Assays and structure of reverse transcriptase of the AIDS virus

101

Clinica Chimicn Acru, 183 (1989) 101-106 Elsevier

CCA 04492

Short Communication

Assays and structure of reverse transcriptase of the AIDS virus Umberto Bertazzoni ‘, Franc0 Lori ‘, Giorgio Achilli * and Ercole Cattaneo * ’ Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R., and ’ L.aboraiorio Virus Oncogeni, Istituto di Malattie Infertive, IRCCS-S. (Received

17 October

Key words: Activity

1988; revision

gel method;

Matteo, Pavia (Italy)

received and accepted

27 March

1989)

Reverse transcriptase;

AIDS virus

Introduction

Reverse transcriptase (RT) is involved in the replication of retroviruses which have RNA as genetic material. The viral cycle starts with the recognition of the target cell receptors and, after fusion with the cellular membrane, the RNA and the RT are released in the cell cytoplasm; the role of RT is to transform RNA into DNA, which is then integrated as a provirus into the host cell genome. In summary, RT uses first RNA as a template to synthesize an RNA-DNA hybrid; then the enzyme degrades the RNA strand using its own RNase H activity; finally, it replicates the remaining strand to form a DNA duplex. An endonuclease activity is also present in the viral particle and is necessary for the integration of the provirus. The integrated viral message is finally transcribed and translated by exploiting the cellular systems, giving origin to the viral proteins and RNA needed for the formation of a new viral particle (for a review see [l]). RT activity represents a useful marker for the identification of retroviral infection, specially in cell cultures. When assaying infected cells or culture supernatants, it is essential to distinguish RT activity from that of the other cellular DNA polymerases. To this end, specific biochemical assays were developed which consider the using of particular template-primer systems and exploit differences in biochemical properties. Recently, a further step in the characterization of RT and its distinction from cellular DNA polymerases has been provided by the development of an activity gel assay [2]. This methodology allows the identification of catalytical polypeptides in situ after polyacrylamide gel electrophoresis under denaturing conditions [3].

Correspondence to: Dr. Umberto Bertazzoni, Istituto C.N.R., via Abbiategrasso 207, I-27100 Pavia, Italy.

0009~8981/89/$03.50

0 1989 Elsevier Science Publishers

di Genetica

Biochimica

B.V. (Biomedical

ed Evoluzionistica

Division)

de1

102

We describe here the biochemical and the activity gel procedures and summarize the physicochemical properties of several retroviral RT. Biochemical assay Reverse transcriptase can be regarded as a DNA polymerase, since it is capable of incorporating a deoxynucleoside triphosphate (dNTP) onto a template-primer system. Therefore, it is necessary to distinguish the RT activity from that of cellular DNA polymerases a, j3, y and S which are present in eukaryotic cells (for a review on cellular DNA polymerases see ref. [4])_ The main biochemical properties and function of cellular and retroviral polymerases are summarized in Table I. The development of a specific assay is based on the utilization of particular template primer systems and ion requirement. In order to measure RT activity and to distinguish it from cellular polymerases it is imperative to use a polyribonucleotide template and an oligodeoxynucleotide primer. The interference with DNA polymerases j3 and y which are both active with this template-primer system is measured by using a d~x~bonucl~tide template which is not read by the RT enzyme. Thus the RT activity is obtained by performing an assay with poly (rA) - {dT),, ~d‘subtracting the counts obtained in the assay with poly (d A) (dV,,-

Heterogeneity of reverse tran~ptase The physicochemical properties of reverse transcriptase differ considerably in different retroviruses [5], as illustrated in Table II. When considering the RT isolated from avian tumor viruses and murine leukemia viruses (C type), some important differences can be appreciated. The avian RT prefers Mg*+ as divalent ion and is composed of an 0: subunit of 63 kDa and of a p subunit of 92 kDa, bound together to form an active holoenzyme. On the other hand, the murine RT is composed of a single unit of 80 kDa that prefers Mn” ions. Both these enzymes derive from a gag-p01 precursor of 180 kDa, which is later cleaved into the different gag proteins, that will give origin to the internal core of the virus, and to the reverse transcriptase (pol). The cleavage is made by a viral protease, coded by the 5’ region of the murine pol gene, or by the 3’ region of the avian murine gag gene. Quite different properties are also observed when comparing RT isolated from two murine retroviruses, the B type mouse rn~~ tumor virus and the C type murine leukemia virus, thus confirming the existence of a wide heterogen~ty among these retroviral enzymes. The HIV-l virus, which is the etiologic agent of AIDS, has a reverse transcriptase which shares properties common to other retroviruses but also presents peculiar characteristics. It has been shown by F. Veronese et al. (ref. [6]) that the mature form of RT from HIV-l is made of two polypeptides of 66 and 51 kDa, but no evidence was provided whether they function as a complex, as it is the case of the

Mg2+

Mn2’

40

160

165

50-160

B

Y

6

RT

W/‘fi

h&z+

Mg2+

180

Q

Ion

f

+++

+

-I-++

+++

Activated DNA

++

+

++

++

dA:dT

Template-primer system

Characteristics of celhtlar and retroviral polymerases

TABLE I

f-4

ff

+

rA:dT

+

-

mRNA

+

Associated exonuclease

Retrovirus

Nuclear

Mitocbondrial

Nuclear

Nuciear

Localization

Retrovirus replication

DNA replication DNA repair mit DNA replication

Function

AZ-TrP

Aphidiwlin; NEM 50 mmol/i K phosphate

Inhibitors

104 TABLE II Heterogeneity of reverse transcriptase Source

Example

Size

Avian

Rous Sarcoma

complex = 92 kDa Mg’+ = 63 kDa

RNaseH Pol Endonucl. 180 kDa 63 kDa 32 kDa

3’ of gag gene

80 kDa Mn*’

