Enzyme Activities in Four Different Forms of Human Immunodeficiency

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 45%-4600, June 1991 Biochemistry Enzyme activities in four different forms of human immunodeficiency virus 1 poi gene products (reverse transcriptase/RNase H/crossover linker mutagenesis/baculoirus expression system) YU-WEN HU AND C. YONG KANG Department of Microbiology and Immunology, University of Ottawa, Faculty of Medicine, Ottawa, ON, Canada K1H 8M5 Communicated by Max D. Summers, February 19, 1991 (received for review December 3, 1990) ABSTRACT Five cassettes of the pol gene of human im- activity (10, 11) but no RNase H activity (3, 7, 12, 13). The munodeficiency virus 1 were constructed and inserted under reverse transcriptase isolated from purified HIV-1 has a the control of the polyhedrin gene promoter of Autographa molecular mass of 95 (14) to 110 (15) kDa, as determined by calfornica nuclear polyhedrosis virus by homologous recom- gel filtration and glycerol gradient centrifugation, respec- bination. The first cassette polF contains the full-length pol tively. However, the 98-kDa nonprocessed polyprotein ex- open reading frame; the second cassette pollOO starts with the pressed in Escherichia coli displayed little or no reverse first AUG codon of the pol gene and deletes 103 amino acids transcriptase activity (16). The p66/pSi heterodimer ex- from the amino terminus of the pol gene product; the third pressed in E. coli has an apparent molecular mass of 120-130 cassette po197 deletes the entire protease coding sequence; the kDa and was active (5, 6). In addition, there is a 165-kDa fourth cassette po166 deletes both the protease and endonucle- putative gag-pol precursor polyprotein that also possesses ase/integrase coding sequences; and the fifth cassette polSl reverse transcriptase activity (10). contains the reverse transcriptase coding sequences plus 39 The enzymatically active forms ofHIV-1 reverse transcrip- 3'-terminal nucleotides of the RNase H coding sequences. We tase have been the subject of much debate. An important have expressed these five forms of the pol gene in Spodoptera question related to the pol gene products is whether only frugiperda SF9 cells and have analyzed for both reverse tran- certain form(s) possesses reverse transcriptase and/or scriptase and RNase H activities. The polF construct expressed RNase H activity or whether many different forms, including several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, processing intermediates, are active. To answer this ques- that were detected only by Western blot. In contrast, pollW, tion, we constructed and expressed several forms of the pol po197, po166, and pol51 products were expressed at high levels gene product in SF9 cells and analyzed them for reverse and were readily detectable in gels by staining. The levels of transcriptase and RNase H activities by using activity gel expression of these four products were estimated to be >150 analysis. We show that four forms of the pol gene product, mg/liter ofculture (5 x 10" cells). Activity gel analyses showed ranging from the unprocessed 100-kDa polyprotein to the that the pol10, po197, po166, and pol5 products possess shortest 51-kDa protein, are independently active for reverse reverse transcriptase activity; however, only po197 and po166 transcriptase; however, only two proteins, p97 and p66, show have RNase H activity. Our results demonstrate that many RNase H activity. forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcrip- MATERIALS AND METHODS tase activity but only certain forms have RNase H activity. Construction of Recombinant Autographa calafornica Nu- clear Polyhedrosis Virus (AcNPV) Containg Different Forms Conversion of the genetic information in human immunode- of the HIV-1 poi Gene. We first constructed the po1100 ficiency virus (HIV) genomic RNA to proviral DNA by cassette that deletes 273 base pairs (bp) from the 5' terminus reverse transcriptase is an essential step in replication of ofthe pol open reading frame [nucleotides (nt) 23%-51311 by HIV. The polymerase (pot) gene of HIV-1 contains within it using the crossover linker mutagenesis method (17-19). The a viral-specific protease, a reverse transcriptase, an RNase Bgl II-Sal I fragment ofplasmid pHXB-2D (1) containing the H, and an endonuclease/integrase in the amino- to carboxyl- HIV-1 pol coding region was isolated and inserted into terminal direction (1). The poi gene is expressed as a poly- BamHI and Sal I sites of pUC18. The resulting recombinant protein precursor that is subsequently cleaved to yield the plasmid (pUC18-Dpol 1) was cut with Sst I and dephosphor- following gene products: the 15-kDa viral protease that in ylated. A synthetic double-strand crossover linker containing turn is responsible for processing the polyprotein precursor; an Sst I cohesive end, a BamHI site, the putative ribosome a 66-kDa protein that contains reverse