptase by affinity chromatography
2010, Protein Expression and Purification
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2 mM Mn²⁺. The RT retained in the column was eluted after soaking overnight in 10 mM EDTA to retrieve the Mn²⁺. In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.
Retroviral reverse transcriptases (other than those of HIV-1 and murine leukemia virus): A comparison of their molecular and biochemical properties
2008, Virus Research
This chapter reviews most of the biochemical data on reverse transcriptases (RTs) of retroviruses, other than those of HIV-1 and murine leukemia virus (MLV) that are covered in detail in other reviews of this special edition devoted to reverse transcriptases. The various RTs mentioned are grouped according to their retroviral origins and include the RTs of the alpharetroviruses, lentiviruses (both primate, other than HIV-1, and non-primate lentiviruses), betaretroviruses, deltaretroviruses and spumaretroviruses. For each RT group, the processing, molecular organization as well as the enzymatic activities and biochemical properties are described. Several RTs function as dimers, primarily as heterodimers, while the others are active as monomeric proteins. The comparisons between the diverse properties of the various RTs show the common traits that characterize the RTs from all retroviral subfamilies. In addition, the unique features of the specific RTs groups are also discussed.
HIV-1 Protease Dimer Interface Mutations that Compensate for Viral Reverse Transcriptase Instability in Infectious Virions
2007, Journal of Molecular Biology
Mature enzymes encoded within the human immunodeficiency virus type 1 (HIV-1) genome (protease (PR), reverse transcriptase (RT) and integrase (IN)) derive from proteolytic processing of a large polyprotein (Gag-Pol). Gag-Pol processing is catalyzed by the viral PR, which is active as a homodimer. The HIV-1 RT functions as a heterodimer (p66/p51) composed of subunits of 560 and 440 amino acid residues, respectively. Both subunits have identical amino acid sequence, but p51 lacks 120 residues that are removed by the HIV-1 PR during viral maturation. While p66 is the catalytic subunit, p51 has a primarily structural role. Amino acid substitutions affecting the stability of p66/p51 (i.e. F130W) have a deleterious effect on viral fitness. Previously, we showed that the effects of F130W are mediated by p51 and can be compensated by mutation T58S. While studying the dynamics of emergence of the compensatory mutation, we observed that mutations in the viral PR-coding region were selected in HIV clones containing the RT substitution F130W, before the imposition of T58S/F130W mutations. The PR mutations identified (G94S and T96S) improved the replication capacity of the F130W mutant virus. By using a trans-complementation assay, we demonstrate that the loss of p66/p51 heterodimer stability caused by Trp130 can be attributed to an increased susceptibility of RT to viral PR degradation. Recombinant HIV-1 PRs bearing mutations G94S or T96S showed decreased dimer stability and reduced catalytic efficiency. These results were consistent with crystallographic data showing the location of both residues in the PR dimerization interface.
Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster
2005, Insect Biochemistry and Molecular Biology
ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies single-stranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg²⁺ or Mn²⁺ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 °C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors.
Proteolytic processing of an HIV-1 pol polyprotein precursor: Insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation
2004, International Journal of Biochemistry and Cell Biology
HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag–pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.

View all citing articles on Scopus

¹: Edited by J. Karn

View full text

Journal of Molecular Biology

Regular articleChimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases1

Abstract

Introduction

Section snippets

Reconstitution, purification and identification of HIV-1/FIV RT heterodimers

Discussion

Chemicals

Buffers

Acknowledgements

Curr. Opin. Biotech.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

Antivir. Res.

Feline immunodeficiency virus reverse transcriptaseexpression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits

J. Virol.

HIV-1 nucleocapsid protein and replication protein A influence the strand displacement DNA synthesis of lentiviral reverse transcriptases

AIDS

Regular article
Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases¹