RNaseH Pol Endonucl. 180 kDa 80 kDa 40 kDa

pol gene

“iNS

Cation Functional required domains

Moloney leuke-

CC57.4 mia

ViNS

Mm-he Mouse mammary (B type) tumor virus

Human

Human immunodeficiency type I (HIV-l)

virus

100 kDa Mg*+

66 kDa 51 kDa Mg2+

N.D.

gag-pol precursor

Protease location

5’of

160 kDa between (candidate) gag and pal genes

Pol RNaseH Endonucl. 167 kDa 31 kDa 66 kDa (putative)

5’of

pol gene

avian enzyme, or are independently active. The possible existence of a gag-pol precursor in HIV-l RT still represents an open question. Its putative size should be of 167 kDa, possibly being composed of a 53 kDa gag precursor, a 66 kDa RT, a 34 kDa endonuclease, and a 14 kDa protease. In order to analyze the structure of HIV-l RT and to answer to these questions we have developed an activity gel procedure which permits visualization of the active bands of the enzyme.

Activity gel analysis of reverse transcriptase

of HIV-1

According to the general procedure previously described [3], extracts of purified HIV-l are incubated at 37” C in the presence of SDS and then loaded onto a polyacrylamide gel in which the substrate poly (rA) . (dT) is embedded. After running the gel in denaturing conditions (SDS-polyacrylamide gel electrophoresis), the proteins are renaturated in situ by washing extensively the gel with a Tris-mercaptoethanol buffer. The RT reaction is carried out on the intact gel allowing the catalytic polypeptide bands to incorporate the labelled nucleotide (32P-d’I”TP) on the hybrid substrate. Non-incorporated material is removed by TCA and the gel is dried. Finally, the active bands are revealed by autoradiography. When the reverse transcriptase of HIV-1 was analysed on activity gel, three active bands with sizes of 165,66 and 51 kDa were observed [3]. Since the 66 and 51 active polypeptides correspond to the known RT mature forms of HIV-l RT, we demonstrated that both these proteins are independently active [3]. The M, of the higher form (165 000) corresponds to that predicted for the gag-pol precursor. At variance, this band could be due to the activity of a cellular DNA

105

Fig. 1. Activity gel analysis of reverse transcriptase from HIV-l. Purified preparations of HIV-l were analyzed by activity gel assay as described in the main text. The RT activity was analyzed on the gel using different reaction conditions. Lane 1: 50 mmol/l Tris-HCl pH 7.8, 10 mmol/l MgCl,, 2 mmol/l dithiothreitol, 20 mmol/l KCl. Lane 2: 50 mmol/l Tri-HCl, pH 8.3, 1 mmoljl MnClr, 2 mmol/l dithiothreitol, 130 mmol/l KCl, 50 mmol/l KPi, pH 8.3.

polymerase y since this enzyme presents a size of 160 kDa and in vitro activity on the same template primer system as the reverse transcriptase (see Table I). In order to exclude this possibility, we have carried out the activity gel reaction according to the conditions preferred for DNA polymerase y, that is in the presence of phosphate buffer and high KC1 concentrations. The results obtained are illustrated in Fig. 1. When the optimal conditions for reverse transcriptase were used (lane l), three active bands were observed on the gel, thus confirming the results previously reported [3]. However, when the activity gel was incubated under the optimal conditions for DNA polymerase y (see legend of Fig. l), the intensity of the signal was strongly reduced (lane 2). These results suggest that the cellular DNA polymerase y could not be responsible for the active bands (in particular the high M, polypeptide) visualized on the activity gel. Therefore, it is likely to conclude that the 165 kDa form could correspond to the gag-pol precursor of the HIV-l reverse transcriptase. It appears that the activity gel system is particularly useful in the analysis of the structure of the RT from HIV-l and can be used to detect the enzyme in cellular preparations infected with the virus. References Lowy DR. Transformation and oncogenesis: retroviruses. In: Fields BN, et al., eds. Virology. New York: Raven Press, 1985;243-251. Bertazzoni U, Scovassi AI, Mezzina M, Sarasin A, Franchi E, Izzo R. Activity gels for analysing DNA processing enzymes. Trends Genet 1986;2:67-71. Lori F, Scovassi AI, Zella D, et al. Enzymatically active forms of reverse transcriptase of the human immunodeficiency virus. AIDS research and human retroviruses 1988;4:393-398.

106 4 Fry M, Loeb LA. Animal cell DNA polymemses. Boca Raton: CRC Press, 1986. 5 Dickson C, Eisemnan R, Fan H, Hunter E, Teich N. Protein biosynthesis and assembly. In: Weiss RA, Teich NM, Varmus HE, Coffin JM, eds. Molecular biology of tumor viruses: RNA tumor viruses. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982;513-648. 6 Di Marzo-Veronese FD, Copeland TD, DeVico AL, et al. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science 1986;231:1289-1291.