transcriptase and binding site, CCTATAAAT that was derived from the nt -9 RNase H activities; a 51-kDa protein that represents a to -1 ofthe polyhedrin gene (20, 21), and 15 nt of "homology truncated form of p66 and is one of the subunits of a p66/pSl searching sequences" that overlap with the 5' terminus ofthe heterodimer; and a 34-kDa endonuclease/integrase that is pollO0 gene was ligated to the Sst I site and the resulting required for integration of the proviral DNA into host DNA. construct was used to transform E. coli. Recombinant plas- p66 contains several catalytic functions including RNA- mid (pUC18-Dpol2) was isolated, digested with Sph I, de- directed DNA polymerase, DNA-directed DNA polymerase, phosphorylated, and ligated with another crossover linker and RNase H activities (2-6). p51 is a cleaved product DNA containing Sph I cohesive end, a BamHI site, and 15 nt representing the amino terminus of p66 (7, 8). Several labo- of "homology searching sequences" that recognized the 3' ratories have reported that pSl contains neither reverse terminus of the pol gene. The resulting recombinant plasmid transcriptase nor RNase H activities (2, 7, 9). However, (pUC18-Dpol3) contains the putative ribosome binding site others have reported that p51 possesses reverse transcriptase followed by the pol open reading frame starting with the first The publication costs of this article were defrayed in part by page charge Abbreviations: AcNPV, Autographa californica nuclear polyhedro- payment. This article must therefore be hereby marked "advertisement" sis virus; HIV, human immunodeficiency virus; DTT, dithiothreitol; in accordance with 18 U.S.C. §1734 solely to indicate this fact. AMV, avian myeloblastosis virus; nt, nucleotide(s). 4596 Downloaded by guest on September 24, 2021 Biochemistry: Hu and Kang Proc. Natl. Acad. Sci. USA 88 (1991) 4597 ATG (nt 2396-2398) in the pol gene and ending with the The infected cell lysate was prepared in denaturing buffer [60 translation termination codon TAG (nt 5132-5134). This mM Tris HCI, pH 7.6/1% SDS/65 mM 2-mercaptoethanol/ pollOO cassette was flanked with BamHI sites. The BamHI 10% (vol/vol) glycerol/0.01% bromophenol blue], incubated fragment was isolated, inserted into the BamHI site of the at 370C for 5 min, and then electrophoresed at 40 mA for 2 hr pAcYM1 baculovirus transfer vector (21, 22), and used to in a Mini-Protean II electrophoresis cell (Bio-Rad). The gel cotransfect SF9 cells along with wild-type AcNPV DNA to was removed and washed by shaking gently in 1 liter of produce recombinant baculovirus as described (19, 22). The renaturing buffer [50 mM Tris HCI, pH 7.5/10 mM MgCI2/1 full-lengthpolgene (polF) was constructed as follows; the Bgl mM dithiothreitol (DDT)/20 mM KCI] at room temperature II-Sal I fragment of plasmid pHXB-2D was isolated and for 1 hr with several changes. The gel was gently washed in ligated with a synthetic double-stranded DNA linker that renaturing buffer at 40C for another 16 hr with three changes provides 12 nt missing immediately upstream from the Bgl II of the buffer. The washed gel was incubated in 20 ml of site plus a translation initiation codon, a putative ribosome reverse transcriptase buffer [50 mM Tris HCI, pH 7.9/6 mM binding site, a BamHI site, and an Sst I cohesive end. The MgCl2/10% glycerol/80 mM KCI/bovine serum albumin (0.4 DNA was inserted into the Sst I and Sal I sites ofpUC18. The mg/ml)/0.01 mM EDTA/1 mM DDT] containing 150,uCi of resulting recombinant plasmid (pUC18-Fpoll) was cut with [a-32P]dTTP (800 Ci/mmol; 1 Ci = 37 GBq; Amersham) and Sst I and EcoRV and a 900-bp fragment was isolated. The (dT)12.18 (20,g/ml) at 370C for 2 hr. The gel was then washed 900-bp Sst I-EcoRV fragment and a 5.4-kbp Sst I-EcoRV with ice-cold 5% (wt/vol) trichloroacetic acid containing fragment ofpUC18-Dpol3 were ligated and used to transform 0.25% sodium pyrophosphate for 1 hr at room temperature E. coli. The resultant plasmid (pUC18-Fpol2) contains the with two changes ofsolution followed with 5% trichloroacetic putative ribosome binding site followed by a newly intro- acid washing at 40C for 20 hr with four changes ofthe washing duced translation initiation codon, the full-length pol open solution. The gel was wrapped and exposed to x-ray film. reading frame (nt 2087-5131), and the translation termination In Situ Gel Assay for RNase H Activity. In situ gel assays for codon and flanked by BamHI sites at either end. This cassette RNase H activity was carried out as modified (23, 24). (polF) was isolated and inserted into the BamHI site of the Briefly, a hybrid of32P-labeled RNA and unlabeled DNA was pAcYMi baculovirus transfer vector and was used to prepared by transcription of single-stranded M13 DNA as a cotransfect SF9 cells along with the wild-type AcNPV to template with [a-32P]CTP and the three remaining ribonucle- produce recombinant baculovirus.

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